MicroRNA PC-5p-1090 Targets SOX9 Gene

The SOX9 gene belongs to the SOX gene family and is closely related to the development of mammalian embryos,sex determination,human genetic syndrome and the occurrence of malignant tumors.MicroRNA PC-5P-1090(PC-1090)is a small RNA newly discovered in antler skin of pilose antler.To verify whether SOX9 is a direct target gene of PC-1090,a wild-type recombinant plasmid containing SOX9 3'UTR(pMIR-Report-SOX9-3'UTR-WT)and a mutant recombinant plasmid(pMIR-Report-SOX9-3'UTR-MUT)were constructed in this study.The pMIR-Report-SOX9-3'UTR-WT,pMIR-Report-SOX9-3'UTR-MUT were transfected with 1090 mimics and control mimics to transfect 293T cells,respectively.Western blot was used to detect the expression of luciferase gene in 293T cells after transfection.Huh-7 cells(expressing endogenous SOX9)were transfected with 1090 mimics and control mimics,respectively,and the expression of SOX9 protein in Huh-7 cells was detected by Western blot.Results:The recombinant vector pMIR-Report-SOX9-3'UTR-WT and pMIR-Report-SOX9-3'UTR-MUT was verified by sequencing.Western blot results showed that the expression level of luciferase was significantly decreased in the 293T cell group co-transfected with PC-1090 mimics and SOX9 3'UTR wild-type recombinant plasmids,but the expression levels of luciferase in other control groups were not significantly changed.Compared with Huh-7 cells transfected with control mimics,the expression of SOX9 protein was significantly reduced in Huh-7 cells transfected with 1090 mimics.Conclusion:These data strongly indicated that PC-1090 has biologically functional,site-specifically bound to SOX9 3'UTR,allowing PC-1090 to negatively regulate the expression of SOX9.Thus,SOX9 is indeed a target of PC-1090.