miRNA-335基因启动子区甲基化与胃癌细胞不良生物学行为的关系

Relationship between methylation of microRNA-335 and poor biological behaviour in gastric cancer

目的:探讨miR-335基因启动子区异常甲基化状态对胃癌细胞系中miR-335表达水平的影响,以及miR-335基因启动子区甲基化状态对胃癌细胞侵袭,迁移,以及增殖能力的影响。方法:1株永生化胃黏膜上皮细胞系(GES-1)和4株胃癌细胞系(SGC-7901,MKN-45,BGC-823和AGS)。实时荧光定量PCR(qRT-PCR)检测胃癌细胞株miR-335及CRKL的表达水平。甲基化特异性PCR(MSP)方法检测胃癌细胞株miR-335的基因启动子区甲基化状态。应用MTT方法检测恢复miR-335表达对胃癌细胞增殖能力的影响,Transwell侵袭迁移实验及划痕愈合实验分析恢复miR-335表达对胃癌细胞系侵袭及迁移能力的影响。结果:MSP实验结果表明,MKN-45、SGC-7901和BGC-823细胞系均存在基因启动子区异常的高甲基化状态,AGS细胞系基因启动子区亦呈部分高甲基化状态。去甲基药物5-aza-2′-deoxycytidine处理后胃癌细胞miR-335的表达水平可升高2~3倍。Transwell侵袭迁移实验及划痕愈合实验表明miR-335表达水平恢复后SGC-7901细胞的侵袭和迁移能力明显降低。MTT实验结果表明5-aza-2′-deoxycytidine处理后的SGC-7901细胞系与对照组相比,增殖能力显著降低。结论:miR-335启动子区的异常高甲基化状态抑制了miR-335在胃癌细胞系中的表达,恢复miR-335的表达水平可以抑制胃癌细胞的增殖,迁移和侵袭能力。miR-335为胃癌的肿瘤抑制因子。

Objective:To investigate the role of miR-335 methylation and expression in gastric cancer(GC) cell lines,the functional impact of miR-335 ectopic expression on gastric cancer cells invasion,apoptopsis and proliferation.Methods:Relative expression of miR-335 in 4 gastric cancer cell lines was confirmed by real-time quantitative reverse transcriptase-PCR(qRT-PCR) compared with controls.Methylation-specifc PCR(MSP) was used to evaluate the DNA methylation status in the CpG islands upstream of miR-335 in gastric cancer cell lines.Gastric cancer cell lines were treated with 5-aza-2′-deoxycytidine and the effects upon cell proliferation,migration and invasion were investigated.Results:We observed aberrant cancer-specific methylation in the upstream CpG-rich regions of miR-335,which dramatically silenced its transcriptional activity in gastric cancer cell lines.Furthermore,we demonstrated that restoration of miR-335 in GC inhibited tumor cell migration,invasion and proliferation.Conclusion:miR-335 may be silenced by promoter hypermethylation and functions as a tumor suppressor and plays a role in inhibiting tumor cell migration,invasion and proliferation.