The Mechanism Of Mir-1a-3p Mediated Fibronectin 1 In Neurogenic Bladder Remodeling

BACKGROUND AND PURPOSEMultiple sclerosis(MS)is a chronic autoimmune disease of the central nervous system,which is the second leading cause of neurogenic bladder.Neurogenic bladder is a lower urinary tract disease caused by damage or disease of the nervous system.In patients with neurological diseases such as multiple sclerosis,Parkinson’s disease,spinal cord injury and spina bifida,neurogenic bladder can cause a range of symptoms and complications,including urinary incontinence,frequent urination,urgency,infection and kidney disease.At present,the molecular mechanism of neurogenic bladder remodeling induced by MS is not clear.Because of the difficulty of restoring bladder function and the complexity of neuropathy,it is necessary to use comprehensive methods to treat patients with neurogenic bladder.Conservative treatment,such as the use of drugs to inhibit overactivity of the bladder,means and ideas of which cover all treatment stages of patients,although the short-term results can be satisfactory,the long-term effect is not good and even accelerate the degree of fibrosis of smooth muscle cells.Fibronectin 1(FN 1)belongs to the fibronectin family,which is the main component of extracellular matrix,participates in cell morphology and cell proliferation,and is a key protein in bladder remodeling after bladder fibrosis.Some literatures have confirmed that there is a close relationship between miRNA and the changes of FN1 in fibrotic diseases.Therefore,in this study,we constructed an experimental autoimmune encephalomyelitis(EAE)model to simulate MS disease to explore the involvement of miRNA-mediated FN1 synthesis in the occurrence and development of neurogenic bladder remodeling.We will provide new research ideas for the control of neurogenic bladder remodeling.METHOD1.Forty 7-week-old female C57BL/6J mice weighed 20g to 28g were brought into the study.The mice were randomly divided into EAE group(n=30)and control group(n=10).Clinical score(CS)was performed according to symptoms.2.Mouse bladder smooth muscle cells were extracted and cultured by multi-enzyme digestion method.Smooth muscle actin was identified by immunofluorescence and cell proliferation was detected by CCK8 method.3.High-throughput sequencing results and bioinformatics websites are used to’predict the miRNA that may combine with FN1.4.The transfection efficiency was examined by transfection of miRNA analogues and antagonists into bladder smooth muscle cells.5.The expression of miRNA and FN1 in bladder tissue was examined by PCR and Western blot.6.The expression of miRNA analogue and antagonist FN1 was examined by Western blot and PCR.7.Double luciferase reporter gene assay was used to examine the targeting relationship between miR-1a-3p and FN1.8.Over-expression and knock-down of miR-1a-3p in vitro,to explore the effect on cell function.RESULTS1.Most of the clinical scores of EAE model mice were 2-3.2.The purity of BSMCs obtained from mice can reach 97%,and the cells have a typical"peak-valley" structure.Immunofluorescence assay showed positive expression of smooth muscle actin(α-SMA)in the cytoplasm.The extracted cells have good proliferation characteristics.3.High-throughput sequencing results and bioinformatics predicted that miR-1a-3p could interact with FN 1.4.PCR and Western Blotting indicate that the high expression of FN1 may be related to the low expression of miR-1a-3p in the process of bladder fibrosis.5.After transfection of BSMCs with miR-1a-3p agonists and antagonists,FN1 decreased when miR-1a-3p was overexpressed(P<0.001),but FN1 increased when miR-1a-3p was knocked down(P<0.001).There was no significant difference in transfection efficiency and knockdown efficiency.6.Double luciferase reporter gene assay confirmed that miR-1a-3p could target the 3 ’UTR region binding to FN1 mRNA.7.That miR-1a-3p could participate in cell function by regulating the synthesis of FN1 protein was detected by CCK8 and cell cycle experiments showed.CONCLUSION1.In the process of neurogenic bladder remodeling induced by MS,bladder remodeling and bladder fibrosis arose in EAE group.FN1 protein may play an important role in promoting bladder fibrosis.2.MiR-1a-3p can specifically inhibit the synthesis of FN1 protein,alleviate the fibrosis progression of bladder smooth muscle cells,and provide a new idea and target for the treatment of neurogenic bladder remodeling.3.In this study,the isolation method of mouse primary bladder smooth muscle cells is simple and reproducible,and the BSMCs cultured in vitro has good proliferation ability.