microRNA启动子区甲基化调控对人肺癌细胞A549增殖、迁移、侵袭的影响

The Effect of Methylation Level of microRNA Promoter on the Expression of microRNAs and on the Proliferation, Migration and Invasion of Lung Cancer Cells

目的研究microRNA启动子区甲基化水平的变化对几种microRNAs(miRNA34a、miRNA34b、miRNA148a、miRNA203a)表达的影响,并进一步研究该甲基化调控对人肺癌细胞A549增殖、迁移以及侵袭能力的影响。方法不同浓度的去甲基化药物5-氮杂-2′-脱氧胞苷(5-Aza-CdR)处理人肺癌细胞A549,分别作用24 h、48 h、72 h,采用CCK8法检测细胞增殖情况,计算增殖抑制率。20μmol/L 5-Aza-CdR处理A549细胞72 h,通过甲基化特异性PCR(MSP)检测A549细胞相关miRNAs启动子甲基化水平的变化;实时荧光定量PCR(real-time PCR)检测A549细胞相关miRNAs表达水平的变化。20μmol/L 5-Aza-CdR处理A549细胞0 h、24 h、48 h,采用划痕实验检测细胞迁移能力(计算细胞在24 h和48 h的划痕愈合率);作用48 h,采用Transwell检测细胞侵袭能力。结果 CCK8结果显示,随着5-Aza-CdR的处理浓度升高、时间延长,A549细胞的增殖抑制率逐渐增加。经5-Aza-CdR去甲基化处理后,MSP结果表明,实验组相对于对照组甲基化引物扩增条带减弱,而非甲基化引物扩增条带增强;Real-time PCR结果显示,相关miRNAs表达水平升高(P<0.05)。由划痕实验和Transwell检测结果揭示,经5-Aza-CdR处理后,A549细胞的生长愈合能力降低(P<0.05),并且侵袭到Transwell小室滤膜下表面的细胞数亦减少(P<0.05)。结论人肺癌细胞A549经5-Aza-CdR去甲基化处理后,miRNAs表达水平升高,进一步抑制肺癌细胞的增殖、迁移以及侵袭能力。

Objective To study the effect of methylation level of microRNA promoter on the expression of microRNAs(miRNA34 a,miRNA34 b,miRNA148 a,miRNA203 a)and on the proliferation,migration and invasion of lung cancer A549 cells.Methods The proliferation of A549 cells treated with different concentrations of demethylated drug 5-aza-2′-deoxycytidine(5-Aza-CdR)was measured by CCK8 assay and calculated the inhibitory rate in 24 h,48 h and 72 h,respectively.After 72 h of treatment with 20μmol/L 5-Aza-CdR,methylation-specific PCR(MSP)was used to detect the methylation level of A549 cells in miRNAs gene promoter regions,and real-time quantitative PCR(real-time PCR)was used to test the expression of miRNAs.The migration abilities of A549 cells treated with 20μmol/L 5-Aza-CdR in 24 h and 48 h were performed with wound healing assay,while the invasion abilities in 48 h were evaluated by Transwell assay,respectively.Results The proliferation inhibition rate of A549 cells gradually increased with the treatment concentration of 5-Aza-CdR increased and the treatment time prolonged.Compared with the control group,the methylated band of the experimental group was weaker and the unmethylated band was stronger,and the miRNAs gene promoter regions methylation level of the experimental group was lower than that of the control group.The expression level of miRNAs was significantly increased in the experimental group(P<0.05).The migration and invasion of the experimental group of A549 cells were inhibited compared with the control group(P<0.05).Conclusion 5-Aza-CdR can reverse methylation levels of miRNAs promoter regions and upregulate the expression level of miRNA34 a,miRNA34 b,miRNA148 a,miRNA203 a,resulting in significantly inhibiting the proliferation,migration and invasion of lung cancer cells.