甲基化转移酶抑制剂SGI-1027对肺腺癌细胞迁移侵袭抑制及SPG20基因甲基化调节作用观察

Inhibitory effects of methyltransferase inhibitor SGI-1027 on migration and invasion of lung adenocarcinoma cells and its regulatory effect on methylation level of SPG20 gene

目的分析甲基化转移酶抑制剂SGI-1027对肺腺癌A549细胞迁移、侵袭能力的抑制作用及痉挛性截瘫-20(SPG20)基因甲基化水平的调节作用,以探讨甲基化转移酶抑制剂对肺腺癌细胞迁移、侵袭的影响及机制。方法以不同浓度甲基化转移酶抑制剂SGI-1027作用于肺腺癌A549细胞,MTT法测算细胞增殖活力,筛选得到SGI-1027最佳作用浓度为7.5μmol/L,作为后续实验的作用浓度。将A549细胞分为SGI-1027组和二甲基亚砜(DMSO)组,SGI-1027组培养基中加入SGI-1027培养,对照组培养基中加入DMSO培养。两组培养48 h时,采用划痕愈合实验观察细胞迁移能力,Transwell小室实验观察细胞侵袭能力;采用qRT-PCR法检测细胞DNA甲基转移酶3b(DNMT3b)、SPG20 mRNA,焦磷酸测序法检测细胞SPG20基因甲基化水平,Western blotting法检测细胞DNMT3b、SPG20蛋白。结果培养48 h时,与DMSO组比较,SGI-1027组细胞迁移距离小、穿膜细胞数少(P均<0.05)。与DMSO组比较,SGI-1027组DNMT3b mRNA及蛋白相对表达量下降,SPG20 mRNA及蛋白相对表达量升高(P均<0.05)。SGI-1027组SPG20基因甲基化率较DMSO组降低(P<0.05)。结论甲基化转移酶抑制剂SGI-1027可抑制肺腺癌A549细胞迁移、侵袭能力,降低细胞SPG20基因甲基化水平;SGI-1027可能通过下调细胞内SPG20基因甲基化水平进而抑制肺腺癌细胞的迁移、侵袭能力。

Objective To analyze the inhibitory effects of methyltransferase inhibitor SGI-1027 on the migration and invasion of lung adenocarcinoma A549 cells and its regulatory effect on the methylation level of spastic paraplegia-20(SPG-20)gene,in order to explore the effect and mechanism of methyltransferase inhibitor on the migration and invasion of lung adenocarcinoma cells.Methods Different concentrations of methyltransferase inhibitor SGI-1027 were used to treat lung adenocarcinoma A549 cells,and the cell proliferation activity was measured by MTT method.The optimal concentration of SGI-1027 was determined as 7.5μmol/L,which was used as the concentration for subsequent experiments.A549 cells were divided into the SGI-1027 group and dimethyl sulfoxide(DMSO)group.Cells in the SGI-1027 group were added with SGI-1027 in the medium,and cells in the DMSO group were added with DMSO.After culturing the cells for 48 h,the cell migration ability was observed by scratch healing test,and the cell invasion ability was observed by Transwell chamber test.DNA methyltransferase 3b(DNMT3b)and SPG20 mRNA were detected by qRT-PCR,SPG20 gene methylation levels were detected by pyrosequencing,and DNMT3b and SPG20 proteins were detected by Western blotting.Results At 48 h of culture,compared with the DMSO group,SGI-1027 group had smaller migration distance and fewer transmembrane cells(both P<0.05).Compared with the DMSO group,the relative expression levels of DNMT3b mRNA and protein decreased,while the relative expression levels of SPG20 mRNA and protein increased in the SGI-1027 group(all P<0.05).The methylation rate of SPG20 gene in the SGI-1027 group was lower than that in the DMSO group(P<0.05).Conclusions Methyltransferase inhibitor SGI-1027 can inhibit the migration and invasion of lung adenocarcinoma A549 cells,and reduce the methylation level of SPG20.SGI-1027 may inhibit the migration and invasion of lung adenocarcino‐ma cells by down-regulating the intracellular SPG20 methylation level.