miR-302 b-3 p靶向AKT1调节皮肤成纤维细胞衰老的作用机制

miR-302b-3p regulating skin fibroblasts senescence via targeting AKT1

目的 探讨miR-302b-3p靶向丝/苏氨酸蛋白激酶(AKT)1调节皮肤成纤维细胞衰老的作用及其分子机制.方法 建立复制性细胞衰老模型后,采用real-time PCR法检测细胞miR-302b-3p的表达情况;采用生物信息学软件分析miR-302b-3p的靶基因;分别将miR-302b-3p模拟物(mimic)及抑制剂(inhibitor)转染皮肤成纤维细胞后,β-半乳糖苷酶染色分析细胞衰老情况,qRT-PCR法检测AKT1 mRNA表达水平,Western印迹法检测AKT1蛋白表达情况.结果 与对照组比较,复制性衰老皮肤成纤维细胞中miR-302b-3p显著升高.转染miR-302b-3p mimic后,细胞衰老阳性染色细胞数目显著增加(P<0.01).AKT1为miR-302b-3p的预测靶基因.当细胞上调miR-302b-3p表达时,靶基因AKT1 mRNA及蛋白水平显著降低(P<0.05).反之,当细胞下调miR-302b-3p表达时,靶基因AKT1 mRNA及蛋白水平显著升高(P<0.05).结论 miR-302b-3p可能通过靶向抑制AKT1表达调节皮肤成纤维细胞的衰老.

Objective To investigate the role of miR-302b-3p in regulating skin fibroblast aging via targeting AKT1.Methods After the replicative senescence fibroblasts model was established,the expression of miR-302b-3p was detected by real-time PCR.Target gene of miR-302b-3p was analyzed by using bioinformatics software.miR-302b-3p mimic/inhibitor were transfected into skin fibroblasts,respectively.β-galactosidase staining cells were performed to evaluate cell senescence.The AKT1 mRNA expression level was detected by RT-PCR and AKT1 protein expression was detected by Western blot.Results miR-302b-3p was significantly increased in senescence skin fibroblasts compared with control group.AKT1 was one of predicted target genes of miR-302b-3p.Transfection of miR-302b-3p mimic or inhibitor could dramatically increase or decrease miR-302b-3p expression in fibroblasts(P<0.05).miR-302b-3p mimic could increase positive cells ofβ-galactosidase staining compared with NC group.What′s more,over-expression of miR-302b-3p significantly inhibited the mRNA and protein expression of AKT1.Otherwise,low-expression of miR-302b-3p dramatically increased the mRNA and protein expression of AKT1(P<0.05).Conclusions miR-302b-3p expression could regulate fibroblasts senescence via targeted inhibiting AKT1 expression.