The Mechanism Of MiR-3682-3p Promoting The Proliferation And Stemness Of Lung Adenocarcinoma

BackgroundsNon-small cell lung cancer,most of which is lung adenocarcinoma,has been a serious threat to people’s health.It have been proved that miRNAs are involved in the proliferation and stemness of lung adenocarcinoma.In this study,we observed that there is higher expression of miR-3682-3p in LUAD cells and tissues,while the higher its expression,the worse the prognosis of LUAD patients.Machanism analyses indicated that miR-3682-3p directly targets ZNF540 and supresses its expression,what’s more,it augments proliferation and stemness of LUAD through a ZNF540-MVP-c-Jun-positive feedback circuit.In conclusion,our studies demonstrated that miR-3682-3p is a significant adverse prognostic factor that promotes the pathogenesis of LUAD.Contents and researchs1.Expression characteristics of miR-3682-3p in lung adenocarcinoma tissues and cells(1)Bioinformatics was used to analyze the expression of miR-3682-3p in lung cancer and lung adenocarcinoma patients in TCGA database.(2)The expression difference of miR-3682-3p between normal human bronchial epithelial cells and lung adenocarcinoma cells was detected by Real-time Quantitative PCR.(3)In situ hybridization was used to determine the differential expression of miR-3682-3p between human lung cancer and adjacent tissues.(4)Analysis of clinicopathological characteristics of miR-3682-3p in patients with lung adenocarcinoma2.miR-3682-3p promoted the proliferation and stemness of lung adenocarcinoma(1)CCK8 and EdU assays were used to detect the effect of miR-3682-3p on the proliferation of lung adenocarcinoma cells.(2)The effects of miR-3682-3p on the stemness of lung adenocarcinoma cells were detected by tumorsphere formation,side population and immunofluorescence assays.(3)The regulation of miR-3682-3p on proliferation and stemness genes was detected by western blotting.(4)The function of miR-3682-3p in vivo growth was detected by subcutaneous tumorigenesis experiment in nude mice,and the effects of miR-3682-3p on tumor tissue of proliferation and stemness genes were detected by immunohistochemistry.3.miR-3682-3p promoted the proliferation and stemness of lung adenocarcinoma by targeting ZNF540.(1)The target genes and binding sites of miR-3682-3p were predicted by data from bioinformatics websites.(2)The regulation of ZNF540 by miR-3682-3p was detected by Real-time quantitative PCR and western blotting;(3)Dual luciferase reporter assays further confirmed that miR-3682-3p targets ZNF540.(4)CCK8 and EdU assays were used to detect the effects of ZNF540 on the proliferation of lung adenocarcinoma cells;(5)The effects of ZNF540 on the stemness of lung adenocarcinoma cells were detected by tumorsphere formation,side population and immunofluorescence assays.(6)Western blotting assays were used to detect the regulatory effect of ZNF540 on proliferation and stem genes,and overexpression of ZNF540 reversed the regulatory effect of miR-3682-3p on proliferation and stemness in lung adenocarcinoma.4.miR-3682-3p promoted the proliferation and stemness of lung adenocarcinoma through ZNF540/MVP/c-Jun pathway(1)Data from Bioinformatics website predicted the interacting proteins of ZNF540;(2)The interaction between ZNF540 and MVP was detected by immunofluorescence and Co-IP assays.(3)The regulatory effects of ZNF540 on MVP was detected by Real-time quantitative PCR and western blotting assays;(4)The interaction between MVP and c-jun and FBXW7 was detected by immunofluorescence and Co-IP assays.(5)The regulatory effects of MVP on c-Jun was detected by Real-time quantitative PCR and western blotting assays;(6)After CHX and MG132 treatment,Co-IP experiment and western blotting assays were used to detect the ubiquitination of FBXW7 recruited by MVP to regulate the expression of c-Jun.(7)The regulatory effect of miR-3682-3p on ZNF540/MVP/c-Jun pathway was detected by western blotting and immunohistochemistry.5.c-Jun binded to the promoter region of miR-3682-3p and promoted its expression(1)The expression level of miR-3682-3p after overexpression of c-jun was detected by Real-time Quantitative PCR;(2)Data from bioinformatics websites were used to predict the transcription factors and binding sites of miR-3682-3p;(3)ChIP,Real-time Quantitative PCR and dual luciferase reporter assays were used to detect the transcriptional binding sites of c-jun to the promoter region of miR-3682-3p;(4)Western blotting assays were used to detect the regulatory effects of knockdown of miR-3682-3p on proliferation and stem genes.Results(1)miR-3682-3p is highly expressed in lung adenocarcinoma tissues and cells,and patients with lung adenocarcinoma with high expression of miR-3682-3p have shorter overall survival time and worse prognosis.(2)miR-3682-3p promotes the proliferation and stemness of lung adenocarcinoma.(3)miR-3682-3p targetes ZNF540 to promotes the proliferation and stemness of lung adenocarcinoma.(4)miR-3682-3p promotes the proliferation and stemness of lung adenocarcinoma through ZNF540/MVP/c-Jun;(5)c-Jun binds to the miR-3682-3p promoter region and promotes its expression.ConclusionmiR-3682-3p promotes the proliferation and stemness of lung adenocarcinoma by regulating ZNF540/MVP/c-Jun feedback loop.