MiR-195 Regulates Lung Adenocarcinoma MTAs Chemoresistance And Metastasis Through TUBB And BIRC5

Background:Lung cancer is the most common type of cancer and is the leading cause of cancer-related deaths worldwide.Non-small cell lung cancer(NSCLC)is the predominant pathological type of lung cancer,comprising about 85%of lung cancer cases.To date,commendable advancements in lung cancer treatment have been made.However,the efficacy of treatment for NSCLC remains unfavorable.Therefore,a comprehensive understanding of NSCLC pathogenesis may shed lights on the identification of potential targets for new treatment strategies.Dysregulated microRNAs(miRNAs)have been reported to act as oncogene or tumor suppressor gene in NSCLC.miR-195 had previously been shown to regulate microtubule-targeting agents(MTAs)related chemoresistance of NSCLC.In present study,a preliminary result showed that tubulin beta class I(TUBB)expression is significantly down-regulated with forced overexpression of miR-195 in NSCLC cell lines.Moreover,we identified a binding site for miR-195 in the 3’ untranslated region(3’UTR)of TUBB by sequence analysis.In addition,miR-195 had been shown to regulate apoptosis and senescence in NSCLC by targeting baculoviral IAP repeat containing 5(BIRC5).Survivin,encoded by the BIRC5 gene,has been shown to regulate the migration and invasion in various cancer types.However,the role of survivin in regulating the metastasis of NSCLC cells hasn’t been fully elucidated.Objectives:1.Explore the effect and mechanism of miR-195/TUBB axis in MTAs chemoresistance of lung adenocarcinoma.2.Explore the effect and mechanism of miR-195/BIRC5 axis in lung adenocarcinoma metastasis in vitro and further validate the role of miR195 in lung adenocarcinoma metastasis in vivo.Methods and results:1.miR-195/TUBB axis and lung adenocarcinoma MTAs chemoresistance:(1)Gene array analysis showed that TUBB was downregulated in miR-195 overexpressed lung adenocarcinomas;the bioinformatics showed that the 3’UTR of TUBB mRNA possessed pairing bases with miR-195 seed region.(2)The cancer genome atlas(TCGA)data analysis showed that TUBB expression was significantly increased in lung adenocarcinoma and lung squamous carcinoma tissues and was negatively correlated with miR195 in lung adenocarcinoma patients.(3)By luciferase reporter assay,we verified that miR-195 directly targeted the 3 ’UTR of TUBB mRNA and inhibited the expression of TUBB mRNA and protein.(4)By cell viability assay,we found that overexpression of TUBB enhanced drug response of lung adenocarcinoma cells to paclitaxel or eribulin,while TUBB knockdown sensitized lung adenocarcinoma cells to paclitaxel or eribulin.(5)Tubulin Immunofluorescence staining showed that miR-195 and TUBB had no effect on microtubule structure of lung adenocarcinoma cells and also had no influence to microtubule destruction of paclitaxel or eribulin.(6)Survival analysis showed that overexpression of TUBB was correlated with worse overall survival,worse chemotherapy response and worse recurrence-free survival in lung adenocarcinoma patients.2.miR-195/BIRC5 axis and lung adenocarcinoma metastasis:(1)Migration and invasion assays showed that miR-195 overexpression significantly impaired migration and invasion of lung adenocarcinoma cells,which,inversely,enhanced by miR-195 knockdown.(2)Migration,invasion and chemotaxis assays showed that BIRC5 knockdown inhibited migration and invasion of lung adenocarcinoma cells;while BIRC5 overexpression was verified to reverse the inhibition effect of miR-195.(3)By E-cadherin and vimentin expression analysis,we verified that miR-195 had no effect on epithelial-mesenchymal transition(EMT)of lung adenocarcinoma cells.(4)By constructing mouse experimental model of lung metastasis,we observed that miR-195 overexpression significantly decreased the ratio of lung metastasis.(5)TCGA data analysis showed that there was no correlation between miR-195/BIRC5 expression and tumor stage both in lung adenocarcinoma and lung squamous carcinoma.Conclusion:Our study demonstrates that:1.miR-195/TUBB axis sensitizes lung adenocarcinoma cells to MTAs.2.miR-195/BIRC5 axis inhibits migration and invasion of lung adenocarcinoma cells in vitro and miR-195 inhibits metastasis of lung adenocarcinoma cells in vivo.