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
miR-145대인각질형성세포계HaCaT세포증식、조망화세포주기적영향
Effects of miR-145 on the proliferation, apoptosis and cell cycle of a human keratinocyte cell line HaCaT
  • 万方数据
    中华皮肤科杂志 중화피부과잡지
    Chinese Journal of Dermatology
    2015年 6期 416-420 ,共5页

    李璟蓉%王建琴%方锐华%曾仁山%莫金雪%郭云龙%贾淑青

    리경용%왕건금%방예화%증인산%막금설%곽운룡%가숙청

    角蛋白细胞%细胞增殖%细胞周期%细胞凋亡%尖锐湿疣%HaCaT细胞%miR-145%NRAS 角蛋白細胞%細胞增殖%細胞週期%細胞凋亡%尖銳濕疣%HaCaT細胞%miR-145%NRAS
    각단백세포%세포증식%세포주기%세포조망%첨예습우%HaCaT세포%miR-145%NRAS
    Keratinocytes%Cell proliferation%Cell cycle%Apoptosis%Condylomata acuminata%HaCaT cells%miR-145%NRAS
    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
    목적 연구miR-145대인각질형성세포계HaCaT세포증식、세포주기급조망적조공효응.방법 화학합성miR-145적모의물,채용순시전염적방법과표체miR-145.채용실시PCR방법검측miR-145적표체.MTS방법검측과표체miR-145대HaCaT세포증식적영향.류식세포의분석과표체miR-145대세포주기급조망적영향.채용형광소매실험,실시PCR화Western인적감정NRAS시부위miR-145적파기인.결과 여음성대조(NC)mimics전염조상비,miR-145 mimics전염조miR-145표체수평상조(85.00±1.21)배,량조차이유통계학의의(t=115.90,P＜ 0.001).전염miR-145 mimics유억제HaCaT세포적증식작용(F=8.76,P=0.008);전염후적시간인소(24、48、72、96 h)대세포유영향(F=17.85,P＜ 0.001),전염mimics화배양시간지간불존재교호작용(F=1.21,P=0.18).여NC mimics전염조상비,miR-145 mimics전염조적조기조망、만기조망세포비례균명현증가,차이유통계학의의(18.9％±4.1％비4.3％±1.2％,t=7.126,P＜0.01;9.3％±2.3％비3.6％±1.6％,f=12.38,P＜ 0.01).여NC mimics전염조상비,miR-145 mimics전염조HaCaT세포적G2급S기세포비례균명현강저,차이균유통계학의의(6.26％±1.2％비19.36％±3.45％,t=7.610,P=0.017;7.91％±1.3％비18.56％±5.23％,t=7.230,P=0.019),이처우G1기적세포비례승고,차이야유통계학의의(85.83％±5.2％비62.08％±6.23％,t=11.78,P=0.007).여NC mimics연합psi-CHECK2-NRAS-wild조상비,재293T세포중공전염miR-145mimics화psi-CHECK2-NRAS-wild질립,기형광소매치명현하강,miR-145가억제함NRAS mRNA 3'UTR보고기인적형광소매표체(t=11.09,P=0.008);이장NRAS mRNA 3'UTR보고기인상miR-145적결합위점진행돌변후,전염miR-145 mimics대함NRAS mRNA 3'UTR보고기인적형광소매표체무명현적영향(P＞0.05).실시PCR화Western인적결과표명,과표체miR-145 mimics후,NRAS mRNA표체수평미출현명현변화(P＞0.05),대NRAS단백수평적표체유명현적억제(1.52±0.07비0.20±0.02,t=28.43,P＜0.01).결론 miR-145가능통과NRAS영향HaCaT세포적주기종이억제세포적증식,동시촉진HaCaT세포적조망
    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.
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