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Aging and disease    2017, Vol. 8 Issue (1) : 71-84     DOI: 10.14336/AD.2016.0530
Original Article |
Long Non-coding RNA H19 Induces Cerebral Ischemia Reperfusion Injury via Activation of Autophagy
Wang Jue, Cao Bin, Han Dong, Sun Miao, Feng Juan*
Department of Neurology, Shengjing Hospital, Affiliated Hospital of China Medical University, Shen Yang, 110004, China
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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 cerebral ischemia reperfusion      lncRNA H19      gene polymorphism      autophagy      apoptosis     
Corresponding Authors: Feng Juan   
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These authors contributed equally to this work

Issue Date: 01 February 2017
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Wang Jue
Cao Bin
Han Dong
Sun Miao
Feng Juan
Cite this article:   
Wang Jue,Cao Bin,Han Dong, et al. Long Non-coding RNA H19 Induces Cerebral Ischemia Reperfusion Injury via Activation of Autophagy[J]. Aging and disease, 2017, 8(1): 71-84.
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http://www.aginganddisease.org/EN/10.14336/AD.2016.0530     OR     http://www.aginganddisease.org/EN/Y2017/V8/I1/71
CharacteristicsCases(n=152)Controls(n=150)p value
Age (years)64.07 ± 11.8165763.68 ±9.5343370.7657
Sex (male/female)107/45100/500.4854
BMI (kg/m2)24.58 ± 3.01473423.17 ±3.6636660.0008
Smoking, n (%)32.216.7< 0.0001
Drinking, n (%)17.119.30.6159
SBP (mmHg)156.5 ± 26.34424128.7 ± 10.91012< 0.0001
DBP (mmHg)96.32 ± 20.778484.93 ± 7.495738< 0.0001
TC (mmol/L)4.619 ± 1.2304244.780 ± 0.9802090.2557
TG (mmol/L)2.181 ± 2.044641.627 ± 1.7854280.0092
HDL-C (mmol/L)1.048 ± 0.2731421.144 ± 0.2899420.0069
LDL-C (mmol/L)3.102 ± 1.0402022.553 ± 0.790455< 0.0001
FBG (mmol/L)5.848 ± 2.2536645.191 ± 1.0809110.0055
Hypertension, n (%)9712< 0.0001
Diabetes, n (%)1040.2004
Hyperlipidaemia, n (%)64570.5589
Table1  Characteristics of study subjects
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.
PolymorphismsCasesControlsP valueCrude OR (95% CI)p valueAdjusted OR§(95% CI)p value
rs217727
Additive model
CC18471-1-
CT63622.653(1.390-5.066)0.00272.381 (1.133-5.005)0.022
TT7141< 0.0014.522(2.324-8.799)< 0.00014.288(2.002-9.181)<0.001
Dominant model
CC18471-1-
CT+TT1341030.05713.397(1.862-6,196)< 0.00013.081(1.563-6.072)0.001
Recessive model
CC+CT811091-1-
TT7141< 0.0012.33(1.442-3.767)0.00052.269(1.344-3.828)0.002
Allele
C99156
T205144< 0.001
rs2067051
Additive model
TT28301-1-
TC54580.9975(0.5288-1.882)0.99391.605(0.529-2.144)0.861
CC70620.71051.21(0.659-2.245)0.54591.359(0.659-2.801)0.406
Dominant model
TT28301-1-
TC+CC1241200.72761.107(0.6242-1.964)0.72761.127(0.648-2.286)0.542
Recessive model
TT+TC82881-1-
CC70620.40841.212(0.7684-1.911)0.40841.319(0.808-2.152)0.268
Allele
T110118
C1941820.4247
rs2251375
Additive model
CC63721-1-
CA69571.383(0.8498-2.252)0.19121.530(0.900-2.602)0.116
AA20210.4161.088(0.5407-2.191)0.81231.257(0.581-2.718)0.562
Dominant model
CC63721-1-
CA+AA89780.25211.304(0.8275-2.055)0.25211.392(0.852-2.273)0.186
Recessive model
CC+CA1321291-1-
AA20210.83080.9307(0.4816-1.799)0.83080.973(0.474-1.997)0.941
Allele
C195201
A109990.4603
rs4929984
Additive model
CC40681-1-
CA62531.989(1.164-3.398)0.01152.275(1.251-4.138)0.007
AA50290.03052.931(1.606-5.350)0.00043.020(1.531-5.959)0.001
Dominant model
CC40681-1-
CA+AA112820.00062.322(1.432-3.766)0.00062.506(1.473-4.262)0.001
Recessive moedel
CC+CA1021211-1-
AA50290.00732.045(1.206-3.468)0.00731.942(1.088-3,464)0.025
Allele
C142189
A162111< 0.001
rs2839698
Additive model
GG80871-1-
GA61501.327(0.8195-2.148)0.24951.563(0.925-2.641)0.095
AA11130.46370.9202(0.3899-2.172)0.84941.086(0.673-1.752)0.736
Dominant model
GG80871-1-
GG+GA72630.34811.243(0.7889-1.958)0.34811.346(0.826-2.195)0.233
Recessive model
GG+GA141137
AA11130.6460.8221(0.3560-1.898)0.6460.665(0.262-1.690)0.391
Allele
G221224
A83760.5827
rs10732516
Additive model
GG69621-1-
GA45470.8603(0.5045-1.467)0.58040.768(0.419-1.409)0.394
AA38410.77170.8328(0.4761-1.457)0.5210.917(0.497-1.689)0.78
Dominant model
GG69621-1-
GA+AA83880.47640.8475(0.5373-1.337)0.47640.822(0.499-1.353)0.441
Recessive model
GG+GA1141091-1-
AA38410.64460.8862(0.5302-1.481)0.64461.060(0.610-1.843)0.836
Allele
G183171
A1211290.425
Table 2  Genotype distribution and allele frequency of the six tested SNPs
[1] Mozaffarian D, Benjamin EJ, Go AS, et al (2016). Executive Summary: Heart Disease and Stroke Statistics-2016 Update: A report from the American Heart Association. Circulation, 133: 447-54.
[2] Amouyel P (2012). From genes to stroke subtypes. Lancet Neurol, 11: 931-3.
[3] Shi X, Sun M, Liu H, Yao Y, Song Y (2013). Long non-coding RNAs: a new frontier in the study of human diseases. Cancer Lett, 339: 159-66.
[4] Gabory A, Jammes H, Dandolo L (2010). The H19 locus: role of an imprinted non-coding RNA in growth and development. Bioessays, 32: 473-80.
[5] Xia T, Liao Q, Jiang X, Shao Y, Xiao B, Xi Y, et al (2014). Long noncoding RNA associated-competing endogenous RNAs in gastric cancer. Sci Rep, 4: 6088.
[6] Matouk IJ, DeGroot N, Mezan S, Ayesh S, Abu-lail R, Hochberg A, et al (2007). The H19 non-coding RNA is essential for human tumor growth. Plos One, 2: e845.
[7] Yu LL, Chang K, Lu LS, Zhao D, Han J, Zheng YR (2013). Lentivirus-mediated RNA interference targeting the H19 gene inhibits cell proliferation and apoptosis in human choriocarcinoma cell line JAR. BMC Cell Biol, 27: 14-26.
[8] DK Han, ZZ Khaing, RA Pollock, CC Haudenschild, G Liau (1996). H19, a marker of developmental transition, is reexpressed in human atherosclerotic plaques andis regulated by the insulin family of growth factors in cultured rabbit smoothmuscle cells. J Clin Invest, 97: 1276-85.
[9] DK Kim, L Zhang, VJ Dzau, RE Pratt (1994). H19, a developmentally regulated gene, is reexpressed in rat vascular smooth muscle cells after injury. J. Clin. Invest, 93: 355-60.
[10] Matouk IJ, Mezan S, Mizrahi A, Ohana P, Abu-Lail R, Fellig Y, et al. (2010). The oncofetal H19 RNA connection: hypoxia, p53 and cancer. Biochim Biophys Acta, 1803: 443-51.
[11] Wang W, Kang J, Li H, Su J, Wu J, Xu Y, et al (2013). Regulation of endoplasmic reticulum stress in rat cortex by p62/ZIP through the Keap1-Nrf2-ARE signaling pathway after transient focal cerebral ischaemia. Brain Inj, 27: 924-33.
[12] Wen YD, Zhang HL, Qin ZQ (2006). Inflammatory mechanism in ischemic neuronal injury. Neurosci Bull, 22: 171-82.
[13] Cuervo AM (2004). Autophagy: in sickness and in health. Trends Cell Biol, 14: 70-7.
[14] Puyal J, Clarke PG (2009). Targeting autophagy to prevent neonatal stroke damage. Autophagy, 5: 1060-1.
[15] Yang C, Tang R, Ma X, Wang Y, Luo D, Xu Z, et al (2015). Tag SNPs in long non-coding RNA H19 contribute to susceptibility to gastric cancer in the Chinese Han population. Oncotarget, 6: 15311-20.
[16] Gao W, Zhu M, Wang H, Zhao S, Zhao D, Yang Y, et al (2015). Association of polymorphisms in long non-coding RNA H19 with coronary artery disease risk in a Chinese population. Mutat Res, 772:15-22.
[17] Hernandez-Valero MA, Rother J, Gorlov I, Frazier M, Gorlova OY (2013). Interplay between polymorphisms and methylation in the H19/IGF2 gene region may contribute to obesity in Mexican-American children. J Dev Orig Health Dis, 4: 499-506.
[18] Tragante V, Barnes MR, Ganesh SK, Lanktree MB, Guo W, Franceschini N, et al (2014). Gene-centric meta-analysis in 87,736 individuals of European ancestry identifies multiple blood-pressure-related loci. Am J Hum Genet, 94: 349-60.
[19] Wang K, Liu CY, Zhou LY, Wang JX, Wang M, Zhao B, et al (2015). APF lncRNA regulates autophagy and myocardial infarction by targeting miR-188-3p. Nat Commun, 6, 6779.
[20] Ge D, Han L, Huang S, Peng N, Wang P, Jiang Z (2014). Identification of a novel MTOR activator and discovery of a competing endogenous RNAregulating autophagy in vascular endothelial cells. Autophagy, 10: 957-71.
[21] Huang S, Lu W, Ge D, Meng N, Li Y, Su L, et al (2015). A new microRNA signal pathway regulated by long noncoding RNA TGFB2-OT1 in autophagy and inflammation of vascular endothelial cells. Autophagy, 11: 2172-83.
[22] Pawar K, Hanisch C, Palma Vera SE, Einspanier R, Sharbati S (2016). Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy. Sci Rep, 6: 19416.
[23] Han W, Fu X, Xie J, Meng Z, Gu Y, Wang X, et al (2015). MiR-26a enhances autophagy to protect against ethanol-induced acute liver injury. J Mol Med (Berl), 93: 1045-55.
[24] Yu LL, Chang K, Lu LS, Zhao D, Han J, Zheng YR, et al (2013). Lentivirus-mediated RNA interference targeting the H19 gene inhibits cell proliferation and apoptosis in human choriocarcinoma cell line JAR. BMC Cell Biol, 27: 14-26.
[25] Ronald M Adkins, Grant Somes, John C Morrison, James B Hill, Erin M Watson, Everett F Magann, et al (2010). Association of Birth Weight with Polymorphisms in the IGF2, H19 and IGF2R Genes. Pediatr Res, 68: 429-34.
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