The Experimental Study On The Mechanism Of ARHI Gene In Ovarian Cancer Cells

Objective: Tumorigenesis is always companied by activation of oncogenes or inactivationof tumor suppressor genes. Studies on the function of these genes promise great help totargeted therapy of cancer. ARHI is a maternal imprinting tumor suppressor gene, whosefunction is abrogated in ovarian cancer. Our work aims to investigate the effect of ARHIconstructive overexpression on cell proliferation in ovarian cancer cells.Methods: In our study, we amplified ARHI gene coding sequence by PCR amplificationusing plasmid pcDNA3.0-ARHI as template. To recombine plasmid, amplified ARHI wasdouble-enzyme digested by EcoRI and BamHI, then inserted into the MCS of eukaryoticvector pIRES2-EGFP by T4ligation. After the reconstruction of ARHI and pIRES2-EGFP,we identified the accuracy of pIRES2-EGFP-ARHI plasmid by enzyme digestion,electrophoresis and gene sequencing. Real-time PCR was performed to detect theexpression of ARHI in different ovarian cancer cell lines. After transfecting thepIRES2-EGFP-ARHI to SKOV3cells with lowest expression of ARHI gene, the inhibitoryrate of cell growth was calculated by CCK-8assay. Cell cycle and apoptosis after ARHIoverexpression were analysed by flow cytometry. In addition, the expression of LC3-Ⅱ,STAT3, p-STAT3, ERK, p-ERK were detected by western blotting approach inARHI-overexpressed SKOV3cells.Results: QPCR results shown that ARHI gene was down-regulated in all ovarian cancercell lines analysed, and SKOV3cells displayed the lowest expression of ARHI. We foundthat the inhibitory rates of cell growth were64.69%,70.17%,67.01%,66.87%,67.70%after the cells in the test group were cultured for24,48,72,96and120h, and weresignificantly higher than the plasmid group (P <0.01). The ratio of S-phase cells was64.18%in the test group,38.43%in the plasmid group and15.15%in untreated group.And the apoptosis rates of three group were47.97%,26.53%and9.33%after the cellswere cultured for48h, respectively. The proportion of S phase cells were43.95%,12.37% and10.89%, and apoptosis rates were51.34%,20.55%and4.39%after the cells for72h,respectively, suggesting the difference remained significant between the test and controlgroups. The expression level of LC3-Ⅱ was greatly increased， but P-STAT3andP-ERK1/2expression levels were largely reduced in test group cultured for48h, comparedwith the control group.Conclusion: These findings suggested that the ARHI gene lost its function in ovariancancer, and constructive overexpression of ARHI inhibits the growth of SKOV3cells,arrests the SKOV3cells at S phase and induces apoptosis and autophagy. The possiblemechanism is that ARHI inhibits phosphorylational activation of ERK1/2and STAT3,thereby blocks proliferative signal and results in apoptosis and autophagy in ovariancancer.