Study On The Mechanism Of Abnormal Expression Of MiR-137 In The Drug Resistance Of Non-small Cell Lung Cancer

BackgroundNon-small cell lung cancer(NSCLC)is the first cause of cancer related death in the world,which occupies 85% of the lung cancers;surgical resection and adjuvant chemotherapy have been reported to improve survival of NSCLC,however,the relapse after surgery is still as high as 20–70%.Most of the NSCLC patients showed chemotherapy resistance after several circles of chemotherapy,although some of them were sensitive to some chemotherapy regimen at the initial stage.Biomarkers to predict the relapse and drug resistance could be extremely useful for a clinical doctor to monitor high risk patients and select rational regimen.Micro-RNAs(miRNAs)are small non-coding RNAs(22–24 nucleotides in length)that negatively regulate RNA translation to protein.miRNAs are essential for regulating cell proliferation,differentiation and maintenance of stemness.Furthermore,miRNAs can be measured from formalin-fixed paraffin-embedded samples and blood samples,which can be easily obtained using non-invasive methods.Mitchell et al.showed that miRNAs are also present in human plasma in a markedly stable form that is protected from endogenous RNase activity.Tumor-derived miRNAs in the serum or plasma are an important method for the blood-based detection of human cancer.Wei et al.found that plasma microRNA-21 could be used as a biomarker for early detection and chemosensitivity in non-small cell lung cancer.Since miRNAs play an important role in lung cancer and detection samples are relatively easy to be obtained,miRNAs could become a promising means of comprehending the oncogenesis and pathogenesis of lung cancer.ObjetivesIn this study,we explored miR-137’s role in the chemosensitivity of lung cancer..We investigate the expression of in tumor tissues and their matched normal tissues,as well as lung cancer A549 cells and their resistant cells strains: A549/Paclitaxel(A549/PTX)and A549/Cisplatin(A549/CDDP)by qReal-Time PCR.We aimed to find the impact of overexpressio or repression of miR-137 on cell proliferation,migration,cell survival and cell cycle in lung cancer cells.We use TargetScan search program to predict targets of miR-137.In addition,we further investigate the role of miR-137 in chemotherapy resistance.Materials and methods 1.Clinical specimens50 fresh tumor samples and 48 paired normal tissue samples were obtained from NSCLC patients who underwent complete surgical resection in the First Affiliated Hospital of Nanjing Medical University,Jiangsu(China),with written informed consent of patients.A part of each tissue sample was immediately snapfrozen in liquid nitrogen while the other part was fixed in formalin for histological examination.All samples were histologically classified and graded according to TNM stage by a blinded clinical pathologist.All experimental protocols were approved by the Institutional Review Committee of the first affiliated hospital of Nanjing Medical University,Nanjing(China).2.Methods 1).The expression level of miR137 in lung cancer tissues and 3 human lung cell lines(A549,A549 / PTX,A549 / CDDP)were examined by Taqman real-time PCR analysis.2).CCK8(Cell Counting Kit-8)assays were conducted to examine the role of miR-137 in the proliferation of lung cancer cells.3).Transwell assays were performed in lung cancer cell lines to determine whether miR-137 could regulate human lung cancer cell migration and invasion.4).Flow cytometry was executed to determine the effects of miR-137 on cell cycle alterations.5).Luciferase reporter assays were performed to further confmn that NUCKS1 is a direct target for these miR-137.6).NUCKS1 expression in lung cancer cells(A549,A549 / PTX,A549 / CDDP)was analyzed by Western blot.7).Ectopic transplantation model of human lung cancer in nude mice was employed to investigate the role of miR-137 in tumor growth in vivo.All experiments were performed three times and data were analyzed with GraphPad Prism 5(La Jolla,CA,USA).The correlation between miR-137 expression and NUCKS1 levels in glioma tissues were analyzed using Spearman’s rank test.Statistical evaluation for data analysis was determined by t-test.The differences were considered to be statistically significant at P< 0.05.Results 1.MiR-137 expression was downregulated in lung cancer tissues compared with adjacent normal tissues.The expression levels of miR-137 in high grade tumors(WHO Grades II and III-IV)showed significantly difference to those in low grade tumors(WHO Grade I)2.A549/PTX and A549/CDDP show stronger activity of proliferation and invasion,lower apoptosis activity as well as stronger activity of cell cycle progression when compared with A549 cells.3.Repression of miR-137 in A549 cells signifcantly promoted cell growth,migration and invasion capability,cell survival and cell cycle G1/S transition.4.Overexpression of miR-137 in A549/PTX and A549/CDDP cells inhibited cell proliferation,migration and invasion capability,induced cell apoptosis and arrest the cell cycle in G1 phase.5.Luciferase activities were significantly reduced in those cells transfected with the wild sequence and miR-137,and significantly induced in those cells transfected with the wild sequence and miR-137 inhibitor.The expression of NUCKS1 protein was downregulated in miR-137 treated cells,but increased in cells transfected with miR-137 inhibitor.6.miR-137 enhances lung cancer paclitaxel and cisplatin sensitivity in nude mice.Moreover,the tumors from A549/PTX/miR-137 plus PTX and A549/CDDP/miR-137 plus CDDP showed an decreased VEGF positive staining ConclusionsmiR-137 was down-regulated in lung cancer tissues and resistant cancer cells strains.Repression of miR-137 in A549 cells signifcantly promoted cell growth,invasion,cell survival and cell cycle G1/S transition.NUCKS1 contains a binding site for miR-137 at 3’-UTR and was a direct target of it.miR-137 inhibited cell malignant behaviors by negatively regulating NUCKS1 and its downstream PI3K/AKT pathways.MiR-137 causes a decrease in tumor volume and weight,enhances lung cancer paclitaxel and cisplatin sensitivity in nude mice.In summary,we have identified a link between miR-137,NUCKS1 and chemoresistance that is a novel constituent of lung cancer tumorigenesis.miR-137 restoration approach may offer a new modulation strategy to overcome chemoresistance.