| Objective:Lung cancer is one of the most common malignant tumors leading to human death.Although significant progress in the early diagnosis and treatment of lung cancer has been made in lung cancer,the 5-year overall survival rate is still relatively low duelargely to the easy metastasis of lung cancer.Since epithelial-mesenchymal transition(EMT)is highly correlated with the ability of cancer cell invasion and metastasis,it has been given important prognostic significance in a variety of tumors.Although smoking has been identified as a cause of lung cancer,how cigarette smoke causes malignant cell transformation,especially the role of epigenetic modification,remains unclear.In this study,high throughput sequencing and bioinformatics analysis were used to screen and identify differentially expressed microRNAs(miRNAs)associated with smoking and lung cancer metastasis.The target genes of miRNAs related to metastasis were predicted and screened through databases,and the function of the target gene of miRNAs in smoking-induced lung cancer and relevant regulatory mechanisms were further explored.The purpose of this study is to provide theoretical basis for the understanding of the pathogenesis of lung cancer and its precise treatment.METHODS:(1)Validation of malignant extent and EMT characteristics of cells.The malignant grade of cigarette smoke-induced malignant transformation cells was verified by soft agar cloning and nude mice tumorigenesis.The ability of cell migration was tested by transwell cell migration assay and wound healing assay.The expression of EMT-related biomarkers,including E-cadherin(CDH1),N-cadherin(CDH2),matrix metalloproteinase 9(MMP9)and vimentin(VIM)was detected by qPCR and western blotting.(2)Screening of the key EMT-related miRNAs and their target genes.High-throughput sequencing was performed to analyze differentially expressed genes(DEGs)and differentially expressed miRNAs between normal passage cells(BEAS-2B,2B)and cigarette smoke-exposed cells(S30).Biological functions and pathways of DEGs were analyzed by function and KEGG pathway enrichment.The expression level of miRNA-106b and miRNA-93(miR-106b/miR-93)in cigarette smoke-exposed cells with different passengers and lung cancer tissues was verified by qPCR.Based on the analysis of lung cancer related data in TCGA database,the correlation between miR-106b/miR-93 and smoking history,metastasis or other clinical information was analyzed;To verify the effect of miR-106b/miR-93 on cell migration,miRNA mimics and inhibitors were transfected to overexpress and knockdown corresponding miRNAs on 2B and S30 cells.the target genes of miR-106b/miR-93 were predicted by miRanda,miDB and miRTarBase databases,and intersect with the significant downregulation of genes in S30 cells;Further analysis of the correlation between target genes and relevant clinical information based on TCGA database,the lung cancer metastasis-related target genes were selected for further analysis;qPCR and Western blot were used to verify the expression of target genes in cigarette smoke-exposed cells and lung cancer tissues.(3)In order to verify the targeting relationship between miR-106b/miR-93 and target genes,we analyzed the correlation between the expression of miRNA-106 b/miRNA-93 and SMAD7 in lung cancer based on TCGA database.Furthermore,change in the expression of SMAD7 and TGF-PRI in transfected cells were detected.Finally,we constructed plasmids containing wild-type and mutant 3'UTR of SMAD7,and verified the targeting regulatory relations of miR-106b/miR-93 by Dual luciferase reporter assay.The prognostic significant in lung cancer patients with or without smoking history was analyzed based on KMplot database by R packages "survminer" and "survival";The single gene enrichment of target gene was further analyzed base on the LUAD and LUSC datasets of TCGA database by GSEA enrichment tool,so as to analyze the possible signal pathways that target gene may affect.Western blot and immunofluorescence assay were used to verify the differential expression of target genes in cigarette smoke-exposed cells.RESULTS:(1)Compared with control BEAS-2B cells(2B),the clony formation rate of S30 cell was significantly increased(t=-12.125,P<0.001).Tumor formation in nude mice showed that S30 cells had strong tumorigenicity,and transwell migration assay and cell wound healing assay results showed that a increased migration ability of S30 cells.qPCR and Western blot experiments showed that CDH1 expression is significantly decreased in cigarette smoke-exposed cells(F=24.604 and 26.462,both P<0.001),while CDH2,MMP9 and VIM significantly increased(qPCR:F=9.926,23.258 and 48.098,all P<0.01;western blot:F=80.512,17.284 and 20.680,all P<0.01).The result indicated that the epithelial-mesenchymal transition of BEAS-2B is gradually enhanced during long-term exposure to cigarette smoke.(2)A total of 755 differentially expressed genes(DEGs)and 203 differentially expressed miRNAs were obtained by high-throughput mRNA and miRNA sequencing.Function and pathway enrichment revealed that DEGs were significantly enriched in multiple EMT-related functions and signaling pathways.Overexpression of miR-106b/microR-93 could significantly enhance the cell migration,and expression level of CDH1(P<0.05),while CDH2 and MMP9 were significantly down-regulated(P<0.05).In addition,the cell migration ability was obviously decreased after knocking down the expression of miR-106b/miR-93,and CDH1 was significantly increased(P<0.05),while CDH2 and MMP9 were significantly decreased(P<0.05).Suggesting that miR-106b/miR-93 can affect cell migration and EMT.The tumor metastasis related target gene SMAD7 was further screened by bioinformatics.The results of qPCR and Western blot showed the significantly down-regulation of SMAD7 in the late stage of exposure(S20 and S30)(P<0.05).Furthermore,the mRNA expression level of SMAD7 was significantly lower in cancer tissues than normal adjacent tissues(t=4.483,P=0.001).Suggesting that miR-106b/miR-93 and its target gene SMAD7 were associated with increased migration ability and EMT activity after malignant transformation.(3)Correlation analysis based on the data from TCGA database showed that the expression of miR-106b/miR-93 in lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)was negatively correlated with SMAD7(LUAD:r=-0.2027,-0.1708,all P<0.001;LUSC:r=-0.2976,0.4036,all P<0.001).The expression of SMAD7 decreased significantly after overexpressing miR-106b/miR-93(t=17.162 and 7.677,P<0.001 and P=0.002).While the expression of SMAD7 increased after knocking down miR-106b/miR-93(t=-4.847 and-12.139,P=0.04 and 0.007).Furthermore,the luciferase activity of SMAD7 decreased significantly after transfecting of mimics(t=27.614 and 20.712,all P<0.01).Survival analysis showed that SMAD7 with high expression had a better prognosis in non-smoking patients(P<0.0001);GSEA enrichment analysis indicated that SMAD7 was involved in TGF-? signaling pathway(NES=2.117,FDR q-value=0.007),tight junctions(NES=1.935,FDR q-value=0.037)and some other signaling pathways.In lung squamous cell carcinoma,SMAD7 are significantly associated with cell adhesion molecules(NES=1.913,FDR q-value=0.050),focal adhesion(NES=1.878,FDR q-value=0.063)and some other signaling pathways;TGF-?RI protein expression was significantly different in cigarette smoke-exposed cells(F=15.385,P<0.001),and was significantly up-regulated in the late stage of cigarette smoke exposure(P<0.01).Conclusion:Long-term cigarette smoke exposure can induce the malignant transformation of BEAS-2B,including tumorigenicity,increased migration ability and epithelial-mesenchymal transition(EMT).miR-106b/miR-93 were significantly up-regulated in smoking-induced malignant transformed cells and lung cancer tissues,while the target gene SMAD7 was significantly decreased.Moreover,miR-106b/miR-93 and SMAD7 were closely related to tumor metastasis;further functional analysis found that SMAD7 was a better prognostic marker in the NSCLC patients of non-smokers.SMAD7 is significantly correlated with several signaling pathways related to tumorigenesis and development. |
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