Regulation Of P21by Sirna Induced Silencing Of HDAC1Gene In Ovairan Cancer SKOV3/DDP Cells

Ovarian cancer is the highest mortality rates malignancy in woman. Platinum-based combination chemotherapy is a standard treatment for ovarian cancer, but the5-year survival of advanced-stage ovarian cancers remains below in the thirty percent,primarily due to the rapid development of resistance. So a new method which is usedfor reversing drug-resistant tumor has become an important issue.Studies have shown that defects of apoptosis programs in tumor cell may berelated to failure of chemotherapy. Drug-resistant tumor cells may have somemechanism to escape apoptosis, by which we speculated that the imbalance ofapoptosis may be related to drug resistance. p~21was the most an inhibitor ofcyclin-dependent kinases, negative regulation of CDKs factor which can regulate cellcycle process.p~21is required for proper cell cycle progresssion and plays a role in celldeath, DNA repair, senescence and aging, and induced pluripotent stem cellreprogramming. The protein of p~21has a number of protein binding sites: N-terminalbind to and inhibit the expression of Cyclin-CDK; C-terminal bind to PCNA, whichlead to cover some of functional domain of PCNA and affect DNA replication.p21play a role in two ways. p~21was first inentified as a p53-inducible gene, and could beactivated in transcription by p53. Another way is p53-independent mechanism. Atpresent the transcription factor which could activate p21, in addition to p53, includeSp1/Sp3, Smads, Ap2, BRCA1, γ-IFN etc. Therefore, p21, influent cell cycleprogression and thus to regulate cell apoptosis by a variety of ways.Studies havefound that the abnormal transcription of p~21gene or protein of p~21abnormalexpression, cause cell proliferation disorders leading to tumorigenesis. Gene silencing associated with tumorigenesis and drug resistance has recentlybeen an area of intense investigation. Studies have shown that gene silencing havecertain correlation with hypoacetylation of histone proteins. Histone acetylation levelsare mainly regulated by histone acetyltransferase (HATs) and histone deacetylases(HDACs). HATs catalyse the addition of acetyl groups to lysine residues of proteins,and HDAC deacetylate histones, thereby facilitating a closed chromatin structure andhence transcriptional repression. Therefore, HDACIs became one of the mostattractive and promising anti-tumor agents. Several studies have shown that HDACIsstrongly activate the expression of p~21through the p~21promoter, which increasedlevel of p~21protein.At present, cisplatin and taxol is recognized as one of the most effectivetreatment of ovarian cancer chemotherapy drugs. Here, we choose cisplatin-sensitiveSKOV3and their cisplatin-resistant clones SKOV3/DDP as the in vitro model toexamine the regulating of p~21by siRNA induced silencing of HDAC1gene. Expectedresults for HDAC1siRNA can improve sensitivity of ovarian cancer chemotherapy,and paly an important role in reversing ovarian cancer cell lines cisplatin resistance.The experiments as follows:The methods of part I: Cell viability was detected by MTT assay and IC50. Thelevel of HDAC1protein was detected by using immunocytochemical staining inovarian cancer cells. Results: SKOV3/DDP cell resistance index is3.5. The level ofHDAC1expression in SKOV3/DDP cells is higher than in SKOV3cells.The methods of part II: Plasmid was sent to the company sequencing and provedsuccessful extraction of plasmid. The expression level of p21, p53and HDAC1mRNA and protein was analyzed by Real-time RCR and immunocytochemicalstaining. Cell viability was detected by MTT assay.Cell cycle was detected by flowcytometry. Results: Recombinant plasmid was successfully extracted, and can be usedfor transfection. The level of p~21mRNA increased, while p53gene expression leveldid not change after transfection of HDAC1reconversion plasmid; Flow cytometryshows that the cycle of transfected cells was arrested in G0/G1phase.Conclusions: 1. There was over expression of HDAC1in ovarian cancer SKOV3andSKOV3/DDP cells, which may be associated with tumor cell resistance to apoptosis.2. HDAC1siRNA can increase the expression level of p~21mRNA, but not affectp53mRNA, so we conclude p~21regulate the cell cycle progression in p53-ndependent manner and promote cells apoptosis.3. HDAC1siRNA increase the sensitivity of SKOV3/DDP cells to cisplatin, soit have paly a role in reversing multidrug resistance of ovarian cancer cells.