Effect Of HDAC2 And Acetylation Of P53^k320 On Ovarian Cancer Cell Line Skov3/ovcar3

Ovarian cancer is one of the most common malignant tumor in female, which ranks first in mortality rate. Ovary hides in the pelvis, because of its insidious onset。Symptoms are vague at the primary stage, and hence early diagnosis is difficult. Approximately, 70% of patients are diagnosed with the International Federation of Gynecology and Obstetrics（FIGO） stage III or IV, with a poor five year survival rate. Epithelial ovarian carcinoma（EOC） is the leading cause of death among gynecological cancers in the western world. Worldwide, approximately 200,000 women are diagnosed with this malignancy, with 125,000 disease related deaths each year. Although the ideal primary cytoreductive surgery and combination chemotherapy with platinum have improved the prognosis of patients with malignant ovarian cancer, the 5-year survival rate remains 40%.Histone acetyltransferases（HATs） and histone deacetylases（HDACs） regulate protein acetylation. Pathological activity and expression of HDACs can lead to severe diseases such as cancer, immunological disturbances and muscular dystrophies. Deacetylases are often dysregulated in cancer and HDAC overexpression disturbs cell cycle control and differentiation. HDAC2 is one of the HDACs most frequently overexpressed in tumors.Moreover, monitoring HDAC2 expression during treatment can serve as a marker for the efficacy of histone deacetylase inhibitors（HDACi）, and HDAC2 expression levels represent an independent prognostic marker in the clinic.The tumor-suppressive transcription factor p53 is currently the most intensely investigated target protein of HDAC2. Numerous target genes of p53 tetramers regulate growth arrest, senescence, and apoptosis to restrict cellular transformation. In addition, p53 affects metabolic processes and the generation of reactive oxygen species. Most tumors have a defective p53 pathway either due to a loss of TP53（the p53-encoding gene in humans）, mutations in TP53, or aberrations in signaling pathways controlling the levels and activity of the p53 protein. PTMs including phosphorylation, acetylation, ubiquitinylation, and sumoylation affect p53 and regulate its functions in vivo. Interactions of p53 with other transcription factors and regulators also dictate p53 expression and activity.Recent data show a tight link between the functions of p53 and HDAC2. Acetylation is indispensable for proper p53 functions. Several lines of evidence suggest that K320 acetylation is relevant for p53 signaling and that HDAC2 targets this site. We could recently show that HDAC2 deacetylates lysine residue K320 of p53 in colon cancer cells. A sumoylation-dependent interaction of HDAC2 and p53 leads to diminished acetylation of p53^k320, reduced DNA binding of p53, and impaired transcriptional regulation of its target genes. K320 is located in the linker/tetramerization domain of p53. It was accordingly found that the C-terminal region of p53 plus the tetramerization domain including K320 interacted more strongly with HDAC2 than the N-terminal part of the protein. Reducing p53^k320 acetylation located in the DNA binding domain and the p53 tetramer-domain produced negactivity of the p53 tetramer which decreased significantly p53 DNA binding capacity, thus strongly affected the expression of p53 target genes,then resulted in cell abnormal proliferation or activation, and ultimately tumor. But the interaction between HDAC2 and acetylated p53^k320 for the development of ovarian cancer remains unclear.ObjectiveIn this study, we did some research about HDAC2, p53 and acetylation of p53^k320 in cellular level, and the effect of expression of HDAC2 and the interaction of HDAC2 with p53 and acetylated p53^k320 on ovarian cancer cell lines were studied.Materials and methods1.Study objects:Ovarian cancer cell line SKOV3 and OVCAR3, the two strains of cells derived from the research center of the Third Affiliated Hospital of Zhengzhou University, two cell lines were cultured by 1640 culture medium（containing 10% inactivated bovine serum, 100u/ml penicillin, 100mg/L streptomycin） in the culture tank temperature of 37 DEG C and 5% CO2 culture. The cells were divided into SKOV3 blank control group, group SKOV3+HDAC2shRNA1, SKOV3+HDAC2shRNA2, SKOV3+HDAC2shRNA3, OVCAR3 blank control group, group OVCAR3+HDAC2shRNA1, OVCAR3+HDAC2shRNA2, OVCAR3+HDAC2 shRNA3 group.2.Methods:Using plasmid transfection method, shRNA was transfected into the cells in the experimental group to make the expression of HDAC2 be silent and HDAC2 protein and mRNA were tested in Real time PCR and Western blot in 10 groups, Western blot was used to detect the expression of p53, acetylation of p53^k320 protein level and the use of CCK8 on cell viability test.3.Statistical analysis: We used statistical software SPSS17.0 to analysis the data.Using single factor analysis of variance to compare the expression levels of HDAC2 mRNA in different experimental groups, LSD-t was used to compare it between two groups.While Using single factor analysis of variance to compare the expression levels of the protein level of HDAC2、p53、acetylation of p53^k320 in different experimental groups, LSD-t was used to compare it between two groups.significant level α=0.05, the comparision between two groups, significant level α’=α/comparision times=0.0167.Result1. After transfection with 48 h, compared with the blank control group and negative control group, HDAC2shRNA1 group and HDAC2shRNA2 group the expression of HDAC2 was decreased, and the difference was significant（P<0.05）.2.48 hours after transfection, compared with the blank control group and negative control group, the expression of p53 in experimental group increased, the difference is significant（P < 0.05）, at the same time, HDAC2 shRNA groups the expression of acetylated p53^k320 decreased, the blank control group compared with negative control group, the difference was significant（P < 0.05）. the experimental group compared with the blank control group and the negative control group, ovarian cancer cell viability decreased, the difference was significant（P<0.05）.Conclusions1.Downregulation of HDAC2 by shRNA leads to upregulation of the expression of p53 protein and acetylated p53^k320.2.Down regulated HDAC2 expression with shRNA, which significantly inhibited the skov3, ovcar3 cell viability.