长链非编码RNA H19对缺血性脑卒中神经元SH-SY5Y细胞活力与自噬影响的探究

Effect of LncRNA H19 on viability and autophagy of neuronal SH-SY5Y cells in ischemic stroke

目的探究长链非编码RNA(LncRNA)H19对缺血性脑卒中神经元系SH-SY5Y细胞活力和凋亡的影响,并探究其和神经元自噬过程的相关性。方法选用神经元SH-SY5Y细胞系,进行氧糖剥夺/复氧建模(OGD/R),依据缺氧时间的不同(4 h,8 h和12 h),分为缺氧4 h组、缺氧8 h组和缺氧12 h组,同时建立未经过缺氧处理的对照组(n=3),测定细胞活力,采用实时PCR检测LncRNA H19表达水平。另外,将细胞在普通培养液中培养24 h,取对数生长期的SH-SY5Y细胞分为正常对照(N)组、OGD/R组、OGD/R+H19 siRNA组以及OGD/R+对照siRNA(NC)组,OGD/R缺氧出时间为8 h,采用Western blot检测微管相关蛋白1转链3(LC3)Ⅱ、Beclin1及P62蛋白定量。结果缺氧8 h组和缺氧12 h组细胞活力明显低于对照组和缺氧4 h组,缺氧8 h组LncRNA H19表达水平明显低于对照组(P<0.05)。与N组比较,OGD/R组、NC组和H19 siRNA组LC3Ⅱ(0.88±0.13、0.90±0.15和0.48±0.05 vs 0.50±0.08,P<0.05)及OGD/R组和NC组Beclin1表达(0.91±0.11和0.86±0.11 vs 0.28±0.04,P<0.05)明显增加,OGD/R组和NC组P62蛋白表达显著降低,差异有统计学意义(0.50±0.09和0.44±0.08 vs 0.88±0.12,P<0.05)。结论LncRNA H19的表达上调;LncRNA H19的上调显著增加细胞活性,降低其死亡率;LncRNA H19参与神经元自噬过程,可能对于缺血性脑卒中起保护作用。

Objective To study the effect of LncRNA H19 on viability and apoptosis of neuronal SH-SY5Y cells and its relationship with neuronal autophagy.Methods An oxygen glucose deprivation/reoxygenation(OGD/R)model was induced by neuronal SH-SY5Y cell line.The neuronal SH-SY5Y cells were divided into 4 h hypoxia group,8 h hypoxia group,12 h hypoxia group and control group.The viability of neuronal SH-SY5Y cells was assayed.The expression of LncRNA H19 was detected by RT-PCR.The neuronal SH-SY5Y cells were cultured in common culture fluid for 24 h.The neuronal SH-SY5Y cells at logarithmic growth phase were divided into normal control group,OGD/R group,OGD/R+H19 siRNA group,and OGD/R+control siRNA group.The OGD/R group was exposed to hypoxia for 8 h.The expressions of LC3Ⅱ,Beclin 1 and P62 proteins were detected by Western blot.Results The viability of neuronal SH-SY5Y cells was significantly lower in 8 h hypoxia group and 12 h hypoxia group than in normal control group and 4 h hypoxia group,the expression level of LncRNA H19 was significantly lower in 8 h hypoxia group than normal control group(P<0.05).The expression level of LC3Ⅱwas significantly higher in OGD/R group,OGD/R+control siRNA group and OGD/R+H19 siRNA group than in normal control group and the expression level of Beclin 1 protein was significantly higher while that of P62 protein was significantly lower in OGD/R group and OGD/R+control siRNA group than in normal control group(0.88±0.13、0.90±0.15 and 0.48±0.05 vs 0.50±0.08,P<0.05;0.91±0.11 and 0.86±0.11 vs 0.28±0.04,P<0.05;0.50±0.09 and 0.44±0.08 vs 0.88±0.12,P<0.05).Conclusion The upregulated expression of LncRNA H19 can significantly increase the viability and reduce the mortality of neuronal SH-SY5Y cells.LncRNA H19 is involved in neuronal autophagy and can thus protect elderly persons against ischemic stroke.