Dact2在肝癌中的表观遗传学变化及功能研究
|
摘要
背景肝细胞癌(hepatocellular carcinoma, HCC)是我国常见的恶性肿瘤之一严重危害人类健康。肝癌的发病机制很复杂,表观遗传学改变调控的wnt/β-catenin信号通路相关基因失活在肝癌发生中发挥了重要作用。Dact (Homo sapiens dapper, antagonist ofβ-catenin)是wnt/β-catenin信号通路的负性调节基因家族。有研究表明Dactl和Dact3的表达分别受DNA甲基化和组蛋白修饰等表观遗传学调控,具有抑制肿瘤发生的作用。但Dact2与肿瘤的关系研究文献报道很少,在肝癌中的表观遗传学变化及功能尚不清楚。
目的
1.检测Dact2在永生化肝细胞系和肝癌细胞系中的表达及甲基化改变。
2.检测Dact2在正常肝组织、癌旁组织及不同分化程度肝癌组织中的表达及意义。
3.检测Dact2在肝癌组织中的甲基化改变。
4.探讨肝癌组织中DNA甲基化与Dact2表达之间的关系。
5.检测Dact2对肝癌细胞生长的影响。
6.探讨Dact2抑制肝癌细胞生长的可能机制。
方法
1. RT-PCR和Western blot检测细胞系中Dact2在甲基转移酶抑制剂5-aza-dc处理前后mRNA及蛋白的表达。
2.甲基化特异性PCR (methylation specific PCR, MSP)检测细胞系和组织标本DNA中Dact2甲基化情况。
3.免疫组织化学方法检测Dact2在正常肝组织、癌旁组织及不同分化程度肝癌组织中的表达分布。
4.脂质体法转染Dact2真核表达载体,流式细胞分选结合G418筛选瞬时转染Dact2的细胞。
5.克隆形成实验及BrdU掺入标记方法分析Dact2对细胞生长的影响
6. Hoechst 33258染色分析Dact2对细胞凋亡的影响。
结果
1. Dact2在肝癌细胞系中的表达及甲基化改变本课题选用5株肝癌细胞系：HepG2、SNU449、SMMC7721、SNU182、PLC-PRF-5和一株永生化的肝细胞系Lo.2。在mRNA水平,Dact2在SNU449细胞系中无表达,在HepG2、SNU182、SMMC7721表达较弱,在Lo.2和PLC-PRF-5细胞中表达正常；5-aza-dc处理后,HepG2、SMMC7721、SNU182细胞中Dact2较未处理时表达上调,而LO.2、PLC-PRF-5和SNU449中Dact2无改变。选取HeoG2和SNU182两株细胞系检测Dact2蛋白的表达,结果显示Dact2蛋白在5-aza-dc处理后上调,与mRNA水平的表达改变一致。MSP检测结果显示lo.2和PLC-PRF-5中Dact2为非甲基化,HepG2、SNU449、SMMC7721、SNU182细胞系中存在甲基化改变,其中,SNU182、SNU449和SMMC7721细胞中同时有甲基化和非甲基化条带。Dact2启动子甲基化改变与基因表达呈对应关系。
2. Dact2在肝癌组织标本中的表达及甲基化情况在表达部位上,Dact2在正常肝组织、癌旁组织和癌组织中表达均在细胞浆；在表达强度上,肝癌组织内Dact2表达较癌旁明显减低,两组比较有统计学意义(P<0.001)；在高分化和中分化肝癌组织中Dact2的表达显著高于低分化组(P＜0.05),而与患者的年龄、姓别、肿瘤大小及HBV相关性肝癌无显著相关。38例肝癌组织中的Dact2甲基化率为60.5%；在16例表达下调的肝癌标本中检测到Dact2甲基化的有11例(68.75%)。
3.恢复Dact2表达对肿瘤细胞生长的影响克隆形成实验结果显示恢复Dact2表达后hepG2细胞的克隆数量明显少于对照转染的空载体,Dact2对细胞长期生长抑制作用大于空载体的2倍。Brdu掺入标记后转染Dact2的阳性细胞为39.63%,明显少于空质粒组48.32%,两组比较有显著差异(P<0.05)。
4. Hoechst 33258染色分析Dact2对细胞凋亡的影响转染Dact2后HepG2的凋亡率为10.9%,而转染空质粒的凋亡率4.9%；SNU449细胞转染Dact2后细胞凋亡率为12.30%,转染空质粒的凋亡率12.10%,提示Dact2对细胞凋亡无明显影响。
结论本课题检测了肝癌中Dact2的表达及DNA甲基化改变,5-aza-dc处理细胞系能恢复dact2的表达,表明在肝细胞癌中Dact2的表达受表观遗传学调控；恢复表达Dact2能够明显抑制肝癌细胞的增殖,但对凋亡无明显影响。以上结果为进一步研究Dact2在肝癌诊断和靶向治疗中的作用奠定了基础。
Background
Hepatocellular carcinoma (HCC) is one of the most common cancer in our country. Its pathogenesis remains unclear. Epigenetic silence of wnt/β-catenin pathway associated genes plays important roles during HCC carcinogensis. Recent studies have demonstrated that Dact1 and Dact3 (homo sapiens dapper, antagonist of P-catenin) were epigenetic regulator of wnt/β-catenin pathway in HCC and colorectal cancer respectively. However, Dact2 epigenetic changes in HCC remains unclear.
Aims
1. To detect Dact2 expression and DNA methylation in immortal liver cell line and HCC cell lines.
2. To examine Dact2 expression in normal liver, HCC and adjacent normal tissues.
3. To detect DNA methylation of Dact2 in HCC tissues.
4. To investigate the relationship of Dact2 expression and DNA methylation.
5. To study the effects of Dact2 on cell growth.
6. To clarify the possible mechanisms of Dact2 on cell growth.
Methods
1. RT-PCR and Western blot were performed to detect Dact2 expression in immortal liver cell line and HCC cell lines before and after 5-aza-dc treatment.
2. Methylation specific PCR (MSP) was used to detect DNA methylation of Dact2 in HCC cell lines and tissues.
3. Immunohistochemisty was performed to examine Dact2 expression in normal liver, HCC and adjacent normal tissues.
4. LipofectamineTM 2000 was used to transfect eukaryotic expression vector into HCC cell lines. FCM and G418 were used to select transient transfection cells.
5. Colony formation assay and BrdU incorporation assay were used to detect cell growth of transfected cell.
6. Apoptosis was analyzed by nuclear staining with Hoechst 33258.
Results
1. HepG2, SMMC7721, SNU449, SNU182, PLC-PRF-5 and LO.2 cell lines were supplied by Gastroenterology Department of PLA General Hospital. Dact2 expression was detected in Lo.2 and PLC-PRF-5, and down regulated in HepG2, SNU182 and SMMC7721. And Dact2 was silenced in SNU449. Promoter region methylation was revealed by MSP in HepG2, SNU449, SMMC7721 and SNU182 cells. Dact2 expression can be reversed in HepG2, SNU449, SMMC7721 and SNU182 cells after treatment with 5-aza-dc.
2. Cytoplastic positive staing of Dact2 was found in normal, adjacent tissues and cancer tissues, but was downregulated in cancer tissues (P<0.001). Significant different of Dact2 expression was found among tumor differentiation levels (P<0.001). Dact2 methylation was exhibited in 23 (60.5%) of 38 HCC cases. Among 19 pairs of tissues,16 were found to lose Dact2 expression compared with their matched normal HCC tissues. Eleven of these 16 cases had Dact2 hypermethylation in tumors (68.75%).
3. Reexpression of Dact2 resulted in significant suppression of long-term cell growth more than 2-folds by colonal formation assays. In BrdU incorporation assay, the percentage of positive cells nuclei is decreased to 36.49% compare with control group (51.96%) (P<0.05).
4. Effects of Dact2 on HCC cell apoptosis. Dact2 has no influence on apoptosis in HepG2 and SNU449 cell lines.
In conclusion, Dact2 expression is silenced by promoter region hypermethylation in HCC. Restoration of Dact2 expression was induced by 5-aza-dc treatment. Forced expression of Dact2 in methylated HepG2 cell line result in growth suppression, but no apoptosis changes was found in HepG2 cell.
目的
1.检测Dact2在永生化肝细胞系和肝癌细胞系中的表达及甲基化改变。
2.检测Dact2在正常肝组织、癌旁组织及不同分化程度肝癌组织中的表达及意义。
3.检测Dact2在肝癌组织中的甲基化改变。
4.探讨肝癌组织中DNA甲基化与Dact2表达之间的关系。
5.检测Dact2对肝癌细胞生长的影响。
6.探讨Dact2抑制肝癌细胞生长的可能机制。
方法
1. RT-PCR和Western blot检测细胞系中Dact2在甲基转移酶抑制剂5-aza-dc处理前后mRNA及蛋白的表达。
2.甲基化特异性PCR (methylation specific PCR, MSP)检测细胞系和组织标本DNA中Dact2甲基化情况。
3.免疫组织化学方法检测Dact2在正常肝组织、癌旁组织及不同分化程度肝癌组织中的表达分布。
4.脂质体法转染Dact2真核表达载体,流式细胞分选结合G418筛选瞬时转染Dact2的细胞。
5.克隆形成实验及BrdU掺入标记方法分析Dact2对细胞生长的影响
6. Hoechst 33258染色分析Dact2对细胞凋亡的影响。
结果
1. Dact2在肝癌细胞系中的表达及甲基化改变本课题选用5株肝癌细胞系：HepG2、SNU449、SMMC7721、SNU182、PLC-PRF-5和一株永生化的肝细胞系Lo.2。在mRNA水平,Dact2在SNU449细胞系中无表达,在HepG2、SNU182、SMMC7721表达较弱,在Lo.2和PLC-PRF-5细胞中表达正常；5-aza-dc处理后,HepG2、SMMC7721、SNU182细胞中Dact2较未处理时表达上调,而LO.2、PLC-PRF-5和SNU449中Dact2无改变。选取HeoG2和SNU182两株细胞系检测Dact2蛋白的表达,结果显示Dact2蛋白在5-aza-dc处理后上调,与mRNA水平的表达改变一致。MSP检测结果显示lo.2和PLC-PRF-5中Dact2为非甲基化,HepG2、SNU449、SMMC7721、SNU182细胞系中存在甲基化改变,其中,SNU182、SNU449和SMMC7721细胞中同时有甲基化和非甲基化条带。Dact2启动子甲基化改变与基因表达呈对应关系。
2. Dact2在肝癌组织标本中的表达及甲基化情况在表达部位上,Dact2在正常肝组织、癌旁组织和癌组织中表达均在细胞浆；在表达强度上,肝癌组织内Dact2表达较癌旁明显减低,两组比较有统计学意义(P<0.001)；在高分化和中分化肝癌组织中Dact2的表达显著高于低分化组(P＜0.05),而与患者的年龄、姓别、肿瘤大小及HBV相关性肝癌无显著相关。38例肝癌组织中的Dact2甲基化率为60.5%；在16例表达下调的肝癌标本中检测到Dact2甲基化的有11例(68.75%)。
3.恢复Dact2表达对肿瘤细胞生长的影响克隆形成实验结果显示恢复Dact2表达后hepG2细胞的克隆数量明显少于对照转染的空载体,Dact2对细胞长期生长抑制作用大于空载体的2倍。Brdu掺入标记后转染Dact2的阳性细胞为39.63%,明显少于空质粒组48.32%,两组比较有显著差异(P<0.05)。
4. Hoechst 33258染色分析Dact2对细胞凋亡的影响转染Dact2后HepG2的凋亡率为10.9%,而转染空质粒的凋亡率4.9%；SNU449细胞转染Dact2后细胞凋亡率为12.30%,转染空质粒的凋亡率12.10%,提示Dact2对细胞凋亡无明显影响。
结论本课题检测了肝癌中Dact2的表达及DNA甲基化改变,5-aza-dc处理细胞系能恢复dact2的表达,表明在肝细胞癌中Dact2的表达受表观遗传学调控；恢复表达Dact2能够明显抑制肝癌细胞的增殖,但对凋亡无明显影响。以上结果为进一步研究Dact2在肝癌诊断和靶向治疗中的作用奠定了基础。
Background
Hepatocellular carcinoma (HCC) is one of the most common cancer in our country. Its pathogenesis remains unclear. Epigenetic silence of wnt/β-catenin pathway associated genes plays important roles during HCC carcinogensis. Recent studies have demonstrated that Dact1 and Dact3 (homo sapiens dapper, antagonist of P-catenin) were epigenetic regulator of wnt/β-catenin pathway in HCC and colorectal cancer respectively. However, Dact2 epigenetic changes in HCC remains unclear.
Aims
1. To detect Dact2 expression and DNA methylation in immortal liver cell line and HCC cell lines.
2. To examine Dact2 expression in normal liver, HCC and adjacent normal tissues.
3. To detect DNA methylation of Dact2 in HCC tissues.
4. To investigate the relationship of Dact2 expression and DNA methylation.
5. To study the effects of Dact2 on cell growth.
6. To clarify the possible mechanisms of Dact2 on cell growth.
Methods
1. RT-PCR and Western blot were performed to detect Dact2 expression in immortal liver cell line and HCC cell lines before and after 5-aza-dc treatment.
2. Methylation specific PCR (MSP) was used to detect DNA methylation of Dact2 in HCC cell lines and tissues.
3. Immunohistochemisty was performed to examine Dact2 expression in normal liver, HCC and adjacent normal tissues.
4. LipofectamineTM 2000 was used to transfect eukaryotic expression vector into HCC cell lines. FCM and G418 were used to select transient transfection cells.
5. Colony formation assay and BrdU incorporation assay were used to detect cell growth of transfected cell.
6. Apoptosis was analyzed by nuclear staining with Hoechst 33258.
Results
1. HepG2, SMMC7721, SNU449, SNU182, PLC-PRF-5 and LO.2 cell lines were supplied by Gastroenterology Department of PLA General Hospital. Dact2 expression was detected in Lo.2 and PLC-PRF-5, and down regulated in HepG2, SNU182 and SMMC7721. And Dact2 was silenced in SNU449. Promoter region methylation was revealed by MSP in HepG2, SNU449, SMMC7721 and SNU182 cells. Dact2 expression can be reversed in HepG2, SNU449, SMMC7721 and SNU182 cells after treatment with 5-aza-dc.
2. Cytoplastic positive staing of Dact2 was found in normal, adjacent tissues and cancer tissues, but was downregulated in cancer tissues (P<0.001). Significant different of Dact2 expression was found among tumor differentiation levels (P<0.001). Dact2 methylation was exhibited in 23 (60.5%) of 38 HCC cases. Among 19 pairs of tissues,16 were found to lose Dact2 expression compared with their matched normal HCC tissues. Eleven of these 16 cases had Dact2 hypermethylation in tumors (68.75%).
3. Reexpression of Dact2 resulted in significant suppression of long-term cell growth more than 2-folds by colonal formation assays. In BrdU incorporation assay, the percentage of positive cells nuclei is decreased to 36.49% compare with control group (51.96%) (P<0.05).
4. Effects of Dact2 on HCC cell apoptosis. Dact2 has no influence on apoptosis in HepG2 and SNU449 cell lines.
In conclusion, Dact2 expression is silenced by promoter region hypermethylation in HCC. Restoration of Dact2 expression was induced by 5-aza-dc treatment. Forced expression of Dact2 in methylated HepG2 cell line result in growth suppression, but no apoptosis changes was found in HepG2 cell.
引文
1 Sherman M. Hepatocellular carcinoma:epidemiology, risk factors, and screening. Semin Liver Seminars in liver disease,2005,25(2):143-54.
2 Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet, 2003,362(9399):1907-17.
3 Kew MC. Epidemiology of chronic hepatitis B virus infection, hepatocellular carcinoma, and hepatitis B virus-induced hepatocellular carcinoma. Pathologie-biologie,2010.
4 Chen CJ, Yu MW, Liaw YF. Epidemiological characteristics and risk factors of hepatocellular carcinoma. Journal of gastroenterology and hepatology, 1997,12(9-10):S294-308.
5 Waddington CH. Preliminary Notes on the Development of the Wings in Normal and Mutant Strains of Drosophila. Proceedings of the National Academy of Sciences of the United States of America,1939,25(7):299-307.
6 Holliday R. The inheritance of epigenetic defects. Science (New York, N.Y.), 1987,238(4824):163-70.
7 Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature, 1983,301 (5895):89-92.
8 Gonzalez-Zulueta M, Bender CM, Yang AS, et al. Methylation of the 5'CpG island of the pl6/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer research, 1995,55(20):4531-5.
9 Herman JG, Merlo A, Mao L, et al. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer research,1995,55(20):4525-30.
10 Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands. Proceedings of the National Academy of Sciences of the United States of America, 1996,93(18):9821-6.
11 Esteller M, Fraga MF, Guo M, et al. DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis. Human molecular genetics, 2001,10(26):3001-7.
12 Esteller M. Cancer epigenomics:DNA methylomes and histone-modification maps. Nature reviews. Genetics,2007,8(4):286-98.
13 Moving AHEAD with an international human epigenome project. Nature, 2008,454(7205):711-5.
14 Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nature genetics,2000,24(2):132-8.
15 Cheyette BN, Waxman JS, Miller JR, et al. Dapper, a Dishevelled-associated antagonist of beta-catenin and JNK signaling, is required for notochord formation. Developmental cell,2002,2(4):449-61.
16 Gloy J, Hikasa H, Sokol SY. Frodo interacts with Dishevelled to transduce Wnt signals. Nature cell biology,2002,4(5):351-7.
17 Katoh M, Katoh M. Identification and characterization of human DAPPER 1 and DAPPER2 genes in silico. International journal of oncology, 2003,22(4):907-13.
18 Polakis P. The many ways of Wnt in cancer. Current opinion in genetics& development,2007,17(1):45-51.
19 Klaus A, Birchmeier W. Wnt signalling and its impact on development and cancer. Nature reviews. Cancer,2008,8(5):387-98.
20 Massague J. TGF-beta signal transduction. Annual review of biochemistry, 1998,67:753-91.
21 Hannigan M, Zhan L, Ai Y, et al. The role of p38 MAP kinase in TGF-betal-induced signal transduction in human neutrophils. Biochemical and biophysical research communications,1998,246(1):55-8.
22 Yau TO, Chan CY, Chan KL, et al. HDPR1, a novel inhibitor of the WNT/beta-catenin signaling, is frequently downregulated in hepatocellular carcinoma:involvement of methylation-mediated gene silencing. Oncogene, 2005,24(9):1607-14.
23 Jiang X, Tan J, Li J, et al. DACT3 is an epigenetic regulator of Wnt/beta-catenin signaling in colorectal cancer and is a therapeutic target of histone modifications. Cancer cell,2008,13(6):529-41.
24 Su Y, Zhang L, Gao X, et al. The evolutionally conserved activity of Dapper2 in antagonizing TGF-beta signaling. The FASEB journal:official publication of the Federation of American Societies for Experimental Biology,2007,21(3):682-90.
25 Waxman JS, Hocking AM, Stoick CL, et al. Zebrafish Dapperl and Dapper2 play distinct roles in Wnt-mediated developmental processes. Development (Cambridge, England),2004,131(23):5909-21.
26 Meng F, Cheng X, Yang L, et al. Accelerated re-epithelialization in Dpr2-deficient mice is associated with enhanced response to TGFbeta signaling. Journal of cell science,2008,121(Pt 17):2904-12.
27 Finn RS, Press MF, Dering J, et al. Estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor expression and benefit from lapatinib in a randomized trial of paclitaxel with lapatinib or placebo as first-line treatment in HER2-negative or unknown metastatic breast cancer. Journal of clinical oncology:official journal of the American Society of Clinical Oncology,2009,27(24):3908-15.
28 Yue W, Sun Q, Dacic S, et al. Downregulation of Dkk3 activates beta-catenin/TCF-4 signaling in lung cancer. Carcinogenesis, 2008,29(1):84-92.
29 Knol JA, Walker SC, Robertson JM, et al. Incorporation of 5-bromo-2'-deoxyuridine into colorectal liver metastases and liver in patients receiving a 7-day hepatic arterial infusion. Cancer research, 1995,55(17):3687-91.
1 Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet, 2003,362(9399):1907-17.
2 Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. The New England journal of medicine, 2003,349(21):2042-54.
3 Shim YH, Yoon GS, Choi HJ, et al. p16 Hypermethylation in the early stage of hepatitis B virus-associated hepatocarcinogenesis. Cancer letters, 2003,190(2):213-9.
4 Esteller M, Fraga MF, Guo M, et al. DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis. Human molecular genetics, 2001,10(26):3001-7.
5 Esteller M. Cancer epigenomics:DNA methylomes and histone-modification maps. Nature reviews. Genetics,2007,8(4):286-98.
6 Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nature genetics,2000,24(2):132-8.
7 Kaneto H, Sasaki S, Yamamoto H, et al. Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut,2001,48(3):372-7.
8 Tannapfel A, Wittekind C. Genes involved in hepatocellular carcinoma: deregulation in cell cycling and apoptosis. Virchows Archiv:an international journal of pathology,2002,440(4):345-52.
9 Teitz T, Wei T, Valentine MB, et al. Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN. Nature medicine,2000,6(5):529-35.
10 Yu J, Ni M, Xu J, et al. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis. BMC cancer,2002,2:29.
11 Sharpless NE, DePinho RA. The INK4A/ARF locus and its two gene products. Current opinion in genetics& development,1999,9(1):22-30.
12 Kubo T, Yamamoto J, Shikauchi Y, et al. Apoptotic speck protein-like, a highly homologous protein to apoptotic speck protein in the pyrin domain, is silenced by DNA methylation and induces apoptosis in human hepatocellular carcinoma. Cancer research,2004,64(15):5172-7.
13 Zhang C, Li H, Zhou G, et al. Transcriptional silencing of the TMS1/ASC tumour suppressor gene by an epigenetic mechanism in hepatocellular carcinoma cells. The Journal of pathology,2007,212(2):134-42.
14 Guilford P, Hopkins J, Harraway J, et al. E-cadherin germline mutations in familial gastric cancer. Nature,1998,392(6674):402-5.
15 Kwon GY, Yoo BC, Koh KC, et al. Promoter methylation of E-cadherin in hepatocellular carcinomas and dysplastic nodules. Journal of Korean medical science,2005,20(2):242-7.
16 Matsumura T, Makino R, Mitamura K. Frequent down-regulation of E-cadherin by genetic and epigenetic changes in the malignant progression of hepatocellular carcinomas. Clinical cancer research:an official journal of the American Association for Cancer Research,2001,7(3):594-9.
17 Wei Y, Van Nhieu JT, Prigent S, et al. Altered expression of E-cadherin in hepatocellular carcinoma:correlations with genetic alterations, beta-catenin expression, and clinical features. Hepatology (Baltimore, Md.), 2002,36(3):692-701.
18 Kanai Y, Ushijima S, Hui AM, et al. The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas. International journal of cancer. Journal international du cancer,1997,71(3):355-9.
19 McConnell BB, Vertino PM. TMS1/ASC:the cancer connection. Apoptosis:
an international journal on programmed cell death,2004,9(1):5-18.
20 Yamada S, Nomoto S, Fujii T, et al. Frequent promoter methylation of M-cadherin in hepatocellular carcinoma is associated with poor prognosis. Anticancer research,2007,27(4B):2269-74.
21 Wong CM, Ng YL, Lee JM, et al. Tissue factor pathway inhibitor-2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma. Hepatology (Baltimore, Md.),2007,45(5):1129-38.
22 Esteller M, Corn PG, Baylin SB, et al. A gene hypermethylation profile of human cancer. Cancer research,2001,61(8):3225-9.
23 Lu GL, Wen JM, Xu JM, et al. [Relationship between TIMP-3 expression and promoter methylation of TIMP-3 gene in hepatocellular carcinoma]. Zhonghua bing li xue za zhi Chinese journal of pathology,2003,32(3):230-3.
24 Wang L, Bani-Hani A, Montoya DP, et al. hMLH1 and hMSH2 expression in human hepatocellular carcinoma. International journal of oncology, 2001,19(3):567-70.
25 Zhang CJ, Li HM, Yau LM, et al. [Methylation of mismatch repair gene (MMR) in primary hepatocellular carcinoma.]. ZZhonghua bing li xue za zhi Chinese journal of pathology,2004,33(5):433-6.
26 Park JH, Cho SB, Lee WS, et al. [Methylation pattern of DNA repair genes and microsatellite instability in hepatocelluar carcinoma]. The Korean journal of gastroenterology= Taehan Sohwagi Hakhoe chi, 2006,48(5):327-36.
27 Matsukura S, Soejima H, Nakagawachi T, et al. CpG methylation of MGMT and hMLHl promoter in hepatocellular carcinoma associated with hepatitis viral infection. British journal of cancer,2003,88(4):521-9.
28 Su PF, Lee TC, Lin PJ, et al. Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma. International journal of cancer. Journal international du cancer,2007,121(6):1257-64.
29 Zhang YJ, Chen Y, Ahsan H, et al. Inactivation of the DNA repair gene 06-methylguanine-DNA methyltransferase by promoter hypermethylation and its relationship to aflatoxin B1-DNA adducts and p53 mutation in hepatocellular carcinoma. International journal of cancer. Journal international du cancer,2003,103(4):440-4.
30 Lee S, Lee HJ, Kim JH, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. The American journal of pathology, 2003,163(4):1371-8.
31 Zhong S, Tang MW, Yeo W, et al. Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas. Clinical cancer research:an official journal of the American Association for Cancer Research,2002,8(4):1087-92.
32 Wang J, Qin Y, Li B, et al. Detection of aberrant promoter methylation of GSTP1 in the tumor and serum of Chinese human primary hepatocellular carcinoma patients. Clinical biochemistry,2006,39(4):344-8.
33 Starr R, Willson TA, Viney EM, et al. A family of cytokine-inducible inhibitors of signalling. Nature,1997,387(6636):917-21.
34 Endo TA, Masuhara M, Yokouchi M, et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature,1997,387(6636):921-4.
35 Okochi O, Hibi K, Sakai M, et al. Methylation-mediated silencing of SOCS-1 gene in hepatocellular carcinoma derived from cirrhosis. Clinical cancer research:an official journal of the American Association for Cancer Research,2003,9(14):5295-8.
36 Niwa Y, Kanda H, Shikauchi Y, et al. Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene, 2005,24(42):6406-17.
37 Miyoshi H, Fujie H, Moriya K, et al. Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma. Journal of gastroenterology,2004,39(6):563-9.
38 Yoshida T, Ogata H, Kamio M, et al. SOCS1 is a suppressor of liver fibrosis and hepatitis-induced carcinogenesis. The Journal of experimental medicine, 2004,199(12):1701-7.
39 Lin CH, Hsieh SY, Sheen IS, et al. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer research,2001,61(10):4238-43.
40 Nagai M, Nakamura A, Makino R, et al. Expression of DNA (5-cytosin)-methyltransferases (DNMTs) in hepatocellular carcinomas. Hepatology research:the official journal of the Japan Society of Hepatology, 2003,26(3):186-191.
41 Goll MG, Kirpekar F, Maggert KA, et al. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science (New York, N.Y.), 2006,311(5759):395-8.
42 Park HJ, Yu E, Shim YH. DNA methyltransferase expression and DNA hypermethylation in human hepatocellular carcinoma. Cancer letters, 2006,233(2):271-8.
43 Saito Y, Kanai Y, Nakagawa T, et al. Increased protein expression of DNA methyltransferase (DNMT) 1 is significantly correlated with the malignant potential and poor prognosis of human hepatocellular carcinomas. International journal of cancer. Journal international du cancer, 2003,105(4):527-32.
44 Choi MS, Shim YH, Hwa JY, et al. Expression of DNA methyltransferases in multistep hepatocarcinogenesis. Human pathology,2003,34(1):11-7.
1 Doench JG, Sharp PA. Specificity of microRNA target selection in translational repression. Genes& development,2004,18(5):504-11.
2 Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet, 2003,362 (9399):1907-17.
3 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993,75(5):843-54.
4 Hagan JP, Croce CM. MicroRNAs in carcinogenesis. Cytogenet Genome Res JT-Cytogenetic and genome research,2007,118(2-4):252-9.
5 Lee Y, Jeon K, Lee JT, et al. MicroRNA maturation:stepwise processing and subcellular localization. The EMBO journal, 2002,21 (17):4663-70.
6 Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell, 2007,129(7):1401-14.
7 Giraldez AJ, Cinalli RM, Glasner ME, et al. MicroRNAs regulate brain morphogenesis in zebrafish. Science (New York, N.Y.), 2005,308 (5723):833-8.
8 Calin GA, Croce CM. MicroRNA signatures in human cancers. Nature reviews. Cancer,2006,6(11):857-66.
9 Caldas C, Brenton JD. Sizing up miRNAs as cancer genes. Nature medicine,2005,11(7):712-4.
10 He X, He L, Hannon GJ. The guardian's little helper:microRNAs in the p53 tumor suppressor network. Cancer research, 2007,67(23):11099-101.
11 Lujambio A, Calin GA, Villanueva A, et al. A microRNA DNA methylation signature for human cancer metastasis. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(36):13556-61.
12 Akao Y, Nakagawa Y, Naoe T. MicroRNAs 143 and 145 are possible common onco-microRNAs in human cancers. Oncology reports, 2006,16 (4):845-50.
13 Varnholt H. The role of microRNAs in primary liver cancer. Annals of hepatology:official journal of the Mexican Association of Hepatology,2008,7(2):104-13.
14 Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature,2005,435(7043):834-8.
15 Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of tissue-specific microRNAs from mouse. Curr Biol JT-Current biology:CB,2002,12(9):735-9.
16 Girard M, Jacquemin E, Munnich A, et al. miR-122, a paradigm for the role of microRNAs in the liver. Journal of hepatology, 2008,48(4):648-56.
17 Roessler S, Budhu A, Wang XW. Future of molecular profiling of human hepatocellular carcinoma. Future oncology (London, England), 2007,3 (4):429-39.
18 Jiang J, Gusev Y, Aderca I, et al. Association of MicroRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival. Clinical cancer research:an official journal of the American Association for Cancer Research, 2008,14 (2):419-27.
19 Varnholt H, Drebber U, Schulze F, et al. MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma. Hepatology (Baltimore, Md.),2008,47(4):1223-32.
20 Michael MZ,O'Connor SM, van Holst Pellekaan NG, et al. Reduced accumulation of specific microRNAs in colorectal neoplasia. Molecular cancer research:MCR,2003,1(12):882-91.
21 Pogribny IP, Tryndyak VP, Boyko A, et al. Induction of microRNAome deregulation in rat liver by long-term tamoxifen exposure. Mutation research,2007,619(1-2):30-7.
22 Murakami Y, Yasuda T, Saigo K, et al. Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene,2006,25(17):2537-45.
23 Gramantieri L, Ferracin M, Fornari F, et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer research,2007,67(13):6092-9.
24 Meng F, Henson R, Wehbe-Janek H, et al. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology,2007,133(2):647-58.
25 Ladeiro Y, Couchy G, Balabaud C, et al. MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology, 2008,47(6):1955-63.
26 Huang YS, Dai Y, Yu XF, et al. Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis. Journal of gastroenterology and hepatology,2008,23(1):87-94.
27 Meng F, Henson R, Lang M, et al. Involvement of human micro-RNA
in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology,2006,130(7):2113-29.
28 Chang TC, Yu D, Lee YS, et al. Widespread microRNA repression by Myc contributes to tumorigenesis. Nature genetics, 2008,40(1):43-50.
29 He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature,2005,435(7043):828-33.
30 Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer research, 2005,65 (16):7065-70.
31 Kong Y, Han JH. MicroRNA:biological and computational perspective. Genomics, proteomics& bioinformatics/Beijing Genomics Institute, 2005,3 (2):62-72.
32 Jopling CL, Yi M, Lancaster AM, et al. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science (New York, N.Y.),2005,309(5740):1577-81.
33 Jacob JR, Sterczer A, Toshkov IA, et al. Integration of woodchuck hepatitis and N-myc rearrangement determine size and histologic grade of hepatic tumors. Hepatology,2004,39(4):1008-16.
34 Krutzfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with'antagomirs'. Nature,2005,438(7068):685-9.
35 Budhu A, Jia HL, Forgues M, et al. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology,2008,47(3):897-907.
2 Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet, 2003,362(9399):1907-17.
3 Kew MC. Epidemiology of chronic hepatitis B virus infection, hepatocellular carcinoma, and hepatitis B virus-induced hepatocellular carcinoma. Pathologie-biologie,2010.
4 Chen CJ, Yu MW, Liaw YF. Epidemiological characteristics and risk factors of hepatocellular carcinoma. Journal of gastroenterology and hepatology, 1997,12(9-10):S294-308.
5 Waddington CH. Preliminary Notes on the Development of the Wings in Normal and Mutant Strains of Drosophila. Proceedings of the National Academy of Sciences of the United States of America,1939,25(7):299-307.
6 Holliday R. The inheritance of epigenetic defects. Science (New York, N.Y.), 1987,238(4824):163-70.
7 Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature, 1983,301 (5895):89-92.
8 Gonzalez-Zulueta M, Bender CM, Yang AS, et al. Methylation of the 5'CpG island of the pl6/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer research, 1995,55(20):4531-5.
9 Herman JG, Merlo A, Mao L, et al. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer research,1995,55(20):4525-30.
10 Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands. Proceedings of the National Academy of Sciences of the United States of America, 1996,93(18):9821-6.
11 Esteller M, Fraga MF, Guo M, et al. DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis. Human molecular genetics, 2001,10(26):3001-7.
12 Esteller M. Cancer epigenomics:DNA methylomes and histone-modification maps. Nature reviews. Genetics,2007,8(4):286-98.
13 Moving AHEAD with an international human epigenome project. Nature, 2008,454(7205):711-5.
14 Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nature genetics,2000,24(2):132-8.
15 Cheyette BN, Waxman JS, Miller JR, et al. Dapper, a Dishevelled-associated antagonist of beta-catenin and JNK signaling, is required for notochord formation. Developmental cell,2002,2(4):449-61.
16 Gloy J, Hikasa H, Sokol SY. Frodo interacts with Dishevelled to transduce Wnt signals. Nature cell biology,2002,4(5):351-7.
17 Katoh M, Katoh M. Identification and characterization of human DAPPER 1 and DAPPER2 genes in silico. International journal of oncology, 2003,22(4):907-13.
18 Polakis P. The many ways of Wnt in cancer. Current opinion in genetics& development,2007,17(1):45-51.
19 Klaus A, Birchmeier W. Wnt signalling and its impact on development and cancer. Nature reviews. Cancer,2008,8(5):387-98.
20 Massague J. TGF-beta signal transduction. Annual review of biochemistry, 1998,67:753-91.
21 Hannigan M, Zhan L, Ai Y, et al. The role of p38 MAP kinase in TGF-betal-induced signal transduction in human neutrophils. Biochemical and biophysical research communications,1998,246(1):55-8.
22 Yau TO, Chan CY, Chan KL, et al. HDPR1, a novel inhibitor of the WNT/beta-catenin signaling, is frequently downregulated in hepatocellular carcinoma:involvement of methylation-mediated gene silencing. Oncogene, 2005,24(9):1607-14.
23 Jiang X, Tan J, Li J, et al. DACT3 is an epigenetic regulator of Wnt/beta-catenin signaling in colorectal cancer and is a therapeutic target of histone modifications. Cancer cell,2008,13(6):529-41.
24 Su Y, Zhang L, Gao X, et al. The evolutionally conserved activity of Dapper2 in antagonizing TGF-beta signaling. The FASEB journal:official publication of the Federation of American Societies for Experimental Biology,2007,21(3):682-90.
25 Waxman JS, Hocking AM, Stoick CL, et al. Zebrafish Dapperl and Dapper2 play distinct roles in Wnt-mediated developmental processes. Development (Cambridge, England),2004,131(23):5909-21.
26 Meng F, Cheng X, Yang L, et al. Accelerated re-epithelialization in Dpr2-deficient mice is associated with enhanced response to TGFbeta signaling. Journal of cell science,2008,121(Pt 17):2904-12.
27 Finn RS, Press MF, Dering J, et al. Estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor expression and benefit from lapatinib in a randomized trial of paclitaxel with lapatinib or placebo as first-line treatment in HER2-negative or unknown metastatic breast cancer. Journal of clinical oncology:official journal of the American Society of Clinical Oncology,2009,27(24):3908-15.
28 Yue W, Sun Q, Dacic S, et al. Downregulation of Dkk3 activates beta-catenin/TCF-4 signaling in lung cancer. Carcinogenesis, 2008,29(1):84-92.
29 Knol JA, Walker SC, Robertson JM, et al. Incorporation of 5-bromo-2'-deoxyuridine into colorectal liver metastases and liver in patients receiving a 7-day hepatic arterial infusion. Cancer research, 1995,55(17):3687-91.
1 Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet, 2003,362(9399):1907-17.
2 Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. The New England journal of medicine, 2003,349(21):2042-54.
3 Shim YH, Yoon GS, Choi HJ, et al. p16 Hypermethylation in the early stage of hepatitis B virus-associated hepatocarcinogenesis. Cancer letters, 2003,190(2):213-9.
4 Esteller M, Fraga MF, Guo M, et al. DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis. Human molecular genetics, 2001,10(26):3001-7.
5 Esteller M. Cancer epigenomics:DNA methylomes and histone-modification maps. Nature reviews. Genetics,2007,8(4):286-98.
6 Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nature genetics,2000,24(2):132-8.
7 Kaneto H, Sasaki S, Yamamoto H, et al. Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut,2001,48(3):372-7.
8 Tannapfel A, Wittekind C. Genes involved in hepatocellular carcinoma: deregulation in cell cycling and apoptosis. Virchows Archiv:an international journal of pathology,2002,440(4):345-52.
9 Teitz T, Wei T, Valentine MB, et al. Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN. Nature medicine,2000,6(5):529-35.
10 Yu J, Ni M, Xu J, et al. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis. BMC cancer,2002,2:29.
11 Sharpless NE, DePinho RA. The INK4A/ARF locus and its two gene products. Current opinion in genetics& development,1999,9(1):22-30.
12 Kubo T, Yamamoto J, Shikauchi Y, et al. Apoptotic speck protein-like, a highly homologous protein to apoptotic speck protein in the pyrin domain, is silenced by DNA methylation and induces apoptosis in human hepatocellular carcinoma. Cancer research,2004,64(15):5172-7.
13 Zhang C, Li H, Zhou G, et al. Transcriptional silencing of the TMS1/ASC tumour suppressor gene by an epigenetic mechanism in hepatocellular carcinoma cells. The Journal of pathology,2007,212(2):134-42.
14 Guilford P, Hopkins J, Harraway J, et al. E-cadherin germline mutations in familial gastric cancer. Nature,1998,392(6674):402-5.
15 Kwon GY, Yoo BC, Koh KC, et al. Promoter methylation of E-cadherin in hepatocellular carcinomas and dysplastic nodules. Journal of Korean medical science,2005,20(2):242-7.
16 Matsumura T, Makino R, Mitamura K. Frequent down-regulation of E-cadherin by genetic and epigenetic changes in the malignant progression of hepatocellular carcinomas. Clinical cancer research:an official journal of the American Association for Cancer Research,2001,7(3):594-9.
17 Wei Y, Van Nhieu JT, Prigent S, et al. Altered expression of E-cadherin in hepatocellular carcinoma:correlations with genetic alterations, beta-catenin expression, and clinical features. Hepatology (Baltimore, Md.), 2002,36(3):692-701.
18 Kanai Y, Ushijima S, Hui AM, et al. The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas. International journal of cancer. Journal international du cancer,1997,71(3):355-9.
19 McConnell BB, Vertino PM. TMS1/ASC:the cancer connection. Apoptosis:
an international journal on programmed cell death,2004,9(1):5-18.
20 Yamada S, Nomoto S, Fujii T, et al. Frequent promoter methylation of M-cadherin in hepatocellular carcinoma is associated with poor prognosis. Anticancer research,2007,27(4B):2269-74.
21 Wong CM, Ng YL, Lee JM, et al. Tissue factor pathway inhibitor-2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma. Hepatology (Baltimore, Md.),2007,45(5):1129-38.
22 Esteller M, Corn PG, Baylin SB, et al. A gene hypermethylation profile of human cancer. Cancer research,2001,61(8):3225-9.
23 Lu GL, Wen JM, Xu JM, et al. [Relationship between TIMP-3 expression and promoter methylation of TIMP-3 gene in hepatocellular carcinoma]. Zhonghua bing li xue za zhi Chinese journal of pathology,2003,32(3):230-3.
24 Wang L, Bani-Hani A, Montoya DP, et al. hMLH1 and hMSH2 expression in human hepatocellular carcinoma. International journal of oncology, 2001,19(3):567-70.
25 Zhang CJ, Li HM, Yau LM, et al. [Methylation of mismatch repair gene (MMR) in primary hepatocellular carcinoma.]. ZZhonghua bing li xue za zhi Chinese journal of pathology,2004,33(5):433-6.
26 Park JH, Cho SB, Lee WS, et al. [Methylation pattern of DNA repair genes and microsatellite instability in hepatocelluar carcinoma]. The Korean journal of gastroenterology= Taehan Sohwagi Hakhoe chi, 2006,48(5):327-36.
27 Matsukura S, Soejima H, Nakagawachi T, et al. CpG methylation of MGMT and hMLHl promoter in hepatocellular carcinoma associated with hepatitis viral infection. British journal of cancer,2003,88(4):521-9.
28 Su PF, Lee TC, Lin PJ, et al. Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma. International journal of cancer. Journal international du cancer,2007,121(6):1257-64.
29 Zhang YJ, Chen Y, Ahsan H, et al. Inactivation of the DNA repair gene 06-methylguanine-DNA methyltransferase by promoter hypermethylation and its relationship to aflatoxin B1-DNA adducts and p53 mutation in hepatocellular carcinoma. International journal of cancer. Journal international du cancer,2003,103(4):440-4.
30 Lee S, Lee HJ, Kim JH, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. The American journal of pathology, 2003,163(4):1371-8.
31 Zhong S, Tang MW, Yeo W, et al. Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas. Clinical cancer research:an official journal of the American Association for Cancer Research,2002,8(4):1087-92.
32 Wang J, Qin Y, Li B, et al. Detection of aberrant promoter methylation of GSTP1 in the tumor and serum of Chinese human primary hepatocellular carcinoma patients. Clinical biochemistry,2006,39(4):344-8.
33 Starr R, Willson TA, Viney EM, et al. A family of cytokine-inducible inhibitors of signalling. Nature,1997,387(6636):917-21.
34 Endo TA, Masuhara M, Yokouchi M, et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature,1997,387(6636):921-4.
35 Okochi O, Hibi K, Sakai M, et al. Methylation-mediated silencing of SOCS-1 gene in hepatocellular carcinoma derived from cirrhosis. Clinical cancer research:an official journal of the American Association for Cancer Research,2003,9(14):5295-8.
36 Niwa Y, Kanda H, Shikauchi Y, et al. Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene, 2005,24(42):6406-17.
37 Miyoshi H, Fujie H, Moriya K, et al. Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma. Journal of gastroenterology,2004,39(6):563-9.
38 Yoshida T, Ogata H, Kamio M, et al. SOCS1 is a suppressor of liver fibrosis and hepatitis-induced carcinogenesis. The Journal of experimental medicine, 2004,199(12):1701-7.
39 Lin CH, Hsieh SY, Sheen IS, et al. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer research,2001,61(10):4238-43.
40 Nagai M, Nakamura A, Makino R, et al. Expression of DNA (5-cytosin)-methyltransferases (DNMTs) in hepatocellular carcinomas. Hepatology research:the official journal of the Japan Society of Hepatology, 2003,26(3):186-191.
41 Goll MG, Kirpekar F, Maggert KA, et al. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science (New York, N.Y.), 2006,311(5759):395-8.
42 Park HJ, Yu E, Shim YH. DNA methyltransferase expression and DNA hypermethylation in human hepatocellular carcinoma. Cancer letters, 2006,233(2):271-8.
43 Saito Y, Kanai Y, Nakagawa T, et al. Increased protein expression of DNA methyltransferase (DNMT) 1 is significantly correlated with the malignant potential and poor prognosis of human hepatocellular carcinomas. International journal of cancer. Journal international du cancer, 2003,105(4):527-32.
44 Choi MS, Shim YH, Hwa JY, et al. Expression of DNA methyltransferases in multistep hepatocarcinogenesis. Human pathology,2003,34(1):11-7.
1 Doench JG, Sharp PA. Specificity of microRNA target selection in translational repression. Genes& development,2004,18(5):504-11.
2 Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet, 2003,362 (9399):1907-17.
3 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993,75(5):843-54.
4 Hagan JP, Croce CM. MicroRNAs in carcinogenesis. Cytogenet Genome Res JT-Cytogenetic and genome research,2007,118(2-4):252-9.
5 Lee Y, Jeon K, Lee JT, et al. MicroRNA maturation:stepwise processing and subcellular localization. The EMBO journal, 2002,21 (17):4663-70.
6 Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell, 2007,129(7):1401-14.
7 Giraldez AJ, Cinalli RM, Glasner ME, et al. MicroRNAs regulate brain morphogenesis in zebrafish. Science (New York, N.Y.), 2005,308 (5723):833-8.
8 Calin GA, Croce CM. MicroRNA signatures in human cancers. Nature reviews. Cancer,2006,6(11):857-66.
9 Caldas C, Brenton JD. Sizing up miRNAs as cancer genes. Nature medicine,2005,11(7):712-4.
10 He X, He L, Hannon GJ. The guardian's little helper:microRNAs in the p53 tumor suppressor network. Cancer research, 2007,67(23):11099-101.
11 Lujambio A, Calin GA, Villanueva A, et al. A microRNA DNA methylation signature for human cancer metastasis. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(36):13556-61.
12 Akao Y, Nakagawa Y, Naoe T. MicroRNAs 143 and 145 are possible common onco-microRNAs in human cancers. Oncology reports, 2006,16 (4):845-50.
13 Varnholt H. The role of microRNAs in primary liver cancer. Annals of hepatology:official journal of the Mexican Association of Hepatology,2008,7(2):104-13.
14 Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature,2005,435(7043):834-8.
15 Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of tissue-specific microRNAs from mouse. Curr Biol JT-Current biology:CB,2002,12(9):735-9.
16 Girard M, Jacquemin E, Munnich A, et al. miR-122, a paradigm for the role of microRNAs in the liver. Journal of hepatology, 2008,48(4):648-56.
17 Roessler S, Budhu A, Wang XW. Future of molecular profiling of human hepatocellular carcinoma. Future oncology (London, England), 2007,3 (4):429-39.
18 Jiang J, Gusev Y, Aderca I, et al. Association of MicroRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival. Clinical cancer research:an official journal of the American Association for Cancer Research, 2008,14 (2):419-27.
19 Varnholt H, Drebber U, Schulze F, et al. MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma. Hepatology (Baltimore, Md.),2008,47(4):1223-32.
20 Michael MZ,O'Connor SM, van Holst Pellekaan NG, et al. Reduced accumulation of specific microRNAs in colorectal neoplasia. Molecular cancer research:MCR,2003,1(12):882-91.
21 Pogribny IP, Tryndyak VP, Boyko A, et al. Induction of microRNAome deregulation in rat liver by long-term tamoxifen exposure. Mutation research,2007,619(1-2):30-7.
22 Murakami Y, Yasuda T, Saigo K, et al. Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene,2006,25(17):2537-45.
23 Gramantieri L, Ferracin M, Fornari F, et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer research,2007,67(13):6092-9.
24 Meng F, Henson R, Wehbe-Janek H, et al. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology,2007,133(2):647-58.
25 Ladeiro Y, Couchy G, Balabaud C, et al. MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology, 2008,47(6):1955-63.
26 Huang YS, Dai Y, Yu XF, et al. Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis. Journal of gastroenterology and hepatology,2008,23(1):87-94.
27 Meng F, Henson R, Lang M, et al. Involvement of human micro-RNA
in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology,2006,130(7):2113-29.
28 Chang TC, Yu D, Lee YS, et al. Widespread microRNA repression by Myc contributes to tumorigenesis. Nature genetics, 2008,40(1):43-50.
29 He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature,2005,435(7043):828-33.
30 Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer research, 2005,65 (16):7065-70.
31 Kong Y, Han JH. MicroRNA:biological and computational perspective. Genomics, proteomics& bioinformatics/Beijing Genomics Institute, 2005,3 (2):62-72.
32 Jopling CL, Yi M, Lancaster AM, et al. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science (New York, N.Y.),2005,309(5740):1577-81.
33 Jacob JR, Sterczer A, Toshkov IA, et al. Integration of woodchuck hepatitis and N-myc rearrangement determine size and histologic grade of hepatic tumors. Hepatology,2004,39(4):1008-16.
34 Krutzfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with'antagomirs'. Nature,2005,438(7068):685-9.
35 Budhu A, Jia HL, Forgues M, et al. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology,2008,47(3):897-907.