Study Of MiRNA-148a-3p On The Relationship With Deer Antler TGF-?2

Antlers are a deer's second sexual characteristics and present upon the forehead upon entry into adolescence.Antlers are a multi-tissue organ that contains blood vessels,skin,nerves,cartilage,and bone.The sika deer pilose antler is the only fully regenerable bone organ in mammals.It has an unmatched growth rate and superior stability.It is an ideal model for studying organ regeneration and wound healing.The cycle of antler regeneration completes through the osteogenesis of specialized cartilage that results from a combination of factors.TGF is predominantly found in fibroblasts and epithelial cells.TGF signaling can cooperate with the Wnt pathway to promote the transformation of epithelial cells into mesenchymal cells and maintain cells and maintain cells in a mesenchymal state.The initial stage of cartilage cell formation in antler tissue involves the rapid proliferation of mesenchymal cells.Therefore,the role of TGF-? signaling as a regulatory mechanism in the rapid formation of velvet cartilage tissue requires further investigation.The research was conducted to obtain the sequence of TGF-?2 by PCR.TGF-?2 was recovered and pMD-18T-TGF-?2 recombinant plasmid was successfully constructed.After the transformation of DH5 a,and PCR reaction and the double enzyme digestion reaction were turned on.The base composition of the final detection sequence was turned on.Immunohistochemistry was used to detect the expression of TGF-?2 protein in different tissues of sika deer pilose antler.Total RNA was extracted with Trizol reagent and deep sequenced via HiSeq.TGF-?2-3'UTR wild type sequence was synthesized.TGF-?2-3'UTR was analyzed by two kinds of bioinformatics software TargetScan predict the miRNA that can interact with it.HiSeq,after which we used to identify miRNAs that diferentially expressed in mesenchyme and cartilage.Differentially expressed miRNAs subsequently verified by qPCR.The most highly expressed miRNA was miRNA-148a-3p.Wild and mutant type pmiR-Report-TGF-?2-3'UTR was constructed.In vitro,miRNA-148a-3p mimic with wild-type and mutant dual luciferase reporter vectors were co-transfected into cartilage.Detection of luciferase activity were conducted to verify the miRNA target genes.qPCR was used to dectect the stability and relative expression level of miRNA-148a-3p mimics in transfected antler cells.Changes of proliferation on antler cell rate was detected by MTT after transfection.We transfected cartilage cells with miRNA-148a-3p mimics and analyzed the abundance of TGF-?2,TGF-?R?and IGF-1 proteins by western blot.The experimental results showed that the sika deer TGF-?2 gene sequence was successfully obtained.The gene is 1245 bp in length,and the nucleotide sequence homology with cattle,sheep,horse and human was 98.55%,98.47%,94.46%,93.90%.The consistency of amino acid composition of sika deer antler TGF-?2 with cattle,sheep,horse and human was 99.38%,99.28%,99.03% and 98.79%,respectively.The results of immunohistochemistry showed that TGF-?2 protein was expressed in the cartilage layer,mesenchyme,dermal layer,the highest level of expression in cartilage.In order to verify the relationship between miRNA-148a-3p and sika deer antler TGF-?2 and its effect on antler cartilage,TGF-?2 3'UTR wild type and mutant sequence containing miRNA-148a-3p binding site were successfully synthesized in this experiment.The natural and mutant luciferase recombinant plasmids were successfully constructed.They were transfected into antler cartilage together with miRNA-148a-3p.The absorbance values were measured.Luciferase activity of wild-type plasmid-transfected cells was reduced,whereas mutant cells had no obvious changes.The results showed that the target gene of miRNA-148a-3p was TGF-?2.Our MTT assay and western blot results demonstrate that the proliferation of cells was inhibited and the relative expression of TGF-?2,TGF-?R?and IGF-1 proteins also decreased.