Research On The Effects Of Androgen Receptor Intervention On Mafb Gene Of Foreskin Fibroblasts And Related Proteins

Background:Hypospadias is one of the most common malformations in children’s genitourinary system.The incidence of epidemiological investigations at home and abroad is 1:300¹:200,and it has an increasing trend year by year.The main clinical manifestations of hypospadias are abnormalities of the urethral opening with penile curvature,and the accumulation of the foreskin on the dorsal side of the penis,which has a great influence on children’s mental health and fertility in adulthood.At present,most of the causes of hypospadias are unknown,but the mainstream view is related to genetic susceptibility and environmental endocrine disruptors,which affect the development of the urethra and eventually lead to this deformity.The masculinization of the urethra depends on the action of androgens,while abnormalities in androgen or androgen receptors can lead to genitourinary developmental disorders in men.Research on androgen receptor-related signaling pathways is still a hot topic.The v-mafmusculoaponeurotic fibrosarcoma oncogene family protein B（Mafb）is an important member of the Maf family and is a basic leucine zipper DNA binding protein,which is a highly conserved one in eukaryotes.Nuclear-like transcription factor.The study found that it is closely related to cell proliferation,differentiation,and organogenesis,and also participates in male genitourinary development,and its mechanism is less studied.This study intends to investigate the effect of Mafb on the expression of Mafb in male genitourinary development through primary cultured foreskin fibroblasts using androgen receptor interfering agents（DHT,Flu）.The study of urethral malformation lays the foundation.Objiective:This study was to investigate the effects of DHT and Flu on the expression of Mafb gene in the body-encapsulated fibroblasts,and to complete the screening of Mafb-related proteins during urethral development,which lays a foundation for the study of urethral malformations.Methods:Primary cultured normal children’s foreskin fibroblasts were treated with different concentrations of DHT and Flu.The expression of Mafb was detected by RT-PCR,immunocytochemistry and Western Blot.Proteins interacting with Mafb were analyzed by immunoprecipitation combined with mass spectrometry（MALDL-TOF/TOF-MS）.Results:The primary foreskin fibroblasts were successfully established.Under the intervention conditions of DHT concentration of 3.0×10^-8mol/L and 3.0×10^-6mol/L,the expression levels of Mafb mRNA were 0.372±0.003and 0.594±0.045,respectively.Compared with the group（0.444±0.007）,the DHT 3.0×10^-6mol/L group was significantly up-regulated（P=0.005）,while the DHT 3.0×10^-8mol/L group was not significantly up-regulated（P=0.145）;Flu 15μmol/L Under the intervention condition,the expression level of Mafb mRNA was 0.221±0.013,and the expression level was significantly decreased compared with the normal group（P=0.0004）.Cellular immunofluorescence analysis showed that the expression of Mafb was significantly up-regulated in the DHT 3.0×10^-8mol/L group（0.046±0.004）and 3.0×10^-6mol/L（0.076±0.003）compared with the normal control group（0.027±0.006）.（P=0.01,P=0.000）,while the Flu group（0.006±0.002）was significantly down-regulated（P=0.009）.Similarly,Western Blot results showed that there was no difference in up-regulation（P=0.96）and DHT3.0×10^-6mol/L（P=0.598）in DHT 3.0×10^-8mol/L group（0.146±0.001）compared with control protein level（0.163±0.001）.The up-regulation of²Mafb expression was significantly different in the group（P=0.0001）,while the Flu group（0.066±0.002）was significantly down-regulated（P=0.04）.Immunoprecipitation combined with mass spectrometry showed that 13proteins interacting with Mafb were FOS,JUN,MAPK8,ATF3,EGR2,HOXB3,KDM6A,NCOA6,CTCF,HOXB1,HOXA3,PKNOX1,RBBP5,according to protein score and match.Sequence results suggest that screening for FOS proteins may be most relevant to MafB.The results of immunofluorescence double labeling confirmed that the expression of FOS and MafB were highly consistent,suggesting that they have close interactions and are consistent with protein network analysis.Conclusion:The expression of Mafb gene is regulated by DHT and Flu in cultured foreskin fibroblasts in vitro.Mafb acts as a downstream regulatory protein of androgen receptors and plays a key role in the physiological mechanisms by which androgens play a role.FOS may be an important synergistic protein for Mafb to function.