siRNA, miRNA AND THEIR POSSIBLE ROLES IN MOLECULAR THERAPY OF CHRONIC HUMAN HEPATITIS B VIRUS INFECTION AND HEPATOCELLULAR CARCINOMA LI YANG (M.Sc ShanDong University, People’s Republic of China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 I ACKNOWLEDGEMENTS This dissertation is dedicated to my family My parents and my brothers have always been encouraging and supporting me no matter how far we are separated They are the source of my intelligence and power I wish to express sincere gratitude to my supervisor Dr Theresa Tan for her serious, responsible supervision on my project all the way during the course of my candidature Her scientific attitude towards research and professionalism has impressed me and will benefit me lifelong I deeply thank my co-supervisors Dr Shanthi Wasser and A/P Lim Seng Gee Their valuable suggestions and continuous support mean a lot to me I thank all the past and current members in our laboratory including: Sherry Neo, Geraldine Yeo, Wu Juan, Yang Fei, Yang Shu, Bai Jing, Bian HaoSheng, Ho Sok Ying, Thomas Neo, Tan WeiQi for their help during the whole course of my study I thank all our collaborators in the National University Hospital I would like to thank Doctor Aung for preparing RNA samples from surgical tissues, Ms Regina Chan for providing technical support for the real-time PCR assays and graduate student Wei Chun for his help in primary hepatocytes culture I would like to thank all my friends who shared the four fruitful years with me: Zhang gang, Li JianHui, Wu BinHui, Hou AiHua, Meng Lei, Dr Zhang Yong, Zhang ShaoChong, Wang PengHua, Ho ZiZhong, Fei WeiHua, Dr Hu ChuangJiong, Ho ZiZhong, Wang Han, WeiWen, Yang Meng, Ding Ying, Song GuangHui, Li Guang, Pradeep, my dear sisters Wang JunZhu, Song Ran, Ding Hui, and lot more Your friendship is one of the most precious things in my life Last but not the least, I would like to thank National University of Singapore for giving me the opportunity for pursuing a higher degree and generously offering me research scholarship II PUBLICATIONS The major part of this work has been published in: Genome-wide expression profiling of RNA interference of hepatitis B virus gene expression and replication Y Li, S Wasser, S.G Lim and T.M.C Tan Cell Mol Life Sci 2004 Aug; 61(16):2113-2124 MicroRNA Expression Profiling in Human Liver Tumors Y Li, WQ Tan, T Neo, MO Aung , S Wasser, S.G Lim and T.M.C Tan Awarded with the Young scientist Award and published in 20th IUBMB International Congress f Biochemistry and Molecular Biology and 11th FAOBMB Congress Abstracts 2006, Kyoto, Japan Page:145 Other publications as co-author: Synthesis and the biological evaluation of 2-benzenesulfonylmethyl-5- substituted -sulfanyl-[1,3,4]-oxadiazoles as potential anti-Hepatitis B virus agents Theresa May Chin Tan, Yu Chen, Kah Hoe Kong, Jing Bai, Yang Li, Seng Gee Lim, Thiam Hong Ang, Yulin Lam Antiviral Res, 2006, 71, 7-14 Manuscripts in preparation: Deregulation of the miR-106b-25 cluster in Human Hepatocellular Carcinoma Y Li, WQ Tan, T Neo, MO Aung , S Wasser, S.G Lim and T.M.C Tan 2007 III SMALL RNA MOLECULES AS POTENTIAL DIAGNOSTIC AND THERAPEUTIC TOOL FOR HEPATIC DISEASES TABLE OF CONTENTS Chapter I: Introduction 1.1 HCC……………………………………………………………………………… 1.1.1 Epidemiology of Hepatocellular Carcinoma…………………………………… 1.1.2 Risk Factors of HCC………………………………………………….……….… 1.1.3 Diagnoses of HCC 1.1.4 Current Treatments of HCC…………………………………………… 1.2 HBV-the Virus and the Disease…………………………………………… 8 10 12 14 14 14 16 16 17 17 19 19 22 24 24 26 30 30 31 32 35 1.2.1 HBV: the Virus…………………………………………………………………… 1.2.1.1 The structure of HBV particles……………………………………………… 1.2.1.2 The HBV genome ………………………………… 1.2.1.3 Life cycle of HBV ………………………………………………….…… 1.2.2 Hepatitis B: the Disease………………………………………………………… 1.2.2.1 Prevalence………………………………………………………….…………… 1.2.2.2 Clinical diagnosis and prevision………………… 1.2.2.3 Therapeutic treatment of hepatitis B…………………………………………… 1.2.2.3.1 Interferon-α…………………………………… 1.2.2.3.2 Nucleoside analogues………………………… 1.2.2.3.3 Sequence-specific approaches… 1.3 RNAi……………………………………………………………………………… 1.3.1 The Mechanism of RNA Interference ……………………….…………… 1.3.2 History of RNAi Study ……………………………………………………… 1.3.3 RNAi and Applications…………………………………………….……… …… 1.3.3.1 RNAi as a tool for functional genomics…………………………………… 1.3.3.2 RNAi as gene-specific therapeutics…………………………………….……… 1.3.3.3 Challenges for the applications of RNAi………………………………………… 1.3.3.3.1 Design of siRNA……………………………………………………………… 1.3.3.3.2 Delivery…………………………………………………………………… 1.3.3.3.3 Specificity of RNAi……………………………………………………….… 1.4 MicroRNA……………………………………………………………………… 1.4.1Biogenesis of miRNA and Mechanisms of miRNA-mediated Gene Regulation…………………………………………………… 35 1.4.2 History of miRNA Study………………………………………………………… 41 IV 1.4.3 Identification of miRNA Genes……………………………………………… 1.4.4 Prediction of miRNA Targets…………………………………………… 1.4.5 miRNAs and Human Cancers …………… 1.4.6 miRNAs deregulated in HCC……………………………………………… … 42 45 46 49 Chapter II Objectives and Significance 2.1 The RNAi Approach for Inhibition of Hepatitis B Viral Gene Expression……………………………………… 52 2.2 miRNAs and HCC……………………………………………………………… 54 Chapter III Materials and Methods 3.1 Materials………………………………………………………………………… 3.1.1 Cell Cultures…………………………………………………………………… 3.1.1.1 Human tumor cell lines………………………………………… …………… 3.1.1.2 Primary hepatocytes………………………………………………………….… 3.1.2 Surgical Tissue Specimens (tumor vs non-tumor)………….………………… 3.1.3 Preparation of Media for Cell Culture…………… ……………………… … 3.1.4 Oligos, Reagents and Special Chemicals……………………………………… 3.2 Methods…………………………………………………………………………… 3.2.1 Inhibition of HBV Gene Expression by siRNAs………………………… …… 3.2.1.1 Cell culture………………………………………………………….………… 3.2.1.2 Synthesis of siRNA…………………………………………………………… 3.2.1.3 Transfection with siRNAs……………………………………………………… 3.2.1.4 Estimation of transfection efficiency…………………………………………… 3.2.1.5 Cell viability assay………………………………………………………….… 3.2.1.6 Quantitative assay of HBsAg…………………………………………………… 3.2.1.7 HBV viral DNA quantification……………………………… ……… 3.2.1.8 RNA extraction using RNezsy mini kit and quantification………………… 3.2.1.9 RNA extraction using TRIzol and quantification……………………………… 3.2.1.10 Denaturing agarose gel electrophoresis of RNA…………………………… 3.2.1.11 Reverse Transcription and PCR…….………………………………………… 3.2.1.12 Microarray procedures and data analysis……………………………… 3.2.2 Analysis of miRNA Expression and Function in HCC………………… 3.2.2.1 Cell culture…………………………………………………….… …… 3.2.2.2 RNA extraction and quantification……………………………… 3.2.2.3 Mature miRNA expression profiling………………………………… … 3.2.2.4 TaqMan® microRNA individual assay………………… .…………… 3.2.2.5 Quantification of miRNA precursors…………………………………………… V 57 57 57 57 57 57 58 59 59 59 59 60 62 62 62 63 65 65 66 67 69 70 70 71 71 74 74 3.2.2.6 Transfection of cell lines with miRNA inhibitors……………………….……… 76 3.2.2.7 Cell viability assay…………………………………………………….….…… 77 3.2.2.8 Soft agar assay for colony formation…………………………….…………… 77 Chapter IV Inhibition of HBV Gene Expression by siRNAs 4.1 Introduction…………………………………………………………….… …… 81 4.2 Selection and Transfection of HBV Specific siRNAs………………… 82 4.2.1 Selection of HBV Specific siRNAs……………………… ……………….…… 82 4.2.2 Selection of Transfection Reagents……………………… ………………….… 83 4.2.3 Transfection Efficiency of HBV Specific siRNA…………………………….… 86 4.3 Inhibition of HBsAg Expression and Reduction in Virion Production……………….…………………….…… 87 4.3.1 Concentration Dependent Inhibition of HBsAg Expression…………… …… 4.3.2 Effect of siRNA on HBsAg Transcripts in PLC/PRF/5 and 2.2.15 Cells……… 4.3.3 Effect of siRNA on HBV Transcripts in 2.2.15 Cells…………………… …… 4.3.4 Effect of siRNA on Virion Production in 2.2.15 Cells……………….….……… 4.3.5 The Time Course of HBsAg Inhibition by siRNA………………………… 4.3.6 Reversal of RNAi………………………………………………………… …… 87 90 92 93 94 96 4.4 Specificity of the Effects of siRNA…………………… ………………… 97 4.4.1 Effects of siRNA on Cell Viability………………………………… …………… 97 4.4.2 Expression Profiling……………………………………………….…………… 98 4.5 Discussion…………………… …………………… …………………… …… 103 4.5.1 Inhibition of HBV Gene Expression by siRNAs……………………………… 103 4.5.2 Microarray Analysis of HBV-induced Changes in Gene Expression………… 105 4.5.3 Specificity of the Effects of siRNAs……………………………………………… 106 4.5.4 Comparison of Studies on siRNA’s effect on HBV…………………………… 108 4.6 Conclusion…………………… …………………… …………………… … 113 Chapter V microRNA and HCC 5.1 Introduction……………………………………………………………… …… 5.2 miRNA Expression in Livers ……… ……… ……… ……… 115 116 5.2.1 Choice of Patient Samples and Validation of RT-real-time PCR Approach… 117 5.2.1.1 Selection of patient RNA samples……………………….….…… 117 5.2.1.2 Quality of RNA preparations………………………………………… ……… 118 5.2.1.3 Validation of RT-real-time PCR approach to quantify the expression of pri- and pre-miRNAs ……………………………………………… 119 5.2.2 Deregulation of miRNA Expressions in HCC………………………………… 122 VI 5.2.2.1 Mature miRNA expression profile……………………………………….…… 5.2.2.1.1 Identification of miRNAs that are deregulated in HCC ……….……… 5.2.2.1.2 miRNA expression in cirrhotic liver samples …………………….….……… 5.2.2.2 Confirmation of the deregulation of miRNAs in 56 paired HCC samples….…… 5.2.2.3 Upregulation of the expression of the miRNA-106b-25 cluster in HCC……… 5.2.2.4 Upregulation of the expression of the miRNA-106b-25 cluster in HCC cell lines 5.3 Functions of Deregulated miRNAs ……… .……… .……… 5.3.1 Effects of the Deregulated miRNAs on Cell Growth…………………………… 5.3.2 Effect of the miR-106b-25 Cluster on Cell Growth……………….…………… 5.3.3 Effect of the miR-106b-25 Cluster on Anchorage-independent Growth………………………………………… ………… 5.3.4 Effect of the Deregulated miRNAs on Anchorage-independent Growth………………………………………… ………… 122 124 125 126 129 130 133 133 135 137 138 5.4 Prediction of the Putative Targets of the miR-106b-25 Cluster…… 140 5.5 Discussion ……… ……… ……… ……… ……… ……… 141 5.5.1 Deregulation of miRNAs in HCC……………………………………… ……… 5.5.2 Validation of the Deregulation of miRNAs in HCC……………….…………… 5.5.3 The miRNA-106b-25 Cluster as an Oncogenic Cluster……………………… 5.6 Conclusion………………… ………………… ………………… …………… 142 146 147 150 Chapter VI Conclusion 6.1 Summary of Important Findings………………………………………… …… 6.2 Suggestions for Future Work………………………………………….… ….…… 153 1555 REFERENCES………………………………………………………………………… 158 SUPPLEMENTARY MATERIALS………………………………………………… 181 VII SUMMARY The study aims mainly to investigate the therapeutic and diagnostic potential of small RNA molecules in hepatic diseases The focus is on hepatitis B infection and hepatocellular carcinoma (HCC) HCC is a major health problem worldwide and chronic infection with hepatitis B virus (HBV) is a major risk factor for HCC This study can be divided into two parts The first part of this study was aimed at investigating the use of the RNA interference (RNAi) approach for inhibition of HBV gene expression while the next was aimed at evaluating the role of microRNAs (miRNAs) in human HCC In the first part of this study, small interfering RNAs (siRNAs) that target HBsAg transcripts were designed and the effects of these siRNAs on the HBsAg producing cell line, PLC/PRF/5 and the HBV producing cell line, 2.2.15 were studied We showed that siRNAs specific for two conserved regions within the HBsAg gene can inhibit the antigen production in the two human liver cell lines which constitutively produce and secrete HBsAg The inhibitory effect was concentration-dependent for the PLC/PRF/5 cells as well as the 2.2.15 cells Decreases in the corresponding viral transcript levels were observed The inhibitory effect was observed within 24 hours and was still evident days after the initial treatment with siRNAs Significant reduction in virion production was also observed for the 2.2.15 cells To address the question on the specificity of the siRNA-mediated inhibition, we first examined the effects on cell growth and viability This was not affected in both cell lines cDNA microarrays were also used to examine genome-wide changes in gene regulation No significant off-target gene regulation was observed in both cell lines Our data also showed that unlike the general interferon VIII response to long dsRNA molecules, there is no single siRNA-induced response to siRNA duplexes in mammalian cells Our findings thus indicate that siRNA can be specific in mediating down-regulation of viral gene expression leading to reduction in virion production In the second part of the study, miRNA expression profiling using reverse transcription (RT)-real-time PCR was carried out in 10 paired HCC tumor and non-tumor samples The expression profiles showed that of the 157 miRNAs examined, 28 were upregulated while 14 were downregulated The deregulation of some of the most frequently upregulated miRNAs (miR-15b, miR-25, miR-93, miR-106b, miR-135a, miR-182 miR-221, miR-222 and miR-224) was confirmed in 56 paired HCC clinical samples and in HCC cell lines It is of interest to note that members of the miR-17-92 cluster and its homology, the miR-106b-25 cluster were among those that were upregulated To date, the miR-17-92 cluster is one of the best characterized miRNAs and is implicated in the tumorgenesis of several types of human cancers including B-cell lymphoma, breast cancer, colon cancer, lung cancer and pancreatic cancer Our data from the knock-down studies in cell lines for the miR-106b-125 cluster showed that the expression of the cluster is necessary for cell proliferation and for the anchorage-independent growth Taken together, these data indicates that the miR-106b-25 cluster, like its homology, the miR-17-92 cluster, may also have oncogenic properties In conclusion, siRNAs can be used as an efficient tool to inhibit HBV gene expression and viral replication in vitro It is possible to design siRNAs which have little off-target effects, IX although 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Inhibition of hepatitis B virus replication and expression by RNA interference in vitro Zhonghua Yi Xue Za Zhi 85(35):2503-2506 180 SUPPLEMENTARY MATERIAL Table S1 Clinical characteristics of 56 patients NO Diagnosis Aetiology Cirrhosis TNM Age Sex Race 23 Poorly differentiated HCC HBV + T3 N0 M0 stage IIIA 49 M Chinese 27 Moderately differentiated HCC HBV + T3 N0 M1 stage IVB 57 M Chinese 47 Moderately differentiated HCC HBV + T3 N0 M0 stage IIIA 47 M Chinese 48 Poorly differentiated HCC HBV - T4 N0 M0 stage IVA 54 F Indonesian 51 Moderately differentiated HCC HCV + T2 N0 M0 stage II 59 M Chinese 52 Moderately differentiated HCC HBV + T3 N0 M0 stage IIIA 55 M Chinese 57 Moderately differentiated HCC HBV - T3 N0 M0 stage IIIA 46 M Chinese 67 Moderately differentiated HCC HBV + T3 N0 M0 stage IIIA 59 F Chinese 77 Well differentiated HCC Cryptogenic + T3 N0 M0 stage IIIA 67 M Indonesian 78 Well differentiated HCC HBV + T3 N0 M0 stage IIIA 56 M Chinese 81 Poorly differentiated HCC Cryptogenic - T3 N0 M0 stage IIIA 71 F Chinese 83 Poorly differentiated HCC HBV + T4 N0 M0 stage IVA 52 M Chinese 96 Moderately differentiated HCC HBV - T3 N1 M0 stage IIIB 71 M Chinese 104 Poorly differentiated HCC HBV + T4 N0 M0 stage IVA 44 M Chinese 108 Moderately differentiated HCC HBV - NIL 65 M Chinese 111 Poorly differentiated HCC HBV - T4 N0 M0 stage IVA 39 M Chinese 117 Poorly differentiated HCC Cryptogenic - T3 N0 M0 stage IIIA 54 M Indian 118 Moderately differentiated HCC HBV + T4 N0 M0 stage IVA 47 M Myanmar 120 Poorly differentiated HCC HBV + T3 N0 M0 stage IIIA 66 M Indian 121 Moderately differentiated HCC HBV + T3 N0 M0 stage IIIA 63 M Chinese 123 Well differentiated clear cell HCC Cryptogenic - 56 M Chinese 125 Moderately differentiated HCC Cryptogenic - T2 N0 M0 stage IIIA 61 M Chinese 126 Poorly differentiated HCC HBV + T3 N0 M0 stage IIIA 53 M Chinese 127 Moderately differentiated HCC HBV - T4 N0 M0 stage IVA 52 M Chinese 129 Moderately differentiated HCC Cryptogenic + T3 N0 M0 stage IIIA 76 F Chinese 131 Moderately differentiated HCC HBV + T3 N0 M0 stage IIIA 32 M Chinese 132 Well differentiated clear cell HCC HCV + 59 M Indian 135 Poorly differentiated HCC Cryptogenic - 62 M Pakistani T2 N0 M0 stage II T3 N0 M0 stage IIIA T4 N0 M0 stage IVA 181 136 Moderately differentiated HCC HBV + T2 N0 M0 stage II 71 M Chinese 137 Moderately differentiated HCC HBV + T4 N0 M0 stage IVA 62 M Myanmar 148 Poorly differentiated HCC HBV + T3 N0 M0 stage IIIA 68 M Chinese 149 Moderately differentiated HCC HBV + T3 N0 M0 stage IIIA 65 M Indonesian 150 Poorly differentiated HCC HBV - T3 N0 M0 stage IIIA 44 M Chinese 151 Moderately differentiated HCC HBV + T4 N0 M0 stage IVA 82 F Chinese 153 Moderately differentiated HCC HBV + T3 N0 M0 stage IIIA 60 M Indonesian 155 Poorly differentiated HCC HBV + T4 N0 M0 stage IVA 63 M Chinese 156 Moderately differentiated HCC HBV - T3 N0 M0 stage IIIA 53 M Chinese 158 Moderately differentiated HCC Cryptogenic + T3 N0 M0 stage IIIA 76 F Malay 160 Poorly differentiated HCC HBV + T3 N0 M0 stage IIIA 56 M Chinese 169 Poorly differentiated HCC HCV + T4 N0 M0 stage IVA 67 M Malay 174 Well differentiated cell HCC Cryptogenic - T2 N0 M0 stage II 68 F Indonesian 186 Moderately differentiated HCC Cryptogenic + NIL 37 F Chinese 187 Poorly differentiated HCC HBV + T3N0M0 61 M Chinese 192 Moderately differentiated HCC Cholangio - NIL 71 F Chinese 193 Moderately differentiated HCC HBV - T4N0M0 38 M Chinese 195 Poorly differentiated HCC HCV + T2 N0 M0 stage II 65 M Indonesian 196 Moderately differentiated HCC HBV + T3N0M0 40 M Chinese 197 Moderately differentiated HCC Cryptogenic - T3N0M0 72 M Indonisian 204 Moderately differentiated HCC Cryptogenic - T2NXMX 74 M Chinese 211 Poorly differentiated HCC Cryptogenic - T3NXMX 57 F Malaysian 222 NIL HBV - NIL NIL NIL NIL 233 Poorly differentiated HCC HBV - T1NXMX 53 M Chinese 240 Moderately differentiated HCC HBV - T2NXMX 70 M Chinese 246 well differentiated HCC HBV - T2NXMX 54 M Chinese 247 well differentiated HCC HBV - T3N0MX 44 F Chinese 248 NIL HBV - NIL NIL NIL NIL 182 ... proteins; TP: terminal protein During HBV infection in human, virus particles are present in a large quantity in patient blood Both infectious and non-infectious particles can be found in the... diagnosis and treatment of HCC 1.2 HBV-the Virus and the Disease HBV is a small enveloped DNA virus which can cause acute and chronic infection of the liver (Robinson, 1994) HBV infection remains a... antigen HBsAg Hepatitis B surface antigen HBV Hepatitis B virus HCC Hepatocellular carcinoma HCV Hepatitis C virus HIV Human immunodeficiency virus IFN Interferon IFN-α Interferon alpha IRES Internal