摘要
研究背景:
肝细胞癌(hepatocellular carcinoma,HCC)是我国最常见的恶性肿瘤之一,多种基因变异与HCC发生相关。EZH2(enhancer of zeste homolog 2)是果蝇zeste基因增强子的人类同源基因,为Polycomb group基因家族的重要成员之一。Ploycomb group(PcG)和Trithorax group(trxG)都是进化过程中保守的染色质修饰基因,通过PcG和trxG蛋白的相互拮抗作用,维持同源异型基因的沉默和活化状态,其中PcG维持同源异形基因的失活状态,trxG蛋白则维持基因的激活状态。近年来,EZH2在多种肿瘤中高表达,具有促进细胞增殖,肿瘤细胞扩散的作用。
近几年来,一类小分子对基因表达的调控作用引起了广大生物研究者的关注,即microRNA (miRNA)。miRNA是一类长度在19~24个核苷酸左右的进化保守的内源性非编码小分子RNA,在动植物中成为一类基因调控子。依据与靶mRNA的互补程度不同通过两种方式负调节靶基因的表达。当miRNA与靶mRNA完全互补结合,作用方式和功能与RNAi (RNA-mediated interference)非常类似,由miRNA相关的多蛋白RNA介导的沉默复合物(the miRNA-associated, multiprotein RNA induced-silencing complex,miRISC)切割靶mRNA,引起靶基因mRNA的降解,这一机制在植物当中比较普遍。然而,大部分动物miRNAs则是通过另一种不牵涉它们的靶mRNA断裂的机制来发挥作用。这些miRNAs与它们的靶mRNA 3′-UTR区以不完全互补的方式结合,在转录后抑制靶基因的翻译,通过这种机制miRNAs降低了靶基因蛋白的表达水平,但该基因的mRNA水平几乎不受影响。
近来的研究结果表明,miRNA突变和异常表达与各种人体肿瘤发生、发展相关,miRNA能抑制多种重要肿瘤相关基因的表达,可作为癌基因或抑癌基因而起作用。深入研究miRNA在人体肿瘤中的表达状况及其功能,对研究肿瘤的发生、发展和转移的机制具有重要意义,并将有利于肿瘤的诊断、治疗及预后判断。
目的:检测EZH2和miR-101在HCC中的表达,探讨miR-101对EZH2表达的调控及其意义。
方法:1.采用本课题组收集的广西医科大学一附院2005年12月-2006年9月期间经病理确诊的HCC患者42例,取其肝癌组织及其癌旁组织,9例血管瘤患者的瘤旁组织作为正常对照。采用RT-PCR方法检测EZH2 mRNA在肝癌、癌旁及正常组织中的表达情况,分析EZH2 mRNA与HCC临床病理特征的关系。
2.采用免疫组织化学方法检测EZH2蛋白在肝癌、癌旁及正常组织中的表达情况,分析EZH2蛋白与HCC临床病理特征的关系。
3.选用国内外最常用也是最权威的三个miRNA预测软件:miRanda,PicTar和Targetscan,预测与EZH2 mRNA 3′-UTR有互补结合位点的miRNA。
4.用Trizol提取总RNA,采用EzomicsTM miRNA q-PCR Detection Primer Set、Real Master Mix(SYBR Green) ,以U6snRNA作为内参,进行实时荧光相对定量RT-PCR,测定18对肝癌及癌旁组织中成熟miR-101的转录水平。
5.应用Western-blot方法检测18对肝癌及癌旁组织中EZH2蛋白表达情况。
结果:1. RT-PCR检测42例肝癌组织EZH2 mRNA阳性率为61.9%(26/42),相对应的癌旁组织阳性率为28.6%(12/42);正常肝组织阳性率为22.2%(2/9)。三组比较有显著差异(P<0.01),其中EZH2 mRNA在肝癌组织中的表达明显高于癌旁组织(P<0.05)和正常肝组织(P<0.05),癌旁组织与正常组织比较,差异无统计学意义(P>0.05)。EZH2 mRNA在肝癌组织中的阳性表达率与HCC分化程度有关(P<0.05),在低分化组的表达高于高-中分化组,而与肿瘤大小、HBV感染、AFP水平、肝内外转移以及是否伴有肝硬化无明显相关(P>0.05)。
2.免疫组化检测EZH2蛋白在肝癌中的阳性率为59.5%(25/42),明显高于癌旁21.4%(9/42)及正常肝组织22.2%(2/9)。三组比较有显著差异(P<0.01),其中,EZH2蛋白在肝癌组织中的表达明显高于癌旁组织(P<0.05)和正常组织(P<0.05),癌旁组织与正常组织比较,差异无统计学意义(P>0.05)。EZH2蛋白在肝癌组织中的阳性表达率与肿瘤大小、分化程度、HBV感染、AFP水平、肝内外转移以及是否伴有肝硬化无明显相关(P>0.05)。
3.三个软件TargetScan、miRanda和PicTar均预测到miR-101与EZH2 mRNA 3′-UTR有2个互补结合位点,提示EZH2 mRNA为miR-101的靶mRNA。
4.在18对肝癌及癌旁组织中均检测到miR-101,癌组织miR-101转录水平明显低于癌旁组织(0.34±0.18vs1.00±0.00),差异显著(P<0.01)。5.在所检测的18对肝癌及癌旁组织均有EZH2蛋白表达,癌组织EZH2表达量明显高于癌旁组织(0.90±0.30vs0.36±0.21),有显著性差异(P<0.01=。
结论:1. EZH2在肝癌组织中呈较高表达,尤其在低分化肝癌中表达更高,提示EZH2在肝癌的发生发展中起重要作用。
2.生物信息学预测EZH2 mRNA为miR-101的靶mRNA。
3. miR-101在肝癌组织中的转录水平明显低于癌旁组织;而EZH2在肝癌组织中的蛋白表达量明显高于癌旁组织,两者呈负相关关系,验证了EZH2为miR-101的靶基因,受miR-101负调控。
Background
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. Many genetic mutations are the major factors to induce the HCC. Enhancer of zeste homologue 2(EZH2) is a critical Polycomb group (PcG) protein homologous to Drosophila Enhancer of Zeste. PcG and Trithorax group (TrxG) proteins are evolutionarily conserved genes of modification in the chromatin, they play a crucial role in maintaining the homeotic genes expression or repression by their antagonistic action. The PcG proteins repress gene expression, while the TrxG proteins promote gene expression. Recently, accumulating evidences indicate that EZH2 overexpresses in the human cancers maybe promote the proliferation and diffusion of cancerous cells.
Recently, a class of small molecules, the microRNAs(miRNAs), evoked researchers much attention because of their regulation of gene expression. miRNAs are evolutionarily conserved, endogenous and non-coding small RNAs about 19~24 nucleotides and constitute a novel class of gene regulators that are found in both plants and animals. They negatively regulate their target mRNAs in two different ways depending on the degrees of complementarity between the miRNA and the target mRNA. First, while miRNAs bind with perfect or nearly perfect complementarity to protein coding mRNA sequences that induce the RNA-mediated interference (RNAi) pathway. The mRNA transcripts are cleaved by ribonucleases in the miRNA-associated, multiprotein RNA induced-silencing complex (miRISC),which results in the degradation of target mRNAs. This mechanism of miRNA-mediated gene silencing is commonly found in plants. However, most animal miRNAs are thought to use a second mechanism of gene regulation that does not involve the cleavage of their target mRNAs. These miRNAs exert their regulatory effects by binding to imperfect complementary sites within the 3′-untranslated regions(3′-UTRs) of their target mRNAs,and they repress target-gene translation post-transcriptionally, miRNAs reduce the protein levels of their target genes by this mechanism, but the mRNA levels of these genes are hardly affected.
Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can funct as tumour suppressors or oncogenes. miRNAs have been shown to repress the expression of important cancer-related genes. Research deep in miRNA expression and function will contribute to understand the mechanisms of tumor genesis, development and metastasis, and might profit in the diagnosis, treatment and prognosis of cancer.
Objective: To detect the expression levels of EZH2 and miR-101 in HCC tissues, and investigate miR-101 regulating EZH2 expression and its significance.
Methods: 1. 42 cases of HCC had been collected and all were diagnosed by the Department of Pathology of the GuangXi Medical University during 2005,12-2006,9. The HCC tissues and surrounding non-cancerous tissues were collected, and 9 cases of adjacent liver tissues from Hepatic hemangiomas were selected for normal control group. Reverse transcription-polymerase chain reaction (RT -PCR) was used to detect the EZH2 mRNA expression in the above tissues, then analyzed the relationship between the EZH2 mRNA expression in HCC and clinicopathological parameters.
2. Immunohistochemistry was used to detect the EZH2 protein expression in the tumor tissues, surrounding non-cancerous tissues and normal liver tissues, and then investigated the correlation between EZH2 protein expression in HCC and various clinicopathological parameters.
3. Three bioinformatics softwares for predicting miRNAs: miRanda,PicTar and Targetscan were utilized to predict the interaction between miRNA and the 3′-UTR of EZH2 mRNA.
4. Total RNA was extracted with Trizol, then the EzomicsTM miRNA q-PCR Detection Primer Set and Real Master Mix (SYBR Green) were used to detect miR-101 transcriptional level in 18 cases of HCC tissues and surrounding non-cancerous tissues by relative real-time quantitative RT-PCR.
5. Western-blot was used to detect the EZH2 protein expression in 18 cases of HCC samples and surrounding non-cancerous liver samples.
Results: 1. RT-PCR was used to detect the transcription of EZH2. The positive rate of EZH2 mRNA in HCC was 61.9% (26/42), the surrounding non-cancerous tissues and the normal liver tissues were at the rates of 28.6% (12/42) and 22.2% (2/9) respectively. The difference was significant among these three groups(P<0.01).The positive rate of EZH2 mRNA in HCC compared with the surrounding tissues of HCC and the normal tissues of liver, the difference was significant (P<0.05). No difference was found between the surrounding tissues of HCC and the normal tissues (P>0.05). Analysed the EZH2 and pathologic parameters, the positive rate of EZH2 mRNA in HCC was significantly correlated with the degree of HCC differentiation(P<0.05), which elevated in poorly differentiated HCC compared with well-moderately differentiated HCC, but were not correlated with the hepatitis B virus, the portal vein tumor thrombus, the presence of metastasis, the level of serum AFP and diameter of tumor(P>0.05).
2. Immunohistochemistry stain revealed a positive rate of 59.5%(25/42) of EZH2 protein in HCC group was higher than their surrounding group21.4% (9/42) and in normal group 22.2%(2/9), the difference was significant among these three groups(P<0.01).The positive rate of EZH2 protein in HCC compared with their surrounding tissues and the normal tissues of the liver, the difference was significant (P<0.05). No difference was found between the surrounding tissues of HCC and the normal tissues (P>0.05). Analysed the EZH2 and pathologic parameters, the expression rate of EZH2 protein in HCC was not correlated with all the parameters ,including the hepatitis B virus, the level of serum AFP, diameter of tumor,the degree of HCC differentiation ,the portal vein tumor thrombus and the presence of metastasis (P>0.05).
3. All of three bioinformatics softwares predicted miR-101 has two complemental binding sites with EZH2 mRNA 3′-UTR, and hinted that EZH2 mRNA was a target mRNA of miR-101.
4. miR-101 was detected in all 18 HCCs and their surrounding non-cancerous liver tissues, The quantity of miR-101 transcript in HCC was much less than that in surrounding non-cancerous tissue(0.34±0.18vs1.00±0.00,P<0.01).
5. EZH2 protein was detected in all 18 HCCs and surrounding non-cancerous liver tissues, The quantity of EZH2 protein in HCC was much more than that in surrounding non-cancerous tissue(0.90±0.30vs0.36±0.21,P< 0.01).
Conclusions: 1. The level of EZH2 expression in HCC was fairly high, especially higher in poorly differentiated HCC, which may play an important role in oncogenesis and development of HCC.
2. Bioinformatics predicted EZH2 mRNA was a target mRNA of miR-101.
3. The quantity of miR-101 transcript in HCC was much less than that in surrounding non-cancerous tissue, but the quantity of EZH2 protein in HCC was much more than that in surrounding non-cancerous tissue. There was a negative correlation between miR-101 and EZH2. These hinted that EZH2 mRNA was a target mRNA of miR-101, miR-101 negatively regulate EZH2 expression.
引文
[1]李连弟,饶克勤,张思维,等.中国12市县1993年～1997年肿瘤发病和死亡登记资料统计分析[J].中国肿瘤. 2002, 11(9): 497-507.
[2]俞顺章.人群中原发性肝癌易感因素的流行病学研究[J].实用肿瘤杂志, 2001, 16(2): 361-363.
[3] Thorgeirsson S S. Hunting for tumor suppressor genes in liver cancer[J]. Hepatology, 2003, 37(4): 739-741.
[4] Hanson R D, Hess J L, Yu B D, et al. Mammalian Trithorax and polycomb- group homologues are antagonistic regulators of homeotic development[J]. Proc Natl Acad Sci U S A, 1999, 96(25): 14372-14377.
[5] Pasini D, Bracken A P, Helin K. Polycomb group proteins in cell cycle progression and cancer[J]. Cell Cycle, 2004, 3(4): 396-400.
[6] Varambally S, Dhanasekaran S M, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer[J]. Nature, 2002, 419(6907): 624-629.
[7] Kleer C G, Cao Q, Varambally S, et al. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells[J]. Proc Natl Acad Sci U S A, 2003, 100(20): 11606-11611.
[8] Arisan S, Buyuktuncer E D, Palavan-Unsal N, et al. Increased expression of EZH2, a polycomb group protein, in bladder carcinoma[J]. Urol Int, 2005, 75(3): 252-257.
[9] Mimori K, Ogawa K, Okamoto M, et al. Clinical significance of enhancer of zeste homolog 2 expression in colorectal cancer cases[J]. Eur J Surg Oncol, 2005, 31(4): 376-380.
[10] Stanley R. Hamilton,Lauri A.Aaltonen.Pathology and Genetics Tumors of the Digestive system.Wold Health Organization Classification of Tumors [M].2000:165-166
[11]许良中,杨文涛.免疫组织化学反应结果的判定标准[J].中国癌症杂志, 1996, 6(4): 229-231.
[12] Francis N J, Kingston R E. Mechanisms of transcriptional memory[J]. Nat Rev Mol Cell Biol, 2001, 2(6): 409-421.
[13] Riising E M, Boggio R, Chiocca S, et al. The polycomb repressive complex 2 is a potential target of SUMO modifications[J]. PLoS One, 2008, 3(7): e2704.
[14] Plath K, Fang J, Mlynarczyk-Evans S K, et al. Role of histone H3 lysine 27 methylation in X inactivation[J]. Science, 2003, 300(5616): 131-135.
[15] Bracken A P, Pasini D, Capra M, et al. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer[J]. EMBO J, 2003, 22(20): 5323-5335.
[16] Kuzmichev A, Jenuwein T, Tempst P, et al. Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3[J]. Mol Cell, 2004, 14(2): 183-193.
[17] Cao R, Wang L, Wang H, et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing[J]. Science, 2002, 298(5595): 1039-1043.
[18] van der Vlag J, Otte A P. Transcriptional repression mediated by the human polycomb-group protein EED involves histone deacetylation[J]. Nat Genet, 1999, 23(4): 474-478.
[19] Cao R, Zhang Y. SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex[J]. Mol Cell, 2004, 15(1): 57-67.
[20] Pasini D, Bracken A P, Hansen J B, et al. The polycomb group protein Suz12 is required for embryonic stem cell differentiation[J]. Mol Cell Biol, 2007, 27(10): 3769-3779.
[21] Cao Q, Yu J, Dhanasekaran S M, et al. Repression of E-cadherin by the polycomb group protein EZH2 in cancer[J]. Oncogene, 2008, 27(58): 7274-7284.
[22] Sasaki M, Yamaguchi J, Itatsu K, et al. Over-expression of polycomb group protein EZH2 relates to decreased expression of p16 INK4a in cholangiocarcinogenesis in hepatolithiasis[J]. J Pathol, 2008, 215(2): 175-183.
[23] Yang X, Karuturi R K, Sun F, et al. CDKN1C (p57) is a direct target of EZH2 and suppressed by multiple epigenetic mechanisms in breast cancer cells[J]. PLoS One, 2009, 4(4): e5011.
[24] Beke L, Nuytten M, Van Eynde A, et al. The gene encoding the prostatic tumor suppressor PSP94 is a target for repression by the Polycomb group protein EZH2[J]. Oncogene, 2007, 26(31): 4590-4595.
[25] Visser H P, Gunster M J, Kluin-Nelemans H C, et al. The Polycomb group protein EZH2 is upregulated in proliferating, cultured human mantle cell lymphoma[J]. Br J Haematol, 2001, 112(4): 950-958.
[26] Tang X, Milyavsky M, Shats I, et al. Activated p53 suppresses the histone methyltransferase EZH2 gene[J]. Oncogene, 2004, 23(34): 5759-5769.
[27] Zeidler M, Varambally S, Cao Q, et al. The Polycomb group protein EZH2 impairs DNA repair in breast epithelial cells[J]. Neoplasia, 2005, 7(11): 1011-1019.
[28] Raaphorst F M, van Kemenade F J, Blokzijl T, et al. Coexpression of BMI-1 and EZH2 polycomb group genes in Reed-Sternberg cells ofHodgkin’s disease[J]. Am J Pathol, 2000, 157(3): 709-715.
[29] van Kemenade F J, Raaphorst F M, Blokzijl T, et al. Coexpression of BMI-1 and EZH2 polycomb-group proteins is associated with cycling cells and degree of malignancy in B-cell non-Hodgkin lymphoma[J]. Blood, 2001, 97(12): 3896-3901.
[30] Raaphorst F M, Meijer C J, Fieret E, et al. Poorly differentiated breast carcinoma is associated with increased expression of the human polycomb group EZH2 gene[J]. Neoplasia, 2003, 5(6): 481-488.
[31] Weikert S, Christoph F, Kollermann J, et al. Expression levels of the EZH2 polycomb transcriptional repressor correlate with aggressiveness and invasive potential of bladder carcinomas[J]. Int J Mol Med, 2005, 16(2): 349-353.
[32] Matsukawa Y, Semba S, Kato H, et al. Expression of the enhancer of zeste homolog 2 is correlated with poor prognosis in human gastric cancer[J]. Cancer Sci, 2006, 97(6): 484-491.
[33] Ding L, Erdmann C, Chinnaiyan A M, et al. Identification of EZH2 as a molecular marker for a precancerous state in morphologically normal breast tissues[J]. Cancer Res, 2006, 66(8): 4095-4099.
[34] Karanikolas B D, Figueiredo M L, Wu L. Polycomb group protein enhancer of zeste 2 is an oncogene that promotes the neoplastic transformation of a benign prostatic epithelial cell line[J]. Mol Cancer Res, 2009, 7(9): 1456-1465.
[35] Li X, Gonzalez M E, Toy K, et al. Targeted overexpression of EZH2 in the mammary gland disrupts ductal morphogenesis and causes epithelial hyperplasia[J]. Am J Pathol, 2009, 175(3): 1246-1254.
[36] Sudo T, Utsunomiya T, Mimori K, et al. Clinicopathological significance ofEZH2 mRNA expression in patients with hepatocellular carcinoma[J]. Br J Cancer, 2005, 92(9): 1754-1758.
[37]龚志斌,厉有名,虞朝辉. EZH2在肝细胞性肝癌中的表达及其对HepG2增殖作用的研究[J].实用肿瘤杂志, 2006, 21(6): 523-526.
[38] Sasaki M, Ikeda H, Itatsu K, et al. The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellular carcinoma[J]. Lab Invest, 2008, 88(8): 873-882.
[39] Bracken A P, Dietrich N, Pasini D, et al. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions[J]. Genes Dev, 2006, 20(9): 1123-1136.
[1] Lai EC. microRNAs: runts of the genome assert themselves[J]. Curr Biol.,2003,13(23):R925-936.
[2] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004,116(2):281-297.
[3] Lai EC. Micro RNAs are complementary to 3’UTR sequence motifs that mediate negative post-transcriptional regulation[J]. Nat Genet, 2002, 30(4): 363-364.
[4] Rhoades MW, Reinhart BJ, Lim LP, et al. Prediction of plant microRNA targets[J]. Cell, 2002 ,110(4):513-520.
[5] Brennecke J, Stark A, Russell R B, et al. Principles of microRNA-target recognition[J]. PLoS Biol, 2005, 3(3): e85.
[6] Enright A J, John B, Gaul U, et al. MicroRNA targets in Drosophila[J]. Genome Biol, 2003, 5(1): R1.
[7] Lewis B P, Shih I H, Jones-Rhoades M W, et al. Prediction of mammalian microRNA targets[J]. Cell, 2003, 115(7): 787-798.
[8] Krek A, Grun D, Poy M N, et al. Combinatorial microRNA target predictions[J]. Nat Genet, 2005, 37(5): 495-500.
[9] Lee R C, Feinbaum R L, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell, 1993, 75(5): 843-854.
[10] Reinhart B J, Slack F J, Basson M, et al. The 21-nucleotide let-7 RNAregulates developmental timing in Caenorhabditis elegans[J]. Nature, 2000, 403(6772): 901-906.
[11] Khvorova A, Reynolds A, Jayasena S D. Functional siRNAs and miRNAs exhibit strand bias[J]. Cell, 2003, 115(2): 209-216.
[12] Schwarz D S, Hutvagner G, Du T, et al. Asymmetry in the assembly of the RNAi enzyme complex[J]. Cell, 2003, 115(2): 199-208.
[1] Chen C Z. MicroRNAs as oncogenes and tumor suppressors[J]. N Engl J Med, 2005, 353(17): 1768-1771.
[2] Zhang B, Pan X, Cobb G P, et al. microRNAs as oncogenes and tumor suppressors[J]. Dev Biol, 2007, 302(1): 1-12.
[3] Lee R C, Feinbaum R L, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell, 1993, 75(5): 843-854.
[4] Reinhart B J, Slack F J, Basson M, et al. The 21-nucleotide let-7 RNA regμlates developmental timing in Caenorhabditis elegans[J]. Nature, 2000, 403(6772): 901-906.
[5] Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs[J]. Science, 2001, 294(5543): 853-858.
[6] Lau N C, Lim L P, Weinstein E G, et al. An abundant class of tiny RNAs with probable regμlatory roles in Caenorhabditis elegans[J]. Science, 2001, 294(5543): 858-862.
[7] Lee R C, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans[J]. Science, 2001, 294(5543): 862-864.
[8] Lee Y, Ahn C, Han J, et al. The nuclear RNase III Drosha initiates microRNA processing[J]. Nature, 2003, 425(6956): 415-419.
[9] Yi R, Qin Y, Macara I G, et al. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs[J]. Genes Dev, 2003, 17(24): 3011-3016.
[10] Castanotto D, Lingeman R, Riggs A D, et al. CRM1 mediates nuclear-cytoplasmic shuttling of mature microRNAs[J]. Proc Natl Acad Sci U S A, 2009, 106(51): 21655-21659.
[11] Hutvagner G, McLachlan J, Pasquinelli A E, et al. A cellμlar function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA[J]. Science, 2001, 293(5531): 834-838.
[12] Lai E C. Micro RNAs are complementary to 3’UTR sequence motifs that mediate negative post-transcriptional regμlation[J]. Nat Genet, 2002, 30(4): 363-364.
[13] Ambros V. The functions of animal microRNAs[J]. Nature, 2004, 431 (7006): 350-355.
[14] Bartel D P. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2): 281-297.
[15] Du T, Zamore P D. microPrimer: the biogenesis and function of microRNA [J]. Development, 2005, 132(21): 4645-4652.
[16] Lewis B P, Burge C B, Bartel D P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets[J]. Cell, 2005, 120(1): 15-20.
[17] Huang G L, Zhang X H, Guo G L, et al. Clinical significance of miR-21 expression in breast cancer: SYBR-Green I-based real-time RT-PCR study of invasive ductal carcinoma[J]. Oncol Rep, 2009, 21(3): 673-679.
[18] Chan J A, Krichevsky A M, Kosik K S. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells[J]. Cancer Res, 2005, 65(14): 6029-6033.
[19] Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival[J]. Cancer Res, 2004, 64(11): 3753-3756.
[20] Johnson S M, Grosshans H, Shingara J, et al. RAS is regμlated by the let-7 microRNA family[J]. Cell, 2005, 120(5): 635-647.
[21] Lu J, Getz G, Miska E A, et al. MicroRNA expression profiles classify human cancers[J]. Nature, 2005, 435(7043): 834-838.
[22] Volinia S, Calin G A, Liu C G, et al. A microRNA expression signature of human solid tumors defines cancer gene targets[J]. Proc Natl Acad Sci U S A, 2006, 103(7): 2257-2261.
[23] Calin G A, Sevignani C, Dumitru C D, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers[J]. Proc Natl Acad Sci U S A, 2004, 101(9): 2999-3004.
[24] Calin G A, Dumitru C D, Shimizu M, et al. Frequent deletions and down-regμlation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia[J]. Proc Natl Acad Sci U S A, 2002, 99(24): 15524-15529.
[25] Kuehbacher A, Urbich C, Zeiher A M, et al. Role of Dicer and Drosha for endothelial microRNA expression and angiogenesis[J]. Circ Res, 2007, 101(1): 59-68.
[26] Lin C J, Gong H Y, Tseng H C, et al. miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellμlar carcinoma cell lines[J]. Biochem Biophys Res Commun, 2008, 375(3): 315-320.
[27] Fornari F, Gramantieri L, Giovannini C, et al. MiR-122/cyclin G1 interaction modμlates p53 activity and affects doxorubicin sensitivity of human hepatocarcinoma cells[J]. Cancer Res, 2009, 69(14): 5761-5767.
[28] Bai S, Nasser M W, Wang B, et al. MicroRNA-122 inhibits tumorigenic properties of hepatocellμlar carcinoma cells and sensitizes these cells to sorafenib[J]. J Biol Chem, 2009, 284(46): 32015-32027.
[29] Li S, Fu H, Wang Y, et al. MicroRNA-101 regμlates expression of the v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) oncogene in human hepatocellμlar carcinoma[J]. Hepatology, 2009, 49(4): 1194-1202.
[30] Su H, Yang J R, Xu T, et al. MicroRNA-101, down-regμlated in hepatocellμlar carcinoma, promotes apoptosis and suppresses tumorigenicity[J]. Cancer Res, 2009, 69(3): 1135-1142.
[31] Wong Q W, Ching A K, Chan A W, et al. MiR-222 overexpression confers cell migratory advantages in hepatocellμlar carcinoma through enhancing AKT signaling[J]. Clin Cancer Res, 2010, 16(3): 867-875.
[32] Meng F, Henson R, Wehbe-Janek H, et al. MicroRNA-21 regμlates expression of the PTEN tumor suppressor gene in human hepatocellμlar cancer[J]. Gastroenterology, 2007, 133(2): 647-658.
[33] Ura S, Honda M, Yamashita T, et al. Differential microRNA expression between hepatitis B and hepatitis C leading disease progression to hepatocellμlar carcinoma[J]. Hepatology, 2009, 49(4): 1098-1112.
[34] Coμlouarn C, Factor V M, Andersen J B, et al. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties[J]. Oncogene, 2009, 28(40): 3526-3536.
[35] Yamamoto Y, Kosaka N, Tanaka M, et al. MicroRNA-500 as a potential diagnostic marker for hepatocellμlar carcinoma[J]. Biomarkers, 2009.
[36] Varambally S, Cao Q, Mani R S, et al. Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer[J]. Science, 2008, 322(5908): 1695-1699.
[37] Friedman J M, Liang G, Liu C C, et al. The putative tumor suppressor microRNA-101 modμlates the cancer epigenome by repressing the polycomb group protein EZH2[J]. Cancer Res, 2009, 69(6): 2623-2629.
[38] Cao Q, Yu J, Dhanasekaran S M, et al. Repression of E-cadherin by the polycomb group protein EZH2 in cancer[J]. Oncogene, 2008, 27(58): 7274-7284.
[39] Sasaki M, Yamaguchi J, Itatsu K, et al. Over-expression of polycomb group protein EZH2 relates to decreased expression of p16 INK4a in cholangiocarcinogenesis in hepatolithiasis[J]. J Pathol, 2008, 215(2): 175-183.
[40] Yang X, Karuturi R K, Sun F, et al. CDKN1C (p57) is a direct target of EZH2 and suppressed by mμltiple epigenetic mechanisms in breast cancer cells[J]. PLoS One, 2009, 4(4): e5011.
[41] Zeidler M, Varambally S, Cao Q, et al. The Polycomb group protein EZH2 impairs DNA repair in breast epithelial cells[J]. Neoplasia, 2005, 7(11): 1011-1019.
[42] Varambally S, Dhanasekaran S M, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer[J]. Nature, 2002, 419(6907): 624-629.
[43] Kleer C G, Cao Q, Varambally S, et al. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells[J]. Proc Natl Acad Sci U S A, 2003, 100(20): 11606-11611.
[44] Weikert S, Christoph F, Kollermann J, et al. Expression levels of the EZH2 polycomb transcriptional repressor correlate with aggressiveness and invasive potential of bladder carcinomas[J]. Int J Mol Med, 2005, 16(2): 349-353.
[45]Sasaki M, Ikeda H, Itatsu K, et al. The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellμlar carcinoma[J]. Lab Invest, 2008, 88(8): 873-882.
[46] Wong C F, Tellam R L. MicroRNA-26a targets the histone methyltrans- ferase Enhancer of Zeste homolog 2 during myogenesis[J]. J Biol Chem, 2008, 283(15): 9836-9843.
[47] Juan A H, Kumar R M, Marx J G, et al. Mir-214-dependent regμlation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells[J]. Mol Cell, 2009, 36(1): 61-74.
[48] Sander S, Bμllinger L, Klapproth K, et al. MYC stimμlates EZH2 expression by repression of its negative regμlator miR-26a[J]. Blood, 2008, 112(10): 4202-4212.
[49] Sander S, Bμllinger L, Wirth T. Repressing the repressor: a new mode of MYC action in lymphomagenesis[J]. Cell Cycle, 2009, 8(4): 556-559.
[50] Chen C, Ridzon D A, Broomer A J, et al. Real-time quantification of microRNAs by stem-loop RT-PCR[J]. Nucleic Acids Res, 2005, 33(20): e179.
[51]王杰,李杰,陈香梅,等.半定量RT-PCR法分析两种microRNAs在肝癌和癌旁组织中的差异表达[J].胃肠病学和肝病学杂志. 2008, 17 (5): 406-409.
[52]Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans[J]. Cell, 1993 ,75(5):855-862.
[53]Olsen PH, Ambros V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation[J]. Dev Biol, 1999,216(2):671-680.
[54]Rhoades MW, Reinhart BJ, Lim LP, et al. Prediction of plant microRNA targets[J]. Cell, 2002 ,110(4):513-520.
[55] Hutvágner G, Zamore PD. A microRNA in a multiple-turnover RNAi enzyme complex[J]. Science, 2002 ,297(5589):2056-2060.
[1] Hanson R D, Hess J L, Yu B D, et al. Mammalian Trithorax and polycomb- group homologues are antagonistic regulators of homeotic development[J]. Proc Natl Acad Sci U S A, 1999, 96(25): 14372-14377.
[2] Pasini D, Bracken A P, Helin K. Polycomb group proteins in cell cycle progression and cancer[J]. Cell Cycle, 2004, 3(4): 396-400.
[3] Abel K J, Brody L C, Valdes J M, et al. Characterization of EZH1, a human homolog of Drosophila Enhancer of zeste near BRCA1[J]. Genomics, 1996, 37(2): 161-171.
[4] Hobert O, Jallal B, Ullrich A. Interaction of Vav with ENX-1, a putative transcriptional regulator of homeobox gene expression[J]. Mol Cell Biol, 1996, 16(6): 3066-3073.
[5] Chen H, Rossier C, Antonarakis S E. Cloning of a human homolog of the Drosophila enhancer of zeste gene (EZH2) that maps to chromosome 21q22.2[J]. Genomics, 1996, 38(1): 30-37.
[6] Cardoso C, Mignon C, Hetet G, et al. The human EZH2 gene: genomic organisation and revised mapping in 7q35 within the critical region for malignant myeloid disorders[J]. Eur J Hum Genet, 2000, 8(3): 174-180.
[7] Hillier L W, Fulton R S, Fulton L A, et al. The DNA sequence of human chromosome 7[J]. Nature, 2003, 424(6945): 157-164.
[8] Riising E M, Boggio R, Chiocca S, et al. The polycomb repressive complex 2 is a potential target of SUMO modifications[J]. PLoS One, 2008, 3(7): e2704.
[9] Plath K, Fang J, Mlynarczyk-Evans S K, et al. Role of histone H3 lysine 27methylation in X inactivation[J]. Science, 2003, 300(5616): 131-135.
[10] Jacobs J J, van Lohuizen M. Polycomb repression: from cellular memory to cellular proliferation and cancer[J]. Biochim Biophys Acta, 2002, 1602(2): 151-161.
[11] Kuzmichev A, Jenuwein T, Tempst P, et al. Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3[J]. Mol Cell, 2004, 14(2): 183-193.
[12] Cao R, Wang L, Wang H, et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing[J]. Science, 2002, 298(5595): 1039-1043.
[13] Kirmizis A, Bartley S M, Kuzmichev A, et al. Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27[J]. Genes Dev, 2004, 18(13): 1592-1605.
[14] Jenuwein T, Laible G, Dorn R, et al. SET domain proteins modulate chromatin domains in eu- and heterochromatin[J]. Cell Mol Life Sci, 1998, 54(1): 80-93.
[15] Cao R, Zhang Y. SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex[J]. Mol Cell, 2004, 15(1): 57-67.
[16] Margueron R, Justin N, Ohno K, et al. Role of the polycomb protein EED in the propagation of repressive histone marks[J]. Nature, 2009, 461(7265): 762-767.
[17] Pasini D, Bracken A P, Jensen M R, et al. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity[J]. EMBO J, 2004, 23(20): 4061-4071.
[18] van der Vlag J, Otte A P. Transcriptional repression mediated by the human polycomb-group protein EED involves histone deacetylation[J]. Nat Genet,1999, 23(4): 474-478.
[19] Varambally S, Dhanasekaran S M, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer[J]. Nature, 2002, 419(6907): 624-629.
[20] Erhardt S, Su I H, Schneider R, et al. Consequences of the depletion of zygotic and embryonic enhancer of zeste 2 during preimplantation mouse development[J]. Development, 2003, 130(18): 4235-4248.
[21] Ohhata T, Tachibana M, Tada M, et al. X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a[J]. Genesis, 2004, 40(3): 151-156.
[22] Caretti G, Di Padova M, Micales B, et al. The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation[J]. Genes Dev, 2004, 18(21): 2627-2638.
[23] O’Carroll D, Erhardt S, Pagani M, et al. The polycomb-group gene Ezh2 is required for early mouse development[J]. Mol Cell Biol, 2001, 21(13): 4330-4336.
[24] Pasini D, Bracken A P, Hansen J B, et al. The polycomb group protein Suz12 is required for embryonic stem cell differentiation[J]. Mol Cell Biol, 2007, 27(10): 3769-3779.
[25] Wang J, Mager J, Schnedier E, et al. The mouse PcG gene eed is required for Hox gene repression and extraembryonic development[J]. Mamm Genome, 2002, 13(9): 493-503.
[26] Mager J, Montgomery N D, de Villena F P, et al. Genome imprinting regulated by the mouse Polycomb group protein Eed[J]. Nat Genet, 2003, 33(4): 502-507.
[27] Wang J, Mager J, Chen Y, et al. Imprinted X inactivation maintained by amouse Polycomb group gene[J]. Nat Genet, 2001, 28(4): 371-375.
[28] Visser H P, Gunster M J, Kluin-Nelemans H C, et al. The Polycomb group protein EZH2 is upregulated in proliferating, cultured human mantle cell lymphoma[J]. Br J Haematol, 2001, 112(4): 950-958.
[29] Bracken A P, Pasini D, Capra M, et al. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer[J]. EMBO J, 2003, 22(20): 5323-5335.
[30] Tang X, Milyavsky M, Shats I, et al. Activated p53 suppresses the histone methyltransferase EZH2 gene[J]. Oncogene, 2004, 23(34): 5759-5769.
[31] Fujii S, Ochiai A. Enhancer of zeste homolog 2 downregulates E-cadherin by mediating histone H3 methylation in gastric cancer cells[J]. Cancer Sci, 2008, 99(4): 738-746.
[32] Sasaki M, Yamaguchi J, Itatsu K, et al. Over-expression of polycomb group protein EZH2 relates to decreased expression of p16 INK4a in cholangiocarcinogenesis in hepatolithiasis[J]. J Pathol, 2008, 215(2): 175-183.
[33] Yang X, Karuturi R K, Sun F, et al. CDKN1C (p57) is a direct target of EZH2 and suppressed by multiple epigenetic mechanisms in breast cancer cells[J]. PLoS One, 2009, 4(4): e5011.
[34] Beke L, Nuytten M, Van Eynde A, et al. The gene encoding the prostatic tumor suppressor PSP94 is a target for repression by the Polycomb group protein EZH2[J]. Oncogene, 2007, 26(31): 4590-4595.
[35] Cha T L, Zhou B P, Xia W, et al. Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3[J]. Science, 2005, 310(5746): 306-310.
[36] Bracken A P, Dietrich N, Pasini D, et al. Genome-wide mapping ofPolycomb target genes unravels their roles in cell fate transitions[J]. Genes Dev, 2006, 20(9): 1123-1136.
[37] Dyson N. The regulation of E2F by pRB-family proteins[J]. Genes Dev, 1998, 12(15): 2245-2262.
[38] Nevins J R. Toward an understanding of the functional complexity of the E2F and retinoblastoma families[J]. Cell Growth Differ, 1998, 9(8): 585-593.
[39] Harbour J W, Dean D C. Rb function in cell-cycle regulation and apoptosis[J]. Nat Cell Biol, 2000, 2(4): E65-67.
[40] Dou Q P, Levin A H, Zhao S, et al. Cyclin E and cyclin A as candidates for the restriction point protein[J]. Cancer Res, 1993, 53(7): 1493-1497.
[41] Sears R C, Nevins J R. Signaling networks that link cell proliferation and cell fate[J]. J Biol Chem, 2002, 277(14): 11617-11620.
[42] Sherr C J, Roberts J M. CDK inhibitors: positive and negative regulators of G1-phase progression[J]. Genes Dev, 1999, 13(12): 1501-1512.
[43] Sherr C J. Cancer cell cycles[J]. Science, 1996, 274(5293): 1672-1677.
[44] Tonini T, Bagella L, D’Andrilli G, et al. Ezh2 reduces the ability of HDAC1-dependent pRb2/p130 transcriptional repression of cyclin A[J]. Oncogene, 2004, 23(28): 4930-4937.
[45] Zeidler M, Varambally S, Cao Q, et al. The Polycomb group protein EZH2 impairs DNA repair in breast epithelial cells[J]. Neoplasia, 2005, 7(11): 1011-1019.
[46] Li G, Margueron R, Ku M, et al. Jarid2 and PRC2, partners in regulating gene expression[J]. Genes Dev, 2010, 24(4): 368-380.
[47] Peng J C, Valouev A, Swigut T, et al. Jarid2/Jumonji coordinates control of PRC2 enzymatic activity and target gene occupancy in pluripotent cells[J].Cell, 2009, 139(7): 1290-1302.
[48] Lessard J, Sauvageau G. Polycomb group genes as epigenetic regulators of normal and leukemic hemopoiesis[J]. Exp Hematol, 2003, 31(7): 567-585.
[49] Raaphorst F M, van Kemenade F J, Blokzijl T, et al. Coexpression of BMI-1 and EZH2 polycomb group genes in Reed-Sternberg cells of Hodgkin’s disease[J]. Am J Pathol, 2000, 157(3): 709-715.
[50] van Kemenade F J, Raaphorst F M, Blokzijl T, et al. Coexpression of BMI-1 and EZH2 polycomb-group proteins is associated with cycling cells and degree of malignancy in B-cell non-Hodgkin lymphoma[J]. Blood, 2001, 97(12): 3896-3901.
[51] Bachmann I M, Halvorsen O J, Collett K, et al. EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast[J]. J Clin Oncol, 2006, 24(2): 268-273.
[52] Karanikolas B D, Figueiredo M L, Wu L. Comprehensive evaluation of the role of EZH2 in the growth, invasion, and aggression of a panel of prostate cancer cell lines[J]. Prostate, 2010, 70(6):675-88.
[53] Saramaki O R, Tammela T L, Martikainen P M, et al. The gene for polycomb group protein enhancer of zeste homolog 2 (EZH2) is amplified in late-stage prostate cancer[J]. Genes Chromosomes Cancer, 2006, 45(7): 639-645.
[54] Karanikolas B D, Figueiredo M L, Wu L. Polycomb group protein enhancer of zeste 2 is an oncogene that promotes the neoplastic transformation of a benign prostatic epithelial cell line[J]. Mol Cancer Res, 2009, 7(9): 1456-1465.
[55] Kleer C G, Cao Q, Varambally S, et al. EZH2 is a marker of aggressivebreast cancer and promotes neoplastic transformation of breast epithelial cells[J]. Proc Natl Acad Sci U S A, 2003, 100(20): 11606-11611.
[56] Raaphorst F M, Meijer C J, Fieret E, et al. Poorly differentiated breast carcinoma is associated with increased expression of the human polycomb group EZH2 gene[J]. Neoplasia, 2003, 5(6): 481-488.
[57] Collett K, Eide G E, Arnes J, et al. Expression of enhancer of zeste homologue 2 is significantly associated with increased tumor cell proliferation and is a marker of aggressive breast cancer[J]. Clin Cancer Res, 2006, 12(4): 1168-1174.
[58] Li X, Gonzalez M E, Toy K, et al. Targeted overexpression of EZH2 in the mammary gland disrupts ductal morphogenesis and causes epithelial hyperplasia[J]. Am J Pathol, 2009, 175(3): 1246-1254.
[59] Ding L, Erdmann C, Chinnaiyan A M, et al. Identification of EZH2 as a molecular marker for a precancerous state in morphologically normal breast tissues[J]. Cancer Res, 2006, 66(8): 4095-4099.
[60] Hinz S, Kempkensteffen C, Christoph F, et al. Expression of the polycomb group protein EZH2 and its relation to outcome in patients with urothelial carcinoma of the bladder[J]. J Cancer Res Clin Oncol, 2008, 134(3): 331-336.
[61] Arisan S, Buyuktuncer E D, Palavan-Unsal N, et al. Increased expression of EZH2, a polycomb group protein, in bladder carcinoma[J]. Urol Int, 2005, 75(3): 252-257.
[62] Raman J D, Mongan N P, Tickoo S K, et al. Increased expression of the polycomb group gene, EZH2, in transitional cell carcinoma of the bladder[J]. Clin Cancer Res, 2005, 11(24 Pt 1): 8570-8576.
[63] Weikert S, Christoph F, Kollermann J, et al. Expression levels of the EZH2polycomb transcriptional repressor correlate with aggressiveness and invasive potential of bladder carcinomas[J]. Int J Mol Med, 2005, 16(2): 349-353.
[64] Sasaki M, Ikeda H, Itatsu K, et al. The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellular carcinoma[J]. Lab Invest, 2008, 88(8): 873-882.
[65] Sudo T, Utsunomiya T, Mimori K, et al. Clinicopathological significance of EZH2 mRNA expression in patients with hepatocellular carcinoma[J]. Br J Cancer, 2005, 92(9): 1754-1758.
[66] Matsukawa Y, Semba S, Kato H, et al. Expression of the enhancer of zeste homolog 2 is correlated with poor prognosis in human gastric cancer[J]. Cancer Sci, 2006, 97(6): 484-491.
[67] Mimori K, Ogawa K, Okamoto M, et al. Clinical significance of enhancer of zeste homolog 2 expression in colorectal cancer cases[J]. Eur J Surg Oncol, 2005, 31(4): 376-380.
[68] Choi J H, Song Y S, Yoon J S, et al. Enhancer of zeste homolog 2 expression is associated with tumor cell proliferation and metastasis in gastric cancer[J]. APMIS, 2010, 118(3): 196-202.
[69] Vire E, Brenner C, Deplus R, et al. The Polycomb group protein EZH2 directly controls DNA methylation[J]. Nature, 2006, 439(7078): 871-874.