Objective To study the effect of targeted inhibition of mitogen-activated protein kinase kinase kinase 1(Map3k1) gene on the cell proliferation and migration abilities of B6 mouse eyelid keratinocytes.Methods An artificial microRNA(amiRNA) interference vector targeting silent Map3k1 gene was constructed in vitro in the test.Lipofectamin 2000 was used to transfect the B6 mouse eyelid keratinocytes(the Ctrl Map3k1 group was transfected with empty vector,while the Map3k1 amiRNA-3 group was transfected with Map3k1 amiRNA-3 interference vector).The Map3k1 mRNA and protein expression levels were respectively de-tected by real-time PCR and Western-blot for determining the interference efficiency.The B6 mouse eyelid keratinocytes prolifera-tion level was detected by MTT.The migration ability of keratinocytes was detected by the scratch experiment.Results After the keratinocytes were transfected with Map3k1 amiRNA interference vector,the levels of Map3k1 mRNA and protein were effectively inhibited,and the interference efficiency was up to 70%(P<0.05).The proliferation level of keratinocytes in the Map3k1 amiRNA-3 group was lower than that in the Ctrl Map3k1 group(P<0.05).The migratory ability of keratinocytes in the Map3k1 amiRNA-3 group was also significantly lower than that in the Ctrl Map3k1 group(P<0.05).Conclusion Targeted inhibition of Map3k1 gene expression in B6 mouse eyelid keratinocytes significantly inhibits cell proliferation and migration,thus influence the cellular bi-ological behaviors.%目的 为了研究靶向抑制M ap3k1基因对B6小鼠眼睑角质形成细胞增殖和迁移能力的影响.方法 通过已构建的靶向沉默Map3k1基因amiRNA干扰载体,Lipofectamin 2000转染B6小鼠眼睑角质形成细胞(Ctrl Map3k1组转染空载体, Map3k1 amiRNA-3组转染Map3k1 amiRNA-3干扰载体);Real-Time PCR和Western blot实验分别检测Map3k1 mRNA和蛋白的相对表达量,以测定干扰效率;四甲基偶氮唑盐(M T T)法检测B6小鼠眼睑角质形成细胞的增殖水平;划痕实验检测角质形成细胞的迁移能力.结果 B6小鼠眼睑角质形成细胞转染Map3k1 amiRNA-3干扰载体后,mRNA和蛋白水平均被有效抑制,干扰效率高达70%(P<0.05);Map3k1 amiRNA-3组在细胞增殖水平上低于Ctrl Map3k1组(P<0.05);Map3k1 amiRNA-3组在迁移能力上亦显著低于Ctrl Map3k1组(P<0.05).结论 靶向抑制B6小鼠眼睑角质形成细胞中Map3k1基因的表达后显著抑制细胞的增殖和迁移,进而影响细胞的生物学行为.
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