The Function And Mechanism Of 1,25（OH）₂D₃ On Inhibiting Proliferation And Apotosis Inducing Hepatocellular Carcinoma Via HDAC2 Down-regulating

Objective: This paper is to explore the impact of 1, 25（OH）₂D₃ on the proliferation and apoptosis of Hep G2 cells, and discuss the impact of 1,25（OH）₂D₃ on the proliferation and apoptosis of Hep G2 cells via regulating HDAC2 mediated molecular pathway, exploring its regulatory mechanism, so as to provide a cellular and molecular biology foundation for 1,25（OH）₂D₃ in clinical application and a theoretical foundation for HDAC2 targeted tumor gene therapy. Method:（1） MTT and flow cytometry are applied to analyse the impact of 1,25（OH）₂D₃ on the proliferation, cycle and apoptosis of Hep G2 cells.（2） The Western blot and Real time PCR are applied to detect the impact of 1,25（OH）₂D₃ on the proliferation and apoptosis of the relevant genes of Hep G2 cells.（3） HDAC2 interference and over-expressed lentiviral vector are constructed and stable cell strains are screened;（4） the gene chips are applied to screen the differential genes relevant to HDAC2 medicated the molecular pathways via regulating 1,25（OH）₂D₃ at the same time.（5） MTT and FCM are appiled to observe the impact of 1,25（OH）₂D₃ on the proliferation and apoptosis of Hep G2 cells by regulating HDAC2.（6） The Western blot and RT-PCR experiment are conducted to observe the mechanism of action in some related protein relevant to the proliferation, cycle and apoptosis signal pathways that can be determined by regulating HDAC2.（7） The xenografts in nude mice model of hepatocellular carcinoma is built, including control group, 1,25（OH）₂D₃ treated group, 1,25（OH）₂D₃ treated group, HDAC2 interfered group and HDAC2 over-expression group respectively. The gross tumor volume among various groups is observed; the HE staining is adopted to observe the histopathologic changes; the TUNEL is applied to detect the tissue cell apoptosis of the transplanted tumors in various groups; and the Western blot is applied to detect the gene expression changes relevant to the proliferation and apoptosis. Results:（1） MTT shows that 1,25（OH）₂D₃ effectively inhibit Hep G2 cell growth and the inhibitition has time-dose dependent effect（P<0.05）. The cell cycle results show that 1, 25（OH）₂D₃ increases the percentage of cells in G1 phase and decreases that in S phase. The apoptosis results also show that 1,25（OH）₂D₃ can effectively induce the cell apoptosis. The cell apoptosis rate rises significantly from 3.2% to 12.4% in the control group, the difference of which is statistically significant（P<0.05）.（2） the results of the Western blot and RT-PCR show that 1,25（OH）₂D₃ significantly up-regulate the expression of p21, PTEN and Bax, and down-regulate that of HDAC2, Cyclin D1, Akt and Bcl-2.（3） The HDAC2 interference and over-expressed lentiviral ventor are constructed with the successful screening the stable cell strains;（4）The gene chips screen 7 genes relevant to HDAC2 medicated the molecular pathways via regulating 1,25（OH）₂D₃ at the same time, which includes 5 down-regulated ones of PTEN, Bax, DR5, Tp53 and caspase 8 respectively and 2 up-regulated ones of Akt and Bcl-2 respectively;（5） the MTT results show that the over-expressed HDAC2 can weaken the effect of 1,25（OH）₂D₃ on inhibiting Hep G2 cell proliferation; the Western blot and RT-PCR results show that in 1,25（OH）₂D₃ treated HDAC2 over-expression group, the expressions of p21, PTEN, Bax, DR5 and caspase 8 are significantly lower than those in 1,25（OH）₂D₃ treated group; and the expressions of Akt and Bcl-2 are significantly higher than those in 1,25（OH）₂D₃ treated group;（6） Compared with the control group, in 1,25（OH）₂D₃ treated group and HDAC interfered group, the transplanted tumor volume in nude mice is significantly inhibited and the apoptosis is induced in the tumor tissue cells; the over-expressed HDAC2 can weaken the inhibiting effect of 1,25（OH）₂D₃ on the transplanted tumor sizes in nude mice and on the ability to induce tissue cell apoptosis. The Western blot results show that the protein expression levels of PTEN, caspase 8, p53, DR5 and Bax in 1,25（OH）₂D₃ treated group and HDAC2 interfered group are significantly higher than those in the negative control group. After HDAC2 gene is over-expressed, the protein expression levels of PTEN, caspase 8, p53, Bax and DR5 are decreased significantly compared with those in 1,25（OH）₂D₃ treated group; the protein expression levels of PI3 K, p-Akt and Bcl-2 in 1,25（OH）₂D₃ treated group and HDAC2 interfered group are significantly down-regulated as compared with those in negative control group（P<0.05）; after HDAC2 gene is over-expressed, protein expression levels of p-PI3 K, p-Akt and Bcl-2 are increased as compared with those in 1,25（OH）₂D₃ treated group（P<0.05）. Conclusion:（1） 1,25（OH）₂D₃ inhibit the proliferation of Hep G2 cells, blocking the cell cycle at G0/G1 phase and inducing the apoptosis. Its mechanism is closely associated with 1, 25（OH）₂D₃ down-regulating HDAC2, up-regulating PTEN, inhibiting activation of downstream PI3K/Akt signaling pathways;（2） 1,25（OH）₂D₃ can up-regulate p53 and regulate its downstream Bax/Bcl-2 mitochondrial pathway and DR5-caspase 8 exogenous death receptor-mediated apoptosis pathway to induce the apoptosis of Hep G2 cells via down-regulating HDAC2;...