Regulation Of Tumor Suppressor Gene Arid2 On Hepatoma Cell Proliferation And Related Molecular Mechanisms

Objective:Arid2 is a tumor suppressor gene, which is newly found located on the long arm of the 12 pairs of chromosomes, containing 21 exons. Arid2 is found associated with SWI/SNF complexes which acted as an ATPase enzymatic subunit. The SWI/SNF complexes uses the energy of ATP hydrolysis to reposition nucleosomes, so as to regulate cell proliferation and differentiation. Arid family can devide into 7 sub-families: Arid 1-5, JAridl and JArid2. SWI/SNF complex was demonstrated to play a tumor suppressive role in human cancer and the possible mechanisms are as following:1) to regulate the immune response via IFN signaling pathway,2) to inhibite the cell proliferation through regulating Rb signaling pathway,3) to regulate cell migration and invasion by modulating the expression of cytoskeletal proteins and transmembrane glycoprotein.It has been reported that the mutation of the Arid family is closely related to a variety of human tumors such as hepatocellular carcinoma, head and neck cancer, melanoma and non-small cell lung cancer. The mutation frequency of Arid2 is nearly 18.2% in HCV related HCC tissue samples. Arid2 mutant embryos exhibited multiple cardiac defects including ventricular septum defect and common atrioventricular valve. So far, the biological function of Arid2 and its role in the occurrence and development of tumor is still poorly understood. In our study, we intended to examine the effect of Arid2 on the proliferation and migration of hepatoma cell lines, and to explore the related molecular mechanism.Methods:RT-PCR and Western blot technology was uesd to detect the expression of Arid2 in hepatoma tissues and different hepatoma cell lines; MTS, Transwell and Flow cytometry were applied to explore the effects of konckdown or overpression of Arid2 on the proliferation and migration of hepatoma cell lines. We determined whether konckdown or overexpression of Arid2 could attentuate the tumor growth and invasiveness in vivo, and further to assess the expression of c-my、β-catenin、ki-67 and cyclinD1 by immunohistochemical staining. MTS and Transwell were uesd to analyze the alternation of Rb-E2F signaling pathway after knock-down or over-expression of Arid2. Immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) were employed to observe the binding of E2F1 and E2F4 in cyclinD1 and cyclinE1 gene promoter regions induced by knock-down or over-expression of Arid2.Result:The expression of Arid2 in tumor tissue was significantly lower than its expression in the adjacent non-tumorous tissues. Compared with control groups, konckdown of Arid2 promoted cell proliferation and migration rate, however cell proliferation and migration rate were inhibited when Arid2 was overexpressed.We found that silencing of endogenous Arid2 significantly promoted the xenograft tumor growth and invasion in nude mice, while exogenous expression of Arid2 could block the tumor formation and inhibite the tumor growth rate. Moreover, the level of cell growth biomarkers:proliferating cell nuclear (ki-67), Wnt effector β-catenin, c-myc, and cell cycle protein(cyclinD1) were effectively up-regulated by silencing of Arid2.With the use of western blot and immunofluorescence assays, Arid2 was found to play a role in regulating inhibit the Rb-E2F signaling effectors, such as pRb, E2F1, cyclinDl and cyclinEl. Co-IP assay revealed that Arid2 could physically interact with transcription factors E2F1 and E2F4. ChIP assay further confirmed that Arid2 significantly decreased the recruitment of E2F1 while enhanced the recruitment of E2F4 to cyclinD1 and cyclinE1 promoter regions,.SiRNA-mediated konck down of Arid2 and E2F1 significantly inhibited cell proliferation and migration, conversely, cell proliferation and migration could be stimulated when overexpression of Arid2 and silencing of E2F4.Conclusion:Arid2 inhibited cell proliferation and the cell cycle transition from G1 phase to S phase via regulating the Rb-E2F signaling pathway. The possible mechanism was to block the bindings of E2F1-pRb complexes to cyclinD1 and cyclinE1 promoters while reversely to enhauce the recuritment of E2F4-p130. Our findings provide new clues for anti-oncogene effects of Arid2, and may suggest a novel therapatic interventions for hepatocellular carcinoma.