miR-30a-5p通过靶向下调ATF1提高非小细胞肺癌A549细胞放射敏感性

miR-30a-5p enhancing the radiosensitivity of A549 NSCLC cell by targeting and down-regulation ATF1

目的:探索miR-30a-5p对非小细胞肺癌(non-small cell lung cancer,NSCLC)A549细胞的放射增敏效应及其相关机制,为提高NSCLC放射敏感性提供理论依据。方法:采用qRT-PCR检测人正常肺上皮细胞株BEAS-2B、人肺癌细胞株A549、H460中miR-30a-5p表达情况;应用miR-30a-5p agomir、antagomir及对照转染A549细胞株,联合放射,检测miR-30a-5p对A549细胞株的放射增敏效应;通过生物信息学预测并筛选miR-30a-5p的靶基因,采用双荧光素酶报告基因检测进行验证;siATF1、miR-30a-5p agomir及siATF1+miR-30a-5p agomir转染A549细胞,通过qRT-PCR、Western Blotting检测靶基因表达与miR-30a-5p的关系,并通过平板克隆形成实验检测其对A549细胞株的放射增敏效应。结果:miR-30a-5p在A549和H460细胞株中表达均低于BEAS-2B细胞株(P<0.001);miR-30a-5p agomir转染联合放射,A549细胞放射生物学参数D_(0)、D_(q)、N较agomir NC组降低(均为P<0.05)。靶基因预测及验证结果显示,miR-30a-5p可以与ATF1的3'UTR特异性结合,ATF1是miR-30a-5p的一个靶基因,过表达miR-30a-5p可下调ATF1表达。siATF1、miR-30a-5p agomir及siATF1+miR-30a-5p agomir转染A549细胞联合照射,A549细胞克隆形成率及放射生物学参数D_(0)、D_(q)、N均低于对照组(均为P<0.05)。结论:miR-30a-5p通过靶向下调ATF1表达,在A549细胞中发挥放射增敏效应。

Objective:To explore the radiosensitizing effect of miR-30a-5p on non-small cell lung cancer(NSCLC)A549 cells and its related mechanism,so as to provide theoretical basis for improving the radiosensitivity of NSCLC.Methods:qRT-PCR was used to evaluate the expression of miR-30a-5p in BEAS-2B normal pulmonary epithelial cell lines and A549,H460 lung cancer cell lines.miR-30a-5p agomir,antagomir,agmir NC and antagomir NC were transfected into A549 cell line.The radiosensitization effect of miR-30a-5p on it was detected after radiation.Bioinformatic analysis was conducted to predict and screen the potential targets for miR-30a-5p.Dual-luciferase report assay was performed to further confirm it.siATF1,miR-30a-5p agomir and siATF1+miR-30a-5p agomir were transfected into A549 cells.The relationship between target gene expression and miR-30a-5p was detected by qRT-PCR and Western Blotting,and the radiosensitizing effect on A549 cell line was detected by plate clone formation assay.Results:miR-30a-5p expression in A549 and H460 were lower than the expression in BEAS-2B cell line(P<0.001).The radiation biological parameters(D_(0),D_(q),N)in miR-30a-5p agomir transfected group were lower than those of agomir NC group after irradiation(P<0.05)in A549 cell line.The results of target gene prediction and verification showed that miR-30a-5p can specifically bind to the 3'UTR of ATF1.ATF1 was a target gene of miR-30a-5p,and overexpression of miR-30a-5p can down-regulate the expression of ATF1.After transfecting with siATF1,miR-30a-5p agomir and siATF1+miR-30a-5p agomir,respectively,the clone formation rate and radiobiological parameters(D_(0),D_(q),N)of A549 cells were lower than those in the control group(P<0.05).Conclusion:miR-30a-5p could enhancing the radiosensitivity of A549 cell by targeting ATF1 and down-regulating its expression.