lncRNA NEAT1激活PI3K/Akt信号通路对Aβ_(25-35)诱导的PC12细胞凋亡的影响及机制

Effect and mechanism of lncRNA NEAT1 activating PI3K/Akt signal pathway on the influence of PC12 cell apoptosis induced by Aβ_(25-35)

目的探讨lncRNA NEAT1通过激活PI3K/Akt信号通路对Aβ_(25-35)诱导的阿尔茨海默病(AD)模型细胞凋亡的影响及可能机制。方法以0、5、10、20μmol/L Aβ_(25-35)诱导PC12细胞,构建AD细胞模型。用5μmol/L Aβ_(25-35)处理PC12细胞,并于24、48、72 h采用qRT-PCR检测lncRNA NEAT1相对表达水平。取PC12细胞,(1)设置对照组、敲降lncRNA NEAT1组、si-NC组、空载体组及过表达lncRNA NEAT1组,对照组不做任何处理,其余4组分别用含si-lncRNA NEAT1、si-NC、pcDNA3.1-NC、pcDNA3.1-lncRNA NEAT1的Opti-MEM培养基培养并转染8 h,然后使用20μmol/L Aβ_(25-35)处理。采用qRT-PCR检测lncRNA NEAT1相对表达水平;CCK-8法检测细胞活力;流式细胞术检测细胞凋亡情况,并以G1和G2期细胞比例反映细胞增殖情况;ELISA法检测细胞上清中炎性因子白细胞介素-1β(IL-1β)、IL-6、IL-18、肿瘤坏死因子-α(TNF-α)水平。(2)设置对照组、空载体组及过表达lncRNA NEAT1组,采用Western blotting检测p-PI3K、p-Akt蛋白相对表达水平。(3)设置对照组、si-NC组、敲降lncRNA NEAT1组、空载体组、过表达lncRNA NEAT1组及过表达lncRNA NEAT1+LY294002组,过表达lncRNA NEAT1+LY294002组细胞转染pcDNA3.1-lncRNA NEAT1后,加入10μmol/L PI3K通路抑制剂LY294002处理,采用流式细胞术检测细胞凋亡情况。结果不同浓度的Aβ_(25-35)均可明显抑制lncRNA NEAT1的表达,且呈浓度依赖性(P=0.001)。与空载体组相比,过表达lncRNA NEAT1组lncRNA NEAT1相对表达水平、细胞增殖率、G2期细胞比例明显增高,细胞凋亡率、G1期细胞比例明显降低(P<0.05);与si-NC组相比,敲降lncRNA NEAT1组lncRNA NEAT1相对表达水平、细胞增殖率、G2期细胞比例明显降低,细胞凋亡率、G1期细胞比例明显升高(P<0.05)。ELISA检测结果显示,与空载体组相比,过表达lncRNA NEAT1组细胞上清中IL-1β、IL-6、IL-18、TNF-α水平明显降低(P<0.05);与si-NC组相比,敲降lncRNA NEAT1组细胞上清中IL-1β、IL-6、IL-18、TNF-α水平明显升高(P<0.05)。Western blotting检测结果显示,与空载体组相比,过表达lncRNA NEAT1组信号通路相关蛋白p-PI3K、p-Akt相对表达水平增高(p-PI3K:0.86±0.05 vs.0.15±0.02,P=0.003;p-Akt:0.86±0.06 vs.0.11±0.04,P=0.000)。与过表达lncRNA NEAT1组相比,过表达lncRNA NEAT1+LY294002组细胞凋亡率明显升高(9.00%±0.10%vs.5.13%±0.21%,P=0.004)。结论lncRNA NEAT1可通过调控PI3K/Akt通路促进Aβ_(25-35)诱导的PC12细胞增殖,抑制PC12细胞凋亡。

Objective To investigate the effect and mechanism of lncRNA NEAT1 activating PI3K/Akt signaling pathway on the influence of Alzheimer's disease(AD)PC12 model cells apoptosis induced by Aβ_(25-35).Methods PC12 cells were induced with Aβ_(25-35) of concentrations 0,5,10 and 20μmol/L to construct AD cell model.PC12 cells were treated with 5μmol/L Aβ_(25-35),and the relative expression level of lncRNA NEAT1 was detected by qRT-PCR at the time points 24,48 and 72 hours,respectively.PC12 cells were divided into:(1)control group,lncRNA NEAT1 knockdown group,si-NC group,empty body group and lncRNA NEAT1 overexpression group.Among them the cells in control group were not treated anyway;in the other four groups were cultured with Opti-MEM medium containing si-lncRNA NEAT1,si-NC,pcDNA3.1-NC and pcDNA3.1-lncRNA NEAT1 and transfected for 8 hours,and then treated with 20μmol/L Aβ_(25-35);qRT-PCR was used to detect the relative expression level of lncRNA NEAT1;CCK-8 was used to detect the cell viability;flow cytometry was used to detect the apoptosis of PC12 cells,and the proliferation of PC12 cells was reflected by the ratio of G1 to G2 phase of cell cycle;the inflammatory factors interleukin-1β(IL-1β),IL-6,IL-18,and tumor necrosis factor-α(TNF-α)in cell supernatant were detected by ELISA.(2)control group,empty body group,and lncRNA NEAT1 overexpression group,Western blotting was used to detect the relative expression levels of p-PI3K and p-Akt.(3)control group,si-NC group,lncRNA NEAT1 knockdown group,empty body group,lncRNA NEAT1 overexpression group,and lncRNA NEAT1 overexpression+LY294002 group.Cells in lncRNA NEAT1 overexpression+LY294002 group were transfected with pcDNA3.1-lncRNA NEAT1,and 10μmol/L P13K pathway inhibitor LY294002 were added,and then flow cytometry was used to detect the apoptosis of PC12 cells.Results Different concentrations of Aβ_(25-35) could significantly inhibit the expression of lncRNA NEAT1 in a concentration-dependent manner(P=0.001).Compared with empty body group,the relative expression level of lncRNA NEAT1,the cell proliferation rate,and the proportion of cells in G2 phase of lncRNA NEAT1 overexpression group increased,the cell apoptosis rate and the proportion of cells in G1 phase decreased(P<0.05).Compared with si-NC group,the relative expression level of lncRNA NEAT1,the cell proliferation rate,and the proportion of cells in G2 phase of lncRNA NEAT1 knockdown group decreased,the cell apoptosis rate and the proportion of cells in G1 phase increased(P<0.05).ELISA results showed that,compared with empty body group,the levels of inflammatory factors IL-1β,IL-6,IL-18,and TNF-αdecreased significantly in lncRNA NEAT1 overexpression group(P<0.05);compared with si-NC group,the levels of inflammatory factors IL-1β,IL-6,IL-18 and TNF-αincreased significantly in lncRNA NEAT1 knockdown group(P<0.05).Western blotting results showed that,compared with empty body group,the expression levels of signal pathway related proteins p-PI3K and p-Akt increased in lncRNA NEAT1 overexpression group(p-PI3K:0.86±0.05 vs.0.15±0.02,P=0.003;p-Akt:0.86±0.06 vs.0.11±0.04,P=0.000).Compared with lncRNA NEAT1 overexpression group,the apoptosis rate in lncRNA NEAT1 overexpression+LY294002 group increased obviously(9.00%±0.10%vs.5.13%±0.21%,P=0.004).Conclusion lncRNA NEAT1 can promote Aβ_(25-35) induced PC12 cell proliferation and inhibit cell apoptosis by regulating PI3K/Akt pathway.