Study On The Targeted Regulation Of TGF-β2 Gene By MiRNA-153 In Velvet Antler Tip Tissue Of Sika Deer

Velvet antler is an accessory organ that male deer can periodically shed and regenerate every year.During the rapid growth period,the division rate of antler cells is about 30 times that of cancer cells,but cancer will not occur.This phenomenon is unique in mammals.Due to its unique biological characteristics,antler has become an ideal biomedical model for people to study the organ regeneration and cancer treatment.Micro RNA is a small molecule RNA with a length of about 20-24 nucleotides produced by the transcription of the non-coding region of the eukaryotic genome,which plays a multiple regulatory role in the life activities of eukaryotes.In the process of antler regeneration and development,miRNA regulates the proliferation of antler cells by regulating the expression of related factors.It has been found that the expression of miRNA-153 is significantly different in different tissues of antler tip.In this study,miRMap,miRcode,Targetscan and miRWalk were used to predict the candidate target genes of miR-153,and a reliable target gene set was screened.It was found that there was a target correlation between TGF-β2 gene which controls cell growth and differentiation and miR-153.Transforming growth factor-β(TGF-β)is a multifunctional growth factor.TGF-β2 gene is widely involved in the regulation of cell proliferation,differentiation,apoptosis and other growth and development processes,which plays an important role in promoting the growth and development of antler.In this study,TGF-β2 wildtype and mutant dual-luciferase reporter gene expression vectors were constructed,and dualluciferase activity detection was performed to verify the regulatory effect of miRNA-153 on TGF-β2 gene in antler tissue cells,and to reveal the effect of miRNA-153 targeting TGF-β2gene transcription on the growth of antler tissue cells.The results are as follows:(1)The relative activity of renilla luciferase was decreased in the cells transfected with wild-type dual luciferase reporter gene expression vector and miRNA-153 mimic,while the relative activity of renilla luciferase was basically unchanged in the cells transfected with mutant dual luciferase reporter gene expression vector and miRNA-153 mimic,indicating that there was a binding site of miRNA-153 on the 3’UTR of TGF-β2;(2)The overexpression level of miRNA-153 in antler chondrocytes and mesenchymal cells was detected by fluorescence quantitative PCR.It was found that the antler chondrocytes and mesenchymal cells transfected with miRNA-153 had a higher level of miRNA expression,indicating that the transfection was successful and the mimic could be stably and efficiently expressed;(3)The proliferation activity of cells transfected with miRNA-153 mimics was significantly lower than that of negative control group;(4)The relative expression level of TGF-β2 in antler chondrocytes transfected with miRNA-153 was detected by Western Blot.It was found that the relative expression level of TGF-β2 protein decreased significantly with time,which was similar to the results of MTT cell proliferation and cytotoxicity assay(MTT).It is concluded that the change of proliferation rate of antler chondrocytes after transfection of miRNA is related to TGF-β2 protein.After transfection of miRNA-153 mimics,the expression of TGF-βRII protein in antler chondrocytes also decreased with time,indicating that there was a certain relationship between them.