Study Of MiRNA-19A And MiRNA-19b On The Relationship With Deer Antler TGF-?R?

Deer antler is born in the top of the frontal bone which the secondary sexual characteristic of the male deer species and the only organ can be completely regenerated in mammals.Antler grows rapidly and can be used as an ideal model for study the regeneration and wound healing of the mammalian organs.Cyclical development of deer antler is regulated by many factors,including growth factors,hormones,light and temperature.MicroRNA(miRNA)is a kind of endogenous non-coding small RNA which is processing generated by Eukaryotic genome non-coding region transcription with apartial hairpin structure,and widely presentsin all eukaryotes,including unicellular algae,and the structure of biological evolution showed a highly conserved.After the combination of miRNA and 3'UTR sequence of the target gene,the expression of the target gene was inhibition.More than half of the noncoding regions with mammalian protein are regulated by mi RNAs.Research has shown that miRNAs can regulate the proliferation of antler cells by regulate the expression of related growth factors in antler cells.TGF-?R ?is a high affinity receptor in the TGF-? pathway,it is a phosphorylated protein which is responsible for receiving and transmitting signals.MiRNA-17-92 cluster is a conserved cluster in vertebrates,including 7 members with miRNA-17-3p ? mi RNA-17-5p ? miRNA-18 a ? miRNA-19 a ?miRNA-19b?miRNA-20 a and miRNA-92 a.Research has shown that miRNA-17-92 cluster has significant effect on the TGF-? pathway.It's not yet reported on study the transcriptional regulation of miRNA-17-92 gene cluster on TGF-?R?at home and abroad.In this study,we collected the deer antler cartilage tissue in vitro cell culture and chondrocyte identification.TGF-?R?-3'UTR was analyzed by two kinds of bioinformatics software TargetScan and miRandato predict the miRNA that can interact with it.Synthesis TGF-?R?-3'UTR and construction wild and mutant type pmiR-Report-TGF-?R?-3,UTR,detection of luciferase activity were conducted to verify the mi RNA target genes.The synthetic mi RNA mimic were transfected into antler chondrocytes,Changes of proliferation on antler cell rate was detected by MTT after transfection;The relative expression level of TGF-?R protein and miRNA on ?the expression of IGF-1?TGF-?2 and other cell growth factors were analyzed with the use of western bloting.Antler chondrocytes identification results showed that the sequence of ColX part of sika deer cartilage specific gene was successfully cloned,the sequence length is 1771 bp and the similarity of ColX sequence reported in GeneBank was 99%.Chondrocytes were identified successfully combined with the observation of chondrocyte morphology,and isolated cells can be used for subsequent experiments.The results of the chip analysis show that mi RNA-19 a and miRNA-19 b were differentially expressed in different tissues of antler,indicating that miRNA-19 a and miRNA-19 b may play an important role in the expression of this mRNA.In order to further investigate the transcriptional regulation of miRNA-19 a and miRNA-19 b on TGF-?R?and the relationship between the growth of velvet cells,TGF-?RII gene 3'UTR wild and mutant double luciferase reporter gene vector which containing miRNA-19 a and mi RNA-19 b binding sites were successfully constructed.Luciferase activity test results showed that transfected with wild-type plasmid and two miRNAs cellular luciferase activity was significantly reduced,and there was no significant change in luciferase activity in transfected mutant plasmids and two miRNAs compared with the negative control group.The results was consistant with previous forecast results that TGF-?RII is the target gene regulated by miRNA-19 a and miRNA-19 b.MTT and western blotting results showed that the proliferation of antler chondrocytes was inhibited,and the relative expression level decreased in TGF-?R ?protein and IGF-1 and TGF-?2 and other cell growth factors with time dependence.