| Objective: Resistance against epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)is one of the biggest obstacles in clinical practice.In this study,according to a series of experiments,we would to illuminate that downregulation of mi R? 125 a expression may induce the activation of the Rab25 and PI3K/Akt signaling pathway,leading to EGFR-TKI resistance.Methods: 1.Expression of Rab25 and mi R-125a-5p in lung adenocarcinoma and its clinical significance: Using real-time RT-PCR to explore the expression of Rab25 and mi R-125a-5p in 3 pair EGFR-TKIs resistant cells and parents(A549/A549 ER,PC9/PC9 ER,HCC827/HCC827ER)and the correlation of EGFR-TKIs resistance.2.Identifity the relationship of mi R-125 a and Rab25:Two public databases including mi Randa and Target Scan were used to predict the targets with search terms of mi R-125a-5p.Rab25 with high predicitve values was selected as candidate target.Then,to construct overexpression vector for mi R-125a-5p,Rab25 gene 3’UTR luciferase reporter vector and mutational vector.Furthermore,Luciferase assay was adopted to investigate whether Rab25 is a target gene of has-mi R-125a-5p.3.The mchanism of mi R-125 a and Rab25 reverse the acquired resistance of PC9 ER cells in vitro experiments: To establish PC9/ER cell lines for stable expression of sh RNA targeting human Rab25 gene and overexpress has-mi R-125a-5p,and Using CCK-8 assay to detect the proliferation rate and resistant index,flow cytometry was used to deteced cell apoptosis and cell cycle distribution,real time RT-PCR,Western blot to detect the expression of EGFR,Rab25,Akt,Caspase-3,Bcl-2,BAX and PARP m RNA and protein level.Results: 1.In vitro,the expression of Rab25 is statistical higher in EGFR-TKIs resistant cells than in the parents,the expression of mi R-125a-5p is statistical lower in EGFR-TKIs resistant cells than in the parents futhermore,the expression of Rab25 and mi R-125a-5p were associated with resistance.2.Bioinformatic analysis shown that mi R-125a-5p could bond Rab25 in 120-126 nt of3’UTR region.Then,3’-UTR of Rab25 gene,mutated gene and overexpression of has-mi R-125a-5p were successfully cloned into the vector,which were vertified by Gel electrophoresis and DNA sequencing methods.Furthermore,luciferase assay revealed that compare with group 2(Rab25 3’ UTR+mi RNA-NC),the luciferase activity of group 4(Rab25 3’ UTR+mi R-125a-5p)is significant reduction,P=0.0489,the luciferase activity of group 6(Rab25 3’ UTR-MU+ mi R-125a-5p)is significant higher than group 4(Rab25 3’UTR+mi R-125a-5p),P<0.00001,anothers did not find significance difference.Those results indicating that mi R-125a-5p inhibits the activity of luciferase through the binding sites of Rab25 3’UTR region.3.Sh RNA-Rab25 and overexpressed mi R-125a-5p can reduce the resistance index of PC9/ER cells,and reversal the drug resistance.The results of flow cytometry showed Sh RNA-Rab25 and overexpressed mi R-125a-5p enhanced the apoptosis induction activity of erlotinib and elevated levels of cell cycle G0/G1 phase arrest in PC9 ER cell.Sh RNA-Rab25 and overexpressed mi R-125a-5p can down-regulated the expression of Rab25,Bcl-2,PARP,Akt m RNA and protein of PC9 ER,especially the phosphorylation level.However,up-regulated the expression of BAX m RNA and protein level,and C-PARP protein level.Conclusion: 1.In general,Rab25 is statistical high express in EGFR-TKIs resistant cells,however,mi R-125a-5p is statistical high express.The expression of Rab25 and mi R-125a-5p were associated with resistance.2.Rab25 is a target gene of has-mi R-125a-5p.In post-transcriptional regulation,mi R-125a-5p inhibits the biological function through the binding sites of Rab25 3’UTR region.3.has-mi R-125a-5p can reverse the acquired resistance of PC9/ER cells,which would associated with induce cell apoptosis,inhibit the expressin of Rab25 and the downstream PI3K/Akt signaling pathway activation. |
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