microRNA-146a通过靶向MAPK4和Myosin Va基因调控羊驼黑色素细胞增殖、迁移

doi:10.11843/j.issn.0366-6964.2021.04.012

摘要/Abstract

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。

关键词: miR-146a, MAPK4, Myosin Va, 增殖, 迁移

Abstract: 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 MAPK4 and Myosin Va were the target genes of miR-146a by co-transfection with MAPK4 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 MAPK4 or Myosin Va, respectively (P<0.001); In alpaca melanocytes, with the overexpression of miR-146a, the mRNA expression of MAPK4 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, Rab27a) 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 MAPK4 and Myosin Va inhibited the proliferation and migration of alpaca melanocytes through downregulating the expression of MEK1, CREB, MITF, MLPH, Rab27a.

Key words: miR-146a, MAPK4, Myosin Va, proliferation, migration

中图分类号:

S813.1

刘学贤, 杜斌, 郭湘, 薛骥轩, 于雷涛, 范瑞文. microRNA-146a通过靶向MAPK4和Myosin Va基因调控羊驼黑色素细胞增殖、迁移[J]. 畜牧兽医学报, 2021, 52(4): 967-975.

LIU Xuexian, DU Bin, GUO Xiang, XUE Jixuan, YU Leitao, FAN Ruiwen. microRNA-146a Regulates the Proliferation and Migration of Alpaca Melanocytes by Targeting MAPK4 and Myosin Va Genes[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4): 967-975.

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