Study On The Mechanism And Function Of MicroRNA PC-3P-40996 Targeting Regulation Of RBPMS Gene Expression In Deer Antler Skin Cells

MicroRNA PC-3P-40996(hereinafter referred to miRNA-40996)is a new small RNA derived from the anlter velvet of red deer at the rapid growth stage.RBPMS is a RNA-binding proteins with multiple splicing forms,which participates in all aspects of RNA metabolism and post-transcriptional regulation of genes.Studies demanstrate that RBPMS can strength the transcriptional activity of Smad2 and Smad3 and contributed to TGF-β pathway signal transduction.TGF-β signaling pathway plays an important role in the regulation of the rapid growth and regeneration of deer antler.Therefore,the purpose of this study is to explore the interaction between miRNA-40996,RBPMS and Smad2/3.In order to study the biological function of miRNA-40996 in anlter velvet,we predicted the targets of miRNA-40996,and found that RBPMS gene may be one of targets.Then,the predicted bind locus in the 3’UTR region of RBPMS was synthesized as the reference sequence.We constructed two types of vectors,included the wild and mutant type vectors,named psi CHECK~TM-2-RBPMs-3’UTR-WT and psi CHECK~TM-2-RBPMs-3’UTR-MUT of RBPMS3’UTR,respectively.After constructed successfully,293 T cells were co-transfected with wildtype and mutant vectors and miRNA mimics miRNA-40996 mimics and NC mimics,respectively.Double lucifase report assay was performed to detect the double lucifase activity in the transfected 293 T cells.The results showed that only the cells co-transfected with RBPMS 3’UTR wild-type vector and miRNA-40996 mimics had significantly decreased luciferase activity.Then miRNA-40996 mimics and NC mimics were transfected into deer skin cells,respectively.Western blot assay was used to detect the relative expression level of endogenous RBPMS protein in the transfected velvet skin cells.The results showed that compared with the NC mimics transfected cell group,The expression level of endogenous RBPMS protein in velvet skin cells transfected with miRNA-40996 mimics was significantly reduced.After confirming that miRNA-40996 can target RBPMS,miRNA-40996 mimics and NC mimics were transfected into deer skin cells to further study the relationship between miRNA-40996 and RBPMS and Smad2 and Smad3.The mRNA levels of Smad2 and Smad3 in the transfected cells were detected by RT-q PCR.The results showed that the mRNA levels of Smad2 and Smad3 in the transfected miRNA-40996 mimics group were significantly lower than those in the NC group.Western blot was used to detect the expression levels of endogenous RBPMS and Smad3 proteins in miRNA-40996 mimics and NC mimics.The results showed that compared with NC mimics,The relative expression levels of RBPMS and Smad3 in miRNA-40996 mimics cells were significantly decreased,and Smad3 protein expression was correlated with RBPMS protein expression.Conclusion: miRNA-40996 has biological function and can bind on the 3’UTR of RBPMS to further regulate the expression of Smad2 and Smad3.