Mesenchymal Stem Cell-derived Exosomal MiR-30b-3p Regulates Cholesterol Metabolism Via MAPK/ERK Signaling Pathway By Targeting CYP7A1 In Renal Cell Carcinoma

Objective: To explore the role of bone marrow mesenchymal stem cell(BMSC)derived exosome in proliferation and migration of renal cancer cell,and to investigate the effect of mBMSC-derived exosomal miRNA on lipid metabolism of renal cancer cell and its mechanism.Method: The exosomes of mBMSCs were extracted and identified by transmission electron microscope.Exosome uptake test was used to validate that mBMSC-derived exosome(mBMSCs-Exo)could be absorbed by Renca cells.Edu assay was used to investigate the proliferation of renal cancer cells.Flow cytometry was used to investigate the apoptosis and cell cycle of renal cancer cells.Transwell assay was used to explore the migration ability of renal cancer cells.Liquid chromatography mass spectrometry(LC MS/MS)was used to identify the differential metabolites.Total cholesterol assay kit was used to evaluate the changes in cholesterol level.Western blot and RT-q PCR were used to investigate the expression of key enzymes of cholesterol metabolism as well as markers of MAPK/ERK pathway.Online databases were used to predict potential miRNAs that could negatively target CYP7A1.RT-q PCR was then used to detect the expression of miRNAs in mBMSC-derived exosome.Renca cell was co-cultured with mBMSC,and the effect of mBMSC-drived exosomal miR-30b-3p on the proliferation,apoptosis and migration of Renca cell was evaluated using methods mentioned above.Result: The results of transmission electron microscope showed that mBMSCs-Exo was round with double-layer vesicle structure;the diameter of mBMSCs-Exo was about 96.23nm;the results of Western blot showed that the surface marker proteins of CD9 and CD63 were positively-expressed,which indicated that mBMSCs-Exo was successfully isolated.Exosome uptake experiments showed that mBMSCs-Exo could be absorbed by Renca cells.Moreover,mBMSCs-Exo could promote the proliferation and migration of Renca cells and reduce their apoptosis in a concentration dependent manner.LC-MS showed that compared with control group,tauroursodeoxycholic acid decreased and cholesterol increased significantly in Renca cells after mBMSC-Exo treatment.WB and RT-q PCR showed that compared with the control group,the expression of CYP7A1 and CYP7B1 in mBMSCs-Exo treatment group was lower,while HMGCR expression was higher,indicating that mBMSCs-Exo may have an impact on cholesterol metabolism as well as proliferation and migration via regulating the expressions of key enzymes HMGCR,CYP7A1 and CYP7B1.In addition,knockdown of CYP7A1 could inhibit the apoptosis and promote the proliferation and migration of renal cancel cells,while overexpression of CYP7A1 had the opposite effect.CYP7A1 could also regulate cholesterol metabolism via MAPK/ERK pathway.The result of online database prediction showed that 99 miRNAs were predicted to negatively regulate CYP7A1.Among them,miR-30b-3p was found to be up-regulated in mBMSC-Exos and mBMSC-Exo-treated renal cancer cells.Conclusion: mBMSCs-Exo derived miR-30b-3p could regulate Renca cell cholesterol metabolism through CYP7A1 and MAPK/ERK signaling pathway,and therefore affects the development of renal cell carcinoma.