miR-27b-3p调控TMBIM6对A549细胞凋亡影响

Effect of miR-27b-3p on apoptosis of A549 cells regulated by TMBIM6

目的 miR-27b-3p在抑制肺癌细胞的增殖中发挥抑癌基因作用,而跨膜Bax抑制剂的母体6(transmembrane Bax inhibitor motif-containing 6,TMBIM6)作为抗凋亡蛋白发挥促癌作用。miR-27b-3p是否调控TMBIM6参与了肺癌细胞凋亡有待进一步探讨。本研究探讨miR-27b-3p靶向调控TMBIM6对非小细胞癌A549细胞的生物学行为的影响。方法细胞实验:以不同肺癌细胞株A549、H1975、H1650和人正常肺上皮细胞BEAS-2B为研究对象;RT-qPCR检测miR-27b-3p表达,双荧光素酶实验检测miR-27b-3p和TMBIM6的靶向关系;脂质体法向A549细胞转染miR-27b-3p mimics和mimics NC后,平板克隆和MTT法检测A549增殖能力,AnnexinⅤ-FITC/PI双染流式细胞术检测对凋亡的影响。动物实验:采用皮下移植方法建立60只A549裸鼠移植瘤模型,瘤内定点注射miR-27b-3p agomir或者agomir NC,3周后统一处死各处理组裸鼠,测量肿瘤质量,并采用TUNEL法检测肿瘤组织细胞凋亡的情况;蛋白质印迹法检测各组细胞和组织中TMBIM6、calpain-2、caspase-12和Bcl-xL蛋白表达。结果细胞实验:miR-27b-3p表达水平在A549(1.44±0.07)、H1975(2.82±0.08)、H1650(4.04±0.11)和Beas2B细胞(14.44±0.20)中差异有统计学意义,F=2 180.00,P<0.05。与人正常肺上皮细胞相比,A549、H1975和H1650中miR-27b-3p表达水平降低,均P<0.05;且A549细胞中miR-27b-3p表达水平低于H1975和H1650细胞,均P<0.05。双荧光素酶实验结果显示,在TMBIM6野生型3′UTR中,miR-27b-3p mimics组荧光素酶的活力值(0.52±0.07)低于mimics NC组(0.98±0.04),差异有统计学意义,t=15.97,P<0.05;提示TMBIM6是miR-27b-3p的直接靶点。与mimics NC和空白对照组相比,miR-27b-3p mimics转染组细胞的增殖率下降,P<0.05;而凋亡率升高,P<0.05。动物实验:与agomir NC组〔(909.30±8.21)mg〕和空白对照组〔(908.20±10.64)mg〕相比,miR-27b-3p agomir组剥离肿瘤的质量〔(398.70±7.54)mg〕减少,F=1 095.00,P<0.05。TUNEL法检测结果显示,miR-27b-3p agomir组组织中细胞凋亡率〔(67.01±3.48)%〕高于agomir NC〔(12.33±1.36)%〕和空白对照组〔(15.97±0.98)%〕,F=188.40,P<0.05。蛋白质印迹结果表明,在A549细胞或者肿瘤组织中过表达miR-27b-3p,calpain-2和caspase-12表达均升高,均P<0.05;而TMBIM6和Bcl-xL表达均降低,均P<0.05。结论 miR-27b-3p靶向抑制TMBIM6表达,激活calpain-2/caspase-12信号通路促进非小细胞肺癌细胞凋亡。

OBJECTIVE miR-27 b-3 p inhibits the proliferation of lung cancer cells and plays the role of tumor suppressor genes,while TMBIM6 as an anti-apoptotic protein plays a role in promoting cancer in cancer.Whether miR-27 b-3 p regulates TMBIM6 and participates in lung cancer cell apoptosis needs further investigation.This study aimed to investigate the effect of miR-27 b-3 p on the apoptosis of non-small cell lung cancer cell A549 and its related mechanism.METHODS Cell experiment:RT-qPCR was used to detect the expression of miR-27 b-3 p in non-small cell lung cancer cell lines A549,H1975,H1650 and human normal lung epithelial cells BEAS-2 B.Bioinformatics software was used to predict the downstream target genes of miR-27 b-3 p,and the targeting relationship between miR-27 b-3 p and TMBIM6 was detected by luciferase reporter assay.After transfection of miR-27 b-3 p mimics and mimics NC to A549 cells by liposome method,plate cloning and MTT method were used to detect A549 proliferation ability,and AnnexinⅤ-FITC/PI dual staining flow cytometry was used to detect apoptosis rate.Animal experiment:subcutaneous transplantation method was used to establish 60 A549 nude mice transplanted tumor models,intra-tumor injection of miR-27 b-3 p agomir or agomir NC was performed.Three weeks later,nude mice of each treatment group were sacrificed to determine the tumor quality and measure the tumor cell apoptosis.RESULTS Cell experiment:miR-27 b-3 p expression level was statistically different in A549 cells(1.44±0.07),H1975 cells(2.82±0.08),H1650 cells(4.04±0.11)and Beas2 Bcells(14.44±0.20;F=2 180.00,P<0.05).Compared with human normal lung epithelial cells,the expression levels of iR-27 b-3 p were lower in non-small cell lung cancer cell line A549 cells,H1975 cells,H1650 cells(all P<0.05),and miR-27 b-3 p expression level in A549 cells was significantly lower than that of H1975 cellsand H1650 cells(all P<0.05).The results of dual luciferase experiments showed that in TMBIM6 wild type 3′UTR miR-27 b-3 p mimics group luciferase activity value(0.52±0.07)was significantly lower than mimics NC group(0.98±0.04;t=15.97,P<0.05).TMBIM6 was a direct target of miR-27 b-3 p.Compared with the mimics NC group and the blank control group,the cell proliferation rate of miR-27 b-3 p mimics-transfected group decreased significantly,while the apoptosis rate increased(all P<0.05).Animal experiment:Compared with the agomir NC group(909.30±8.21)mg and the blank control group(908.20±10.64)mg,the mass of tumor dissection(398.70±7.54)mg in miR-27 b-3 p agomir group was significantly reduced(F=1095.00,P<0.05).The results of TUNEL method showed that the apoptosis rate in the miR-27 b-3 p agomir group(67.01±3.48)% was significantly higher than that in the agomir NC group(12.33±1.36)% and the blank control group(15.97±0.98)%(F=188.40,P<0.05).Overexpression of miR-27 b-3 p in A549 cells or tumor tissues,calpain-2 and caspase-12 were significantly increased,while TMBIM6 and Bcl-xL expression were significantly reduced(all P<0.05).CONCLUSION MiR-27 b-3 p promotes apoptosis of non-small cell lung cells by activating calpain-2/caspase-12 signaling via targeting TMBIM6.