实用医学杂志 ›› 2023, Vol. 39 ›› Issue (6): 672-678.doi: 10.3969/j.issn.1006⁃5725.2023.06.004

• 基础研究 • 上一篇    下一篇

lncRNA KCNQ1OT1靶向miR⁃129⁃5p调控帕金森病细胞模型炎症和细胞凋亡的实验研究 

张卓尔1 高筱雅2 余娟3 王玉理3    

  1. 1 南方医科大学第二临床医学院(广州 510515);2 南方医科大学珠江医院神经内科(广州510282); 3 深圳市第二人民医院医学影像科(广东深圳518000)

  • 出版日期:2023-03-25 发布日期:2023-03-25
  • 基金资助:

    基金项目:广东省基础与应用基础研究基金项目(编号:2021A1515220131)
    通信作者:高筱雅 E⁃mail:tracygxy@126.com

Experimental study of lncRNA KCNQ1OT1 targeting miR⁃129⁃5p to regulate inflammation and apoptosis in a Parkinson′s disease cell model

ZHANG Zhuoer*,GAO Xiaoya*,YU Juan,WANG Yuli.   

  1. The Second Clini⁃ cal Medical College of Southern Medical University,Guangzhou 510515,China

  • Online:2023-03-25 Published:2023-03-25
  • Contact: GAO Xiaoya E⁃mail:tracygxy@126.com

摘要:

目的 探讨 lncRNA KCNQ1OT1 靶向 miR⁃129⁃5p 调控帕金森病(PD)细胞模型炎症和细胞凋亡的机制。方法 体外培养人 SK⁃N⁃SH 细胞,用 1、2、4 mmol/L MPP+ 处理 SK⁃N⁃SH 构建PD细胞损伤模 型。将si⁃NC、si⁃lncRNA KCNQ1OT1、miR⁃NC、miR⁃129⁃5p、si⁃lncRNA KCNQ1OT1anti⁃miR⁃NC、si⁃lncRNA KCNQ1OT1与anti⁃miR⁃129⁃5p分别转染至MPP+处理SK⁃N⁃SH构建PD细胞中,记为si⁃NC+MPP+组、si⁃lncRNA KCNQ1OT1+MPP+组、miR⁃NC+MPP+组、miR⁃129⁃5p+MPP+组、si⁃lncRNA KCNQ1OT1+anti⁃miR⁃NC+MPP+组、si-lncRNA KCNQ1OT1+anti⁃miR⁃129⁃5p+MPP+组。酶联免疫吸附法(ELISA)检测细胞 TNF⁃α、IL⁃6、IFN⁃γ 水平;实时荧光定量 PCR(RT⁃qPCR)检测细胞lncRNA KCNQ1OT1和miR⁃129⁃5p表达情况;双荧光素酶报告 实验检测 lncRNA KCNQ1OT1 miR⁃129⁃5p 的靶向关系。结果 NC 组比较,1、2、4 mmol/L MPP+ 组细胞 凋亡率、Cleaved⁃caspase⁃3 蛋白表达、TNF⁃α、IL⁃6、IFN⁃γ 水平显著升高(P < 0.05)。与 NC 组比较,1、2 4 mmol/L MPP+ 组细胞lncRNA KCNQ1OT1表达,其水平明显较高,而miR⁃129⁃5p表达,明显更低(P < 0.05)。 si⁃NC+MPP+ 组比较,si⁃lncRNA KCNQ1OT1+MPP+组细胞凋亡率、Cleaved⁃caspase⁃3蛋白表达、TNF⁃α、IL⁃6 IFN⁃γ水平显著降低(P < 0.05)。与miR⁃NC+MPP+组比较,miR⁃129⁃5p+MPP+组细胞凋亡率、Cleaved⁃caspase⁃3 蛋白表达、TNF⁃α、IL⁃6、IFN⁃γ水平显著降低(P < 0.05)。si⁃lncRNA KCNQ1OT1+anti⁃miR⁃129⁃5p+MPP+ 细胞 凋亡、Cleaved⁃caspase⁃3 蛋白表达、TNF⁃α、IL⁃6、IFN⁃γ 水平显著升高(P < 0.05)。结论 lncRNA KCNQ1OT1 下调miR⁃129⁃5p表达抑制PD 细胞模型炎症和细胞凋亡。

关键词:

lncRNA KCNQ1OT1, miR?129?5p, 帕金森病, 凋亡, 炎症

Abstract:

Objective lncRNA KCNQ1OT1 targets miR⁃129⁃5p to regulate inflammation and apoptosis in a Parkinson disease(PD)cell model. Methods Human SK⁃N⁃SH cells were cultured in vitro,and SK⁃N⁃SH was treated with 1,2,4 mmol/L MPP+ to construct a PD cell injury model. Transfect si⁃NC,si⁃lncRNA KCNQ1OT1 miR⁃NC,miR⁃129⁃5p,si⁃lncRNA KCNQ1OT1 and anti⁃miR⁃NC,si⁃lncRNA KCNQ1OT1 and anti⁃miR⁃129⁃5p into MPP+treated SK,respectively⁃N⁃SH constructs PD cells,denoted as si⁃NC+MPP+group,si⁃lncRNA KCNQ1OT1+ MPP+group,miR ⁃NC+MPP+group,miR ⁃129⁃5p+MPP+group,si ⁃lncRNA KCNQ1OT1+anti ⁃miR ⁃NC+MPP+ group,si⁃lncRNA KCNQ1OT1+anti⁃miR⁃129⁃5p+MPP+group. Flow cytometry to detect cell apoptosis;Western Blot method to detect cell Cleaved⁃caspase⁃3 protein expression;enzyme⁃linked immunosorbent assay(ELISA)to detect cell TNF⁃α,IL⁃6,IFN⁃γ levels;real⁃time Fluorescence quantitative PCR(RT⁃qPCR)was used to detect the expression of lncRNA KCNQ1OT1 and miR⁃129⁃5p in cells;the dual luciferase reporter experiment was used to detect the targeting relationship between lncRNA KCNQ1OT1 and miR ⁃129⁃5p. Results Compared with the NC group,the apoptosis rate,Cleaved⁃caspase⁃3 protein expression,TNF⁃α,IL⁃6,and IFN⁃γ levels in the 1,2 4 mmol/L MPP+ group were significantly increased(P < 0.05). Compared with the NC group,the expression level of lncRNA KCNQ1OT1 in the 1,2,4 mmol/L MPP+ group was significantly increased,and the expression level of miR⁃129⁃5p was significantly decreased(P < 0.05). Compared with the si⁃NC+MPP+ group,the apoptotic rate,Cleaved ⁃caspase ⁃3 protein expression,TNF ⁃α,IL ⁃6,and IFN ⁃ γ levels in the si ⁃lncRNA KCNQ1OT1+MPP+ group were significantly reduced(P < 0.05). Compared with the miR⁃NC+MPP+group,the apoptosis rate,Cleaved⁃ caspase⁃3 protein expression,TNF⁃α,IL⁃6,and IFN⁃γ levels in the miR⁃129⁃5p+MPP+group were significantly reduced(P < 0.05). Compared with the si⁃lncRNA KCNQ1OT1+anti⁃miR⁃NC+MPP+group ,the si⁃lncRNA KCNQ1OT1+anti⁃miR⁃129⁃5p+MPP+group cell apoptosis rate,Cleaved⁃caspase⁃3 protein expression,TNF⁃α,IL⁃ 6. The level of IFN ⁃ γ increased significantly(P < 0.05). Conclusion lncRNA KCNQ1OT1 down ⁃ regulates the expression of miR⁃129⁃5p to inhibit inflammation and apoptosis in PD cell models.

Key words:

lncRNA KCNQ1OT1, miR?129?5p, Parkinson′s disease, apoptosis, inflammation