Expression Of EZH2 Gene In Hepatocellular Carcinoma And To Be Regulated By MIR-101

BackgroundHepatocellular 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.