miR-1293促进非小细胞肺癌耐药分子机制探讨

目的microRNA与非小细胞肺癌耐药性密切相关,但miR-1293在非小细胞肺癌中的作用机制仍不清楚。本研究探讨miR-1293调控Akt信号通路促进非小细胞肺癌耐药的分子机制。方法培养人肺癌A549与耐药细胞株A549/DDP,RT-qPCR检测miR-1293和抑癌基因RUNX3 mRNA表达,蛋白质印迹检测RUNX3蛋白表达。采用脂质体法将miR-1293抑制物(miR-1293inhibitor)和阴性对照RNA(miR-NC)转染A549/DDP。MTT检测细胞IC50;流式细胞仪检测细胞凋亡;蛋白质印迹检测凋亡相关蛋白Bcl-2和RUNX3;荧光素酶报告基因检测miR-1293能否调控RUNX3;qPCR检测miR-1293和RUNX3的mRNA表达;蛋白质印迹检测MDR1/ABCB1、ABCC1、RUNX3、Akt和p-Akt蛋白表达。结果A549/DDP细胞对DDP的耐药指数为7.23。miR-1293在耐药细胞株A549/DDP的表达(3.89±0.08,W=0.946,P=0.698)与A549组(1.01±0.05,W=0.938,P=0.622)相比升高,差异有统计学意义,t=74.78,P<0.001;而RUNX3 mRNA和RUNX3蛋白在耐药细胞株A549/DDP的表达与A549组相比降低,tmRNA=7.74,t蛋白=20.04,均P<0.001。与miR-NC比较,miR-1293 inhibitor组IC50降低,t=94.49,P<0.001。miR-1293 inhibitor组MDR1/ABCB1、ABCC1蛋白表达低于miR-NC,tMDR1/ABCB1=10.61,tABCC1=39.57,均P<0.001。与miR-NC比较,miR-1293 inhibitor组细胞凋亡增加,t=7.15,P<0.001。促凋亡蛋白RUNX3在miR-1293 inhibitor组表达更高,t=33.63,P<0.001;凋亡抑制蛋白Bcl-2表达更低,t=11.62,P<0.001。荧光素酶报告实验显示,在野生型RUNX33′UTR中,miR-1293 inhibitor的荧光素酶活性(6.24±0.04,W=0.911,P=0.399)高于miR-NC(3.21±0.07,W=0.879,P=0.221),t=92.06,P<0.001;提示RUNX3是miR-1293靶基因。miR-1293 inhibitor组RUNX3蛋白表达高于miR-NC,t=17.75,P<0.001;Akt1、p-Akt低于miR-NC组,tAkt1=67.55,tp-Akt=16.98,均P<0.001。结论在耐药细胞株A549/DDP中miR-1293高表达,抑制细胞凋亡,促进细胞耐药,其机制可能与靶向下调RUNX3,促进Akt信号通路激活有关。

Effect of miR-1293 on drug resistance of non-small cell lung cancer

OBJECTIVE MicroRNA is closely related to drug resistance in non-small cell lung cancer,and the role of miR-1293 in non-small cell lung cancer remains unclear.This study aimed to investigate the molecular mechanism of miR-1293 regulating Akt signaling pathway to promote drug resistance in non-small cell lung cance.METHODS A549 and DDP-resistant cell line A549/DDP were used.The mRNA expression of miR-1293 and tumor suppressor gene RUNX3 in A549 and drug-resistant A549/DDP cells was detected by RT-qPCR,and the expression of RUNX3 protein was detected by Western Blot.The miR-1293 inhibitor(miR-1293 inhibitor)and the negative control RNA(miR-NC)were transfected into A549/DDP using the liposome method.The half-inhibitory concentration(IC50)was tested by the MTT assay.Cell apoptosis was detected by flow cytometry;The expression of apoptosis-related protein Bcl-2,RUNX3 was detected by Western Blot.The information website predicted that RUNX33′UTR has a sequence that binds to miR-1293.Dual luciferase reporter gene assay was used to verify the interaction between miR-1293 and RUNX3;the mRNA expression miR-1293 and RUNX3 was detected by RT-qPCR;the protein expression of MDR1/ABCB1,MRP1/ABCC,RUNX3,Akt and p-Akt was detected by Western Blot.RESULTS The resistance index of A549/DDP cells to DDP was7.23.The expression of miR-1293 in drug-resistant cell line A549/DDP(3.89±0.08,W=0.946,P=0.698)was significantly higher than that in A549 group(1.01±0.05,W=0.938,P=0.622;t=74.78,P<0.001).The expression of RUNX3 mRNA and RUNX3 protein was significantly lower in the drug-resistant cell line A549/DDP than in the A549 group(tmRNA=7.74,P<0.001;tprotein=20.04,P<0.001).Compared with miR-NC,IC50 was significantly lower in miR-1293 inhibitor group(t=94.49,P<0.001).The protein expression of MDR1/ABCB1 and ABCC1 in miR-1293 inhibitor group was significantly lower than that of miR-NC(tMDR1/ABCB1=10.61,P<0.001;tABCC1=39.57,P<0.001).Compared with miR-NC,the apoptosis was significantly increased in the miR-1293 inhibitor group(t=7.15,P<0.001),the proapoptotic protein RUNX3 expression was higher in the miR-1293 inhibitor group(t=33.63,P<0.001),and apoptosis inhibition Protein Bcl-2 expression was lower in the miR-1293 inhibitor group(t=11.62,P<0.001).Luciferase reporter assay showed that the luciferase activity of miR-1293 inhibitor(6.24±0.04,W=0.911,P=0.399)was higher than that of miR-NC(3.21±0.07,W=0.879,P=0.221;t=92.06,P<0.001)in wild-type RUNX33′UTR.Indicating miR-1293 can be combined with RUNX33′UTR.The protein expression of RUNX3 in miR-1293 inhibitor group was significantly higher than that in miR-NC(t=17.75,P<0.001);the protein expression of Akt1 and p-Akt was significantly lower than that in miR-NC group(tAkt1=67.55,P<0.001;tp-Akt=16.98,P<0.001).CONCLUSION High expression of miR-1293 in drug-resistant cell line A549/DDP inhibits apoptosis and promotes cell resistance may be related to down-regulation of RUNX3 and activation of Akt signaling pathway.