The Study On The MicroRNA-148 Which Could Inhibit The Key Factor Of Pigementation In The Alpaca's Melanocyte

The color of the fur is one of the most important characteristics for the economic animals,which mainly affected by the melanin content.It's great important that studying the regulation mechanism of relative genes synthesize melanin for breeding rich and high quality animals by are of great importance.In recent years,related research about microRNA regulated the target gene mRNA after the transcriptional has been concerned by more and more researchers.The relevant mechanisms have gradually become clear.In order to study the regulation mechanism of miR-148 on the transcription factor MITF in alpaca melanocytes,The optimal conditions just as the optimal cell density and trypsin digestion time for culturing alpaca melanocytes in vitro were selected.Based on the method of miR-148a-3m-mimic transfection.miR-148 was overexpressed in alpaca melanocytes cultured in vitro.Finally,qRT-PCR and Western blot were used to analysis the relative expression of mitf from the level of mRNA and protein.Make further efforts to analysis the effect of miR-148 on the alpacas melanocytes genes,the transcribed group of alpacas melanocytes was sequenced by transcriptome sequencing(RNA-seq).Then functional annotation analysised by KEGG,COG,KOG,GO and so all.The results showed that the growth rate of alpaca melanocytes initial seeding density was 5×105/mL.The cells were growing best when the trypsin digestion time was 5min,and were significant difference between the digestive time of 3min and 8min.The results of Quantitative real-time PCR showed that miR-148 did overexpressed in alpacas melanocytes,which was 15 times than the original expression level and no change for mitf mRNA expression.Western blot results showed that the expression of mitf protein was decreased after overexpression of miR-148,and the expression of mitf protein was increased after the expression of endogenous miR-148 was inhibited.The results of RNA sequencing showed that 51.42Gb of data and 333,884 Unigene were obtained,in which 48,993 were annotaed.Among them,216 genes were related to melanin synthesis.The results of FPKM showed that there were no differences in MITF expression,but the expression of tyrosinase(TYR)and tyrosinase related protein 2(TYRP2)in the tyrosinase protein family related to melanin synthesis were decreased.KEGG function notes found that there are any differentially expressed genes in the MAPK signal pathway,Ras signal pathway and cGMP-PKG signal pathway,and these three pathways are involved in the transcription factor MITF metabolism,but also found a number of different expression gene related to human disease,pridicted may be related to overexpressed of miR-148,can be used as the future research content.Conclusion:(1)5×105/mL initial seeding density was the most favorable for alpaca melanocyte growth;(2)trypsin digestion for 5min was the best digestion time;(3)miR-148 could binding in the MITF mRNA,thereby inhibiting the expression of alpaca melanocytes genes related to melanin synthesis,leading to melanin synthesis is inhibited.