KDM2B Regulates P21 Expression In Ovarian Cancer And Influences Proliferation And Migration Ability

BackgroundOvarian cancer(OC)is a common gynecological malignant tumor.In recent years,although the treatment of OC has been continuously improved,the prognosis of patients is still not satisfactory.Therefore,studying the pathogenesis of OC and finding new targets for early diagnosis and treatment of OC has become an urgent problem to be solved.Lysine-specific Demethylase2B(KDM2B)exerts an epigenetic effect on the expression of its target gene by affecting the methylation status of histones,and promotes cancer in a variety of tumors.P21(p21waf1/Cip1),a key negative regulator of cell cycle progression,is closely related to tumor suppression.Studies have found that KDM2 B can regulate the expression of P21 involved in tumorigenesis,and the relationship between the two in ovarian cancer remains unclear.ObjectiveTo investigate whether KDM2 B gene alteration affects P21 expression in ovarian cancer.the effect of KDM2 B gene alteration on the biological behavior of ovarian cancer A2780/SKOV3 cell line and the effect of KDM2 B gene silencing on ovarian cancer xenograft model in tumor growth,The correlation between KDM2 B and P21 expression was further clarified.Provide a theoretical basis for studying the pathogenesis of ovarian cancer.Method1.Construction of lentiviral vector packaging KDM2 B shRNA and infection of ovarian cancer A2780,SKOV3 cells(shKDM2B infection group),while infecting empty vector lentivirus as negative control(shNC infection group),uninfected A2780,SKOV3 cells as blank control group The puromycin was screened to obtain a stably transfected cell line in which KDM2 B was silenced.Real-time quantitative PCR and Western blotting were used to detect the expression of KDM2 B mRNA and protein,and the expression of P21 mRNA and protein after KDM2 B silencing was detected.The proliferation of A2780 and SKOV3 cells was detected by CCK-8 at 24,48,72 and 96 h.The number of colonie formed by A2780 and SKOV3 cells after 15 days of colony formation assay,scratch test and Transwell chamber assay for 24 and 48 h.A2780/SKOV3 cell migration ability.2.The SKOV3 cell line in the logarithmic growth phase was inoculatedsubcutaneously into BALB/c nude mice according to the experimental group(shKDM2B group)and the control group(shNC group)to establish a subcutaneous transplantation tumor model of ovarian cancer.The tumor growth was observed,the tumor growth curve was drawn,and the expression levels of KDM2 B and P21 in shKDM2 B group and shNC group were detected by real-time fluorescent quantitative PCR and immunohistochemistry.Results1.KDM2 B silenced lentiviral vector was successfully constructed.Compared with the blank control group and shNC group,shRNA targeting KDM2 B gene significantly inhibited the expression of KDM2 B mRNA and protein in ovarian cancer cell line A2780/SKOV3,and the silencing KDM2 B could be up-regulated.The mRNA and protein levels of P21;the shKDM2 B group compared with the blank control group and shNC group,the proliferation activity of A2780/SKOV3 cells decreased at 48,72,96,120h(P<0.05);the number of colony formation of cells decreased(P<0.05);the scratch area mobility of A2780/SKOV3 cells decreased at 24 and 48 h,and the number of transmembrane cells decreased at 24 h(P<0.05).2.The KDM2 B silenced ovarian cancer subcutaneous xenograft model was successfully constructed.The tumor volume was smaller in the shKDM2 B group than in the shNC group within 4 weeks after tumor formation,and the difference was statistically significant(P<0.05).Real-time quantitative PCR showed that the expression of KDM2 B in the experimental group was lowerthan that in the control group(P<0.05).On the contrary,the expression of P21 in the experimental group was increased compared with the control group(P<0.05).The immunohistochemical results showed that the positive rate of KDM2 B expression in the experimental group was 62% lower than that in the control group.The positive rate of p21 expression in the experimental group was also increased by 38% compared with the control group,and the difference was statistically significant(P<0.05).conclusion1.KDM2 B silencing lentiviral vector was successfully constructed and stably expressed in human ovarian cancer cells.Silencing KDM2 B up-regulated the expression of P21 mRNA and protein,and silencing KDM2 B inhibited the proliferation and migration of ovarian cancer cells.2.KDM2 B silenced ovarian cancer subcutaneous xenograft model was successfully constructed.KDM2 B knockdown at the animal level can inhibit the growth of ovarian cancer tumors.P21 expression was up-regulated in KMD2 B knockdown tumors,,further verifying that KDM2 B may regulate P21 expression.