Cigarette Smoke Promote Chronic Obstructive Pulmonary Disease(COPD)through The MiR-130a/WNT1 Axis

BackgroundCigarette smoke(CS)is a crucial factor in chronic obstructive pulmonary disease(COPD).Wnt/?-catenin signaling deregulation may further contribute to COPD progression.The deregulation and dysfunction of microRNAs(miRNAs)in COPD have been reported.Investigating the deregulated miRNAs and their potential role in COPD progression may provide novel strategies for COPD treatment.ObjectiveTo explore the miRNA related to the pathogenesis of COPD,the human bronchial epithelial cells(BEAS-2B)were treated with cigarette smoke extract(CSE).It was concluded that miR-130a plays an important role in COPD.We investigated the relationship between miR-130a and Wnt/?-catenin pathway.Thepurpose of this study was to explore the mechanism of chronic obstructive pulmonary disease(COPD)induced by cigarette smoke by affecting miRNA and downstream signaling pathways,and to provide theoretieal basis for prevention and treatment of COPD induced by smoking.MethodsCell experiments(1)GSE 44531 was downloaded from NCBI GEO website,and the changes of differential expression of miRNAs in non-disease and COPD samples were analyzed.Ten miRNAs,related to COPD were selected to treat BEAS-2B cells with cigarette smoke extract(CSE)and compared with those treated with Phosphate buffer saline(PBS).The expressions of these 10 kinds of miRNA in two groups of cells were confirmed by RT-PCR assay,and the most up-regulated miRNA is miR-130a.(2)The cells were treated with PBS and CSE respectively and transfected with miR-130a mimics,miR-130a inhibitor and corresponding negative control.The effects of over-expression or inhibition of miR-130a on cell proliferation and migration were investigated by MTT assay and scratch test.(3)The cells were divided into 4 groups and transfected with mimics-NC,miR-130a mimics,inhibitor-NC,miR-130a inhibitor.The expression of three important proteins in the Wnt/?-catenin pathway,including WNT1,?-catenin and LEFI,were detected by immunoblotting.Then the cells were treated with PBS and CSE respectively and transfected with miR-130a inhibitor and inhibitor-NC.MNT1,?-catenin and LEF1 protein were detected by immunoblotting.(4)We searched the sequence of human target genes in GenBank and predict the binding site of miR-130a and WNT1 3'UTR.The binding site was mutated.The HEK 293 cells were divided into group wt-WNT1(wild group)and group mut-WNT1(mutation group).And the luciferase reporting experiment was carried out to verify the binding site of miR-130a and WNT1 mRNA.It was further verified by RIP test that miR-130a down-regulated WNT1 through this binding site.(5)To further explore and verify the dynamic role of miR-130a and Wnt/?-catenin signals in CSE-induced cell damage,CSE-treated BEAS-2B cells were co-transfected with si-WNT1/si-NC and miR-130a inhibitor/inhibitor-NC at the same time,and the proliferation and migration ability of the cells were detected by MTT assay and scratch test.Animal experimentsThe mice model of emphysema induced by cigarette smoke exposure was established.The mice were given specific anti miR-130a or PBS into nasal cavity.After the mice were killed,the lung tissue was taken to make paraffin fixed sections for HE staining and morphological observation.The expression of miR-130a and Wntl mRNA in the lung tissue was detected by PCR,and the other lung was lavaged.The bronchoalveolar lavage(BAL)was detected by flow cytometry(FCM)for total cells,macrophages,neutrophils,dendritic cells,CD4+T cells and CD8+T cells counts.Results(1)Screening and verification of miRNA related to COPD:the up-regulation of expression of miR-13Oa was the highest in the cells treated with CSE.(2)miR-130a inhibitor could alleviate the inhibitory effect of CSE on migration and proliferation of BEAS-2B cells.(3)The overexpression of miR-130a could block Wnt/?-catenin signalling pathway.Inhibition of miR-130a could reduce the inhibitory effect of CSE on Wnt/?-Catenin pathway.(4)It was predicted that there was binding site between miR-130a and WNTI 3'UTR,and the luciferase activity in group wt-WNT1 3'UTR could be significantly inhibited by the overexpression of miR-130a.After inhibiting miR-130a,luciferase activity of reporter vector was increased,and the change of luciferase activity in mutated group was cancelled.Rip test showed that the expression of WNT1 in group miR-130a mimies was significantly higher than that in group mimics-NC.Therefore,miR-130a regulates WNT1 mRNA through expected binding sites.(5)WNT1 silencing can inhibit cell migration and proliferation,and miR-130a inhibitor can significantly promote cell migration and proliferation.WNT1 silencing can partially reverse the effect of miR-130a inhibitor on cell migration and proliferation.(6)The expression of miR-130a in group CS was significantly higher than that in group Air,and the expression of Wnt1 mRNA in group CS was significantly lower than that in group Air.There was a negative correlation between the expression of miR-130a and Wntl mRNA in BAL.(7)Exposure to CS significantly increased the number of total cells,macrophages,neutrophils,dendritic cells,CD4+ T cells and CD8+ T cells in BAL.The use of miR-130a inhibitor significantly reduced the increase of the above-mentioned cells in BAL induced by CS.ConclusionIn this study,both cellular and animal experiments were carried out to explore and verify that cigarette smoke can inhibit the Wnt/?-catenin signaling pathway by up-regulating miRNA-130a targeting on WNT1,and thus inhibit the repair of lung tissue.MIR-130a/WNT1 axis may be a new target for the treatment of CS-induced COPD.