Effect Of MiR-146a On Melanogenesis Of Mouse Melanoma Cells

MicroRNAs(miRNAs)are endogenous small non-coding RNAs,which play crucial roles in numerous biological processes and are related to the occurrence of tumor and cancer.It has been reported that many miRNAs regulate the production of melanins in melanocytes.However,the effects of miR-146a on the melanogenesis have not been reported.Here,miR-146a mimic was transfected by liposome in mice melanocytes in vitro.PCR methed was used to amplify TYRP1-3'UTR,which was ligated into PGLO vector to construct pmirGLO-TYRP1-3'UTR.Following pmirGLO-TYRP1-3'UTR and miR-146a mimic were co-transfected into 293T cells,the luciferase activity was measured;qRT-PCR and western blotting were used to measure the mRNA and protein of TYR,TYRP1 and TYRP2 expressed in melanocytes of mice which were transfected by miR-146a mimic and miR-146a inhibitor.The main research contents and results were as follows:1?Dual Fluorescence Report vector of TYRP1 3'-UTR was constructed and pmirGLO-TYRP13'UTR plasmid and miR-146a mimic were co-transfected into 293T cells.The results showed that the luciferase activity of experimented group was decreased to 0.70 folds compared with the control group,suggesting that the targeting relationship between miR-146a and TYRP1.2.The melanocytes were transfected with the miR-146a mimic,and then the assay of melanin was measured.The results showed that melanin content of melanocytes in the test group was significantly reduced to 0.74 times of the control group and was increased 1.2 times in inhibitor group.3.The expression of miR-146a,TYR,TYRP1 and TYRP2 were tested by qRT-PCR method.The results showed that:compared with the NC group,the expression of miR-146a was significantly increased 80 times for the NC group,mRNA expression of TYR gene was significantly reduced to 0.583 times,mRNA expression of TYRP1 gene was significantly lower to 0.808 times,mRNA expression of TYRP2 gene was significantly reduced to 0.663 times.4.The protein expression of TYR,TYRP1 and TYRP2 in miR-146a transfected group and NC,inhibitor group were tested by western blotting method.The results show that TYR,TYRP1 and TYRP2 protein expression of the test group were significantly lower,respectively,0.443 times,0.468 times and 0.705 times to NC group.These results indicated that miR-146a affected gene TYR,TYRP1 and TYRP2 which were important relevant genes in melanogenesis synthesis pathway,by regulating the expression of TYRP1 and miR-146a effected the assay of melanin.The results supported that miR-146a could participate in regulating the synthesis of melanin at the molecular level.