The Role And Mechanism Of MiRNA-21 In Partial Bladder Outlet Obstruction Of Rats

Background and ObjectiveAfter partial bladder outlet obstruction(pBOO),detrusor is hypertrophic and hyperplastic under the effect of chronic pressure load.Fibroblasts derived from mesenchymal cells are activated which can secrete much extracellular matrix.It will lead to bladder tissue thickness,stiffness,and collagen deposition increased and seriously affect the contractile ability of the bladder detrusor muscle.Even after the patient who are relieved of the obstruction by surgery.Due to the reduced contractility of the detrusor muscle,the patient's postoperative urination symptoms cannot be fully recovered,which seriously affects the patient's quality of life.During the progress of pBOO,many miRNAs were changed in expression and have participated in bladder tissue remodeling.miRNA-21 was involved in the progress of heart,liver,skin scar fibrosis.It has been reported that miRNA-21 was up-regulated in pBOO,but whether it was involved in bladder tissue remodeling after pBOO remains unclear.This study aimed to explore the role and mechanism of miRNA-21 in partial bladder outlet obstruction.Methods(1)The animal model of pBOO rat was established by partial ligation of the bladder neck and urethral transition in 8-week-old Wistar rats,and the obstruction time gradient was set.After the modeled,the weight and morphological changes of bladder tissue after obstruction were observed.The pathological and collagen fibers changes of the bladder were respectively observed by HE and Masson staining.The expression of Col1a1 was detected by real-time fluorescence quantification PCR(RT-qPCR)and the protein expression levels of Col1a1 was detected by Western blot.(2)The expression of miRNA-21 was analyzed by RT-qPCR in the pBOO model.The miRNA-21 target genes were predicted by online miRNA target gene prediction databases(targetscan,miRDB,miRTarBase).The mRNA and protein levels of smad7 were examined by RT-qPCR and Western blot,respectively.And the relationship between miRNA-21 and smad7 was analyzed.(3)Isolated and cultured the primary bladder fibroblasts of six-week-old Wistar rats,and stimulated the primary bladder fibroblasts with TGF-?1 in vitro.After the stimulation,the expression levels of miRNA-21,smad7,Col1a1 were examined.(4)Transferred the miRNA-21 Agomir and Antagomir into primary bladder fibroblasts by the primary cell small nucleic acid transfection reagent reFlect.After overexpression and inhibition of miRNA-21,the expression of miRNA-21 and smad7 and Col1a1 mRNA was analyzed by RT-qPCR,and the expression of smad7 and Col1a1 protein was analyzed by Western blot.Results(1)After the establishment of pBOO model in Wistar rats,the bladder weight and bladder wall thickness increased with the prolongation of obstruction time.HE staining showed that detrusor was mainly hypertrophic,and the longer the obstruction time,the more pronounced the hyperplasia.Masson staining showed that the expression of collagen in bladder tissue was obvious with the extension of the obstruction time.RT-qPCR and Western blot showed that the Col1a1 expression increased after obstruction.(2)RT-qPCR analysis showed that miRNA-21 was highly expressed after obstruction.The miRNA online target gene database predicts that smad7 was the potential target gene of miRNA-21.After obstruction smad7 expression was lowly,which was negatively related to miRNA-21 expression.(3)After primary bladder fibroblasts were stimulated by TGF-?1,the levels of miRNA-21 and Col1a1 were increased while smad7 were decreased.(4)In primary bladder fibroblasts,when overexpression of miR-21,smad7 expression was reduced as well as Cola1 levels increased.what's more,when miRNA-21 was inhibited,smad7 expression was up-regulated and Cola1 was decreased.ConclusionIn Wistar rat pBOO model,the expression of miRNA-21 increased,which was negatively correlated with smad7 expression.Vitro experiments confirmed that miRNA-21 may regulate bladder tissue fibrosis by targeting inhibition of smad7.