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Universit degli Studi di Napoli Federico II Dipartimento di Biologia e Patologia Cellulare e

Molecolare L. Califano

Doctorate School in Molecular Medicine

PhD Program in

Genetics and Molecular Medicine Coordinator: Prof. Lucio Nitsch


Alterations of microRNAs expression profile

induced by oncogenic Ras

Supervisor: PhD student: Ch.mo Prof. Roberto Di Lauro Dr. Valentina DAmato Co-Supervisor: Dott.ssa Gabriella De Vita







































INDEX Abbreviations 5Abstract 61. Introduction 8

1.1 MicroRNAs 81.2 Genomic localization of microRNAs 81.3 MicroRNA biogenesis 91.4 microRNA function 121.5 microRNAs: tumour suppressors and oncogenes 141.6 Thyroid cancer 151.7 miRNA in Thyroid Cancer 161.8 Role of Ras oncogenes 191.9 Ras genes 201.10 Ras proteins signal transduction pathways 221.11 The FRTL-5 thyroid cell line 241.12 Transformation of FRTL-5 cells by Ras oncogene 24

2. Aim of the Study 263. Materials and methods 27

3.1 Cell culture. 273.2 Vectors. 273.3 Transfections. 283.4 Chromatin Immuno-Precipitation (ChIP) 29

3.4.1 Crosslinked chromatin preparation. 293.4.2 Transcription rate measurement. 29

3.5 RNA extraction and Real-Time PCR. 303.6 Protein studies. 323.7 Immunoblot. 323.8 Proliferation analysis. 323.9 Motility assay. 33

4. Results and Discussion 344.1 Screening of microRNA regulated by Ras. 354.2 Generation of miR135a over expressing clones. 384.3 Functional studies of miR135a. 39

4.3. 1. miR135a and Proliferation 404.3. 2. miR135a and Differentiation. 424.3. 3. miR135a and Cell Motility. 44

4.4 Regulation of miR-21 is cell-type specific. 474.5 miR21 induction by Ras in FRTL-5 is mediated by transcriptional regulation. 484.6 miR21 promoter is responsive to oncogenic Ras expression. 504.7 Identification of miR21 regulatory regions responsive to Ras. 51

5.Conclusion 54


6. Bibliography 557. Acknowledgement 64


The abbreviations used are: microRNA, miRNA; Papillary carcinoma, PC; Follicular carcinoma, FC; poorly differentiated carcinoma, PDC; anaplastic carcinoma, AC; Medullary carcinoma, MC; Follicular thyroid cell, TFC; Follicular Rat thyroid epithelial cell line, FRTL-5; Oncogenic human h-ras (G12V) fused to the ERTM Ligand binding domain, ER-RasV12; FRTL5 derived stable cell line expressing ER-RasV12, Cl11; FRTL5 derived stable cell lines expressing oncogenic human h-ras (G12V), RasV12 clones (V27, V29 or V39); Tamoxifen, 4OHT; Thyroperoxidase, TPO; Sodium-Iodide symporter, NIS; TSH receptor, TSHr; thyroglobulin, Tg; V29 derived stable cell lines expressing miR135a, miR135a clones.



Literature emphasizes the key role of miRNAs in cancer development. Moreover it has been demonstrated that oncogenes and tumorsuppressors can act also by regulating the expression of specific miRNAs. Previous results published by Landgraf et al., using the FRTL-5 ER-H-RasV12 inducible system highlighted that Ras is able to induce aberrant expression of microRNAs during the transformation of thyroid epithelial cells. Taking advantage of the inducible system FRTL-5 ER-H-RasV12 we decided to study the miRNAs involved in Ras-induced transformation. The aim of my project was to identify Ras-regulated miRNAs and study their functional or transcriptional role in transformation of thyroid epithelial cells. We decided first to validate the Landgrafs libraries data by Real-Time PCR, later we selected microRNAs of interest to study their functional role in Ras-induced transformation. We measured the level of expression of miR15b, miR16, miR27a, miR27b, miR29b, miR99b, miR135a, miR135b, miR324-5p in inducible systems FRTL-5 ER-Ras clone (Cl11) treated with 4OHT for 24h, 48h, 72h, 96h and 7days, in FRTL-5 parental cell line and in FRTL-5 clones constitutively expressing human H-RasV12. From the screening by Real-Time PCR we identified two microRNAs particularly regulated by oncogenic Ras: miR135a resulted down-regulated in chronically transformed cells by Ras oncogene meanwhile miR21 resulted up-regulated by Ras oncogene in the inducible system. We over expressed miR135a in the chronically transformed FRTL-5 H-RasV12 clone V29 to test the possibility that miR135a rescue could interfere with at least one of the phenotypes induced by oncogenic Ras. We proceeded to analyze proliferation, differentiation and cell motility of FRTL-5 RasV12 miR135a cells. We observed a little reduction of proliferation and migration in miR135a clones compared to FRTL-5 RasV12. It is known that Ras is able to promote miR21 expression in an epithelial system we asked if this regulation was cell-type specific. The Real-Time PCR results highlights that even though the two inducible systems analyzed (FRTL-5 ER-Ras and NIH ER-Ras) have a different basal level of miR21, in NIH ER-Ras cells 4OHT treatment do not lead to a significant increase of miR21, suggesting that Ras-induced miR21 up-regulation is cell-type specific. Once established the effects of oncogenic Ras on mature miR21 expression the following step was to study the mechanism through which this regulation takes place. Our data obtained by Real-Time PCR and chromatin immuno-precipitation (ChIP) suggests that miR21 induction by Ras in FRTL-5 cells is a transcriptional phenomenon. By luciferase assay we analyzed the miR21 promoter responsive to Ras oncogene and we verified if also its activation was cell-type specific. To this purpose we used FRTL-5, NIH3T3 and Rat2 cell


lines. The data obtained suggests that even though oncogenic Ras is able to lightly regulate the activity of miR21 promoter in fibroblasts, the activation displayed in FRTL-5 is much stronger and significant. The final step was the identification of potential regulatory regions, aside from the promoter already identified, responsive to oncogenic Ras.


1. Introduction

1.1 MicroRNAs MicroRNAs (miRNAs) are endogenous non-coding RNAs of about 22 nucleotides, regulating genes in animals and plants by pairing to the 3UTR regions of messenger RNAs (Zeng et al. 2002) of target genes and specifying mRNA cleavage or repression of protein synthesis. There is increasing evidence that miRNAs have an important regulatory role in a broad range of biological processes, including cellular differentiation, proliferation, apoptosis and cancer development (Bartel 2004). After the initial genetic discovery in nematodes (Lee et al. 1993), it was soon confirmed that all investigated higher eukaryotes, including plants and mammals, contain miRNA genes (Ambros et al. 2003). miRNA genes represent only a small part of the genome (3%), but they regulate approximately from 20% to 30% of all human genes and there is an average of 200 predicted targets per miRNA (Carthew 2006). The microRNAs discovery has been an exciting breakthrough in Biological Sciences of the past decade, culminating in Nobel Prize in Physiology or Medicine awarded to Andrew Fire and Craig Mello. Building on previous work mainly in plants (Lindbo et al. 1993), Fire discovered that exogenous double-stranded RNA can be used to specifically interfere with gene function. This phenomenon was called RNA interference (RNAi) (Fire et al. 1998). They also speculated that organisms might use double-stranded RNA naturally as a way of silencing genes. It was then shown that RNA interference was mediated by 22 nucleotide single-stranded RNAs termed small interfering RNAs (siRNAs) derived from the longer double-stranded RNA precursors (Zamore et al. 2000). Computational predictions of miRNA targets suggest that up to 30% of human protein coding genes may be regulated by miRNAs (Rajewsky 2006). This makes miRNAs one of the most abundant classes of regulatory genes in humans. Mic


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