* > ď1 í/í MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA ÚSTAV BIOCHEMIE STUDIUM EPITELIÁLNĚ-MEZENCHYMÁLNÍ TRANZICE NA BUNĚČNÝCH MODELECH RENÁLNÍHO KARCINOMU Diplomová práce Táňa Macháčková Vedoucí práce: doc. RNDr. Ondřej Slabý, Ph.D. Brno 2016 Bibliografický záznam Autor: Název práce: Studijní program: Studijní obor: Vedoucí práce: Táňa Macháčková Přírodovědecká fakulta, Masarykova univerzita Ústav biochemie Studium epiteliálně-mezenchymální tranzice na buněčných modelech renálního karcinomu Biochemie Analytická biochemie doc. RNDr. Ondřej Slabý, Ph.D. Akademický rok: 2015/2016 Počet stran: 75 Klíčová slova: renální karcinom; epiteliálně-mezenchymální tranzice; mikroRNA Bibliographic Entry Author: Title of Thesis: Degree programme: Táňa Macháčková Faculty of Science, Masaryk University Department of Biochemistry Study of epithelial-mesenchymal transition on cellular models of renal cell carcinoma Biochemistry Field of Study: Analytical Biochemistry Supervisor: Assoc. Prof. RNDr. Ondřej Slabý, Ph.D. Academic Year: 2015/2016 Number of Pages: 75 Keywords: renal cell carcinoma; epithelial-mesenchymal transition; microRNA Abstrakt Renální karcinom je nejčastějším nádorovým onemocněním ledvinného parenchymu a představuje přibližně 3 % všech zhoubných nádorových onemocnění dospělé populace. Renální karcinom se vyznačuje vysokou chemo- a radio-rezistencí a časným relapsem po nefrektomii. Jedním z klíčových procesů v progresi renálního karcinomu je proces epiteliálně-mezenchymální tranzice, který je zodpovědný za přeměnu nemotilních a neinvazivních epiteliálních buněk na motilní a invazivní mezenchymální buňky. MikroRNA jsou 18-25 nukleotidů dlouhé nekódující RNA schopné post-transkripční regulace genové exprese a jejich deregulace je spojena i s procesem epiteliálně-mezenchymální tranzice u renálního karcinomu. Cílem této studie bylo ověřit účinky miR-429 in vitro. Jednalo se o sérii experimentů založených na in vitro indukci epiteliálně-mezenchymální tranzice u stabilních buněčných linií odvozených od renálního karcinomu. V průběhu experimentů byla sledována exprese epiteliálně-mezenchymálních markerů a to zejména E-kadherinu. Abstract Renal cell carcinoma is the most common neoplasm of renal parenchyma that accounts for about 3 % of all adult malignancies. Renal cell carcinoma is highly chemo- and radioresistant tumour with occurence of early relapse after nephrectomy. Process of epithelial-mesenchymal transition plays crucial role during progression of renal cell carcinoma. Epithelial-mesenchymal transition is responsible for conversion of non-motile and non-invasive epithelial cells to motile and invasive mesenchymal cells. MicroRNAs are 18-25 nucleotides long non-coding RNAs which function in post-transcriptional regulation of gene expression. Deregulation of miRNA expression was also observed during epithelial-mesenchymal transition in renal cell carcinoma. The goal of this study was to evaluate effects of miR-429 in vitro. We performed series of experiments based on in vitro induction of epithelial-mesenchymal transition in stable cell lines derived from renal cell carcinoma. Expression levels of epithelial-mesenchymal markers, especially E-cadherin, were observed during experiments. MASARYKOVA UNJVERZITA Prrodovědecká faJcufta zadaní diplomové prace Akademicky rok: 2Cľlfl/2th5 Ustav; Ústav j ;<.-■■ —k Studetitka: Bc. TWa MatháŕkcvA Program Bioctiemie Ober: Analytická biochemie ŔedlW (Jstóťi1 ŕroílrtemve Přf MU Vám ve smyslu Studijního a zkušebního řádu MU urruje diplomovou prád s tématem: Téma prara; Studium epiLdialnr-mczcncrrymalni tranzite na btineĎnýcri modelech renilnihn kartiraimu Těma prác* anglicky: Study or epithelial-messníliymal translůon on MlMar modeJí Of .'eoal ůíll íaldmrna Ql i "■ 'i11 i zatfáni: RflriáJní kílKirlom jf ■ncjčasr.ěj&irn nádorovým onemocnärkm ledvinnčho parenchymu a představuje približne 3 % zhou.b-ných nádorů dospřié populace. Z jrolopckých malijjnřt dotahuje nejvy&i letalily. Jednim : Mirových okamžiků v parooge-nezi nádorových onemocnění, včetně renelnŕho karcinomu, je proces epitelLálne-ríiezůnchvmjrrii tranz^e CÉMT), V1 rámci ÉMT dochjiík přemřnĚ epitdiAlnííh bunŕk na více rnotitai a invazi vri bunky mezenchymalni. Narušená regulace exprese m'troRNAÍrniRNA) je jednou z kauzálních událostí v karKeragetiezl a.získií\ráni InvazivrHch vlasfrtňsrí P.C.C a sprintere dr-regulace miRNA byfy popsŕíjiy I v souvislostí procesŕtn EMT u RCC Cilrm trto pľace bude srne rxprrimentu založených na in v'ilro Indukci EMT u stabilních buněčných linjidi odvezených od R-CC. DalSľm dlem ie i": >■■ r-.-sr prípadné zrn^rry hladín e-uprese- mlRNA ovllvŕljjítích tento prtK« (miR-20Obr miR-200r.r miR-141. miR-192 a miR-215]_ Sooíastľ praoe bude také měřeni exprese epiteiiilnich a mezenchymálnldi rnarkerů íľEBlrZEB2, F-itadherin. N-kadherin, vimen&rti, Jazyk záverečné práce: angličtina Vedoucí práíír doc. RNtír, Ondřej Slabý. Ph.D. Datum zadáni práosr 14. 10. 2014 V Brne dna: 27,11.2014 Souhlasím se zadáním (podpis, datum}: flor. RNDr. Oŕdilch Janiczok. CSc zástupce ředitele Ústavu biochemie pra pcdagTtigkkě záležitosti studentka vedouci práce Poděkování Na tomto místě bych ráda poděkovala vedoucímu své diplomové práce doc. RNDr. Ondřeji Slabému, Ph.D. za odborné vedení, cenné připomínky a vstřícný přístup. Dále děkuji své odborné konzultantce Mgr. Haně Mlčochové za vedení při práci v laboratoři po celou dobu mého studia, trpělivost a korekci práce. Za rady ohledně statistického zpracování dat děkuji Mgr. Jaroslavu Juráčkovi. Na závěr bych ráda poděkovala celému kolektivu skupiny Molekulární onkológie II-solidní nádory za podporu a přátelskou atmosféru. Prohlášení Prohlašuji, že jsem svoji diplomovou práci vypracovala samostatně s využitím informačních zdrojů, které jsou v práci citovány. Brno 12. května 2016 Táňa Macháčková Table of contents List of abbreviations................................................................................................9 Introduction............................................................................................................12 1. Theoretical part.................................................................................................13 1.1 Renal cell carcinoma........................................................................................................................13 1.1.1 Incidence........................................................................................................................................................13 1.1.2 Mortality.........................................................................................................................................................14 1.1.3 Risk factors....................................................................................................................................................14 1.1.4 Sex and Age...................................................................................................................................................15 1.1.5 Pathogenesis of clear cell renal cell carcinoma..............................................................................15 1.2 Epithelial-mesenchymal transition............................................................................................16 1.2.1 Types of EMT................................................................................................................................................16 1.2.2 E-cadherin.....................................................................................................................................................18 1.2.3 Cytoskeleton rearrangement.................................................................................................................20 1.2.4 Regulation of EMT......................................................................................................................................20 1.3 MicroRNA.............................................................................................................................................22 1.3.1 MicroRNA biogenesis................................................................................................................................22 1.3.2 MicroRNAs associated with EMT.........................................................................................................25 2. Objectives............................................................................................................28 3. Matherial and Methods...................................................................................29 3.1 Chemicals.............................................................................................................................................29 3.2 Instruments.........................................................................................................................................31 3.3 Patients.................................................................................................................................................31 3.4 Tissue homogenization...................................................................................................................32 3.5 RNA isolation from native frozen tissue..................................................................................32 3.6 RNA isolation from cell lines........................................................................................................33 3.7 MicroRNA specific reverse transcription................................................................................34 3.8 Gene expression reverse transcription....................................................................................35 3.9 Quantitative real time polymerase chain reaction..............................................................36 3.10 Analysis of the expression data................................................................................................38 3.11 In vitro functional analyses........................................................................................................39 7 3.11.1 Cell lines.......................................................................................................................................................39 3.11.2 EMT induction...........................................................................................................................................40 3.11.3 Transfection...............................................................................................................................................40 3.11.4 MTT assay...................................................................................................................................................41 3.11.5 Scratch wound assay..............................................................................................................................41 3.12 Statistical evaluation.....................................................................................................................42 4. Results...................................................................................................................43 4.1 Expression of miR-429 in patient samples.............................................................................43 4.2 E-cadherin expression in patient samples..............................................................................45 4.3 Effects of TGFß on RCC cell lines.................................................................................................47 4.3.1 Effects of TGFß on E-cadherin expression.......................................................................................47 4.3.2 Effects of TGFß on expression of other EMT-related genes and miR-429 in 786-0 cell line...............................................................................................................................................................................50 4.3.3 Effects of TGFß on cell morphology....................................................................................................51 4.3.4 Effects of TGFß on metabolic activity of cells.................................................................................53 4.4 Effects of miR-429 and TGFß on E-cadherin expression levels......................................55 4.5 Effects of miR-429 and TGFß on cell migratory capacity..................................................59 5. Discussion............................................................................................................63 Summary...................................................................................................................67 References................................................................................................................69 8 List of abbreviations 3'UTR Three prime untranslated region AGO Argonaute protein BC Breast carcinoma BMP Bone morphogenic protein ccRCC Clear cell renal cell carcinoma ChRCC Chromophobe renal cell carcinoma CRC Colorectal carcinoma CSC Cancer stem cell Ct Cycle threshold DGCR8 DiGeorge Syndrome Critical Region Gene 8 DNA Deoxyribonucleic acid dsRNA Double strand ribonucleic acid ECM Extracellular matrix EMT Epithelial-mesenchymal transition EMT-TF Epithelial-mesenchymal transition associated transcription factor EPO Erythropoietin HIFI Hypoxia-inducible factor 1 LOH Loss of heterozygosity MET Mesenchymal-epithelial transition MDR1 Multidrug ressistance protein 1 miRISC MicroRNA-induced silencing complex 9 mRNA Mediator ribonucleic acid miRBase MicroRNA database miRNA MicroRNA miR MicroRNA MRE MiRNA recognition element MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MYC Myelocytomatosis oncogene ncRNA Non-coding ribonucleic acid NSCLC Non-small cell lung cancer p53 Tumour protein 53 PDGF Platelet derived growth factor PPIA Peptidylprolyl isomerase A pRCC Papillary renal cell carcinoma pre-miRNA Precursor miRNA pri-miRNA Primary miRNA PTM Post translation modification pVHL Von Hippel-Lindau protein qRT-PCR Quantitative real time polymerase chain reaction RBM RNA binding motif RCC Renal cell carcinoma RNA Ribonucleic acid RNApolII RNA polymerase II RT Reverse transcription 10 TF Transcription factor TGF Transforming growth factor TLDA TaqMan Low Density Arrays TRBP TAR RNA binding protein VEGF Vascular endothelial growth factor VHL Von Hippel-Lindau syndrome VHL Von Hippel-Lindau gene Wnt Wingless-type MMTV integration site family XP05 Exportin 5 ZEB1 Zinc finger E-box binding homebox 1 ZEB2 Zinc finger E-box binding hmebox 2 ZFC Zinc finger cluster 11 Introduction Renal cell carcinoma is the most common urological cancer that accounts for about 3% of all adult malignancies. This type of cancer is specific by its high chemo- and radio-resistance and high risk of relapse after surgical treatment. All patients with RCC undergo total or partial resection of the kidney with the tumor. Unfortunately, about 40% of all RCC patients experience recurrence of the disease, even after this radical intervention. This could be happening due to presence of process called epithelialmesenchymal transition. Epithelial-mesenchymal transition is a biological process that allows a cell of an epithelial phenotype change to a cell of a mesenchymal phenotype. Mesenchymal phenotype is connected to higher motility and invasivness of the cells and it is initial step of the metastatic cascade. The transition is driven by many molecules and microRNAs are one of them. Since the discovery of non-coding RNAs and the fact that they have important role in regulation of gene expression, number of studies about this topic has grown continually. Deregulation of microRNAs is often associated with various diseases and cancers. Between microRNAs involved in regulation of epithelial-mesenchymal transition are the most described miRNAs that belong to miR-200 family. Members of miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) cooperatively regulate translation of E-cadherin transcriptional repressors ZEB1 and ZEB2. These microRNAs often create regulation loops with their targets so they can function like molecular on-off switches. The aim of this diploma thesis was to evaluate impact of microRNA-429 on process of epithelial-mesenchymal transition in stable cell lines derived from RCC. This was done by in vitro induction of epithelial-mesenchymal transition using transforming growth factor ß followed by functional analyses. 12 1. Theoretical part 1.1 Renal cell carcinoma Kidney and renal pelvis cancer ranges between top ten most common cancers in the world. The most frequent type of renal neoplasm is renal cell carcinoma (RCC), which accounts for 85% of all renal malignancies. This malignity arises from the various parts of the nephron and can be classified into several subtypes [1]. The most common subtype is clear cell renal cell carcinoma (ccRCC), (70%) followed by less common papillary renal cell carcinoma (pRCC), (10-15%) and chromophobe renal cell carcinoma (ChRCC), (5%), [2]. These subtypes differ not only histologically but they also have different clinical features and genetic determinants [3]. 1.1.1 Incidence Incidence of RCC varies internationally and Czech Republic ranks with the highest incidence of RCC in the world (22/100,000 in men and 9.9/100,000 in women), (Figure 1). Developed countries have higher incidence than less developed [4]. This fact could be caused by higher chance of incidental diagnosis of RCC due to better technique in developed countries and risk factors connected with western society [2]. C64-C66 - Kidney, renal pelvis and ureter Comparison of incidence in the Czech Republic world standard Czech Republic Latvia United States of America Lithuania Iceland Slovakia Estonia Germany Albania Belarus Israel Croatia Slovenia Belgium Austria 1 1 1 1 1 MU 1 1 1 _ij 1 1 1 1 1 MS 1 1 4 6 8 http: //www. svod. cz 10 12 18 14 16 Source of data: GLGBÜCAN 2008 Place of the Czech Republic: 1 Figure 1: Comparison of incidence in the Czech Republic and other countries [4] 13 1.1.2 Mortality Over the decades mortality rate decreased. Cause of this phenomenon is probably improvement of detection techniques. This improvement leads to a higher chance of incidental diagnosis and diagnosis at early stages of RCC. In contrast to that, incidence rate increases (Figure 2). However, mortality rate between countries varies and there is no satisfactory explanation for this fact till today [5]. 2D 15 10 0 C64 - halignant neoplasn of kidney,.. Tine trend Incidence Mortality l—i—i—i—i—i—r Analysed data: N(inc) = 72393. N(rmor) =34683 Source of data: UZIS CR l—i—i—I—I—I—I—I—I—I—i—i—i—i http://www.si/od.cz Figure 2: Incidence and mortality of RCC in the Czech Republic 1977-2013 [4]. 1.1.3 Risk factors Based on the current observations - smoking, obesity, and hypertension are the most well-estabilished risk factors for developing RCC. From the evidence, tobacco exposure is the biggest risk factor. There are multiple mechanisms that describe how smoking promotes development of RCC. Cigarette smoke induces oxidative stress and injury in the kidney and causes renal impairment. Also the free radicals contained in cigarette smoke cause oxidative DNA damage, which may lead to the development of cancer [2,6]. Several studies have demonstrated an increased risk for RCC in association with obesity [5]. 14 1.1.4 Sex and Age RCC is globally as twice as common in males as in females. Age also plays role in incidence. Occurence of RCC increases rapidly after fifth decade of life [4]. 1.1.5 Pathogenesis of clear cell renal cell carcinoma Clear cell renal cell carcinoma is the most common subtype of RCC and accounts for 70% of all RCCs. Cells of the ccRCC appear translucent under the microscope and this is how this subtype got its name. RCC is mostly sporadic tumor but there are also several hereditary and familial forms. The most described is the Von-Hippel Lindau (VHL) syndrome. VHL syndrome is associated with occurrence of ccRCC, hemangiomas and pancreatic tumours. The VHL gene is an evolutionarily well conserved tumour-suppressor gene composed of at least three exons [7]. Product of VHL gene is VHL protein (pVHL) that plays important role in regulation of hypoxia pathway. The VHL protein (pVHL) functions as a part of an E3 ubiquitin ligase which ubiquitylates protein called Hypoxia Inducible Factor 1 (HIF1) and targets this protein for degradation by proteasome. This complex is consisted of elongin B, elongin C, cullin and pVHL. HIF is a heterodimeric protein which consist of two subunits (HIF-a and HIF-(3) and plays major role in cellular response to oxidative stress. Early event in ccRCC pathogenesis is a loss of VHL gene function due to its deletion on chromosome 3p, loss of heterozygosity (LOH), promoter methylation or missense mutation. The lack of functional pVHL or low concentration of oxygen cause that subunits of HIF can translocate to the nucleus and function as transcriptional factors. HIF proteins transcriptionally target and promote expression of many oncogenic molecules including Vascular Endothelial Factor (VEGF), Platelet Dervied Growth Factor (PDGF), Multidrug Resistance Protein 1 (MDR-1) and Erythropoietin (EPO), (Figure 3), [8,9]. Mutation, loss or methylation of both VHL alleles, has been reported in sporadic ccRCC and in the inherited VHL syndrome [9]. Additionally, hypoxia and HIF-a are linked to transcriptional factor TWIST. Upregulation of TWIST cause higher expression of pro-EMT molecules and supports promotion of EMT. The kidney is mesenchymal in origin and develops through mesenchymal-epithelial transition. In ccRCC this transition is reversed and results in EMT and dedifferentation. This transition requires presence of transcription factors like Snail, Slug, ZEB1 and ZEB2 [10]. 15 VHL function Nomnoxia VHL t ürn pie x HIF1i Proteosorne Hypoxia (gr mutated VHL) VHL complex HIFlj HIF1/I Nucleus leus HRb Figure 3: Function of VHL protein [11]. 1.2 Epithelial-mesenchymal transition Epithelial mesenchymal transition is a biologic process that allows cells of epithelial phenotype to undergo series of biochemical processes that result in mesenchymal phenotype. Mesenchymal phenotype includes enhanced migratory capacity, invasivness, elevated resistance to apoptosis and increased production of extracellular matrix (ECM) components. Characteristic event during EMT is a loss of E-cadherin and its replacement by another type of Cadherin. This process is called Cadherin switch. With the loss of E-cadherin expression of mesenchymal markers increases. The most well known EMT markers are vimentin, fibronectin, ZEB1, ZEB2, Snail, Slug, Twist, N-cadherin, E-cadherin, zonula occludens and clusterin [12,13]. 1.2.1 Types of EMT Process of EMT is mainly associated with embryonic development and organ formation. However, EMT dysfunction in normal cells leads to diseases such as cancer and fibrosis. EMT occurs in three different biological settings - embryogenesis, organ fibrosis and carcinogenesis (Figure 4), [14]. 16 Embryos Fibrosis Tumor progression Renal or hepatic Primary epithelial tumor cell ECM EMT inducers Physiologcal expression EMT inducers Aberrant activation EMT inducers Aberrant activation Cancer associated fibroblast ■■^ Invasive migratory tumor eel Mesodermal cell Epithelial cells Activated fibroblast or epithelial cell after EMT Mesenchymal cells ECM accumulation Figure 4: Types of epithelial-mesenchymal transition [12]. During embryogenesis EMT plays a critical role in generating the first set of mesenchymal cells, which are known as the primary mesenchyme. This primary mesenchyme gives rise to a secondary epithelia via mesenchymal-epithelial transition (MET). Whole process of organ formation during embryogenesis is dependent on EMT and MET [15]. Fibrosis or scarring is a feature of many chronic inflammatory diseases. This process is characterized by overexpression of ECM components which results in tissue scarring. Epithelial tissue has ability to heal itself after trauma by generating cells of mesenchymal phenotype and activation of fibroblasts. Mesenchymal cells can generate components of ECM, molecules important for healing. In healthy organism production of mesenchymal cells stops when the tissue is healed. In organism affected by fibrosis production of these cells lasts and results in total destruction of function and structure of the tissue. This production of mesenchymal-like cells from epithelial tissue is possible due to EMT [16]. Third type of EMT is associated with carcinogenesis. Cells of carcinomas generated by EMT lose apical-basal polarity, connection to basal lamina and to each other, and achieve mesenchymal features as enhanced migratory capacity, invasivness, resistance to apoptosis, ability to escape from immune system and expression of metalloproteinases. 17 Cells with mesenchymal features are capable of degradation of ECM, intravassation and extravassation followed by MET and thus formate secondary tumour (Figure 5). There are two hypotheses that are trying to explain EMT and metastasis. In the first hypothesis cancer progenitor cells present in a tumour do not undergo EMT simultaneously, so the cancerous population contains cells at different stages of differentiation. Cancer progenitor cells can undergo EMT to achieve further stage of differentation and develop into advanced stage of cancer. Although these grades are different, they arise from the same progenitor cell and undergo differential EMT at different time points. The second hypothesis predicts cancer progenitor cells to undergo EMT and then metastatize following by clonal expansion [17]. EMT is also considered as a process capable of generating cancer stem cells (CSCs). Last step of metastasis is adaptation and colonisation of foreign tissue by cancer cells. It seems unlikely that these adapative steps are enabled only by EMT programs and thus may require additional changes to cells. It seems that the most important of these adaptive changes is self-renewal. Self-renewal enables progenitor cell to generate cells that are copies of itself. CSCs possess this ability of self-renewal and are linked to many different types of cancer [18]. PARTIAL EMT PRIMARY TUMOR EMT MESENCHYMAL » CIRCULATING TUMOR CELLS MET EPITHELIAL/ MESENCHYMAL CIRCULATING TUMOR CELLS MET METASTATIC SITE "^"'"^^^^ MESENCHYMAL MESENCHYMAL _______----- EPITHELIAL ^**"*,**«^>^ __--■------' EPITHELIAL STEMNESS Figure 5: EMT, MET and sternness during carcinogenesis [19]. 1.2.2 E-cadherin Epithelial cells display cell to cell adhesions, adhesions to ECM and apical-basal polarity. Cell to cell adhesions are realised through adherens junctions, tight junctions, gap junctions and desmosomes at lateral surfaces. Apical-basal polarity is maintained due to connection of cells to basal lamina. During EMT these juncions are deconstructed and 18 junction proteins are relocalised or degradated. E-cadherin is one of the most important proteins in cell to cell adhesion and EMT initiation is accompanied by decrease of E-cadherin expression. Both EMT and MET are dependent on E-cadherin expression levels. E-cadherin is a calcium-dependent homodimeric transmembrane glycoprotein present in epithelial cells and can be regulated on both mRNA and protein levels [20]. E-cadherin ectodomains are linked to actin cytoskeleton by a-catenine and (3-catenine and its endodomains bind homotypically with each other via Ca2+ bridges (Figure 6). Figure 6: Adherens junction represented by E-cadherin homotypical binding [21]. Cells undergoing EMT often present switch from E-cadherin to another type of cadherin. Mesenchymal cells express various types of cadherins including N-cadherin, R-cadherin and cadherin-11. It is well known that cadherin switching allows cells to segregate population of cells from their neighbours by expression of certain type of cadherin. However, there is no satisfactory explanation of how cadherin switch promotes migratory capacity and invasivness which are essential for metastasis. There are three potential underlying mechanisms: the capacity of E-cadherin to regulate (3-catenin signaling in the canonical Wnt pathway, its potential to inhibit mitogenic signaling through growth factor receptors and the possible links between cadherins and the 19 molecular determinants of epithelial polarity. The term cadherin switching usually refers to a switch from E-cadherin to N-cadherin [22,23]. 1.2.3 Cytoskeleton rearrangement The cell cortex is composed of microfilament cytoskeleton, microtubule network and intermediate filaments. Microfilament cytoskeletone influeneces cell morphology, migration capacity and invasivness. Microtubule network is the driving force of cell migration. In epithelial cells intermediate filaments are at least ten times more abundant than microfilaments and microtubules, and their main role is to reinforce cells and to reorganize cells into tissue. It is clear that rearrangement of epithelial cytoskeleton is necessary during EMT [24]. 1.2.4 Regulation of EMT EMT is regulated at several levels. At transcriptional level by wide range of transcriptional factors (TFs), at post-transcriptional level by non-coding RNAs and at post-translational level by post-translational modifications (PTMs). Transcriptional regulation is initiated by microenvironmental (hypoxia) or/and biological factors (transforming growth factor (TGF), bone morphogenic protein (BMP)) and is orchestrated by a network of EMT associated transcription factors (EMT-TFs) that interact with epigenetic regulators to control the expression of proteins involved in cell to cell connection, ECM degradation and cytoskeleton rearrangement. E-cadherin is a key molecule in EMT and its promotor is a target of majority of EMT-TFs [25]. There are three families of EMT-TFs that have central role in regulation of EMT - Snail, ZEB and TWIST (Figure 7). EMT-TFs have potential to transcriptionally repress expression of epithelial markers (e.g. E-cadherin, Occludin, Desmoplakin, Plakophilin and Zonula Occludens) and to enhance expression of mesenchymal markers (e.g. N-cadherin, a-smooth muscle actin, vimentin, fibronectin, vitronectin). Funciton of these TFs is further enhanced by close cooperation with the epigenetic machinery [26,27]. Transcription factors from Snail family are zinc-fnger TFs and are capable of direct repression of E-cadhehn transcription. The most well known TFs from Snail family are Snail encoded by SNAI1 gene and Slug encoded by SNAI2 gene [28]. ZEB family includes two zinc-finger transcription factors known under names zinc finger E-box binding homebox 1 (ZEB1) 20 and zinc finger E-box binding homebox 2 (ZEB2). ZEB1 and ZEB2 function as E-cadherin repressors as well. Structurally are ZEB proteins comprised of two zinc finger clusters (ZFCs) located towards the N- and C- terminal ends of the protein. These zinc-finger motifs bind to ZEB boxes in the regulatory regions of target genes. Towards the centre of ZEB proteins there is an extra zinc finger that also helps to bind to the DNA. ZEB proteins are highly modular with independent regions mediating their binding to DNA, to other TFs and to proteins with activator or repressor activity but lacking a DNA binding motif on their own [29]. The TWIST family includes two members - Twist-1 known as Twist and Twist-2 known as Dermo-1. Both Twist and Dermo-1 are basic helix-loop-helix TFs that have important regulatory functions during embryo development. These proteins may function either as transcriptional activators or repressors through both direct and indirect mechanisms. Twist acts as an oncogene and is negatively associated with protein p53. High expression of Twist is correlated with numerous types of carcinomas, sarcomas, glioblastomas, neuroblastomas, and melanomas [30,31]. MAPK Degradation (JWJST) PKD1 (J) (SNAILI) SNAIL factors Repression Claudins Occludin E-cadherin Desmoplakin Plakophilin Crumbs3 PALS1 PATJ Cytokeratins Activation Fibronectin Vitronectin N-cadherin Collagen MMPs TWIST and IDs ZEBl and ZEB2 bHLH factors Repression Activation Claudins Fibronectin Occludin Vitronectin E-cadherin N-cadherin Desmoplakin SPARC Plakoglobin 0(5 integrin PRC2 CZEB2, I ZEB factors Repression Activation ZOl Vitronectin E-cadherin N-cadherin Plakophilin MMPs Crumbs3 Nature Reviews | Molecular Cell Biology Figure 7: Transcription factors involved in EMT [26]. 21 Post-transcriptional regulation of EMT is provided by microRNAs. The most well-known microRNAs involved in EMT regulation are members of miR-200 family. These miRNAs target mRNAs of EMT-TFs that repress E-cadherin and almost all of them are tumor suppressors. EMT-TFs and miRNAs often create reciprocal regulation loops. Post-translational modifications are covalent modificiations of a newly translated protein from mRNA. Protein is during PTMs modified by introduction of new functional groups into the peptide side amino acid chains. PTMs can either occur on a single residue or on multiple residues. Chemical modifications present during process of EMT include hydroxylation, phosphorylation, SUMOylation and glycosylation. PTMs can either have stabilisating or destabilisating effect on the modified protein [32]. 1.3 MicroRNA MicroRNAs (miRNAs) are a class of small non-coding RNAs that function as guide molecules in RNA silencing. They are approximately 18-25 nucleotides long and capable of post-transcriptional regulation of gene expression in metazoa and plants. At 5'termini miRNAs have 2-8 nucleotides long sequence that is essential for pairing with their targets. This sequence is called seed sequence and is complementary to the miRNA recognition elements (MREs) within 3' termini untranslated region (3'UTR) of mRNAs. MiRNAs act as guides by partial or perfect base pairing with they target mRNAs. These small RNAs are closely connected to Argonaute (AGO) family proteins. AGO proteins serve as effectors by recruiting factors that induce translational repression, mRNA deadenylation and mRNA decay [33]. 1.3.1 MicroRNA biogenesis MiRNA genes are localized through the whole genome including intronic, exonic and protein coding regions. In humans majority of miRNAs is encoded by introns of non-coding and coding transcripts. They are often present in clusters and are transcribed as polycistronic transcripts [34]. Their genes usually have their own promotors but if they arise from protein-coding gene, they can share promotor of the host gene. The latest release of the miRNA database (miRBase) has catalogued 2,588 genes for human miRNAs. The most accepted model of miRNA biogenesis is canonical biogenesis and it is predicted that majority of miRNAs is processed this way (Figure 8). According to the 22 cannonical biogenesis the miRNA genes are transcribed by Polymerase II (RNApolII) as long double strand RNA (dsRNA) primary transcripts called primary miRNA (pri-miRNA). Within long primary transcript is a local hairpin structure where miRNA sequences are embedded. Transcription is positively or negatively regulated by RNApolII-associated factors (p53, MYC, ZEB1, ZEB2) and epigenetic regulators (DNA methylation, histone modification), [33]. In the nucleus pri-miRNA is cleaved into 70 nucleotides long precursor-miRNA (pre-miRNA) by a complex of proteins. This complex is called microprocessor complex and is comprised of RNAselll Drosha and dsRNA binding protein Pasha (DGCR8), [35]. Pre-miRNA structure is then exported to the cytoplasm, where undergoes another cleavage. The transport from the nucleus to the cytoplasm is carried by a karyopherin Exportin 5 (XP05) and small Ran protein with GTPase activity. Cytoplasmic cleavage of stem loop structured pre-miRNA into 18-25 nucleotides long miRNA/miRNA* duplex is realised by RNAselll Dicer and TAR RNA binding protein (TRBP). Consequently, miRNA/miRNA* duplex is divided into two separate strands. Leading strand (strand with less stable 5' termini) is incorporated into a complex of proteins and the other strand is degradated [36,37]. Leading strand of miRNA is called mature miRNA. This protein/miRNA complex is called microRNA induced silencing complex (miRISC). MiRISC complex consists of Argonaute proteins (AG01-4), proteins GEMIN3 and GEMIN4 and mature miRNA. Incorporated miRNA is able to bind to the complementary sequences in 3'UTR of target mRNAs and thus initiate translational repression, deadenylation or degradation of mRNA. Translational repression, deadenylation and degradation is provided by proteins from miRISC complex. The fate of the mRNA depends on the degree of miRNA/mRNA hybridization and due to this one miRNA is able to target up to hundreds of mRNAs [33]. Canonical biogenesis pathway requires microprocessor complex for processing mature miRNA from pri-miRNA and pre-miRNA structures. There are several alternative pathways of miRNA biogenesis that do not require components of the microprocessor complex. One of these pathways is biogenesis from mirtrons. Mirtrons are miRNAs that arise from the introns of the mRNA coding genes. These miRNAs are spliceosome-excised and are direct substrates for Dicer. However, there are also mirtrons that are not dependent on spliceosome. They are called splicing-independent-mirtron-like miRNAs (simtrons). Simtrons occur by a pathway that involves Drosha but does not require DGCR8 or Dicer [38]. 23 Prt-miRNA V Processing inside nucleus •»»--» ^v».». DGCR8 Droste I ' (Microprocessor) Pri-miRNA with Microprocessor complex Processing inside cytoplasm (Pie-miR processing complex) Dtc*t —TRBP miRNA miRNA' duplex (20-24 nil Strand selection miRNP assembly Ayol -4 RNP Degradation miRNA' Ago1-« Ma1ure-r«RNA, guide strand, 1-1
99.5% (Sigma-Aldrich, USA) Direct-zol mini prep kit (ZymoResearch, USA) Qiazol Lysis Reagent (Qiagen, Germany) Mitomycin C from Streptomyces caespitosus (Sigma-Aldrich Inc., Saint Louis, MO, USA) GlutaMAX™ (Gibco, USA) HyClone Sodium pyruvate (GE Healtcare Cell Culture, USA) HyClone Penicilin/Streptomycin (GE Healtcare Cell Culture, USA) HyClone Non-Essential Amino Acids (NEAA) (GE Healtcare Cell Culture, USA) Dulbecco's phosphate buffered saline (PBS),(Sigma-Aldrich Inc., Saint Louis, MO, USA) OPTI-MEM® Reduced Serum Medium lx (Invitrogen, Carlsbad, CA, USA) Trypan Blue Stain 0.4% (Invitrogen, Carlsbad, CA, USA) Lipofectamine® RNAiMAX (Invitrogen, Carlsbad, CA, USA) DEPC-Treated water (Ambion INC, Austin, Texas) Acid Phenol: CHC13 (Ambion INC, Austin, Texas) MTT Formazan powder (Sigma-Aldrich Inc., Saint Louis, MO, USA) 29 mirVanaTM miRNA Isolation Kit (Ambion INC, Austin, Texas) TaqMan® MicroRNA Reverse Transcription Kit, 1000 Reactions (Applied Biosystems, CA, USA) TaqMan® High Capacity Reverse Transcription Kit, 1000 Reactions (Applied Biosystems, CA, USA) TaqMan® MicroRNA Assays - hsa-miR-429 ,and RNU48 (Applied Biosystems, CA, USA), (Table I). Table I: List of miRNA expression qPCR assays used in the study. miRBa se ID Releas miRBase Accession No. AB assay name AB assa yID Mature miRNA Sequence versio n21 hsa-miR-429 MIMAT0001 536 hsa-miR-429 102 4 UAAUACUGUCUGGUAAAACCGU RNU48 NCBI Accession Number: NR_002745 RNU4 8 100 6 GATGACCCCAGGTAACTCTGAGTGTGTCGCTGATGCCATCACCGCAG CGCTCTGACC TaqMan® Assays - CDH1, CDH12, VIM, ZEB1, ZEB2 ,and PPIA (Applied Biosystems, CA, USA), (Table II). Table II: List of gene expression qPCR assays used in the study. Gene symbol Entrez Gene ID Gene name Assay name Assay ID CDH1 999 Cadherin 1, type 1, E-cadherin (epithelial) CDH1 Hs01023894_ml CDH12 1010 Cadherin 12, type 2 (N-cadherin 2) CDH12 Hs00362037_ml VIM 7431 vimentin VIM Hs00958111_ml ZEB1 6935 zinc finger E-box binding homeobox 1 ZEB1 Hs00232783_ml ZEB2 9839 zinc finger E-box binding homeobox 2 ZEB2 Hs00207691_ml PPIA 5478 peptidylprolyl isomerase A (cyclophilin A) PPIA Hs99999904_ml 30 Pre-miR™ miRNA Precursor Molecules - pre-miR-429 and pre-miR-Negative Control #1 (Ambion INC, Austin, Texas) 3.2 Instruments Microcentrifuge 5424 (Eppendorf, Germany) Thermal cycler T100 (Biorad, USA) Quant Studio 12k flex (Applied Biosystems, USA) FLUOstar Omega (BMG LABTECH, Germany) Microscope CKX31, CKX41 (Olympus, Japan) Camera DS126491 (Canon, Japan) Nanodrop ND-1000 (Thermo Fisher Scientific, USA) MagNA Lyser (Roche, Switzerland) 3.3 Patients In this study expressions of miR-429 and E-cadherin were measured in 231 native frozen tissue samples (186 primary tumor tissues, 45 renal parenchyma tissues). Samples were divided into four groups - renal parenchyma (n=45), no progression (n=109), progression (n=29) and stage IV (n=48), (Table III). All patients underwent radical nephrectomy. Samples were obtained from Masaryk Memorial Cancer Institute in Brno, Czech Republic. All patients signed an informed consent form. Table III: Characterization of patients involved in study Overall Renal parenchyma No progression Progression Stage IV Number of patients 231 45 109 29 48 Age Range 31-86 39-80 31-86 42-84 34-81 (years) Median 63 61 64 63 64 Sex Female 84 17 35 9 16 Male 159 28 74 20 32 31 3.4 Tissue homogenization For homogenization of samples was used mechanical homogenization by ceramic beads. 1. Add 500 \iL of Lysis/Binding Buffer into the homogenization tube. 2. Place tissue into the homogenization tube with buffer. Keep on ice. 3. Place tube into MagnaLyzer and homogenise 2x for 60 sec at 6500 rpm. 4. Place the tube on ice. 5. Centrifuge for 5 min at 13400 rpm. 6. Replace supernatant into new microtube. 7. Immediatly perform RNA isolation. 3.5 RNA isolation from native frozen tissue Total RNA enriched with small RNAs was isolated by commercial mirVana™ miRNA Isolation Kit. This kit combines extraction of nucleic acids by acidic phenol and RNA capture in glass fibre filter. RNA captured by filter is then washed and eluted. 1. Add 50 \iL of miRNA Homogenate Additive to the supernatant. Incubate for 10 min on ice. 2. Add 500 \iL of Acid-Phenol: Chloroform to the mixture. 3. Vortex for 15 sec. 4. Centrifuge for 5 min at 13 400 rpm. 5. Transfer aqueous phase into new tube. 6. Add 1.25 volumes of absolute ethanol. Mix by pipetting. 7. Place a filter cartridge into a collection tube. 8. Pipette 650 \iL of ethanol/aqueous phase mixture onto the filter catridge. 9. Centrifuge for 30 sec at 13 400 rpm. 10. Discard filtrate from collection tube. Reuse collection tube. 11. Repeat steps 8. and 9. till whole volume of ethanol/aqueous phase mixture passes through the cartridge. 12. Pipet 700 \iL of Wash Solution 1 onto the filter cartridge. 13. Centrifue for 30 sec at 13 400 rpm. 32 14. Remove filtrate form collection tube. 15. Pipette 500 \iL of Wash Solution 2/3 onto the filter cartridge. 16. Centrifuge for 30 sec at 13 400 rpm. 17. Discard filtrate from collection tube. 18. Repeat steps 15. and 16. one more time. 19. Centrifuge empty cartridge for 1 min at 13 400 rpm. 20. Transfer the cartridge into new clean microtube. 21. Add 50 \iL of Elution Solution (pre-heated to 95°C) onto the cartridge. 22. Centrifuge for 30 sec at 13 400 rpm 23. Discard the cartridge. 24. Measure concentration and purity of total RNA using NanoDrop® ND-1000 25. Store at-80°C. 3.6 RNA isolation from cell lines For isolation of total RNA enriched with small RNAs from cell lines was chosen commercial kit Direct-zol RNA miniPrep. Lysis of cells was done by Qiazol reagent. Lysis of cells 1. Discard media from well where cells are seeded. 2. Add 200 \iL of Qiazol into the well. 3. Incubate for 5 min at room temperature. 4. Transfer the lysate into new microtube. Isolation 1. Spin microtube with lysate for 5 min at 13 400 rpm. 2. Transfer supernatant into new clean microtube. 3. Add 200 \iL of absolute ethanol. Mix by pipetting. 4. Pipette mixture onto the cartridge placed in collection tube. 5. Centrifuge for 1 min at 13 400 rpm. 6. Discard filtrate. 33 7. Pipette 400 \iL of Direct-zol RNA pre Wash buffer onto the cartridge. 8. Centrifuge for 1 min at 13 400 rpm . 9. Discard filtrate. 10. Pipette 700 [ih of RNA Wash Buffer onto the cartridge. 11. Centrifuge for 1 min at 13 400 rpm. 12. Discard filtrate. 13. Centrifuge empty cartridge for 2 min at 13 400 rpm. 14. Transfer cartridge to new clean microtube. 15. Pipette 30 \iL of RNAse free water onto the cartridge. 16. Centrifuge for 1 min at 13 400 rpm. 17. Discard the cartridge. 18. Measure concentration and purity of total RNA using NanoDrop® ND-1000. 19. Store at -80°C. 3.7 MicroRNA specific reverse transcription Since microRNAs are 18-25 nucleotides long there is a problem with use of conventional primers for reverse transcription. This problem is solved by use of microRNA specific stem loop primers. We used TaqMan® MicroRNA Reverse Transcription Kit. 1. In clean microtube prepare master mix according to table below (Table IV). Table IV: Composition of miRNA RT master mix Component Reagent volume [ uL ] per lOul reaction lOOmM dNTPs fwith dTTP] 0,10 MultiScribe Reverse Transcriptase, 50 U/ul 0,67 lOx Reverse Transcription Buffer 1,00 Rnase Inhibitor, 20 U/uL 0,13 Nuclease-free watter 2,77 MiRNA specific primer 2,00 Total volume 6,67 2. Shortly wortex and keep on ice. 34 3. Dilute sample RNAto 6,67 ng/3,33 \iL 4. Pipette 6,67 \iL of RT master mix into strip well per reaction. 5. Add 3,33 [ih of diluted RNA. 6. Shake the strip and then shortly spin down. 7. Incubate for 5 min on ice. 8. Set thermocycler according to table below (Table V). Table V: Thermocycler conditions for RT. Temperature [°C 1_Time [ min 1 hold 16_30_ hold 42_30_ hold 85_5_ hold 4_oo_ 9. Set reaction volume to 10 \iL. 10. Run reverse transcription. 11. Store cDNA at-20°C. 3.8 Gene expression reverse transcription Reverse transcription of mRNA was performed using High capacity cDNA reverse transcription kit. In contrary to miRNA specific reverse transcription this method uses pool of universal random primers. 1. In clean microtube prepare master mix according to the table below (Table VI). Table VI: Composition of gene expression RT master mix. Component Reagent volume [ ul ] per 20uL reaction lOOmM dNTPs (with dTTP) 0,80 MultiScribe Reverse Transcriptase, 50 U/ul 1,00 lOx Reverse Transcription Buffer 2,00 lOx Reverse Transcription random primers 2,00 Nuclease-free watter 4,20 Total volume 10,00 2. Shortly vortex. Keep on ice. 35 3. Dilute sample RNA to 400 ng/10 |iL for RNA from cell lines and to 750 ng/10 |iL for RNA from native frozen tissue. 4. Pipet 10 [ih of master mix per reaction. 5. Add 10 nL of diluted RNA. 6. Shake the strip and spin down. 7. Incubate for 5 min on ice. 8. Set cycler according to table below (Table VII]. Table VII: Thermocycler conditions for RT _Temperature [°C ]_Time [ min ] hold 25_10_ hold 37_120_ hold 85_5_ hold 4_co_ 9. Set reaction volume to 20 |iL. 10. Run reverse transcription 11. Store cDNA at-20°C. 3.9 Quantitative real time polymerase chain reaction Relative gene/miRNA expression was measured by quantitative real time polymerase chain reaction (qRT-PCR). We used hydrolysis TaqMan™ assays. Analysis was done on qRT-PCR instrument Quant studio 12k flex. Measurment was performed in duplicates for each sample. microRNA expression 1. Prepare master mix according to the table on next page (Table VIII). 36 Table VIII: Composition of miRNA qRT-PCR master mix Component Reagent volume [ ul ] per 15uL reaction Universal no UNG Master Mix 7,50 Nuclease-free water 5,75 Probe 0,75 Total volume 14,00 2. Prepare 384 well optical reaction plate. 3. Pipette 14 \iL of master mix per sample into a well. 4. Add 1 uX of cDNA to the well. 5. Pipette 1 |j.L of Nucleas-free water into two wells as non-template control. 6. Place adhesive optical foil onto the reaction plate. 7. Centrifuge the well plate for 1 min at 900 rpm. 8. Set cycler according to the table below (Table IX). Table IX: Thermocycler conditions for miRNA qRT-PCR Temperature f°C ] Time T s 1 Cycles 50 120 hold 95 600 hold 95 15 45 65 60 9. Set reaction volume to 15 |j.L. 10. Place the plate into the cycler. 11. Start run. 12. Analyze the results using Applied Biosystems software. 13. Gene Expression 1. Prepare master mix according to the table on next page (Table X). 37 Table X: Composition of gene expression master mix Component Reagent volume [ ul ] per 15uL reaction TaqMan Gene Expression Master Mix 7,50 Nuclease-free water 5,75 Probe 0,75 Total volume 14,00 2. Prepare 384 well optical reaction plate 3. Pipette 14 uL of master mix per sample into a well. 4. Add 1 uL of cDNA to the well. 5. Pipette 1 uL of Nucleas-free water into two wells as non-template control. 6. Place adhesive optical foil onto the reaction plate. 7. Centrifuge the well plate for 1 min at 900 rpm. 8. Set the cycler according to the table below (Table XI). Table XI: Thermocycler conditions for gene expression qRT-PCR Temperature f°C ] Time T s 1 Cycles 50 120 hold 95 600 hod 95 15 45 65 60 9. Set reaction volume to 15 uL. 10. Place the plate into the cycler. 11. Start run. 12. Analyze the results using Applied Biosystems software. 3.10 Analysis of the expression data For analysis of expression data, 2"ACt method was used. MiRNA expression was normalized to RNU48 and gene expression data to PPIA. 38 ACT(target gene) = Ctftarget gene) - Ctfendogenous control) RQ*(target gene) = 2"ACt(target gene) *Relative quantity 3.11 In vitro functional analyses For in vitro analyses were chosen three ccRCC cell lines. Cell lines were obained from ATCC and cultivated in recommended media. 3.11.1 Cell lines ACHN - Derived from metastatic site (malignant pleural effusion) of renal cell adenocarcinoma, male, Caucasian, 22 years old. - Cultivated in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), 1% sodium pyruvate, 1% GlutaMAX, 100 \ig/ ml Streptomycin and 100 U/ml penicilin. Cells were cultivated under these conditions - temperature 37°C, 95% humidity and atmosphere with 5% CO2. Caki-2 - Derived from primary clear cell renal cell carcinoma, male, Caucasian, 69 years old. - Cultivated in McCoy's 5A medium supplemented with 10% FBS, 1% GlutaMAX, 100 [ig/ ml Streptomycin and 100 U/ml penicilin. Cells were cultivated under these conditions -temperature 37°C, 95% humidity and atmosphere with 5% CO2. 786-0 - Derived from primary renal cell adenocarcinoma, male, Caucasian, 58 years old. - Cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 1% GlutaMAX, 1% sodium pyruvate, 100 ug/ml Streptomycin and 100 39 U/ml penicilin. Cells were cultivated under these conditions - temperature 37°C, 95% humidity and atmosphere with 5% CO2. 3.11.2 EMT induction In this study we induced EMT using transforming growth factor (3 (TGFfB). One day after the cells were seeded, standard medium was changed to a medium with addition of TGFfB. Used concentration of TGFfB was 10 ng/|il. Effects of TGFfB were observed after two and four days after addition of TGFfB. 3.11.3 Transfection Method of transient transfection was used for artificial overexpression of miR-429 in RCC cell lines. During transient transfection nucleic acids exist in cells only for limited time. Depending on the construct used, generally transiently expressed transgene can be detected from 1 to 7 days. Transiently transfected cells are typically harvested from 24 to 96 hours after transfection. The RCC cells were transfected by oligonucleotides pre-miR-429 and pre-miR negative control #1. As a transfecting reagent we used Lipofectamine RNAiMax. Concentration of oligonucleotides used in experiments was 33,3 nM per well (24 well plate). Amounts of reagents were multiplied by 5 for experiments performed in 6 well plates. Cell seeding 1. Prepare 24 well plate. 2. Seed cells in concentration - 30*104 cells/well (Caki-2), 25*104 cells/well (ACHN), and 20*104 cells/well (786-0). Use complete relevant medium without antibiotics. 3. Leave the cells in incubator for 24 h. Transfection 1. Prepare two clean microtubes. 2. Pipette 50 \iL of opti-MEM medium into each tube. 40 3. Add 4 |j.L of oligonucleotide (5 pmol/ui) to the one tube. 4. Add 1 |j.L of Lipofectamine RNAiMAX to the another one. 5. Incubate for 5 min at room temperature. 6. Pipette the content of tube with oligonucleotide into the tube with lipofectamine. 7. Incubate for 20 min. 8. Pipette 100 \iL of mixture into the well with cells. 9. Put the plate with cells into the incubator. 3.11.4 MTT assay This method is based on conversion of soluble MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to insoluble formazan. Conversion can be done only by living cells. The exact cellular mechanism of MTT reduction into formazan is not well understood, but likely involves reaction with NADH or similar reducing molecules that transfer electrons to MTT. The quantity of produced formazan is measured photometrically. Changes in absorbance reflect the metabolic activity of cells. Strenght of the signal is dependent on several factors including metabolic activity of cells, number of cells in well and incubation time. Absorbance maximum of formazan is at 570 nm. 1. Pipette 50 |j.L of MTT solution into a well (24 well plate) where cells are seeded. 2. Shake the plate and put it into the incubator. 3. Incubate for 2 hours. 4. Discard medium. 5. Add 500 uX of DMSO into the well. 6. Shake the plate. 7. Measure absorbance at 570 nm using FLUOstar Omega photometer. 3.11.5 Scratch wound assay Scratch wound assay is a method for in vitro evaluation of cell migratory capacity. Method is based on creating a "scratch" into the cell monolayer and wound is then observed in time. Scratch wound assay is usually performed on 6 well plates. We compared differences in cell free area after 12 h (Caki-2, 786-0) and after 24 h (ACHN). 41 1. Seed cells on 6 well plate in following concentrations, 30*104 cells/well (ACHN, Caki-2) and 20*104 cells/well (786-0). 2. Cultivate for 24 h. 3. Add 50 |j.L of mitomycin (0,5 mg/ml) into the well. 4. Place the plate into the incubator. 5. Incubate for 1 hour. 6. Use a 1000 liL pipette tip for creating a scratch into the cell monolayer. 7. Discard the medium. 8. Rinse the cells with 2 ml of PBS. 9. Discard the PBS. 10. Pipette 2,5 ml of medium. 11. Take a picture of the scratch area at Oh, 12h and 24h. 12. Analyze the data using TS scratch software. 3.12 Statistical evaluation Statistical analysis of the data was performed using GraphPad Prism 5 software. For validation phase of study was used Kruskal-Wallis one-way analysis of variance, Mann-Whitney non parametric test, ROC analysis and Kaplan-Meier survival analysis. In vitro experiments were evaluated by a two-tailed non-parametric t-test. P values lower than 0.05 were considered as statistically significant. 42 4. Results 4.1 Expression of miR-429 in patient samples Based on previous data measured in our laboratory we decided to validate miR-429 in independent 231 ccRCC samples. Samples in this validation cohort included 186 primary tumor tissues and 45 renal parenchyma tissues. MiR-429 expression was significantly downregulated in primary tumor tissues in comparison to renal parenchyma (p<0.0001), (Figure 11). Tumor tissue samples were further divided into three subgroups (no progression, progression, stage IV). We found out that miR-429 was significantly downregulated in primary tumor tissues of stage IV patients when compared to primary tumor tissues of patients without progression (p<0.0001) and primary tumor tissues of patients with progression (p=0.0115), (Figure 12). Further, we evaluated influence of miR-429 expression levels on disease-free survival (DFS) of ccRCC patients. Patients with higher expression levels of miR-429 showed significantly longer time to recurrence of ccRCC after radical nephrectomy than patients with lower expression levels of miR-429 (p=0.0105), (Figure 13). As endogenous control was used RNU48. miR-429 p<0.0001 Figure 11: Expression of miR-429 in renal parenchyma vs. ccRCC. 43 CO 3 O cvi 1 0.1 0.01 0.001 0.0001 v<2> miR-429 _p< 0,0001 p=0,0115 l—-1 TTT 'It"' ■ 'ttt ■ '''t'"T t'ttJ' p<0.0001 Figure 12: Expression of miR-429 in renal parenchyma and ccRCC samples. RCC samples were divided into three subgroups (no progression, progression, stage IV). 100-. 80- > ■> 60- c d) u 40- a) Q. 20- 0- 50 100 DFS (months) high expression low expression * p=0.0105 —I 150 Figure 13: Kaplan-Meier survival analysis. Patients with higher miR-429 expression vs. patients with lower miR-429 expression in primary tumor tissue. 44 4.2 E-cadherin expression in patient samples E-cadherin is the most well-known EMT marker. We measured expression of E-cadherin in 231 ccRCC samples. Cohort included 186 primary tumor tissues and 45 renal parenchyma tissues. E-cadherin expression was significantly downregulated in primary tumor tissues in comparison to renal parenchyma tissues (p<0.0001), (Figure 14). Tumor tissue samples were further divided into three subgroups (no proression, progression, stage IV). We found out that E-cadherin was significantly downregulated in primary tumor tissues of stage IV patients when compared to primary tumor tissues of patients without progression (p<0.0001) and primary tumor tissues of patients with progression (p=0.0100), (Figure 15). Further, we evaluated influence of E-cadherin expression levels on DFS of ccRCC patients. Patients with higher expression levels of E-cadherin did not showed any significant differences in DFS when compared to patients with lower expression levels of E-cadherin (Figure 16). E-cadherin Q. Q. • O < 0.01- 0.1- •w:.-j,;.,v.w p<0.0001 0.001 ■ 0.0001 ■ 0.00001 Figure 14: Expression of E-cadherin in renal parenchyma vs. ccRCC. 45 E-cadherin < Q. Q. 1| 0.1-0.01- 7 0.001 i CM 0.0001 ■ 0.00001 G p<0,0001 p=0,0100 " ' T p<0.0001 Figure 15 : Expression of E-cadherin in renal parenchyma and ccRCC samples. RCC samples were divided into three subgroups ( no progression, progression, stage IV). As endogenous control was used PPIA. E-cadherin 100 80H TO > I 60H § 40- a> o. -^L'' ------ 20- 50 100 DFS (months) -■- high expression -■- low expression p=0.2307 —I 150 Figure 16: Kaplan-Meier survival analysis. Patients with high E-cadherin expression vs. patients with low E-cadherin expression in primary tumor tissue. 46 4.3 Effects of TGFp on RCC cell lines Three RCC cell lines (ACHN, Caki-2, 786-0) were treated with lOng/ml TGFp. Cytokine was added into the media one day after seeding of cells. 4.3.1 Effects of TGF|3 on E-cadherin expression ACHN and Caki-2 cell lines did not show any significant differences in E-cadherin expression between cells treated with TGFp and control cells (MOCK). However, trend in E-cadherin downregulation was observed in TGFp treated cells in cell lines-ACHN (Figure 17, Figure 18) and Caki-2 (Figure 19, Figure 20) for both day 2 and day 4 after the TGFfB treatment. Cell line 786-0 showed significant downregulation of E-cadherin expression in cells treated with TGFp when compared to control cells both on day 2 (p=0.0054) and day 4 (p=0.0053), (Figure 21, Figure 22). As endogenous control was used PPIA. Presented data are from three independent biological replicates. ACHN ACHN (DAY 2) o 0.010 0.008- 0.006 o 'in in a) 0.004- £ 0.002- > » 0.000 Figure 17: E-cadherin expression in ACHN cells treated with TGFp vs. MOCK. Day 2. 47 ACHN (DAY 4) I 0.05-1 I 0.04- .c ■S 0.03- 0 1 0.02- V) 0) 1_ 2 0.01- > I 0.00- Figure 18: E-cadherin expression in ACHN cells treated with TGFfB vs. MOCK. Day 4. Caki-2 Caki-2 (DAY 2) o 0.004 5 0.003^ ■a re o I 0.002 0 'in in 1 0.001 H 0) 0) > » 0.000 Figure 19: E-cadherin expression in Caki-2 cells treated with TGFfB vs. MOCK. Day 2. 48 Caki-2 (DAY 4) g 0.003 c g 0.002H o 111 o c o 'in in a) 0.001 H | 0.000 Figure 20: E-cadherin expression in Caki-2 cells treated with TGFfB vs. MOCK. Day 4. 786-0 CM C T3 (0 O ill •♦— O c o (0 V) 0) 786-0 (DAY 2) o 0.0004 £ 0.0003 H 0.0002 H 0.0001 H I 0.0000 p=0.0054 Figure 21: E-cadherin expression in 786-0 cells treated with TGFfB vs. MOCK. Day 2. 49 786-0 (DAY 4) o O.OOO6-1 Figure 22: E-cadherin expression in 786-0 cells treated with TGFfB vs. MOCK. Day 4. 4.3.2 Effects of TGF|3 on expression of other EMT-related genes and miR-429 in 786-0 cell line Since the decreasing trend in E-cadherin expression after TGFfB treatment was significant only in 786-0 cell line, we decided to measure expression of other EMT-related genes (N-cadherin, vimentin, ZEB1, ZEB2) and miR-429 in this cell line. Cells were treated with 10 ng/ml TGFfB and expressions of genes and miR-429 were measured 2 days after the treatment. Any of the measured genes did not show significant differences in expression between cells treated with TGFfB and control cells (MOCK), (Figure 23). N-cadherin expression was not present at all. As endogenous controls were used RNU48 for miRNA expression and PPIA for gene expression. Presented data are from three independent biological replicates. 50 Figure 23: Expression of EMT-related genes and miR-429 in 786-0 cell line treated with TGFp and 786-0 control cells (a. ZEB1, b. ZEB2, c. Vimentin, d. miR-429). 4.3.3 Effects of TGF|3 on cell morphology ACHN, Caki-2, and 786-0 cell lines were treated with 10 ng/ml TGFp. Images (lOOx magnified) of the cells were taken 2 and 4 days after the addition of TGFp into the media. Ideally, cells treated with TGFp should change their shape from cuboidal to spindle-shaped. Since evaluation by observation is highly subjective method, it can not be said that any of the cell lines showed significant differences in morphology between cells treated with TGFp and control cells (MOCK). Subjectively were observed slight changes in ACHN morphology (Figure 24), no changes in Caki-2 morphology (Figure 25), and the biggest change in morphology was observed in 786-0 cell line (Figure 26). 51 ACHN MOCK TGFP . o < DAY 2 DAY 4 Figure 24: Images of ACHN cell line without and with TGF(3 treatment. TGF(3 treated cells appear slighty longer than the controls. Caki-2 MOCK TGFP DAY 2 DAY 4 $1 v \ - ' , ■ ■ v )f. '■ f1 * -i off #> • fl ■ 0 o v | o , * - * 5 ■ j (j'.' 0 Figure 25: Images of Caki-2 cell line. No change in morphology was observed. 52 786-0 MOCK TGFP DAY 2 DAY 4 ' J •' rr ? ° 'it b % * , s 1 : . (J Figure 26: Images of 786-0 cell line without and with TGFfB treatment. TGFfB treated cells appear more spindle-shaped than the controls. 4.3.4 Effects of TGF|3 on metabolic activity of cells Metabolic activity of cells was measured by MTT test. TGFfB was added into the media one day after seeding. Incubation time with MTT was 2 hours and absorbance was measured for three days. MTT test was done in three technical replicates and in three independent biological replicates. No difference in metabolic activity was observed in cells treated with TGFfB in comparison to control cells (NC, MOCK) in all three cell lines-ACHN (Figure 27), Caki-2 (Figure 28) and 786-0 (Figure 29). TGFfB does not have any significant influence on metabolic activity of RCC cell lines ACHN, Caki-2 and 786-0. 53 ACHN Figure 27: MTT test of ACHN cell line. Cells treated with TGFp vs. MOCK vs. NC. Figure 28: MTT test of Caki-2 cell line. Cells treated with TGFp vs. MOCK vs. NC. 54 786-0 Figure 29: MTT test of 786-0 cell line. Cells treated with TGFp vs. MOCK vs. NC. 4.4 Effects of miR-429 and TGFp on E-cadherin expression levels Cells were transfected with 33 nM of pre-miR-429 and negative control #1 (MOCK) and treated with 10 ng/ml of TGFfB one day after seeding. Expression levels of E-cadherin were measured 2 and 4 days after transfection and treatment. Any significant differences in E-cadherin expression between cells transfected with pre-miR-429 + treated with TGFfB and cells treated with TGFfB were not present in ACHN cells (Figure 30, Figure 31), Caki-2 cells (Figure 32, Figure 33) and 786-0 cells(Figure 34, Figure 35). Decresing trend in E-cadherin expression was observed in all three cell lines after TGFfB treatment, on the other hand cells transfected with pre-miR-429 + treated with TGFfB had very similar expression of E-cadherin as control cells. MiR-429 has probably capacity to restore E-cadherin expression in TGFfB treated RCC cells. Presented data are from three independent biological replicates. As endogenous control was used PPIA. 55 DAY 2 ACHN Figure 30: E-cadherin expression in ACHN cells treated with TGFp and cells transfected with pre-miR-429 + treated with TGFp. Day 2. Figure 31: E-cadherin expression in ACHN cells treated with TGFp and cells transfected with pre-miR-429 + treated with TGFp. Day 4. 56 DAY 2 Caki-2 o ■a 73 ta o 1l 0.1 J 0.01 0.001 g 0.0001 J? Figure 32: E-cadherin expression in Caki-2 cells treated with TGFfB and cells transfected with pre-miR-429 + treated with TGFp. Day 2. o ■a ■a ta o ill •*— o c o in in a) 1l 0.1-J 0.01-J I 0.001 DAY 4 Caki-2 Figure 33: E-cadherin expression in Caki-2 cells treated with TGFp and cells transfected with pre-miR-429 + treated with TGFp. Day 4. 57 DAY 2 786-0 o ■a ■a CO o ill o c g in 1 0.1 0.01 0.001 ■ 0.0001 ■ 0.00001 ■ £ 0.000001 Figure 34: E-cadherin expression in 786-0 cells treated with TGFfB and cells transfected with pre-miR-429 + treated with TGFp. Day 2. 1 0.1 0.01- o ■a c\i c i_ "D ra o ill ° 0.001 o 2 0.0001 Z 0.00001 I 0.000001 DAY 4 786-0 Figure 35: E-cadherin expression in 786-0 cells treated with TGFfB and cells transfected with pre-miR-429, transfected with pre-miR-429 + treated with TGFfB. Day 4. 58 4.5 Effects of miR-429 and TGFp on cell migratory capacity Migratory capacity was measured by scratch wound assay. Cells were transfected with negative control #1 (MOCK), pre-miR-429 and treated with TGFp one day after seeding. One day after transfection and treatment the scratch was made. Images (40x magnified) were taken after 12 h (Caki-2, 786-0) and 24 h (ACHN). ACHN cell line did not show any differences in relative cell migration independently on transfection and treatment (Figure 36, Figure 37). Caki-2 cell line did not show any differences in relative cell migration between transfected/treated cells and control cells as well (Figure 38, Figure 39). 786-0 cell line showed significantly increased relative cell migration of cells treated with TGFp in comparison to control cells (p=0.0011) and cells transfected with pre-miR-429 + treated with TGFp (p<0.0001). This observation proves ability of miR-429 to reverse effects of TGFp on cell migration in 786-0 cell line (Figure 40, Figure 41). Presented data are from three independent biological replicates. ACHN Figure 36: Relative cell migration of ACHN cells transfected with negative control #1 (MOCK), cells treated with TGFp and cells transfected with pre-miR-429 + treated with TGFp. 59 MOCK TGFp pre-miR-429+TGFp Figure 37: Images of ACHN cells transfected with negative control #1 (MOCK), cells treated with TGFp and cells transfected with pre-miR-429 + treated with TGF p. Oh vs. 24h. Caki-2 1.5n Figure 38: Relative cell migration of Caki-2 cells transfected with negative control #1 (MOCK), cells treated with TGFp and cells transfected with pre-miR-429 + treated with TGFp. 60 MOCK TGFp pre-miR-429+TGFp ■ Figure 39: Images of Caki-2 cells transfected with negative control #1 (MOCK), cells treated with TGFp and cells transfected with pre-miR-429 + treated with TGF p. Oh vs. 12h. 786-0 Figure 40: Relative cell migration of 786-0 cells transfected with negative control #1 (MOCK), cells treated with TGFp and cells transfected with pre-miR-429 + treated with TGFp. 61 MOCK TGFp pre-miR-429+TGFp Figure 41: Images of 786-0 cells transfected with negative control #1 (MOCK), cells treated with TGFfB and cells transfected with pre-miR-429 + treated with TGF (3. Oh vs. 12h. 62 5. Discussion RCC is the most common neoplasm of renal parenchyma with the highest mortality rate of all genitourinary cancers. Czech Republic ranks with the highest incidence of RCC in the world [55]. RCC is a heterogeneous disease both in cellular morphology and clinical course of the disease. Heterogeneity is the problem that has been frequently associated with treatment failure [56]. RCC is classified into several subtypes from which ccRCC is the most common. Unfortunately, this disease is characterized by high chemo- and radio-resistance and early relapse after nephrectomy. Approximately 40% of all RCC patients with localized disease undergo relapse of the disease after surgical removal of the tumor. This high rate of recurrence for clinically localized disease after nephrectomy underscores the importance of post-surgical surveillance [57]. EMT is a multistep dynamic cellular phenomenon in which epithelial cells lose their cell-cell adhesion and gain migratory and invasive traits that are typical of mesenchymal cells. Other mesenchymal features include increased production of ECM components, resistance to apoptosis and ability to escape from immune system [58]. Initial step of EMT is a loss of E-cadherin, cell adhesion molecule. E-cadherin loss ostensibly promotes metastasis by enabling the first step of the metastatic cascade: the disaggregation of cancer cells from one another [59]. Since the first choice treatment of RCC is surgical removal of the tumor and then follow up of the patient, there is an urgent need for biomarkers that would be able to detect and predict metastasis in RCC patients. In recent years biomarker research has been oriented especially to non-coding RNAs and miRNAs among them. MicroRNAs are short 18-25 nucleotides long ncRNAs with outstanding stability even after several cycles of freezing and thawing. They can be detected by conventional methods like qRT-PCR, PCR, Northern Blot. MiRNAs have wide range of regulatory functions in cells and their expression is very often deregulated in tumor tissue in comparison to healthy tissue [60]. Moreover, miRNAs have been detected in body fluids (plasma, urine, saliva, tears, semen, cerebrospinal fluid, bronchial lavage, peritoneal fluid, pleural fluid, colostrum and breast milk). These circulating miRNAs have great potential as biomarkers in liquid biopsy [61,62]. Process of EMT is also accompanied by deregulation of several miRNAs. MiR-200 family includes five members (miR-200a, miR-200b, miR-200c, miR-141, 63 miR-429) and all of them are tumor suppressors targeting ZEB1 and ZEB2, transcriptional repressors of E-cadherin [63]. MiR-429 was chosen for validation after previous analyses in our laboratory using TLDA. EMT+ samples showed significantly downregulated miR-429 expression in comparison to EMT- samples. EMT+/EMT- status was determined immunohistochemically. In this study we validated expression of miR-429 in independent 231 samples (186 primary tumor tissues, 45 renal parenchyma tissues). Primary tumor tissue samples were further divided into three subgroups (no progression, progression and stage IV). MiR-429 was found to be significantly downregulated in primary tumor tissues in comparison to renal parenchyma tissues (p<0.0001). Moreover, miR-429 was significantly downregulated in primary tumor tissues of stage IV patients when compared to primary tumor tissues of patients with no progression (p<0.0001) and primary tumor tissues of patients with progression (p=0.0115) of RCC. Further, Kaplan-Meier survival analysis of miR-429 expression in primary tumor samples with/without progression showed that expression levels of miR-429 correlate with time to progression of the disease. Patients with higher expression levels of miR-429 in primary tumor tissue had significantly longer disease free survival than patients with lower expression levels of miR-429 in primary tumor tissue (p=0.0105). MiR-429 was previously reported to be downregulated in colorectal carcinoma (CRC) tissues and cell lines [64]. Another study reported significant downregulation of miR-429 expression in serum of patients with non-small lung cancer (NSLC) when compared to healthy serum. In addition, serum levels of miR-429 were associated with poor overall survival of NSCLC patients. Both univariate and multivariate analyses showed that serum miR-429 level was an independent prognostic predictor for NSCLC [65]. We can state that our observations correlated with results of previous studies of CRC and NSLC. E-cadherin expression was measured in 231 RCC samples (186 primary tumor tissues, 45 renal parenchyma tissues) and primary tumor tissue samples were further divided into three subgroups (no progression, progression and stage IV). E-cadherin expression was found to be significantly downregulated in primary tumor tissues when compared to renal parenchyma tissues (p<0.0001). Downregulation of E-cadherin expression was also observed in primary tumor tissues of stage IV patients when compared to primary 64 tumor tissues of patients without progression (p<0.0001) and primary tumor tissues of patients with progression (p=0.0100). Kaplan-Meier survival analysis of E-cadherin expression in primary tumor tissues with/without progression did not prove any correlation between E-cadherin expression levels and DFS. E-cadherin is considered as a key molecule in EMT process; however, its expression is not consistent among various cancers. Different expression of E-cadherin was observed in primary breast cancer cells (BC) and invasive BC. Carcinomas in situ showed strong expression of E-cadherin while invasive carcinoma tissues without axillary lymph node metastases showed low expression of E-cadherin [66]. Induction of EMT by TGFp was done in three RCC cell lines (ACHN, Caki-2, 786-0). Based on literature we used concentration 10 ng/ml of TGFp. We evaluated effects of TGFp on E-cadherin expression, cell metabolic activity and cell morphology. After TGFp treatment decrease of E-cadherin expression was observed in all three cell lines. However, significant downregulation in E-cadherin expression was observed only in 786-0 cells treated with TGFp in comparison to control cells both on day 2 (p=0.0054) and day 4 (p=0.0053). Downregulation of E-cadherin was previously reported in pancreatic cell lines PANC-1 and BXPC3 treated with TGFp [67]. Another study reported upregulation of N-cadherin without change in E-cadherin expression after TGFp treatment in ovarian cancer cell line NIH-OVCAR3 [68]. In this study we did not observe any relevant changes in cell morphology of ACHN and Caki-2 cell lines after TGFp addition into the media. 786-0 cells appeared to be visibly longer and more spindle-shaped after TGFp treatment. Previous evidence of TGFp influence on cell morphology was observed in study of mouse mammary epithelial cells EpH4. In this study treatment of EpH4 cells by TGFp resulted in unordered cell strands and cords with spindle-like cellular morphology [69]. Using MTT test we measured metabolic activity of ACHN, Caki-2 and 786-0 cells. Any significant differences in cell metabolic activity were not observed in all three cell lines. We can state that TGFp does not affect metabolic activity of RCC cell lines ACHN, Caki-2 and 786-0. Effects of TGFp on cell proliferation were described in several articles. For example in study from 2007 RCC bone metastasis (RBM) cells displayed either no change or decrease of metabolic activity after TGFp treatment [70]. Another study of rat placental cells HRP-1 and RCHO-1 showed similar results. Decrease in proliferation was observed in HRP-1 cell line. On the other hand, no such a inhibitory effect was observed in RCHO-1 cell line [71]. 65 Another task in functional studies was to find out the potential of miR-429 to repress effect of TGFp on E-cadherin expression. We compared E-cadherin expression in cells transfected with negative control #1, cells transfected with pre-miR-429 + treated with TGFp and cells treated with TGFp. Any significant changes in E-cadherin expression between transfected/treated cells were not present. However, all three cell lines showed decreasing trend in E-cadherin expression in cells treated with TGFp when compared to cells transfected with negative control #1 and most importantly to cells transfected with pre-miR-429 + treated with TGFp. This results indicate effects of miR-429 on maintenance of E-cadherin expression during TGFp-induced EMT. Effect of miR-200 family members on mainteinance of E-cadherin expression level during TGFp-induced EMT were studied in several studies. Results vary dependently on miRNA and cell line [43,72,73]. Finally, we studied effect of miR-429 and TGFp on cell migratory capacity. Cell lines ACHN and Caki-2 did not show any differences in relative cell migration after transfection with pre-miR-429/TGFp treatment. Significant differences in relative cell migration were observed in 786-0 cell line. Cells treated with TGFp migrated more than cells transfected with negative control #1 (p=0.0011) and cells transfected with pre-miR-429 + treated with TGFp (p<0.0001). MiR-429 proved its potential to inhibit migratory capacity of 786-0 cells during TGFp-induced EMT. Inhibition of cell migration by miR-200 members was previously reported. In study from 2008, overexpression of each member of the miR-200 clusters strongly reduced growth factor-induced directional migration of the mouse mammary tumor 4T07 cells [74]. The above mentioned results prove that miR-429 is a tumor suppressor molecule involved in EMT regulation in RCC. MiR-429 and E-cadherin were significantly downregulated in RCC tissues in contrast to renal parenchyma tissues. Significant downregulation of miR-429 and E-cadherin was also observed in primary tumor tissues of stage IV patients in comparison to primary tumor tissues of patients without metastasis. Moreover, higher expression of miR-429 positively correlated with longer DFS of RCC patients.. Functional analyses on RCC cell lines showed that miR-429 has capacity to reverse E-cadherin repression and suppress enhanced cell migration during TGFp-induced EMT in 786-0 cell line. 66 Summary Introduction Renal cell carcinoma is the most common neoplasm of renal parenchyma and accounts about 3% of all adult malignancies. Czech Republic ranks with the highest incidence of RCC in the world. RCC is characterized by high chemo- and radio-resistance and early relapse after nephrectomy. EMT is initial step of metastasis and cancer progression. Molecules involved in EMT regulation include, e.g. E-cadherin, ZEB1, ZEB2, TGFfB and miR-200 family. Methods Determination of miR-429 and E-cadherin expressions in 231 ccRCC samples was performed using qRT-PCR. In vitro analyses on three RCC cell lines (ACHN, Caki-2, 786-0) included TGFfB treatment, transient transfection (pre-miR-429), MTT test and scratch wound assay. Results Both miR-429 and E-cadherin were significantly downregulated in primary RCC tissues in comparison to renal parenchyma tissues (p<0.0001 and p<0.0001). Downregulation of miR-429 and E-cadherin was also observed in primary tumor tissues of stage IV patients when compared to primary tumor tissues of patients without progression (p<0.0001, p<0.0001) and with progression (p=0.0115, 0.0100). Higher expression of miR-429 positively correlated with longer DSF of RCC patients (p=0.0105). In vitro analyses showed that TGFfB treatment significantly decreases E-cadherin expression in 786-0 cell line (0.0053) and that miR-429 has ability to restore E-cadherin expression levels in RCC cell lines. Finally, miR-429 was able to suppress TGFfB-enhanced cell migratory capacity of 786-0 cell line (p<0.0001). Conclusion MiR-429 function as a tumor suppressor and is involved in regulation of EMT in RCC. In this study we found out that miR-429 and E-cadherin are significantly downregulated in RCC tissues in comparison to renal parenchyma tissues and significantly downregulated in primary RCC tissues of patients with metastasis in comparison to primary RCC tissues 67 of patients without metastasis. Furthermore, we upregulated miR-429 expression in three RCC cell lines and described its effects during TGF(3-induced EMT. We found out that miR-429 regulates E-cadherin expression levels and has capacity to repress cell migration of 786-0 cells during TGFfB-induced EMT. However, it would be needed to use stable transfection in order to analyze long term impact of miR-429 on other cellular processes. 68 References [I] Ljungberg B, Campbell SC, Cho HY, Jacqmin D, Lee JE, Weikert S, et al. 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