The Effects Of LSD1Gene Down-regulation Induced By SiRNA On The Proliferation And Cell Cycle Of Ovarian Cancer Cells

Background:Tumor morbidity and mortality increased year by year and has become one of themajor challenges of harm to human health[1], ovarian cancer mortality rate ranks firstin gynecological malignancies[2], which is a serious threat to women’s health, thoughthe cytoreductive surgery associate with chemotherapy can effectively improve thesurvival time of patients with ovarian cancer，the advanced five-year survival ratehovers around30%. Therefore, searching for novel cancer therapeutic targets hasbecome an urgent need to reality.The proliferation of tumor cells is an important feature of tumor development[3].Histone methylation is an important epigenetic reversibility, involved in regulation ofchromatin structure and gene expression, which is regulated by histonemethyltransferase (histone methyltransferases, HMTs) and histone demethylationenzymes (histone demethylases, HDMS). Previous study has found that imbalancebetween histone methylation and demethylation was closely related to tumor development[4]. Lysine specific histone demethylase1(LSD1),which is the firsthistone demethylase[5],can regulate many genes transcription and be highly expressedin various tumor tissues[6].It was negatively correlated with tumorprognosis[6].Inhibition of LSD1can inhibit tumor cell proliferation[7],which indicatesthat LSD1plays a role in promoting tumor cell proliferation in the tumor developmentprocess. Because epigenetic modification mechanism can lead to gene silencing butnot accompanied by changes in the gene sequence,so it is important to study it for thetreatment of disease. A variety of histone modifying enzymes has become importanttargets for cancer treatment[8], LSD1is expected to be an new target for anti-tumor.Here,we study the effect of LSD1knockdown induced by the siRNA on proliferationand cell cycle and potential mechanisms in ovarian cancer cells. It maybe provide thetheoretical basis for the new target for ovarian cancer therapy.Objective:To study the effect of LSD1knockdown induced by the siRNA on theproliferation and cell cycle of ovarian cancer cells line and investigate the potentialmechanisms.Methods:1. Screening of ovarian cancer cell line with high expression of LSD1Western blot were used to detect the protein quantity of LSD1in ovarian cancercell lines ES-2, HO-8910, SKOV-3and OVCAR-3.2.Cells transfectionThe siRNA was transfected into ovarian cancer cells by lipofectamine2000.Theexpression of LSD1mRNA and protein were examined by real time fluorescencequantitative PCR(FQ-PCR) and Western blot.3. Effect of LSD1knockdown on the proliferation ability of ovarian cancer cellsThe MTT was performed to detect proliferation ability; The EdU kit was used todetermine the capacity of cellular DNA synthesis．the FCM was performed to detectthe cell cycle distribution.4. Effect of LSD1knockdown on the lever of H3K4me2The Western blot was performed to detect level of H3K4me2after LSD1 knockdown.5. Mechanism of LSD1on proliferation and cell cycleFQ-PCR was used to detet mRNA expression of p21and CCNA2in ovariancancer cells,and then explore the potential mechanisms of LSD1regulation of cellproliferation.6.Statistical analysisAll experiments were performed at least in triplicate and data were compiled fromthree separate experiments. The results were calculated as means±SD. All statisticalanalyses were determined by one-way ANOVA using the SPSS18.0software. AP-value<0.05was considered significant.Results:1.The expression was detected in all of the four ovarian cancer cell line,but thehighest expression of LSD1was in cell line OVCAR-3.2.After transfection of LSD1-siRNA in ovarian cancer OVCAR-3cells, FQ-PCRand Western blot were used to detect the LSD1mRNA and proteinexpression.Compared to the blank control group, The expressions of LSD1mRNAand proteins were all decreased markedly in the LSD1-siRNA transfected cells, theaverage inhibition rates were78.98%and48.72%(P <0.05).3.In the LSD1-siRNA transfection cells, growth of OVCAR-3cells slowed down,DNA synthesis of cells reduced an，the percentage of cells G2/M phase was decreasedand the percentage of cells G2/M phase became higher compared to the blank controlgroup (P <0.05).4. The Western blot results showed that the level of H3K4me2was elevated by1.57-fold compared to the blank control group (P <0.05).5.The FQ-PCR results showed that p21mRNA expression levels was elevated by1.36-fold,however the CCNA2mRNA expression levels was decreased with68.73%of the rate of decline compared to the blank control group (P <0.05).Conclusion:1. After LSD1konkdown, the ovarian cancer cells grew slowly and were arrestedat the G2/M phase. 2. Silencing of LSD1was shown to inhibit ovarian cancer cells growth byregulating the transcription of genes involved in cell proliferation and cell cycle.