Correlation Between Mafb And Cell Biological Behavior Of Hypospadias

Objective:To study the expression difference of transcription factor Mafb in hypospadias and normal penile tissues and the effect of low Mafb expression on the biological behavior of foreskin fibroblasts.Methods: 1.Collection of clinical specimens: The prepups were obtained from children with hypospadias who underwent surgical repair and children who underwent circumcision in the affiliated Children’s Hospital of Chongqing Medical University.Patients diagnosed with cryptorchidism,gender dysplasia,or endocrine abnormalities were excluded from the study.25 cases of hypospadias(average 3.5 years old)and 15 cases of circumcision(control group,average 5 years old)were included.The expression area of Mafb was observed by immunohistochemistry,Real-time fluorescent quantitative polymerase chain reaction and western blot were used to detect the expression level of Mafb.2.Cell experiment: UseRNA interference siRNA to inhibit the expression of Mafb in foreskin fibroblasts,the main role of Mafb in prepuce fibroblasts was also studied.Small interferingRNA transfects Mafb-targeted siRNA into foreskin fibroblasts.After successful transfection,the cell proliferation was tested by CCK-8 experiment.Changes in the cell cycle are detected by flow cytometry.QPCR and WB were used to detect the changes of cell cycle related genes and proteins.3.Clinical sample experiment: The expression of cell cycle-associated proteins in the prepuce of 25 children with hypospadias and 15 children with circumcision(control group)was detected by WB experiment.Results: 1.In patients with hypospadias,the expression level of Mafb protein in the foreskin mesenchymal cell layer was also significantly reduced and the level of Mafb protein in hypospadias patients was significantly reduced [(1.932±0.1139),(1.006±0.03312)] and the expression of Mafb mRNA was significantly lower than that of the control group [(1.179±0.1275),(0.6652±0.07506)].However,there was no significant difference in the expression of Mafb in subjects with mild and severe hypospadias.2.Using qPCR and WB to detect the Mafb gene expression in the siRNA group and the Negative Control(NC)group,the results showed that the Mafb mRNA(0.4436±0.07228%;)and protein(0.4276±0.1691%;)expression levels in the siRNA group Significantly reduced(p<0.05).The results of CCK-8 experiment showed that the growth of foreskin fibroblasts transfected with Mafb siRNA group was significantly increased.After 48 hours of transfection with Mafb siRNA,the results of flow cytometry showed that the cells in the G1 phase of the Mafb siRNA group decreased significantly,and the cells in the S phase increased significantly,the difference was statistically significant(p<0.05).The results of qPCR and WB showed that the mRNA and protein levels of cell cycle-related proteins CDK2,cyclin E and PCNA in the Mafb siRNA group increased significantly,indicating that inhibition of Mafb expression can promote the proliferation of foreskin fibroblasts by regulating cell cycle-related proteins.3.WB results showed that compared with the control group,the hypospadias group CDK2,cyclin E,PCNA protein levels were significantly increased(p<0.01)and the cell cycle related protein changes after the decrease of Mafb expression in the cell experiment.Conclusion: 1.Mafb is low in the hypospadias group.Therefore,we conclude that the abnormal expression of Mafb gene is closely related to the occurrence of hypospadias,which may be the cause of abnormal urethral development in embryonic stage.2.The study confirmed for the first time that the low expression of Mafb can promote the proliferation of foreskin fibroblasts.It is related to proteins that regulate the cell cycle,including Cylin E,CDK2 and PCNA.These findings help explain the clinical manifestations and mechanisms of hypospadias.