摘要
目的分析组蛋白去甲基化酶(KDM2B)对卵巢癌细胞骨架排列中的调控作用,并探讨KDM2B作用的可能分子机制。方法以人卵巢癌细胞系HO8910为研究模型,采用慢病毒pLKO.1-puro-shRNA表达系统建立KDM2B稳定敲减细胞系(KDM2B敲减组);采用KDM2B慢病毒质粒包装慢病毒,并感染上述人卵巢癌细胞,建立KDM2B过表达细胞系(KDM2B过表达组),以感染慢病毒空载组的HO8910细胞作为对照组。RT-qPCR和western blot检测各组KDM2B mRNA及蛋白质的表达水平;F-actin荧光染色法检测卵巢癌细胞骨架排列变化;对照组及KDM2B敲减组进行转录组测序分析;RT-qPCR检测黏着斑激酶(FAK)通路上游基因ITGB1、ITGA6 mRNA的表达水平;免疫荧光染色试验检测FAK蛋白的表达水平;使用GEPIA肿瘤数据库分析FAK在卵巢癌组织样本中的表达水平,并分析FAK与KDM2B的相关性。结果RT-qPCR结果证实,与对照组相比,KDM2B敲减组KDM2B mRNA水平降低,KDM2B过表达组KDM2B mRNA水平升高,差异有统计学意义(P<0.05);western blot结果表明,与对照组相比,KDM2B敲减组KDM2B蛋白水平下降,KDM2B过表达组KDM2B蛋白水平上升,差异具有统计学意义(P<0.05)。F-actin荧光染色法结果证实,与对照组相比,KDM2B过表达组F-actin的表达量增加,排列显示相同且规则的方向,而KDM2B敲减组F-actin的表达量降低,且排列无序。转录组测序结果表明,KDM2B调控FAK信号通路。GEPIA肿瘤数据库分析结果显示,FAK在卵巢癌组织中的表达升高,且与KDM2B呈正相关(r=0.37,P<0.01)。结论KDM2B促进F-actin的表达,使卵巢癌细胞骨架排列更加规则有序,其作用机制与FAK通路激活有关。
Objective To investigate the effect of Lysine(K)-specific demethylase 2B(KDM2B)on cytoskeleton rearrangement in ovarian cancer cells,and explore the possible molecular mechanism of KDM2B.Methods Using human ovarian cancer cell line HO8910 as the research model,the KDM2B stable knockdown cell line was established by the lentiviral pLKO.1-puro-shRNA expression system(KDM2B knockdown group),use KDM2B lentiviral plasmid to package lentivirus,infect HO8910 ovarian cancer cells,and establish KDM2B overexpression cell line(KDM2B overexpression group).The expression level of KDM2B was detected by RT-qPCR and western blot.The F-actin fluorescent staining was used to detect cytoskeletal arrangement,transcriptome sequencing analysis of HO8910 control group and knockdown group was conducted by Shanghai Kangcheng Company,use RT-qPCR to detect the mRNA levels of the upstream genes ITGB1 and ITGA6 in the FAK(Focal adhesion kinase,FAK)pathway;the expression of FAK was evaluated by immunofluorescences.The expression levels of FAK in ovarian cancer tissues and the correlation of FAK with KDM2B was analysed via GEPIA tumor database.Results RT-qPCR results confirmed that compared with the control group,the mRNA level of KDM2B was decreased in the knockdown group,and the mRNA level of KDM2B was increased in the overexpression group.The difference was statistically significant(P<0.05).Compared with the control group,the expression of F-actin was increased,and the arrangement displayed more spear-shaped elongation in identical and regular orientation in the KDM2B overexpression group.However,the F-actin in KDM2B knockdown group was decreased,and the F-actin arrangement lost their identical and regular orientation compared to the control group.Moreover,transcriptome sequencing showed that KDM2B activated the FAK signaling pathway.GEPIA database analysis showed that the expression of FAK was increased in ovarian cancer tissues,and was positively correlated with KDM2B(r=0.37,P<0.01).Conclusion KDM2B upregulates the expression of F-actin a
作者
张国平
顾秀玉
翟光华
闫美娜
ZHANG Guoping;GU Xiuyu;ZHAI Guanghua;YAN Meina(Department of Laboratory Medicine,The Affiliated Suzhou Hospital of Nanjing Medical University,North of Suzhou Municipal Hospital,Gusu School,Nanjing Medical University,Suzhou 215008,Jiangsu,China)
出处
《临床检验杂志》
CAS
2021年第12期886-891,共6页
Chinese Journal of Clinical Laboratory Science
基金
江苏省卫健委面上项目(H2019064)
苏州市卫健委“科教兴卫”青年项目(KJXW2019039)。
