The Functions Of EZH2, Bmi-1and MiR-203on HCC Recurrence And Metastasis After Liver Transplantation

Part OneThe role of EZH2, Bmi-1and miR-203for hepatocellular carcinomarecurrence and metastasis after liver transplantation【Objective】To detect the expression levels of EZH2, Bmi-1and miR-203in HCC tissues, and investigate thesignificance of EZH2and Bmi-1coordinate their functions through miR-203in HCC.【Methods】64cases of cancer tissue had been collected from the Pathology Department of FuzhouGeneral Hospital of Nanjing Military Area Command2007,01-2011,01. Data including age,tumor size, number, TNM classification, pathological classification, whether vascularmetastasisa, AFP index, liver cirrhosis background, time to recurrence, survival time were alsocollected. By RT-PCR, Western blot and immunohistoehemistry, the expression ofEZH2,Bmi-1in HCC was detected, respectively, and the expression of miR-203was tested byreal-PCR. Then statistical analyses were applied to test the relationship between expression ofthe three molecular biological index and clinicopathologic features and prognosis.【Results】（1）The expression of Bmi-1, EZH2of mRNAand protein in cancer tissue of relapse were higher thanthat in non recurrence after transplantation (P<0.0001). The expression of miR-203inhepatocellular carcinoma(HCC) tissues of non recurrence was higher than in recurrence (P<0.0001). The lower expression of Bmi-1, EZH2, or higher expression of miR-203patients hadbetter survival（P<0.05).(2)Analysed the EZH2,Bim-1and pathologic parameters, the rate of HCC recurrence after livertransplantation(LT) were significantly correlated with the expression of EZH2(P＜0.05),Bmi-1(P＜0.05),miR-203(P＜0.05),the degree of HCC differentiation(P＜0.05), portal vein invasion(P＜0.05). The expression of EZH2, Bmi-1and miR-203in HCC tissue and the degree of HCC differentiation is independentrisk factors for HCC recurrence.(3) There was a negative correlation between miR-203and EZH2or Bim-1, while the expression ofEZH2and Bim-1was positive.【Conclusions】(1) miR-203, EZH2, Bim-1and tumor size, grade, portal vein invasion and preoperativetreatment is closely related to the prognosis of LT for HCC.(2) The recurrence risk factors after LT for HCC included the expression EZH2. Bim-1and miR-203, the degree of HCC differentiation and portal vein invasion. The expression ofEZH2, Bmi-1and miR-203in HCC tissue and the degree of HCC differentiation areindependent risk factors for HCC recurrence, so they can be prognostic markers for HCCpatients who have undergone LT.(3) The expression of miR-203and EZH2, Bim-1expression is closely related to,participate in the development and metastasis of regulating tumor. Part TwoThe effects on invasion and proliferation of Hep3B cell of the theCoordinated Regulation of EZH2and Bmi-1through miR-203【Objective】to investigate the role of coordinated regulation of apoptosis and invasion of EZH2,Bim-1and miR-203on the proliferation of Hep3B cells,Expression of EZH2, Bim-1andmiR-203and Hep3B cell proliferation, apoptosis and invasion of tumor was detected afterEZH2gene expression in Hep3B cells had been reduced,or miR-203gene had beenoverexpressed.【Methods】(1)Detection of the expression of EZH2, BMI-1andMicroRNA203gene in four lines ofHCC cells and normal hepatic cells.(2)After Construction of EZH2gene targeted shRNA plasmid was transfected to Hep3Bcells, expression of EZH2, Bim-1mRNA and protein was detected by RT-PCR and Westernblot, respectively, and miR-203by RT-PCR.Using methyl thiazolyl tetrazolium (MTT) assayand flow condition of the proliferatAn of Hep3B cells was observed and transwell forapoptosis of tumor invasion.(3) After transfection Overexpression of miR-203gene, detection of miR-203, EZH2,Bmi-1, apoptosis of cell line, Transwell detection of tumor invasion was done by the sameway of above.【Results】(1) EZH2, Bmi-1and Mir-203expressed in both HCC cells and normal liver cells, whilethe expression EZH2, Bmi-1in four HCC cell lines (PLC, Huh7, Hep3B, HepG2) werehigher than the expression in normal cells in LO2high (P <0.05); the expression level of Mir-203in four lines of HCC cells normal liver cells are relatively low (P <0.05).(2) targeting shRNA gene plasmid EZH2successfully inhibited the expression of EZH2gene. Cell growth was significantly suppressed (P <0.001). The expression of MiR-203wassignificantly increased (p<0.05), while Bmi-1significantly lowered (p<0.05).(3) Over-expression of miR-203significantly inhibited the expression of EZH2，Bmi-1inHep3B cells (p <0·01), suppressed Hep3B cells invasion and proliferation, and promotedapoptosis.【Conclusions】(1) down-regulating the expression of EZH2, suppressed the expression both of Bim-1protein and mRNA,,but promoted MiR-203expressed, suppressed Hep3B cells invasionand proliferation, and promoted apoptosis.(2) Over-expression of miR-203significantly inhibited the expression of EZH2，Bmi-1inHep3B cells, suppressed Hep3B cells invasion and proliferation, and promoted apoptosis.(3)there may be a kind of coordinated regulation of PcG proteins EZH2, Bmi-1throughmiR-203which regulated invasion and proliferation of HCC cell.