摘要
目的 探讨长链非编码RNA核富集转录本1(lncRNA NEAT1)对膜性肾病(MN)中自噬的影响及其机制.方法 利用白蛋白诱导HK-2细胞构建体外MN模型,将HK-2细胞分为5组:对照组、模型组、LY294002(PI3K抑制剂)组、OV-NEAT1(NEAT1过表达)组和OV-NEAT1+LY294002组.CCK-8检测细胞增殖情况,流式细胞术检测细胞凋亡情况,qRT-PCR和Western blot检测自噬和PI3K/Akt/mTOR通路相关mRNA和蛋白的表达水平.结果 与对照组相比,模型组细胞增殖能力及细胞中LC3-Ⅱ、Beclin 1 mRNA水平和LC3-Ⅱ/Ⅰ、Beclin 1蛋白表达水平显著降低(均P＜0.01),细胞凋亡率及细胞中LC3-Ⅰ、PI3K、Akt、mTOR mRNA和p-PI3K、p-Akt、p-mTOR蛋白表达水平显著升高(均P＜0.01);与模型组相比,OV-NEAT1组细胞增殖能力、细胞中LC3-Ⅱ、Beclin 1 mRNA和LC3-Ⅱ/Ⅰ、Beclin 1蛋白表达水平显著降低(均P＜0.05),细胞凋亡率及细胞中LC3-Ⅰ、PI3K、Akt、mTOR mRNA和p-PI3K、p-Akt、p-mTOR蛋白表达水平显著升高(均P＜0.01),LY294002组趋势相反;与LY294002组相比,O V-NEAT1+LY294002组细胞增殖能力及细胞中LC3-Ⅱ、Beclin 1 mRNA和LC3-Ⅱ/Ⅰ、Beclin 1蛋白表达水平显著降低(均P＜0.05),细胞凋亡率及细胞中LC3-Ⅰ、PI3K、Akt、mTOR mRNA和p-PI3K、p-Akt、p-mTOR蛋白表达水平显著升高(均P＜0.05).结论 lncRNA NEAT1能够抑制白蛋白诱导的HK-2细胞的自噬,其机制可能与PI3K/Akt/mTOR信号通路的激活有关.
Abstract
Objective To investigate the effect of long noncoding RNA nuclear paraspeckle assembly transcript 1(lncRNA NEAT1)on autophagy in membranous nephropathy(MN)and its mechanism.Methods HK-2 cells were induced by albumin to construct MN model in vitro.HK-2 cells were divided into 5 groups:control group,model group,LY294002(PI3K inhibitor)group,OV-NEAT1(NEAT1 overexpression)group and OV-NEAT1+LY294002 group.Cell proliferation was detected by CCK-8.Cell apoptosis was detected by flow cytometry.Expressions of autophagy and PI3K/Akt/mTOR pathway-related mRNA and protein were detected by qRT-PCR and Western blotting.Results Compared with control group,the cell proliferation ability,LC3-Ⅱ and Beclin 1 mRNA levels as well as LC3-Ⅱ/Ⅰ and Beclin 1 protein expression levels in the model group were signifi-cantly decreased(all P＜0.01).The apoptosis rate,LC3-Ⅰ,PI3K,Akt,mTOR mRNA and p-PI3K,p-Akt,p-mTOR protein ex-pression levels were significantly increased(all P＜0.01).Compared with model group,the cell proliferation ability,LC3-Ⅱ and Beclin 1 mRNA and LC3-Ⅱ/Ⅰ and Beclin 1 protein expression levels in OV-NEAT1 group were significantly decreased(all P＜0.05).The apoptosis rate,LC3-Ⅰ,PI3K,Akt,mTOR mRNA and p-PI3K,p-Akt,p-mTOR protein expression levels were sig-nificantly increased(all P＜0.01).LY294002 group witnessed the opposite trend.Compared with LY294002 group,cell prolifer-ation ability,LC3-Ⅱ and Beclin 1 mRNA and LC3-Ⅱ/Ⅰ and Beclin 1 protein expression levels in OV-NEAT1+LY294002 group were significantly decreased(all P＜0.05).The apoptosis rate,LC3-Ⅰ,PI3K,Akt,mTOR mRNA and p-PI3K,p-Akt,p-mTOR protein expression levels were significantly increased(all P＜0.05).Conclusion lncRNA NEAT1 can inhibit albumin-induced autophagy in HK-2 cells,and the mechanism may be related to the activation of PI3K/Akt/mTOR signaling pathway.
基金项目
武汉市卫生健康委员会科研计划项目(WX21A16)
NETL
NSTL
万方数据