miR-605对非小细胞肺癌细胞放射敏感性的影响

Downregulating miR-605 enhances radiosensitivity of non-small cell lung cancer cells

目的探讨miR-605对非小细胞肺癌(NSCLC)细胞放射敏感性的影响及机制。方法人肺腺癌细胞A549常规培养,使用Lipofectamine 2000转染试剂转染,分为miR-605模拟物组(miR-605 mimic)、miR-605抑制剂组(miR-605 inhibitor)和空白对照组(NULL),通过qRT-PCR实验检测3组细胞miR-605的表达,细胞克隆法检测3组细胞的细胞活力,细胞凋亡实验检测各组细胞凋亡水平;生物信息学算法确定肿瘤坏死因子α诱导蛋白3(tTNFAIP3)是miR-605的潜在靶点,Western blot实验检测各组细胞TNFAIP3蛋白含量;将NFAIP33′-UTR报告质粒(pRL-TNFAIP3)转染进修饰过miR-605的A549细胞,采用双荧光素酶检测系统检测细胞中的荧光素酶活性,并观察其辐射敏感性。结果与空白对照组比较,miR-605模拟物(miR-605 mimic)组中miR-605的表达水平明显升高,miR-605抑制剂(miR-605 inhibitor)组中miR-605的表达水平明显降低,差异有统计意义(P<0.05);细胞克隆法实验结果显示,照射后miR-605模拟物组的细胞活力水平高于空白对照组,miR-605抑制剂组的细胞活力水平低于空白对照组,miR-605模拟物组的细胞凋亡水平低于空对照组,miR-605抑制剂组的细胞凋亡水平高于空白对照组,差异有统计意义(P<0.05);照射后miR-605模拟物组TNFAIP3蛋白表达低于空白对照组(P<0.05),miR-605抑制剂组TNFAIP3蛋白表达高于空白对照组;miR-605模拟物能抑制TNFAIP33′-UTR报告基因的荧光素酶活性,miR-605抑制剂能提高TNFAIP33′-UTR报告基因的荧光素酶活性;照射后(6Gy)pcDNA-TNFAIP3组的细胞活力水平低于空白对照组,差异有统计学意义(P<0.05)。结论下调miR-605后能够增强非小细胞肺癌细胞A549的放射敏感性,其机制可能是上调了TNFAIP3的表达。

Objective To investigate the mechanism by which miR-605 regulates the radiosensitivity of non-small cell lung cancer(NSCLC)cells.Methods Human NSCLC cell line A549 was cultured and transfected with miR-605 mimic(mimic group),or miR-605 inhibitor(inhibitor group)or blank control(blank group)with the help of Lipofectamine 2000 transfection reagent,respectively.The expression of miR-q605 was detected by RT-PCR.Cologenic formation assay was used to measure cell proliferation of three groups.Apoptosis level of different groups was detected by apoptosis experiment.TNFAIP3 was identified as a potential target of miR-605 using bioinformatics algorithm.TNFAIP3 protein expression was detected by Western blot.A549 cells were co-transfected with luciferase reporter vector carrying TNFAIP33′-UTR reporter,with miR-605 mimic,or miR-605 inhibitor or blank.Luciferase activity and radio-sensitivity were examined.Results Compared with the blank control group,miR-605 expression was increased in miR-605 mimic group and decreased in miR-605 inhibitor group(P<0.05).Cologenic assay results showed that cell proliferation of miR-605 mimic group was higher than that of blank control group after irradiation,while cell proliferation was lower in miR-605 inhibitor group than in blank control group(P<0.05).The apoptosis level was lower in miR-605 mimic group in blank control group(P<0.05),while the apoptosis level was higher in miR-605 inhibitor group than that in blank control group(P<0.05).After irradiation,TNFAIP3 protein expression in miR-605 mimic group was lower than that in blank control group(P<0.05),while TNFAIP3 protein expression in miR-605 inhibitor group was higher than that in blank control group(P<0.05).In addition,miR-605 mimic decreased the luciferase activity of cells transfected with TNFAIP33′-UTR reporter gene(P<0.05),while miR-605 inhibitor increased the luciferase activity(P<0.05).After irradiation(6 Gy),cell viability of cells transfected with pcDNA-TNFAIP3 was lower than that of blank control group(P<0.05).Conclusion Downregulating miR-605 can enhance the radio-sensitivity of NSCLC A549 cells.Its mechanism may be related to upregulation of TNFAIP3 expression.