The Role Of MiR-125a-5p In Erlotinib Acquired Resistance Of Human Lung Adenocarcinoma Xenografts

Objective:To study the effect of miR-125a-5p overexpression on the erlotinib acquired resistance of human lung adenocarcinoma xenografts in nude mice by constructing animal model,and to investigate the underlying molecular mechanism of miR-125a-5p involved in erlotinib acquired resistance in vivo.Methods:1.Culture of erlotinib acquired drug-resistant cell PC9/ER and resistance detection:PC9and PC9/ER cells were cultured and the inhibition rate of erlotinib on them was detected by CCK-8.2.miR-125a-5p lentiviral transfection of PC9/ER cells and the diffetntial expression detection of miR-125a-5p:PC9/ER cells were infected by lentivirus carrier andthe changes of miR-125a-5p expression in tumor cells were detected by real-time RT-PCR.3.The effect of erlotinib on the nude mouse xenograft growth of PC9,PC9/ER and PC9/ER with mi R-125a-5p lentivirus cells:the nude mouse xenograft models were established and the mice were treated with erlotinib or saline.The wight of nude mice was wighed,and the long and short diameter of tumor were measured every 2 days.Observe the growth state of nude mice and the situation of erlotinib anti-tumor.Nude mice were sacrificed after gavage treatment for 2 weeks and the tumor tissues were dissected.4.The molecular mechanism of miR-125a-5p overexpression and erlotinib inhibiting the growth of nude mice xenografts of PC/ER cells:real-time RT-PCR and Western blot were used to detect the mRNA and related protein expression level of miR-125a-5p,EGFR,Rab25,PARP,Caspase3 and PI3K/AKT signaling pathway.Rab25 protein expression in tumor tissue was deteted by immunohistochemistry.Results:1.PC9 cells showed a short spindle or polygon shape,while PC9/ER cells with erlotinib presented a long spindle shade.After 48 hours of erlotinib treatment,the IC50 of PC9 and PC9/ER cells were(0.16±0.06)μmol/L,(6.14±0.27)μmol/L respectively.The resistance index of PC9/ER was 38.37 times.2.The infection efficiency of lentiviral carrier infecting PC9/ER cells was over 90%.The results showed that miR-125a-5p was stably overexpressed in PC9/ER-mi R-125a-5p cells,and it was significantly higher than PC9,PC9/ER and PC9/ER-Vector cells.3.The tumor formation rate of nude mice was 100%.The tumor growh rate of erlotinib group was slower than that of the saline group after gavage treatment.The tumor volume of saline and erlotinib group in PC9,PC9/ER,PC9/ER-Vector,PC9/ER-miR-125a-5p nude micewere(581.24±48.07),(111.95±54.30),(1099.98±37.92),(450.63±65.75),(1064.55±81.09),(486.21±29.01),(848.21±50.08),(345.90±69.60)mm~3 respectively.Compared with the saline group,the tumor volume of erlotinib group was significantly reduced.Of them,PC9 group had the smallest tumor volume,while the tumor volume of PC9/ER-miR-125a-5p group was smller than PC9/ER and PC9/ER-Vector groups.All xenografts in the saline and erlotinib groups were tumor tissues.4.In tumor tissues of nude mice,overexpression of miR-125a-5p decreased the mRNA expresson level of Rab25,PI3K and AKT,and also decreased the protein expression level of Rab25 and p-AKT,while the level of p-AKT reduced observedly after erlotinib treatment.At the same time,overexpression of miR-125a-5p increased the protein expression level of active Caspase 3,and cleaved PARP protein expression had no change,while active Caspase 3 was cut down and cleaved PARP protein increased after erlotinib treatment.Howerver,miR-125a-5p overexpression had no significant effect on the mRNA and protein expression level of EGFR,PARP and Caspase 3.Conclusion:1.Overexpression of miR-125a-5p can enhance the sensitivity of xenografts bearing PC9/ER cell to erlotinib to some degree.2.miR-125a-5pmayaffectRab25/PI3K/AKTpathwayandexpressionof apoptosis-associated protein to involve the erlotinib acquired resistance of NSCLC.