The Mechanism Of Mir-219c-5p Mediated FN1 In Neurogenic Bladder Fibrosis Caused By Multiple Sclerosis

Background and purposeMultiple sclerosis(MS)is a chronic autoimmune central nervous system disease.It is mainly caused by multiple inflammatory changes in the white matter of the nervous system.It is mainly manifested by focal inflammatory infiltration,demyelination of nerve cells,nerve.Axonal injury,glial fibrosis,and calcification is the second leading causes of neurogenic bladder.According to literature statistics,more than 75%of MS patients may have different degrees of urinary system symptoms.Mild patients show frequent urination and urgency,while severe patients show urinary retention,hydronephrosis,and bladder fibrosis,which seriously affects patients.Physical and mental health.The molecular mechanisms of MS-induced neurogenic bladder fibrosis have not been fully elucidated.As far as the current methods of diagnosing and treating MS-induced neurogenic bladder,previous treatment methods such as the use of drugs that inhibit excessive bladder activity to control the involuntary contraction of bladder smooth muscle,although there are certain effects in the short term,but the long-term effect is not good,And bladder smooth muscle cells continued to fibrous degeneration.As a major protein in the extracellular matrix,fibronectin 1(FN1)may be involved in bladder fibrosis.A lot of literature has confirmed that miRNA is closely related to the occurrence and development of fibrosis diseases.Therefore,this study intends to construct an experimental autoimmune encephalomyelitis(EAE)model to simulate the occurrence and progress of MS disease.The EAE model was used to explore the molecular mechanism of miRNA-mediated FN1 synthesis and participate in neurogenic bladder fibrosis induced by MS,to find targets for molecular targeted therapy,and to provide new therapeutic direction for controlling neurogenic bladder fibrosis.Method1.Forty C57BL/6J female 7-week-old mice weighing 20-22 g.They were randomly divided into EAE group and NC group,of which 30 were in EAE group and 10 in NC group,and clinical scores(Clinical score,CS)were performed according to the severity of neurological symptoms in mice.2.After the clinical symptoms of the mice are stable,divide the mass into CS1,CS2,CS3,and NC groups according to clinical score(CS).Select 3 mice from each group and observe in a metabolic cage for 24 hours.Single micturition volume and mictourition time of the mice was recorded.The mice were subsequently weighed,and the bladder tissue was taken after the mice were sacrificed by isoflurane anesthesia and the wet weight of the mice was measured in the empty state of the bladder.3.The mouse bladder tissue was stained with HE and Masson.4.Use a database such as Target Scan(http://www.targetscan.org)to predict miRAN that can regulate FN1 synthesis.5.The expression of miRNA and FN1 in bladder tissue was detected by PCR.6.Western blot technology and immunohistochemistry were utilized to detect the expression of FN1 protein in bladder tissue.7.The miRNA analogs and antagonists were transfected,and their transfection efficiency and FN 1 expression were measured.8.Double luciferase reporter assay was used to detect the targeting relationship between miR-219c-5p and FN 1.9.MiR-219c-5p was overexpressed or knocked down in vitro and subjected to cell proliferation,cell cycle,and apoptosis experiments.Results1.The CS scores of EAE mouse models were mostly 2-3.2.Compared with the NC group,the mice in the EAE model group had fewer single urinations,increased urination time,and increased the ratio of bladder wet weight to body weight,which increased with the CS score.3.Compared with the NC group,the smooth muscle components in the bladder wall of the CS3 group were significantly thickened,and the Masson staining results showed that the collagen fiber components were substantially deposited in the bladder wall.4.After miRNA was transacted into bladder smooth muscle cells,it was found that in the case of no statistical difference in transfection efficiency and knockdown efficiency,only the miR-219c-5p group achieved the expected effect.That is,when miR-219c-5p is overexpressed,FN1 decreases(P<0.001),and when miR-219c-5p is brought down,FN1 increases(P<0.0001).5.Double luciferase reporter gene experiments and Western blot experiments confirmed that miR-219c-5p can target the 3 'UTR region of FN1 mRNA and regulate FN1 synthesis.6.Cell proliferation,cell cycle,and apoptosis experiments show that miR-219c-5p can participate in cell functional activities by regulating the synthesis of FN1 protein.Conclusion1.During the process of neurogenic bladder fibrosis induced by MS,bladder remodeling and bladder fibrosis occurred in the mice of the EAE group,and the FN1 protein is one of the key molecules of bladder fibrosis.2.miR-219c-5p can target the inhibition of FN1 protein synthesis and alleviate fibrosis of bladder smooth muscle cells to a certain extent,providing a new idea and target for the treatment of neurogenic bladder fibrosis in MS patients.