Role of microRNAs in Hepatocarcinogenesis DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Bo Wang, M.S. Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2012 Dissertation Committee: Dr. Jacob T. Samson, Advisor Dr. Kalpana Ghoshal Dr. SaÏd Sif Dr. Thomas D. Schmittgen Copyright by Bo Wang 2012 Abstract MicroRNAs are conserved, small (20-25 nucleotide) noncoding RNAs that negatively regulate expression of mRNAs at the post-transcriptional level. MicroRNA signature is altered in different disease states including cancer and some microRNAs act as oncogenes or tumor suppressors. To identify microRNAs that may play a causal role in hepatocarcinogenesis we used an animal model in which C57BL/6 mice fed choline deficient and amino acid defined (CDAA) diet develop nonalcoholic steatohepatitis (NASH)-induced hepatocarcinogenesis after 70 weeks. Microarray analysis identified 30 hepatic microRNAs that are significantly (P≤0.01) altered in mice fed CDAA diet for 6, 18, 32 and 65 weeks compared to those fed choline sufficient and amino acid defined diet (CSAA). Real-time RT-PCR analysis demonstrated upregulation of oncogenic miR-155, miR-181b, miR-221/222 and miR-21 and downregulation of the most abundant liver specific miR-122 at early stages of hepatocarcinogenesis. Western blot analysis showed reduced expression of hepatic PTEN, a target of miR-21, and C/EBPβ, a target of miR-155, in these mice at early stages. DNA binding activity of NF-κB that transactivates miR-155 gene was significantly (P=0.002) elevated in the liver nuclear extract of mice fed CDAA diet. Further, the expression of miR-155, as measured by in situ hybridization and real-time RT- ii PCR, correlated with diet-induced histopathological changes in the liver. Ectopic expression of miR-155 promoted growth of hepatocellular carcinoma (HCC) cells whereas its depletion inhibited cell growth. Notably, miR-155 was significantly (P=0.0004) upregulated in primary human HCCs with concomitant decrease (P=0.02) in C/EBPβ level compared to matching liver tissues. The expression of tissue inhibitor of metalloprotease 3 (TIMP3), a tumor suppressor and a validated miR-181 target, was markedly suppressed in the livers of mice fed CDAA diet. Upregulation of hepatic transforming growth factor β (TGFβ) and its downstream mediators Smad 2, 3 and 4 and increase in phospho-Smad2 in the liver nuclear extract correlated with elevated miR-181b/d in mice fed CDAA diet. The levels of the precursor and mature miR-181b were augmented on exposure of hepatic cells to TGFβ and were significantly reduced by small interference RNA-mediated depletion of Smad4, showing the involvement of TGFβ signaling pathway in miR-181b expression. Ectopic expression of miR-181b showed that miR-181b enhanced matrix metallopeptidases 2 (MMP2) and MMP9 activity and promoted growth, clonogenic survival, migration and invasion of HCC cells that could be reversed by modulating TIMP3 level. Further, depletion of miR-181b inhibited tumor growth of HCC cells in nude mice. miR-181b also enhanced resistance of HCC cells to the anticancer drug doxorubicin. Conclusion: Temporal changes in microRNA profile occur at early stages of CDAA diet-induced hepatocarcinogenesis. Reciprocal regulation of specific iii oncomirs and their tumor suppressor targets implicate their role in NASH-induced hepatocarcinogenesis and suggest their use in the diagnosis and prognosis of liver cancer. iv Dedicated to my parents, my wife Jian, and my son David. v Acknowledgements First of all, I would like to extend my sincere gratitude to my advisor, Dr. Samson T. Jacob, for his supporting and mentoring during my graduate study. Dr. Jacob’s illuminating insight and great view in science inspire me all the way along my study. Dr. Jacob has taught me not only science but also how to become an independent scientist. I have benefited a lot from the experience working in his lab, and will carry with me throughout my career. I am sincerely grateful to Dr. Kalpana Ghoshal, my co-advisor. It has been a privilege to work with her, a great person with genuine care and an excellent scientist with persistent motivation and enthusiasm in science. Without her instructive advice and constant guidance, I cannot achieve all the success in my research. I owe special thanks to Dr. Sarmila Majumder for her invaluable discussion and enormous help. I also would like to thank Dr. Jharna Datta, Dr. Huban Kutay and Dr. Tasneem Motiwala for their discussion and technical help. My thanks go to my classmate and friend, Shuhao Hsu, for the productive collaboration and generous help with mouse techniques. I enjoy all the chatting and discussions with Dr. Yuanzhi Lu, a diligent scientist and intimate friend, who has helped me a lot in the lab as well as in life. vi It is my great honor to work with all the great members in Dr. Jacob’s lab, who made this lab such a wonderful and friendly place to work. I really appreciate all their help and discussions. This project would not be possible without all the collaborators: Dr. Stefano Volinia and Dr. Carlo M. Croce, who helped analyze the microarray data; Dr. Thomas D. Schmittgen, who showed me the real-time PCR analysis of microRNA expression; Dr. Gerard Nuovo, who helped with the pathological analysis and microRNA in situ hybridization; Drs. Stefan Costinean and Wendy Frankel for help with pathological analysis. I am deeply grateful to all my committee members, Drs. Thomas D. Schmittgen, SaÏd Sif, and Tushar Patel for their exceptional guidance, critiques and timely help. Finally, I would like to thank my parents, my wife, my son and my brother. It is their constant support that makes me achieve this. This study was supported by grants from National Institute of Health. vii Vita 2002 ....................................................... Bachelor of Medicine, Shandong University 2005 ....................................................... M.S. Peking Union Medical College Chinese Academy of Medical Science 2006 to present ...................................... Graduate Research Associate, Molecular, Cellular and Developmental Biology, The Ohio State University Publications Wang B, Hsu SH, Frankel W, Ghoshal K, Jacob ST. Stat3-mediated activation of miR-23a suppresses gluconeogenesis in hepatocellular carcinoma by downregulating G6PC and PGC-1α. Hepatology. 2012; in press Wang B, Jacob ST. Role of cancer stem cells in hepatocarcinogenesis. Genome Medicine. 2011; 3:11. Majumder S, Roy S, Kaffenberger T, Wang B, Costinean S, Frankel W, Bratasz A, Kuppusamy P, Hai T, Ghoshal K, Jacob ST. Loss of metallothionein predisposes mice to diethylnitrosamine-induced hepatocarcinogenesis by activating NF-kappaB target genes. Cancer Res. 2010; 70(24):10265-76. Wang B, Hsu SH, Majumder S, Kutay H, Huang W, Jacob ST, Ghoshal K. TGFbeta-mediated upregulation of hepatic miR-181b promotes hepatocarcinogenesis by targeting TIMP3. Oncogene. 2010; 29(12):1787-97. Xiong X., Chen M, Lu Y, Zhang L, Wang B, Zhao Y, Wang X.-J., Liang, Z. Discovery of novel cell proliferation-enhancing gene by random siRNA library based combinatorial screening. Combinatorial Chemistry and High Throughput Screening, 2010; 13 (9): 798-806. viii Cao W., McMahon M., Wang B., O'Connor R., Clarkson M. A case report of spontaneous mutation (C33 > U) in the iron-responsive element of l-ferritin causing hyperferritinemia-cataract syndrome. Blood Cells, Molecules, and Diseases, 2010; 44 (1): 22-27. Wang B, Majumder S, Nuovo G, Kutay H, Volinia S, Patel T, Schmittgen TD, Croce C, Ghoshal K, Jacob ST. Role of microRNA-155 at early stages of hepatocarcinogenesis induced by choline-deficient and amino acid-defined diet in C57BL/6 mice. Hepatology. 2009; 50(4):1152-61. Bai S*, Nasser MW*, Wang B*, Hsu SH, Datta J, Kutay H, Yadav A, Nuovo G, Kumar P, Ghoshal K. MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib. J Biol Chem. 2009; 284 (46):32015-27. * Equal contribution Datta J, Kutay H, Nasser MW, Nuovo GJ, Wang B, Majumder S, Liu CG, Volinia S, Croce CM, Schmittgen TD, Ghoshal K, Jacob ST. Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis. Cancer Res. 2008; 68(13):5049-58. Nasser MW, Datta J, Nuovo G, Kutay H, Motiwala T, Majumder S, Wang B, Suster S, Jacob ST, Ghoshal K. Down-regulation of micro-RNA-1 (miR-1) in lung cancer. Suppression of tumorigenic property of lung cancer cells and their sensitization to doxorubicin-induced apoptosis by miR-1. J Biol Chem. 2008; 283(48):33394-405. Chen MH, Zhang LS, Zhang HY, Xiong XH, Wang B, Lu B, Wahlestedt C, Liang ZC. A Universal Plasmid Library Encoding All Permutations of siRNA. Proc. Natl. Acad. Sci.USA 2005; 102 (7): 2356-2361 Wang B, Chen MH. Progress in the studies of antisense technologies. China Biotechnology 2004; 24(12): 43-47 Fields of Study Major Field: Molecular, Cellular and Developmental Biology ix Table of Contents Abstract…….. ...................................................................................................... ii Dedication.. ......................................................................................................... v Acknowledgments ............................................................................................. vi Vita……….. ......................................................................................................