摘要
旨在研究microRNA-146a(miR-146a)对羊驼黑色素细胞增殖和迁移的调控及其分子机制。本研究使用双荧光素酶试验验证MAPK4和Myosin Va是miR-146a的靶基因;利用荧光定量PCR和蛋白质免疫印迹试验检测在羊驼黑色素细胞中过表达miR-146a对相关下游基因表达的影响;利用CCK8和Transwell检测miR-146a过表达对羊驼黑色素细胞增殖和迁移的影响。结果显示,与对照组相比,将miR-146a和MAPK4或Myosin Va共转染293T细胞,双荧光素酶活性分别极显著下降36%和30%(P<0.001);MAPK4和Myosin Va基因转录水平分别极显著下调67%和47%(P<0.001,P<0.01);蛋白质水平的表达量分别显著或极显著下调38%和69%(P<0.05,P<0.01);增殖和迁移相关的基因CREB、MITF、MLPH和Rab27a在转录水平和蛋白水平的表达均极显著下调(P<0.01,P<0.001);CCK8和Transwell结果显示,过表达miR-146a使羊驼黑色素细胞的增殖和迁移能力极显著下调(P<0.01)。综上所述,miR-146a通过靶向调控MAPK4和Myosin Va,使增殖和迁移相关的基因MEK1、CREB、MITF、MLPH和Rab27a的表达下调,从而对羊驼黑色素细胞的增殖和迁移起抑制作用。
This study aimed to explore the regulatory role of miR-146a on the proliferation and migration of alpaca melanocytes and its molecular mechanism.The dual luciferase assay was used to verify that MAPK 4 and Myosin Va were the target genes of miR-146a by co-transfection with MAPK 4 or Myosin Va in 293T cells;Quantitative real-time PCR and Western blotting were used to detect the expression of downstream genes in alpaca melanocytes after overexpressing miR-146a;CCK8 and Transwell were used to detect the proliferation and migration of alpaca melanocytes after overexpressing miR-146a.The results showed that,compared to the negative control,dual luciferase activity extremely significantly decreased by 36%or 30%in 293T cells co-transfected with miR-146a and MAPK 4 or Myosin Va,respectively(P<0.001);In alpaca melanocytes,with the overexpression of miR-146a,the mRNA expression of MAPK 4 and Myosin Va were extremely significantly down-regulated by 67%and 47%(P<0.001,P<0.01),and the protein expression of MAPK4 and Myosin Va were significantly down-regulated by 38%and 69%(P<0.05,P<0.01),respectively;The expression of genes related to proliferation and migration(CREB,MITF,MLPH,Rab 27a)were extremely significantly down-regulated at the transcriptional and translational levels(P<0.01,P<0.001);CCK8 and Transwell results showed that overexpression of miR-146a extremely significantly down-regulated the proliferation and migration ability of alpaca melanocytes(P<0.01).In summary,miR-146a targeting MAPK 4 and Myosin Va inhibited the proliferation and migration of alpaca melanocytes through downregulating the expression of MEK 1,CREB,MITF,MLPH,Rab 27a.
作者
刘学贤
杜斌
郭湘
薛骥轩
于雷涛
范瑞文
LIU Xuexian;DU Bin;GUO Xiang;XUE Jixuan;YU Leitao;FAN Ruiwen(College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2021年第4期967-975,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
山西省研究生创新项目(2019BY064)
山西省“青年三晋学者”人才专项
山西省回国留学人员科研资助(2017-072)。
