Roles And Molecular Mechanism Of MiR-1293 In Promoting Non-small Cell Lung Cancer Cisplatin Resistance

Objective To study the relative expression of miR-1293 in non-small cell lung cancer（NSCLC）cell line A549 and cisplatin resistant cell line A549/DDP,and study the role and molecular mechanism of miR-1293 in the process of cisplatin resistance in NSCLC.Methods Cisplatin induced lung cancer drug-resistant cell lines A549/DDP cells were established,IC50 values were detected by MTT assay,and drug resistance index was calculated.A549 and A549/DDP cell were transfected with Mir-1293 inhibitor（miR-1293 inhibitor）and negative control RNA（miR-NC）by lipofection transfection.Flow cytometry method was used to measure cell apoptosis ability.Dual-luciferase reporter assay was used to clear whether RUNX3 was the target gene of miR-1293.The m RNA expressions of miR-1293 and RUNX3 were detected by q PCR assay.Western Blot was used to detect the protein expressions of MDR1/ABCB1,MRP1/ABCC1,Bcl-2,RUNX3,Akt1 and P-Akt.Results A549/DDP cell lines were established and the resistance index to cisplatin was7.23.The expression level of miR-1293 in drug-resistant cell line A549/DDP（3.89±0.08）was significantly higher than that in drug-resistant cell line A549（1.01±0.05）.The expression of RUNX3 m RNA level and protein level in drug-resistant cell line A549/DDP were significantly lower than that in drug-resistant cell line A549(t_{m RNA}=7.74,P<0.05;t _protein=20.04,P<0.01).Compared with miR-NC group,IC50value of miR-1293 inhibitor group was significantly decreased（t=94.49,P<0.05）.The protein expression levels of MDR1/ABCB1 and MRP1/ABCC1 in miR-1293 inhibitor group were significantly lower than those in miR-NC group(t _MDR1/ABCB1=10.61,P<0.05;t _MRP1/ABCC1=39.57,P<0.05).Meanwhile,the apoptosis level of miR-1293inhibitor group was significantly higher than that of miR-NC group（t=7.15,P<0.05）.The expression of BCL2-Associated X protein（Bax）was increased in the miR-1293inhibitor group（t=33.63,P<0.05）,while apoptosis inhibitor Bcl-2 decreased significantly（t=11.62,P<0.05）.The dual-luciferase reporter assay showed that luciferase activity in the wild-type RUNX3 3’UTR+miR-1293 group was significantly lower than that in the other groups,while the luciferase activity in the mutant RUNX33’UTR+miR-1293 group was not significantly different from that in the control group.Results of WB showed that RUNX3 protein expression of miR-1293 inhibitor group was much higher than that of miR-NC group（t=17.75,P<0.05）;The expression levels of Akt and P-Akt were significantly lower than those of miR-NC group(t _Akt=67.55,P<0.05;t_p-Akt=16.98,P<0.05).Conclusions Mi R-1293 promotes the cisplatin resistance in non-small cell lung cancer,and its mechanism is:miR-1293 targets Runx3,activates Akt signaling pathway,and inhibits A549 cell apoptosis,thereby promoting A549/DDP cell resistance.