The Inhibitory Role And Mechinsm Of MiR-32 In NSCLC Growth And Metasiasis By Targeting ITGA6

Objectives:In this study we analyzed the correlation between miR-32 and ITGA6 expression,and the clinicopathological parameters of NSCLC patients.Furthermore,we investigated the role and mechanism of miR-32 in NSCLC growth and metastasis by targeting ITGA6 expression in vitro and in vivo,so as to provide new molecular markers and potential important targets for clinical diagnosis and treatment of NSCLC.Methods:1.Quantitative polymerase chain reaction(qPCR)was used to detect the expression of miR-32 in cancer tissues and paired distal control lung tissues of NSCLC patients And clinicopathological parameters of NSCLC patients were collected.2.NSCLC cells were transiently transfected with miR-32-5p mimics,miR-32-5p inhibitors and pGpU6/GFP/Neo-miR-32 using lipofectamine.The transfection efficiency was evaluated by fluorescence microscopy and qPCR.CCK assay,colony formation assay,scratch assay,and transwell invasion assay were performed to evaluate the influence of miR-32 expression on NSCLC cell proliferation,tumorigenesis,migration and metastasis.3.Bioinformatics analysis software and online database(PITA,RNA22,miRmap,microT,miRanda,picTar,TargetScan)were used to identify candidate target genes.The wild-type and mutant 3'UTR of ITGA6 were constructed.297T cells were co-transfected with-type or mutant 3'UTR of ITGA6,miR-32-5p mimimics or miR-32-5p inhibitors.The luciferase activities of individual cells were detected by dual-luciferase reporter assay system.The expression levels of miR-32 and ITGA6 in A549 and XWLC-05 cells were detected by qPCR and Western blot after transient transfection with miR-32-5p mimics and inhibitors using lipofectamine.We also analyzed the correlation between the expression levels of miR-32 and ITGA6 in NSCLC patients at mRNA level.The expression levels of ITGA6 and ITGB4 in cancer tissues and paired distal control lung tissues of NSCLC patients were detected by immunohistochemistry,and the clinicopathological parameters were collected.MTS assay,soft agar colony formation assay,and transwell migration/invasion assay were used to study the effects of integrin ?6?4 inhibition on proliferation,tumorigenesis,migration and metastasis of NSCLC cells.We then investigated the role of integrin ?6?4 in the growth and metastasis of NSCLC in vivo via subcutaneous transplantation in nude mice and orthotopic lung transplantation in SCID mice.4.GFP-labeled and LUC-labeled A549 cells stably expressing miR-32 and miR-NC were achieved via lentiviral-mediated transduction.The expression levels of miR-32 and ITGA6 were detected by qPCR and Western blot to evaluate the efficiency of miR-32 overexpression and ITGA6 silencing.Nude mice were injected subcutaneously with GFP-labeled A549 cells stably expressing miR-32 and miR-NC.The general situation of the nude mice was observed,and the weight and tumor size of the nude mice were measured.The nude mice were killed up to 22 days after inoculation,and the weight and size of the tumor were measured.Tumor tissues were frozen to detect the expression of mir-32 by qPCR,whereas parts of the tumor tissues were fixed by 10%neutral formaldehyde to carry out HE staining.The effect of stable overexpression of miR-32 on lung metastasis of A549 cells in SCID mice was observed by injecting the luc-labeled miR-32-overexpressed A549 cells into tail vein In vivo imaging of small animals was performed up to 30 days after inoculation.Then mice were sacrificed and lung tissue sections were stained with hematoxylin and eosin.5.The expression levels of miR-32,ITGA6,FAK,pFAK,E-cadherin,and Vimentin were detected by qPCR and Western blot in A549 and XWLC-05 cells after transient transfection of miR-32-5p mimics,miR-32-5p inhibitors and the negative controls using lipofectamine.Furthermore,we preliminarily determined the mechanisms of miR-32 in NSCLC progression using rescue experiments.The expression levels of miR-32,ITGA6,FAK,pFAK,AKT,p-AKT,ERK1/2,p-ERK1/2,E-cadherin,Vimentin and SLUG were detected by qPCR and Western blot.Results:1.The relative expression of miR-32 in 94 NSCLC tumor tissues was 0.79(0.14,1.71),whereas that in paired distal control lung tissues was 1.00(0.37,3.65),the difference was statistically significant(P<0.01).The relative expression of miR-32 in 37 Xuanwei NSCLC tumor tissues was significantly lower than that in paired distal control lung tissues(P<0.05),but had no significant difference compared to non-Xuanwei NSCLC tumor tissues(P>0.05).The expression of miR-32 was not correlated with age,sex,stage,tumor size,lymph node metastasis and histological types(P>0.05).2.qPCR data showed that the expression level of miR-32 in NSCLC cell lines was lower than that in normal bronchial epithelial cells 16HBE,the difference was statistically significant(P<0.01 respectively).The expression levels of miR-32 in A549,XWLC-05,H157 and YTMLC cells transfected with miR-32-5p mimics,pGpU6/GFP/Neo-miR-32 were significantly increased compaired to negative controls(P<0.001 respectively).CCK assay showed that the proliferation rates of A549 and XWLC-05 cells transfected with miR-32-5p mimics were significantly inhibited(P<0.001 respectively),whereas the proliferation rates of A549 and XWLC-05 cells transfected with miR-32-5p inhibitors were significantly higher than those of the negative control inhibitors NC(P<0.001 respectively).Colony formation assay showed that the numbers of clones in A549,XWLC-05 and H157 cells transfected with miR-32-5p mimics were significantly lower than those of the negative control groups(P<0.001 respectively).Scratch test showed that the rates of wound healing in A549,XWLC-05,H157 and YTMLC cells transfected with pGpU6/GFP/Neo-miR-32 were significantly slower than that of negative control groups(P<0.001 respectively).Transwell invasion assay showed that the numbers of invasive cells in A549,XWLC-05,H157 and YTMLC cells transfected with pGpU6/GFP/Neo-miR-32 were significantly higher than that of negative control groups(P<0.001 respectively).3.Four databases(PITA,TargetScan,microanda,microT)predicted that ITGA6 had a binding site of miR-32.Moreover,dual-luciferase reporter assay showed that the luciferase activities of 293T cells co-transfected with ITGA6 3'UTR wild-type plasmid and miR-32-5p mimics were significantly inhibited(P<0.001),whereas the luciferase activities of 293T cells co-transfected with ITGA6 3'UTR mutant plasmid miR-32-5p mimics had no significant change(P>0.05).On the contrary,the luciferase activities of 293T cells co-transfected with ITGA6 3'UTR wild-type plasmid and miR-32-5p inhibitors were significantly increased(P<0.001),qPCR data showed that the expression levels of miR-32 in A549 and XWLC-05 transfected with miR-32-5p mimics were significantly higher than those in negative control groups(P<0.001,P<0.05 respectively),while the expression levels of ITGA6 were significantly lower than those in negative control groups(P<0.01 respectively).The expression levels of miR-32 in A549 and XWLC-05 transfected with miR-32-5p inhibitor were significantly lower than those in negative control groups(P<0.001,P<0.01 respectively),while the expression levels of ITGA6 were significantly higher than those in negative control groups(P<0.001,P<0.05 respectively).Western blot data showed that the expression levels of ITGA6 proteins in A549 and XWLC-05 were lower than those in negative control groups after transfection of miR-32-5p mimics.The relative expression of miR-32 in NSCLC tumor tissues was negatively correlated with ITGA6 expression(r=-0.34,P<0.001).Moreover,the expression levels of integrin ?6?4 in NSCLC tumor tissues was significantly higher than that in paired distal control lung tissues(P<0.05 respectively).The expression levels of integrin?6?4 were closely related to the recurrences of NSCLC patients(P<0.01 respectively),and also dysexpressed in NSCLC cells.The proliferation rates,the numbers of colonies,the migration and invasion rates of H460SM cells which the expression levels of ITGA6 and ITGB4 were inhibited in the experimental group were significantly lower than those in the negative control groups(P<0.05 respectively).The volumes of subcutaneous transplant tumors in ITGA6 and ITGB4 shRNA experimental mice were significantly smaller than those in the negative control groups,and the metastatic rates that developed in the mouse lungs were significantly reduced in ITGA6 and ITGB4 shRNA orthotopic lung cancer SCID mice(P<0.05 respectively).4.The expression levels of miR-32 in the experimental group(A549/LV-miR-32)were significantly higher than those in the control group(A549/LV-miR-NC),while the expression levels of ITGA6 were significantly lower than those in the control group(P<0.001 respectively).Compared with the control groups(A549/LV-miR-NC),the subcutaneous transplanted tumor growth in the experimental mice(A549/LV-miR-32)was significantly inhibited(P<0.01).The weight and volume of transplanted tumor in the experimental mice were lower than those in the control group(P<0.01,P<0.05,respectively).qPCR data showed that the expression levels of mir-32 in the experimental mice were significantly higher than those in the control group(P<0.01).In vivo experiment was performed by injecting cells via the lateral tail vein into SCID mice.Our data showed that the number of pulmonary tumor nodules in control group mice was significantly higher than that in experimental mice(P<0.001).5.The expression levels of ITGA6,pFAK(Y397),pFAK(Y925)and Vimentin in A549 and XWLC-05 cells transiently transfected with miR-32-5p mimics were significantly lower than those in negative control groups,but the expression leves of E-cadherin were significantly higher than those in negative control groups(P<0.05 respectively).On the contrary,the expression levels of ITGA6,pFAK(Y397),pFAK(Y925),and Vimentin in A549 and XWLC-05 cells transfected with miR-32-5p inhibitors were significantly higher than those in negative control groups,while the expression levels of E-cadherin were significantly lower than those in negative control groups(P<0.001,P<0.01 respectively).A549 and XWLC-05 cells transiently co-transfected with miR-NC and overexpression control plasmid exhibited the decreased expression levels of ITGA6,pFAK(Y397),pFAK(Y925),p-AKT and p-ERK1/2,Vimentin and SLUG,but the increased expression levels compared to those co-transfected with miR-NC and overexpression control plasmid(P<0.05 respectively).Furthermore,the inhibition of ITGA6 protein expression caused by miR-32-5p mimics could partially be reversed by ITGA6 overexpression in A549 and XWLC-05 cells,resulting in enhancing the levels of downstream pFAK(Y397),pFAK(Y925),p-AKT(ser473),p-ERK 1/2(pT202/Y204),increasing the expression of Vimentin and SLUG,and decreasing the expression of E-cadherin expression compared with those co-transfected with miR-32-5p mimics and overexpression control plasmid.Conclusions:1.The expression levels of miR-32 were lower in NSCLC tumor tissues,suggested that miR-32 might played an anti-cancer role in the development of NSCLC.2.Overexpresion of miR-32 could inhibit the growth and metastasis of NSCLC.3.Integrin alpha6 beta4 signaling pathway could promote the growth and metastasis of NSCLC.ITGA6 was a novel target gene of miR-32.miR-32 could negatively regulate the expression of ITGA6,and then inhibited the activation of integrin alpha6 beta4 signaling pathway,activated AKT and ERK,decreased SLUG expression,suppressed EMT process,and suppressed invasion and metastasis of NSCLC.