miR-329通过负性调节KDM1A抑制胃癌细胞增殖及侵袭的机制研究

The inhibitory mechanism of mir-329 inhibiting the proliferation and migration of gastric cancer cells by negatively KDM1A

目的探讨miR-329通过负性调节KDM1A抑制胃癌细胞增殖及侵袭的机制。方法人胃腺癌细胞系(BGC-823)分为BGC-823癌细胞组、miR-329 mimics组、miR-329 inhibitor组,MTT测定BGC-823胃腺癌细胞增殖情况,结晶紫染色测定癌细胞单克隆形成数目,流式细胞仪测定胃癌细胞凋亡情况及细胞周期,Transwell细胞迁移实验测定胃癌细胞侵袭情况,RT-PCR法测定胃腺癌细胞miR-329、KDM1A mRNA水平,WEST-BLOT法测定癌细胞Caspase-3、Caspase-6、Caspase-9蛋白表达水平。结果miR-329 mimics组OD值、存活率、克隆形成数目、穿膜数低于BGC-823癌细胞组(P<0.05),miR-329 inhibitor组OD值、存活率、克隆形成数目、穿膜数高于BGC-823癌细胞组和miR-329 mimics组(P<0.05)。miR-329 mimics组细胞凋亡率、G1期、Caspase-3、Caspase-6、Caspase-9蛋白表达水平高于BGC-823癌细胞组(P<0.05),miR-329 inhibitor组细胞凋亡率、G1期、Caspase-3、Caspase-6、Caspase-9蛋白表达水平低于BGC-823癌细胞组和miR-329 mimics组(P<0.05)。miR-329 mimics组miR-329 mRNA表达水平高于BGC-823癌细胞组,KDM1A mRNA蛋白表达水平低于BGC-823癌细胞组(P<0.05),miR-329 inhibitor组miR-329 mRNA表达水平低于BGC-823癌细胞组和miR-329 mimics组,KDM1A mRNA蛋白表达水平高于BGC-823癌细胞组和miR-329 mimics组(P<0.05)。miRNA-329与KDM1A呈负相关,与Caspase-3、Caspase-6、Caspase-9呈正相关(P<0.05)。结论miR-329可抑制胃癌细胞增殖及侵袭,其机制与miR-329可能通过抑制KDM1A的表达导致Caspase-3、Caspase-6、Caspase-9高表达有关。

Objective To investigate the inhibitory mechanism of mir-329 inhibiting the proliferation and migration of gastric cancer cells by negatively regulating lysine specific demethylase-1(LSD1/KDM1A).Methods Human gastric cancer cell line(BGC-823)was divided into BGC-823 cancer cell group,miR-329 mimics group,and miR-329 inhibitor group.Then the proliferation rate,the number of monoclonal formation,apoptosis rate,cell cycle,migration,and expression levels of miR-329 mRNA and KDM1A,as well as the expression levels of caspase-3,caspase-6 and caspase-9 were detected by MTT assay,crystal violet staining,flow cytometry,Transwell assay,RT-PCR,and Western Blot,respectively.Results The OD value,survival rate,number of clone formation and transmembrane number ranking among the three groups in a descending order were miR-329 inhibitor group,BGC-823 cancer cell group,and miR-329 mimics group,with signficant difference(P<0.05).The apoptosis rate,the cells arrested at G1 phase,and expression levels of caspase-3,caspase-6 and caspase-9 protein ranking in an ascending order were miR-329 inhibitor group,BGC-823 cancer cell group,and miR-329 mimics group,with signficant difference(P<0.05).The expression levels of miR-329 mRNA ranking in an ascending order were miR-329 inhibitor group,BGC-823 cancer cell group,and miR-329 mimics group,with signficant difference(P<0.05).The expression levels of KDM1A mRNA ranking among three groups in a descending order were miR-329 inhibitor group,BGC-823 cancer cell group,and miR-329 mimics group,with signficant difference(P<0.05).The expression levels of miRNA-329 were negatively correlated with those of KDM1A,however,which were positively correlated with those of caspase-3,caspase-6 and caspase-9(P<0.05).Conclusion Mir-329 can inhibit the proliferation and migration of gastric cancer cells,and its action mechanism may be related to the high expressions of caspase-3,caspase-6 and caspase-9 caused by the inhibitory effects of miR-329 on KDM1A.