Study On The Role And Mechanism Of MicroRNA-383 In Regulating Cisplatin Sensitivity In Non-small Cell Lung Cancer Cells

According to the world health organization(WHO),lung cancer has the highest morbidity and mortality worldwide and is the leading cause of death among male cancer patients.According to the classification of pathological tissue,lung cancer includes squamous cell carcinoma,adenocarcinoma,adenosquamous carcinoma,undifferentiated carcinoma,carcinoid and bronchial adenocarcinoma.However,due to the different manifestations of malignancy,it is used to be roughly divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC)according to their malignant degree in clinical,in which non-small cell lung cancer accounts for 85% of cases.In recent decades,a variety of treatments for NSCLC have been developed,including surgical resection,radiotherapy,chemotherapy,targeted therapy,and combined immunotherapy.Among them,chemotherapy is the main treatment method for tumors of all stages.Platinum-based drugs are the basic drugs for chemotherapy.However,due to the individual differences of patients and the heterogeneity of tumor cells,some patients will develop multidrug resistance(MDR)after chemotherapy,which is an urgent problem to be solved in clinical practice.MicroRNAs(miRNAs)are small non-coding RNAs,usually containing 18-22 nucleic acids,which can directly bind to the 3’-UTR of target mRNA and play important roles in regulating of gene expression.Nearly 50% of human genes are regulated by miRNAs,and they are involved in the development of many human diseases including cancer.In recent years,MicroRNA-383(miR-383)has shown abnormally expressed in various cancers such as malignant melanoma,colorectal cancer,hepatocellular carcinoma,and glioma.Increasing evidences indicate that miR-383 is involved in various cellular biological processes such as cell proliferation,apoptosis,invasion and metastasis.Although downstream targets such as CCND1,LDHA,VEGF,and IGF have been confirmed,the role of miR-383 in tumor multidrug resistance is still unclear.RBM24(RNA Binding Motif Protein 24)is a post-transcriptional regulatory factor,which belongs to the RNA-binding protein family,and plays an important role in regulating RNA metabolism and post-transcriptional expression.Its post-transcriptional regulation function mainly affects the target genes expression by regulating the target mRNAs stability or their alternative splicing.In this study,we used epidemiological control and cohort studies in clinical samples to investigate the correlation between the miR-383,RBM24 expression and the poor prognosis in patients with lung adenocarcinoma.Also,we used methods like bioinformatics analysis,lentiviral stable overexpression,si RNA/miRNA transient transfection and plasmidtransient overexpression to explore the mechanism of mi R-383 regulating RBM24 expression,as well as the mechanism of miR-383-RBM24 signal axis promoted drug resistance.The main experimental results are as follows:(1)miR-383 is down-regulated in NSCLC clinical tissue samples,and its low expression is associated with the poor prognosis of patients;compared to lung epithelial cells,miR-383 is down-regulated in NSCLC cell lines and cisplatin-resistant A549 cell(A549/CDDP)is further down-regulated than the parental A549 cells.(2)Stable overexpression of miR-383 in parental A549 cells and A549/CDDP cells enhances cells sensitivity to cisplatin treatment.(3)miR-383 targets the 3’-UTR of RBM24 mRNA;RBM24 mRNA is up-regulated in clinical tissue samples of NSCLC.In cellular level and in clinical samples.It is confirmed in cell lines and clinical samples that the expression of miR-383 is negatively correlated with the expression of RBM24 mRNA.(4)The expression of RBM24 protein is up-regulated in clinical tissue samples of NSCLC and the high expression of RBM24 is related to the poor prognosis of patients.(5)Compared with A549 cells,RBM24 protein is up-regulated in A549/CDDP cells;silencing RBM24 in A549/CDDP cells enhances the sensitivity of cells to cisplatin;on the contrary,stability overexpression of RBM24 or inhibition of miR-383 expression in A549 cells can reduce the sensitivity of cells to cisplatin.While silencing the expression of RBM24 in A549 cells can neutralize the cisplatin insensitivity effect which caused by the suppression of miR-383.(6)In both parent A549 cells and drug-resistant A549/CDDP cells,overexpression of miR-383 or silencing RBM24 expression can down-regulate the amount of phosphorylated p65 and phosphorylated IκBα,which inhibited the NF-κB signaling pathway and thereby preventing the expression of anti-apoptotic protein Bcl-2 and Bcl-xL.In miR-383 stably overexpressed A549 cells,the recovery of RBM24 expression can significantly increase the amount of phosphorylated p65 and IκBα protein,and the expressions of anti-apoptotic proteins Bcl-2 and Bcl-xL also increased accordingly.Based on our results,we propose the miR-383-RBM24-NF-κB signaling axis,which affects the sensitivity of NSCLC cells to cisplatin treatment by regulating the expression of RBM24.This topic deeply studies the role and mechanism of miR-383 in cisplatin resistance of NSCLC,expands the theoretical basis of multidrug resistance of NSCLC.The research results suggest that restoration of mi R-383 or interference of RBM24 may provide potential therapeutic approaches for reversing the chemotherapy resistance of NSCLC.