长链非编码RNA TUG1参与氧糖剥夺所致Neuro-2a细胞凋亡的作用研究

目的 探讨长链非编码RNA牛磺酸上调基因1(Taurine up-regulated gene 1,TUG1)对小鼠脑神经瘤细胞(Neuro-2a,N2a)糖氧剥夺(Oxygen and glucose deprivation, OGD)后细胞凋亡的影响。方法 利用CCK-8检测OGD处理后N2a细胞的增殖能力; 用Annexin V-FITC/PI双染处理后检测细胞凋亡情况,qRT-PCR检测LncRNA TUG1的表达水平; 设计LncRNA TUG1特异性siRNA片段(si-TUG1)及阴性对照(Negative control); 脂质体转染N2a细胞24 h后将细胞分成Control,OGD,Negative control组、si-TUG1组,除Control组外其他各组进行氧糖剥夺处理24 h; CCK-8评价细胞的增殖能力,流式细胞术检测细胞凋亡,Western blot检测凋亡相关蛋白Bad,Bcl-2,Cleaved-Caspase-3及PI3K/Akt通路激活情况。结果 OGD显著抑制N2a细胞增殖,促进细胞凋亡; LncRNA TUG1表达水平随着OGD处理时间延长而逐步升高(F=134.100,P<0.01); si-TUG1显著降低OGD处理引起的细胞凋亡,并促进细胞增殖; Western blot检测发现,与OGD组比较,si-TUG1能够抑制Bad表达(t=9.409,P<0.01)、促进Bcl-2表达(t=5.067,P<0.01),抑制Cleaved Caspase-3表达(t=5.757,P<0.01),进而抑制细胞凋亡; si-TUG1能够显著上调p-Akt(t=3.850,P<0.01),p-PI3Kp65(t=4.690,P<0.01)的表达,促进通路激活。结论 LncRNA TUG1 促进氧糖剥夺所致的N2a细胞凋亡,其机制可能与抑制PI3K/Akt通路激活有关。

The effects of long non-coding RNA TUG1 on the oxygen and glucose deprivation induced cell apoptosis in N2a cells

ObjectiveTo investigate the effects of long non-coding RNA(lncRNA)TUG1(Taurine up-regulated 1)on the cell apoptosis in mouse brain neuroma cells Neuro-2a(N2a)after oxygen and glucose deprivation(OGD)treatment.Methods CCK-8 assay was employed to test the proliferation of N2a cells after OGD treatment. The Annexin V-FITC/PI double strain analysis was used to explore the effects of OGD on the cell apoptosis. Lnc RNA TUG1 expressive level was tested by qRT-PCR. The unique siRNA for LncRNA TUG1(si-TUG1)and the negative control siRNA was designed and produced. After 24h of transfection, cells were divided into four groups(Control group, OGD group, Negative control group, and si-TUG1 group), then treated with OGD for 24 h. the cell proliferation was tested by CCK-8 assay. The cell apoptosis was tested by flow cytometry. Altered expressions of cell apoptosis related genes(Bad/Bcl-2,Cleaved-Caspase-3)and the activation of PI3K/Akt pathway were detected by western blotting analysis.Results The OGD significantly reduced the proliferation and induced apoptosis of N2a cells. LncRNA TUG1 expressive level was increased after OGD treatment with time dependent manners(F=134.100, P<0.01). Compared with OGD group, si-TUG1 could significantly reduce the OGD induced cell apoptosis, and promote cell proliferation. The expression of Bad and Cleaved Caspase-3 were decreased, and the expression of Bcl-2 was increased. Furthermore, the p-Akt and p-PI3Kp65 expressive levels were increased.Conclusion LncRNA TUG1 could promote the ODG induced apoptosis in N2a cells, and might be related with the suppression of PI3K/Akt pathway.