抑制miR-31表达对人角质形成细胞增殖、凋亡的影响研究

目的探讨miR-31对人角质形成细胞系HaCaT细胞增殖和凋亡的影响以及可能机制。方法培养HaCaT细胞，利用瞬时转染的方法，对不同组细胞进行转染。反义寡核苷酸技术（ASO）组:转染微小RNA（miR）-31 ASO；对照ASO组:转染对照ASO；空白对照组:转染空白质粒。MTT法检测不同转染组细胞增殖情况，Annexin V-FITC/PI双染色检测不同转染组细胞凋亡情况，PI单染色法检测不同转染组细胞周期，Western blotting检测不同转染组细胞中Rho相关结构域BTB蛋白质1（RhoBTB1）蛋白表达。结果ASO组细胞中miR-31表达量明显低于对照ASO组和空白对照组（P < 0.01）；ASO组细胞在24、48、72、96 h时吸光度值均低于对照ASO组和空白对照组（P < 0.05~P < 0.01），且3组细胞随着时间推移，吸光度值均较之前逐渐增加（P < 0.01）；ASO组细胞凋亡率、G₀/G₁期细胞比例、RhoBTB1蛋白表达量明显高于对照ASO组和空白对照组（P < 0.01）；而S期和G₂期细胞比例明显低于对照ASO组和空白对照组（P < 0.01）。结论miR-31可能参与了人角质形成细胞系HaCaT细胞增殖、凋亡过程，可能通过调控RhoBTB1蛋白表达而实现这一生物学过程。

Effect of inhibiting the miR-31 expression on the proliferation and apoptosis of human keratinocyte cells

ObjectiveTo investigate the effects of inhibiting the miR-31 expression on the proliferation and apoptosis of human keratinocyte cells, and its mechanism.MethodsThe HaCaT cells were cultured, and transfected using transient transfection method.These cells were divided into the antisense oligonueleotide(ASO) group(treatment with miR-31 ASO), control ASO group(treatment with control ASO) and blank control group(treatment with blank plasmid).The cell proliferation, apoptosis, cell cycle and Rho related domain BTB protein 1(RhoBTB1) expression in three groups were detected using MTT assay, Annexin V-FITC/PI double staining, PI single staining and Western blotting, respectively.ResultsThe expression level of miR-31 in ASO group were significantly lower than that in ASO control group and blank control group(P < 0.01).The absorbance values in ASO group at 24 h, 48 h, 72 h and 96 h were significantry lower than that in ASO control group and blank control group(P < 0.05 to P < 0.01), and the absorbance values in three groups gradually increased as time went on(P < 0.01).The apoptotic rate, proportion of G₀/G₁ phase cells and expression level of RhoBTB1 protein in ASO group were significantly higher than those in ASO control group and blank control group(P < 0.01), while the proportion of S/G₂ phase cells in ASO group were significantly lower than those in ASO control group and blank control group(P < 0.01).ConclusionsmiR-31 may be involved in the proliferation and apoptosis of human keratinocyte cell lines HaCaT cells, which may achieve this biological process by regulating the expression of RhoBTB1 protein.