| PART1 THE REGULARATION OF THE SUPPRESSORGENE ARID2 TO PROLIFERATION AND MIGRATIONABILITIES OF HEPATOCELLULAR CARCINOMACELLSObjective:To construct the adenovirus of ARID2 CDS plasmid and investigate the potential mechanisms of ARID2 regulating cell proliferation and migration of hepatocellular carcinoma.Methods: Recombinant p Ad-track-ARID2 plasmids were packed up Adenovirus in HEK293 cells. Hepatoma cell lines SK-Hep1, Hep G2 were transfected with p Gl3-cyclin D1 WT or p Gl3-cyclin D1 Mut, p Gl3-cyclin E1 WT or p Gl3-cyclin E1 Mut, p Gl3-CD44, respectively, then infected with adenovirus Ad Arid2 or Ad GFP. Dual luciferase assays were used to determine relative lucifearse activities of reporter plasmids. Immunohistochemistry assay and Western-Blot technology were used to detect cell location of ARID2 and the protein expression of ARID2. Western blot technique was used to detect the influence of Arid2 on the expression of transmembrane glycoprotein CD44. Transwell assays were employed to observe the impact of over-expression of Arid2 on the invasion and migration abilities of tumor cells. The sizes of transplanted tumors were recorded to observe the growth of subcutaneous transplanted tumors in nude mice. Statistical significance was analyzed by one-way ANOVA for multiple comparisons, and independent-samples t-test was utilized to compare two groups.Results:Dual luciferase activities assay showed the relative luciferase activities of GFP groups were no significant difference, compared with vector p GL3-Basic(p>0.05). Compared with GFP groups, the luciferase activities of p Gl3-cyclin D1/E1 were significantly repressed after over-expression of Arid2. The inhibited rates were seperately up to 51.72%±1.4%(t=88.46, p<0.05,Hep G2),22.06%±1.1%(t=38.15, p<0.05, SK-Hep1). After changing the binding region of Arid2 and cyclin D1/E1 promoter, the inhibited rates were no significant difference. Luciferase assay of human CD44 promoter detected in Hep G2 cells or Huh7 cells showed Hep G2 cells or Huh7 cells were transfected with different length of CD44 reporter plasmids(p GL3-CD44-791~+224bp or p GL3-CD44-400~+224bp), and their relative luciferase activities were improved to different degrees, compared with p GL3-Basic control.. Meanwhile, the mean luciferase activities of p GL3-CD44-791~+224bp reporter plasmids were significantly repressed by the overexpression of Arid2 which inhibition rates were up to 73.83%±0.92%(t=116.24, p<0.05)in Hep G2 cells or 48.99%±1.37%(t=42.56, p<0.05)in Huh7 cells, compared with Ad-GFP control. Western blot results showed that CD44 protein expression was obviously decreased by over-expression of Arid2.We detected nuclear staining of Arid2 was significantly positive and the expression of Arid2 was lower by Immunohistochemistry in hepatocellular carcinoma tissues, compared with normal hepatic tissues and Hepatocellular peritumorial tissues. Cell migration assays confirmed that the invasion and migration abilities were inhibited to 66.95%±0.59%(t=30.815, p<0.05)in Hep G2 cells, and 73.86%±0.49%(t=111.276, p<0.05) in Huh7 cells after over-expression of Arid2, respectively. The animal experiment indicated that Arid2 could obviously delay or hinder the subcutaneous transplanted tumors growth in nude mice, which was declined by 98.57%±0.34%(t=18.123, p<0.05).Conclusions: Luciferase activities of PGl3-cyclin D1/E1 were significantly repressed with the over-expression of Arid2. They were no difference after mutation of Arid2 and cyclin D1/E1 promoter binding region. The expression of Arid2 in hepatocellular carcinoma tissue was lower, compared with normal hepatic tissues and hepatocellular peritumorial tissues. CD44 promoter activities and protein expressions were significantly down-regulated by Arid2 in vitro. The tumors’ growth and metastasis were obviously restrained in the hepatocellular carcinoma cells and nude models. In brief, the research indicates cyclin D1/E1 and CD44 may play important roles in the process of invasion and metastasis of hepatocellular carcinoma cells which are under the control of Arid2. These experiments pointed out cyclin D1/E1 and CD44 were important in regulating the biological functions of hepatocellular carcinoma and the biological functions of hepatocellular carcinoma were inhibited by Arid2, and further provide a new research direction for searching new therapeutic target of hepatocellular carcinoma.PART2 THE MOLECULAR MECHANISMS OFSUPPRESSOR GENE ARID2 REGULATING APOPTOSISIN HEPATOMA CELLSObjective: To explore whether ARID2 could regulate cell apoptosis in Human hepatocellular carcinoma cells and potential molecular mechanisms.Methods: SK-Hep1, Hep G2 cells were transfected with ARID2 plasmids,after 96 h transfection. Flow cytometry were used to detected cell apoptotic rates. Cell apoptotic rates, intracellular ROS levels and mitochondrial membrane potential were analyzed after Annexin V-FITC/7-AAD, DHE, JC-1 staining by Flow cytometry, respectively. Meanwhile, the morphological changes were observed in fluorescence microscope. RT-PCR and Real-Time PCR Assays were applied to observe the m RNA expression in mitochondrial apoptotic pathways relative gene after over-expression ARID2. Western Blot technology was further used to observe protein expression.Results: Compared with control groups, cell apoptosis rates of hepatocellular carcinoma cells Hep G2 and SK-Hep1 were increased singly by 1.59±0.17 times(p<0.05) and 1.58±0.56 times(p<0.05) after over-expression ARID2 for 96 hrs transfection. ARID2 could apparently increase intracellular ROS levels of hepatocellular carcinoma cells. Hep G2 and SK-Hep1 cells were improved severally by 3.41±0.34 times(p<0.05) and 3.50±0.52 times(p<0.05). Mitochondrial membrane potential were obviously decreased by 21.69%±1.2%, Hep G2(p<0.05) and 41.38%±1.9%,SK-Hep1(p<0.05) by ARID2. RT-PCR, Real-Time PCR and Western Blot technologies showed that the expression level of anti-apoptosis gene Bcl-w and Mcl-1 was reduced and pro-apoptosis gene Bax was significantly increased by the over-expression of ARID2. The protein expression of Caspase9 and Caspase3 were obviously increased. Besides, we could become conscious of the protein expressiom of cleaved-Caspase3 and cleaved-PARP.Conclusions: We preliminary observed exogenous over-expression of ARID2 may promote hepatocellular carcinoma cell apoptosis by mitochondrial apoptotic signaling pathway. However, further studies are warranted to further explore the molecular targets of ARID2 in hepatocellular carcinoma. |
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