miR-145对人角质形成细胞系HaCaT细胞增殖、凋亡和细胞周期的影响

Effects of miR-145 on the proliferation, apoptosis and cell cycle of a human keratinocyte cell line HaCaT

目的 研究miR-145对人角质形成细胞系HaCaT细胞增殖、细胞周期及凋亡的调控效应.方法 化学合成miR-145的模拟物,采用瞬时转染的方法过表达miR-145.采用实时PCR方法检测miR-145的表达.MTS方法检测过表达miR-145对HaCaT细胞增殖的影响.流式细胞仪分析过表达miR-145对细胞周期及凋亡的影响.采用荧光素酶实验,实时PCR和Western印迹鉴定NRAS是否为miR-145的靶基因.结果 与阴性对照（NC）mimics转染组相比,miR-145 mimics转染组miR-145表达水平上调（85.00±1.21）倍,两组差异有统计学意义（t=115.90,P＜ 0.001）.转染miR-145 mimics有抑制HaCaT细胞的增殖作用（F=8.76,P=0.008）;转染后的时间因素（24、48、72、96 h）对细胞有影响（F=17.85,P＜ 0.001）,转染mimics和培养时间之间不存在交互作用（F=1.21,P=0.18）.与NC mimics转染组相比,miR-145 mimics转染组的早期凋亡、晚期凋亡细胞比例均明显增加,差异有统计学意义（18.9％±4.1％比4.3％±1.2％,t=7.126,P＜0.01;9.3％±2.3％比3.6％±1.6％,f=12.38,P＜ 0.01）.与NC mimics转染组相比,miR-145 mimics转染组HaCaT细胞的G2及S期细胞比例均明显降低,差异均有统计学意义（6.26％±1.2％比19.36％±3.45％,t=7.610,P=0.017;7.91％±1.3％比18.56％±5.23％,t=7.230,P=0.019）,而处于G1期的细胞比例升高,差异也有统计学意义（85.83％±5.2％比62.08％±6.23％,t=11.78,P=0.007）.与NC mimics联合psi-CHECK2-NRAS-wild组相比,在293T细胞中共转染miR-145mimics和psi-CHECK2-NRAS-wild质粒,其荧光素酶值明显下降,miR-145可抑制含NRAS mRNA 3＇UTR报告基因的荧光素酶表达（t=11.09,P=0.008）;而将NRAS mRNA 3＇UTR报告基因上miR-145的结合位点进行突变后,转染miR-145 mimics对含NRAS mRNA 3＇UTR报告基因的荧光素酶表达无明显的影响（P＞0.05）.实时PCR和Western印迹结果表明,过表达miR-145 mimics后,NRAS mRNA表达水平未出现明显变化（P＞0.05）,对NRAS蛋白水平的表达有明显的抑制（1.52±0.07比0.20±0.02,t=28.43,P＜0.01）.结论 miR-145可能通过NRAS影响HaCaT细胞的周期从而抑制细胞的增殖,同时促进HaCaT细胞的凋亡。

Objective To investigate the regulatory effects of miR-145 on the proliferation,cell cycle and apoptosis of a human keratinocyte cell line HaCaT.Methods miR-145 mimics and negative control （NC） mimics were chemically synthesized and then transiently transfected into HaCaT cells respectively.After additional culture for different durations,real-time PCR was performed to determine the expression level of miR-145,MTS assay to estimate cell proliferation,and flow cytometry to detect cell apoptosis and cycle.Luciferase assay,real-time PCR and Western blot were conducted to determine whether NRAS was the target gene of miR-145.Results The miR-145 expression level in miR-145 mimic-transfected cells increased by 85.00 ± 1.21 folds compared with NC mimic-transfected cells （t =115.90,P 〈 0.0001）.The transfection with miR-145 mimics significantly inhibited the proliferation of HaCaT cells （F =8.76,P =0.008）,and the inhibitory effect significantly varied with the duration （24-96 hours） of culture after transfection,with no interaction effect between the transfection with miR-145 mimics and culture duation （F =1.21,P =0.18）.Compared with NC mimic-transfected cells,those transfected with miR-145 mimics showed a significant increase in the proportion of early apoptotic cells （18.9% ± 4.1% vs.4.3% ± 1.2%,t =7.126,P 〈 0.01）,late apoptotic cells （9.3% ± 2.3% vs.3.6% ± 1.6%,t =12.38,P 〈 0.01）,G1-phase cells （85.83% ± 5.2% vs.62.08% ± 6.23%,t =11.78,P =0.007）,but a significant decrease in the percentage of G2-phase cells （6.26% ± 1.2% vs.19.36% ± 3.45%,t =7.610,P =0.017） and S-phase cells （7.91% ± 1.3% vs.18.56% ± 5.23%,t =7.230,P=0.019）.As luciferase assay showed,luciferase activity was significantly lower in HaCaT cells cotransfected with miR-145 mimics and a recombinant luciferase reporter vector psi-CHECK2-NRAS-wild carrying the wild-type 3＇UTR of NRAS than in those cotransfected with NC mimics and the vector psi-CHECK2-NRAS-wild （t =11.09,P =0.008）,but similar between cells cotransfected with miR-145 mimics and a recombinant luciferase reporter vector psi-CHECK2-NRAS-mut carrying the mutant-type 3＇UTR of NRAS and those cotransfected with NC mimics and the vector psi-CHECK2-NRAS-mut （P 〉 0.05）.Real-time PCR and Western blot revealed that the overexpression of miR-145 mimics had no significant effect on NRAS mRNA expression （P 〉 0.05）,but significantly inhibited NRAS protein expression （1.52 ± 0.07 vs.0.20 ± 0.02,t =28.43,P〈 0.01）.Conclusion miR-145 might inhibit proliferation and promote apoptosis of HaCaT cells by influencing cell cycle via NRAS.