Long Non-coding RNA H19 Induces Cerebral Ischemia Reperfusion Injury via Activation of Autophagy

Abstract

Long non-coding RNA H19 (lncRNA H19) was found to be upregulated by hypoxia, its expression and function have never been tested in cerebral ischemia and reperfusion (I/R) injury. This study intended to investigate the role of lncRNA H19 and H19 gene variation in cerebral I/R injury with focusing on its relationship with autophagy activation. Cerebral I/R was induced in rats by middle cerebral artery occlusion followed by reperfusion. SH-SY5Y cells were subjected to oxygen and glucose deprivation and reperfusion (OGD/R) to simulate I/R injury. Real-time PCR, flow cytometry, immunofluorescence and Western blot were used to evaluate the level of lncRNA H19, apoptosis, autophagy and some related proteins. The modified multiple ligase reaction was used to analyze the gene polymorphism of six SNPs in H19, rs217727, rs2067051, rs2251375, rs492994, rs2839698 and rs10732516 in ischemic stroke patients. We found that the expression of lncRNA H19 was upregulated by cerebral I/R in rats, as well as by OGD/R in vitro in the cells. Inhibition of lncRNA H19 and autophagy protected cells from OGD/R-induced death, respectively. Autophagy activation induced by OGD/R was prevented by H19 siRNA. Autophagy inducer, rapamycin, abolished lncRNA H19 effect. Furthermore, we found that lncRNA H19 inhibited autophagy through DUSP5-ERK1/2 axis. The result from blood samples of ischemic patients revealed that the variation of H19 gene increased the risk of ischemic stroke. Taken together, the results of present study suggest that LncRNA H19 could be a new therapeutic target of ischemic stroke.

Keywords: apoptosis; autophagy; cerebral ischemia reperfusion; gene polymorphism; lncRNA H19.

Figure 1.

LncRNA H19 expression in rat cerebral ischemia reperfusion model (I/R) and cellular oxygen glucose deprivation/reperfusion (OGD/R) model. A) 2, 3, 5-triphenyltetrazolium chloride (TTC) staining evaluation of rat cerebral infarct volume induced by I/R (n=6). The representative images of rat brain slices in different groups are displayed on the left and the quantification of infarct volume on the right. B) LncRNA H19 level determined by real-time PCR, normalized to the expression of GAPDH in the sham and I/R group (n=6). C) Cell viability induced by different conditions of OGD/R. D) LncRNA H19 level determined by real-time PCR, normalized to the expression of 18S rRNA in the normal and OGD/R groups. N represents normal group, OGD/R 4 hr, OGD/R 8 hr, and OGD/R 12 hr represent s the cells subjected to 4, 8, and 12 hr oxygen glucose deprivation, respectively, followed by 24 hr reperfusion. Bar represents the mean value ± SD. *p < 0.05 vs. the sham-operated or normal group.

Figure 2.

The effect of inhibition of lncRNA H19 and autophagy on cell viability and apoptosis in OGD/R model. A) The cell viability in different groups. B) Percentage of apoptotic cells in different groups. C-G) The result of Annexin V/PI staining in different groups measured by flow cytometry. N, normal group; OGD/R, OGD 8 hr and reperfusion 24 hr; OGD/R + H19 siRNA and OGD/R + N.C, transfecting the cells with H19 siRNA and normal control siRNA, respectively, before OGD/R treatment. OGD/R+3MA, 3MA was added to the medium 10 min before OGD/R. Bar represents the mean value ± SD. *p<0.05 vs. the normal group, ^#p<0.05 vs. OGD/R group, ^&p<0.05 vs. OGD/R + H19 siRNA group.

Figure 3.

The effect of H19 siRNA on autophagy in OGD/R model. A) Immunofluorescence of LC3II in different conditions. B) Immunofluorescence of Beclin1 in different conditions. C) Immunofluorescence of P62 in different conditions. D) The change of lncRNA H19 expression level induced by H19 siRNA. E. Western blot of LC3II, LC3I, Beclin1, and P62 in different groups. F, G, and H) Statistical analysis of the expression of LC3II, Beclin1 and P62, respectively. N, normal group; OGD/R, OGD 8 hr and reperfusion 24 hr; OGD/R + H19 siRNA and OGD/R + N.C, transfecting the cells with H19 siRNA and normal control siRNA, respectively, before OGD/R treatment. Bar represents the mean value ± SD. *p<0.05 vs. normal group, ^#p<0.05 vs. OGD/R group, ^&p<0.05 vs. OGD/R + H19 siRNA group. White arrows indicate the co-localized positive cells.

Figure 4.

LncRNA H19 impairs cells in OGD/R via activation of autophagy. A) The cell viability in different conditions. B) Western blot of LC3I, L3II, Beclin1, and P62 in different groups. C, D and E) Statistical analysis of the expression of LC3II, Beclin1, and P62. F) Western blot of LC3I, L3II, Beclin1, and P62 in different groups with or without rapamycin treatment. G) H and I) Statistical analysis of the expression of LC3II, Beclin1, and P62. N, normal group; OGD/R, OGD 8 hr and reperfusion 24 hr; OGD/R + H19 siRNA and OGD/R + N.C, transfecting the cells with H19 siRNA and normal control siRNA, respectively, before OGD/R treatment; OGD/R + H19 siRNA + RAP, the cells transfected with H19 siRNA and administration of rapamycin before OGD/R; OGD/R + vehicle, treating the cells with vehicle before OGD/R. Bar represents the mean value ± SD. *p<0.05 vs. the normal group, ^#p<0.05 vs. OGD/R group, ^&p<0.05 vs.OGD/R + H19 siRNA group.

Figure 5.

LncRNA H19 induces autophagy through DUSP5-ERK1/2 axis. A) The cell viability in different conditions. B) Western blot and statistical analysis of DUSP5 in different conditions. C) Western blot of LC3I, L3II, Beclin1, P62 and DUSP5 in different groups. D-G) Statistical analysis of the expression of LC3II, Beclin1, P62 and DUSP5 in different groups. H) Western blot of ERK1/2 and p-ERK1/2 in different groups. I and J) Statistical analysis of the expression of ERK1/2 and p-ERK1/2 in different groups. N, normal group; OGD/R, OGD 8 hr and reperfusion 24 hr; OGD/R + H19 siRNA, OGD/R + N.C and OGD/R+DUSP5, transfecting the cells with H19 siRNA, normal control siRNA and DUSP5 siRNA, respectively, before OGD/R treatment; OGD/R + H19 siRNA + DUSP5 siRNA, the cells transfected with H19 siRNA and DUSP5 siRNA before OGD/R. Bar represents the mean value ± SD. *p < 0.05 vs. the normal group, ^#p 0.05 vs. OGD/R group, ^&p<0.05 vs.OGD/R + H19 siRNA group.