Objective: To investigate the effect of a new tumor-suppressor gene Arid2 on the expression level of CD44 and the mechanism that Arid2 regulates the invasion and metastasis in human hepatocellular carcinoma cells HepG2 and Huh7.
Methods: Recombinant pGL3-CD44 reporter plasmids were transfected into hepatoma cell lines HepG2 and Huh7 cells infected with adenovirus Arid2(Ad-Arid2). Dual luciferase assays were used to determine the relative luciferase activities of reporter plasmids. Western blot technique was used to detect the influence of Arid2 on the expression of transmembrane glycoprotein CD44. Cell migration assays of tumor cells were employed to observe the impact of overexpression of Arid2 on the invasion and metastasis 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: Luciferase assay showed cells were transfected with different length of CD44 reporter plasmids, and their relative luciferase activities were improved to different degrees, compared with pGL3-Basic control. Meanwhile, the mean luciferase activities of pGL3-CD44 -791~+224bp reporter plasmids were significantly repressed by the overexpression of Arid2 which inhibition rates were up to 73.83%±0.92%(P< 0.05, HepG2) or 48.99%±1.37% (P <0.05, Huh7), compared with Ad-GFP control. Western blot results showed that CD44 protein expression was obviously decreased by overexpression of Arid2. Cell migration assays confirmed that the invasion and metastasis abilities were inhibited by increasing Arid2 expression in Human HepG2 or Huh7 cells, which inhibition rates were 66.95%±0.59%(P< 0.05)in HepG2 cells and 73.86%±0.49%(P< 0.05) in Huh7 cells respectively. The animal experiment results indicated that Arid2 could obviously delay or restrict the subcutaneous transplanted tumors growth in nude mice, which was declined by 98.57%±0.34%(P< 0.05).
Conclusion: CD44 promoter activities and protein expressions were significantly down-regulated by Arid2 in vitro. The growth and metastasis of tumors were obviously restrained in the hepatocellular carcinoma cells and nude models. In brief, these researches indicate CD44 may play important roles in the process where the invasion and metastasis of hepatocellular carcinoma cells are under the control of Arid2.The studies introduce and evaluate the relationships between Arid2 and CD44, and further provide a new research direction with the occurrence and development of hepatocellular carcinoma.