The Expression And Regulation Mechanisms Of Exosometransmitted MicroRNA-133b In Bladder Cancer

Background: Bladder Cancer(BC)is the ninth most common tumor in the world and one of the most common malignant tumors of the urinary system.In the past three decades,the diagnosis,treatment and 5-year survival rate of BC have not improved.Therefore,it is the key to study the pathogenesis of BC,providing new targets and new ideas for clinical diagnosis and treatment.Exosome is a kind of extracellular vesicles secreted by most cells.It plays an important role in cell-to-cell information exchange and is closely related to tumorigenesis,invasion and metastasis.Compared with normal cells,tumor cells secrete more exosomes,which can be used as effective markers for diagnosis and monitoring of prognosis.In recent years,it has been found that exosomes produced by BC cells can regulate cell pathways and affect tumor development.Micro RNAs(miRNAs)are a class of important,highly conserved,non-coding smallmolecule single-stranded RNAs that have been shown to be closely related to tumorigenesis and development.Mature miRNAs are widely involved in the regulation of gene expression by binding to the complementary sequence of the target messenger RNA,and play a role similar to oncogenes or tumor suppressor genes.At the same time,studies have confirmed that miRNAs metastasize from cancer cells to normal cells in the tumor microenvironment through exosomal pathways,causing the latter to proliferate abnormally and become cancerous.miR-133b(micro RNA-133b)is first discovered in cardiomyocytes,and studies have found that its abnormal expression in tumors such as kidney cancer,breast cancer,and prostate cancer,which is closely related to tumor progression.Some studies reported that miR-133 b expression is reduced in bladder cancer,but whether it can play a role through exosome pathway and the underlying mechanism has not been studied.Objective: To study the expression pattern of exosome-derived miR-133 b in BC;To clarify the effects of miR-133 b overexpression and exosome-derived miR-133 b on BC cell proliferation,apoptosis,and growth of nude mice transplanted tumors;To explore the role of exosome-derived miR-133 b in the development of BC.Methods:(1)Quantitative Real-time PCR(q RT-PCR)to verify the expression of miR-133 b in tissues and serum exosomes,as well as the stability in exosomes;(2)KaplanMeier Survival curve was used to analyze the relationship between miR-133 b expression and survival time in patients with BC;(3)Cell supernatants were treated with RNase alone or in combination with Triton X-100 to analyze the main ways of miR-133b;(4)PKH67 fluorescently labeled exosomes were traced,to figure out cellular sublocalization;(5)Transfection of miR-133 b mimics to obtain BC cell lines with high expression of miR-133 b and exosomes with high expression of miR-133b(miR-133b-EXO),clone formation and apoptosis experiments were used to analyze the biological function of exosomal-derived miR-133 b in BC;(6)agomir-miR-133 b reagent and miR-133b-EXO injection in nude mice transplant tumors,to figure out the effects of tumor growth;(7)Target genes that can interact with miR-133 b were predicted by bioinformatics;(8)q RT-PCR to detect target gene expression in BC cancer tissues and adjacent tissues;(9)q RT-PCR and Western blot to explore the effects of miR-133 b on target gene m RNAs and protein expression.Results:(1)The expression of miR-133 b in BC cancer tissues was significantly lower than that in adjacent tissues,P <0.05.(2)The expression of exosomal miR-133 b in serum of BC patients was significantly down-regulated,and the expression was stable,and did not change significantly with changes in temperature.(3)The survival curve showed that the overall survival time of BC patients with low expression of miR-133 b was significantly shortened,P <0.01.(4)When treated with RNase alone,the level of miR-133 b remained almost unchanged,but when treated with RNase and Triton X-100 at the same time,the level of miR-133 b was significantly reduced,indicating that miR-133 b was mainly coated on the membrane structure.(5)PKH67 showed that exosomes are mainly localized in the cytoplasm after co-culture of exosomes and cells.(6)miR-133 b mimics transfected into BC cells or fusion culture of miR-133b-EXO and BC cell,both can inhibit cell proliferation,reduce the ability of cell clone formation,and induce BC cell apoptosis in vitro.(7)agomir-miR-133 b and miR-133b-EXO injection in nude mice transplant tumors can inhibit tumors growth in vivo.(8)miR-133 b was compatible with two adjacent sequences in the Dual-specific protein phosphatase 1(DUSP1)promoter.(9)The expression of DUSP1 m RNA in BC cancer tissues was significantly lower than that in adjacent tissues,P <0.05.(10)miR-133 b mimics transfected BC cells or fusion culture of miR-133b-EXO and BC cells,the expression of DUSP1 m RNA and protein decreased.(11)Injection of agomir-miR-133 b reagent or miR-133b-EXO in nude mice transplanted tumor tissue,DUSP1 m RNA and protein expression also decreased.Conclusion: miR-133 b was lower expressed in BC tissues and is positively correlated with overall survival time.The expression of exosome-derived miR-133 b was significantly down-regulated in the serum of BC patients.Exosomal miR-133 b could inhibit BC cell proliferation,promote apoptosis,and affect the formation of xenograft tumors in nude mice.Further research found that miR-133 b can regulate the expression of DUSP1 through exosomes,thereby inhibiting the development of BC.The study provided a possible pathogenesis of BC from the exosome pathway,and also provided new targets for the diagnosis and treatment of BC by exosomes targeting miRNAs.