Exosomal Micro RNA-9-3p Secreted From BMSCs Downregulates ESM1 To Suppress The Malignant Progression Of Bladder Cancer

Objective:Bladder cancer(BCa)is one of the most common malignant tumors of the urinary system.Previous studies have shown that exosomes derived from bone marrow mesenchymal stem cells(BMSC)are carriers for the transfer of endogenous molecules and play an important role in the occurrence and development of BCa.This study aims to explore the mechanism of miR-9-3p in the malignant progression of BCa in exosomes derived from BMSCs.Methods:Part one:the differentially expressed genes(DEGs)in BCa were screened from gene expression database(GEO)by bioinformatics analysis,and the expression of DEGs was verified by qRT-PCR in BCa tissues and paired normal tissues adjacent to cancer,and the most significant gene ESM1was selected for follow-up study.MiRNAs that may regulate ESM1 were predicted in miRDB、TargetScan and mirDIP,and their expression was verified in BCa cells(UMUC-3,BIU-87,EJ,T24 and 5637)and normal bladder epithelial cells(SV-HUC-1).Part two:The secretion of BMSCs was identified by transmission electron microscope,and the expression of surface marker protein was analyzed by Western blot.Subsequently,the exosomes secreted by BMSCs were co-cultured with UMUC-3 cell,and the uptake of BMSCs derived exosomes by UMUC-3 cell was observed by confocal microscopy.The effects of BMSCs on the proliferation,migration,invasion and apoptosis of BCa cells were investigated by MTT cell proliferation assay,scratch assay,Transwell invasion assay and flow cytometry.By adding DMSO and exosome inhibitor GW4869 into the co culture system,it was verified that the effect of BMSCs on the biological function of BCa cells was exerted by exosomes.Part three:MiR-9-3p mimics was used to overexpress miR-9-3p in BMSCs,the exosomes of BMSCs were isolated and purified,and the expression of miR-9-3p in exosomes was verified by qRT-PCR.After the transfection efficiency was verified,the effect of miR-9-3p overexpression in BMSCs on the biological function of BCa was verified by cell function test.The protein expression levels of proliferation related factors(Ki67 and PCNA)and invasion related factors(MMP-2 and MMP-9)were detected by Western blot.In addition,the expression of miR-9-3p was enhanced and inhibited by miRNA mimics and inhibitors in UMUC-3 cells to explore their effects on the biological function of UMUC-3 cells.Part four:In this part,we knocked down ESM1 in UMUC-3 cells and explored its effect on the biological function of UMUC-3 cells.The binding sites of ESM1 and miR-9-3p were verified by dual luciferase reporter gene analysis.Then,knockdown of ESM1 and transfection of miR-9-3p inhibitors were performed in UMUC-3 cells to explore the effect of si-ESM1+miR-9-3p inhibitors on the biological function of UMUC-3 cells.The effect of knockdown of ESM1 on tumorigenesis was studied by subcutaneous injection of UMUC-3 cells in nude mice.The expression of ESM1 was detected by qRT-PCR,western blot and immunohistochemistry.Immunofluorescence staining was used to detect the expression of invasion related proteins in tumor tissues,and he staining was used to detect liver metastasis.In addition,UMUC-3 cells treated with exo-miR-9-3p for injection were used to study the effects of exo-miR-9-3p on tumor formation related indicators in nude mice.The expression of MMP-2 and MMP-9 was detected by immunofluorescence staining,and the effect of exo-miR-9-3p on liver metastasis was studied by HE staining.Finally,the expression of ESM1 was detected by immunohistochemistry and Western blot.Results:1.Through bioinformatics analysis and RT-PCR technology,it was screened that ESM1 was highly expressed in BCa,and it was predicted that miR-9-3p might be the miRNA that targeted to regulate ESM1.2.Transmission electron microscopy,nanoparticle tracking analysis and western blot analysis confirmed that the secretions of BMSCs were exosomes.Exosomes derived from BMSCs can be taken up by UMUC-3 cells,and inhibit the proliferation,invasion and migration of UMUC-3 cells,and promote cell apoptosis.3.Overexpression of miR-9-3p in BMSCs can significantly inhibit the proliferation,migration and invasion of UMUC-3 cells,and promote cell apoptosis.4.Knockdown of ESM1 in UMUC-3 cells can inhibit the proliferation and migration of UMUC-3 cells,and promote cell apoptosis.The dual luciferase reporter gene verified that ESM1 was the target gene of miR-9-3p.In addition,overexpression of exosomal miR-9-3p or knockdown of ESM1 can inhibit the proliferation,migration and invasion of BCa cells,induce cell apoptosis,and inhibit tumor growth and metastasis in vivo.Conclusion:MiR-9-3p in exosomes secreted by BMSCs may suppress the progression of BCa by down-regulating the expression of ESM1,providing a new therapeutic target for the treatment of BCa.