Bladder Cancer Cell-derived Exosomal Microrna-21 Promotes Cancer Metastasis By Polarizing Tumor-associated Macrophages

Objective: Bladder cancer is one of the most common urological malignant tumors.The clinical features are multiple tumors and high recurrence rate.The treatment is based on radical surgery,but it does not bring breakthrough improvement to patients’ survival.The main cause of bladder cancer death is tumor metastasis and recurrence,and the mechanism of onset and recurrence is not fully understood.Exosomes are vesicle-like structures present in multivesicular bodies(MVBs).Because exosomes contain genetically related factors,they can transmit important genetic information to recipient cells,which is important for cell-cell signal transmission.Tumor-associated macrophages(TAMs)are the major components of the tumor microenvironment and form pre-metastatic sites in the microenvironment prior to cancer metastasis,promoting tumor metastasis.However,the detailed mechanism of tumor reconstruction of TAMs has not been clearly defined before bladder cancer metastasis.Methods: Macrophage models were constructed and identified by flow cytometry.Exosomes were collected from conditioned medium of human bladder cancer cells by optimized differential centrifugation.Electron microscopy,Western blot and nanoparticle size tracking analysis were used to detect exosome separation efficiency.Confocal microscopy showed uptake of fluorescently labeled exosomes by macrophages.Q-PCR was used to detect M2 type macrophage-specific gene expression.The effect of macrophages treated with exosomes derived from T24 cells on bladder cancer cell migration was analysed by Transwell assays.The expression levels of endogenous and exosomal miR-21 were examined by Q-PCR,while the expression level of the target protein was analysed by western blot.Luciferase reporter plasmids and its mutant were used to confirm direct targeting.The effect of miR-21 on the migration of bladder cancer cells was analysed by Transwell assays after transfection with miR-21.Protein expression of target protein and possible signaling pathway were analyzed by western blot.Results: In this study,we showed that exosomes derived from bladder cancer cells polarized the M2 phenotype of THP-1 cell-derived macrophages and determined TAM-mediated pro-migration activity.Moreover,we found that miR-21 is highly expressed in exosomes derived from bladder cancer cells as well as in macrophages treated with exosomes.In addition,macrophages transfected with miR-21 showed M2 polarization and promoted the migration of bladder cancer cells.Mechanistically,exosomal miR-21 derived from bladder cancer cells inhibits PTEN activation of the PI3K/AKT signalling pathway in macrophages and enhances the expression of STAT3 to promote M2 phenotypic polarization.Conclusions: Bladder cancer cell-derived exosomes are capable of polarizing TAMs to affect tumor metastasis and enhance the M2 phenotype of macrophages by inhibiting the target gene PTEN by exosomal miR-21.