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Silencing of long noncoding RNA AK139328 attenuates ischemia/reperfusion injury in mouse livers.

Chen Z, Jia S, Li D, Cai J, Tu J, Geng B, Guan Y, Cui Q, Yang J - PLoS ONE (2013)

Bottom Line: Eleven of the most significantly deregulated LncRNAs were further validated by quantitative PCR assays.Furthermore, knockdown of AK139328 also reduced macrophage infitration and inhibited NF-κB activity and inflammatory cytokines expression.In conclusion, these findings revealed that deregulated LncRNAs are involved in liver ischemia/reperfusion injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Peking University School of Basic Medical Sciences, Beijing, China ; MOE Key Laboratory of Molecular Cardiovascular Science, Peking University, Beijing, China.

ABSTRACT
Recently, increasing evidences had suggested that long noncoding RNAs (LncRNAs) are involved in a wide range of physiological and pathophysiological processes. Here we determined the LncRNA expression profile using microarray technology in mouse livers after ischemia/reperfusion treatment. Seventy one LncRNAs were upregulated, and 27 LncRNAs were downregulated in ischemia/reperfusion-treated mouse livers. Eleven of the most significantly deregulated LncRNAs were further validated by quantitative PCR assays. Among the upregulated LncRNAs confirmed by quantitative PCR assays, AK139328 exhibited the highest expression level in normal mouse livers. siRNA-mediated knockdown of hepatic AK139328 decreased plasma aminotransferase activities, and reduced necrosis area in the livers with a decrease in caspase-3 activation after ischemia/reperfusion treatment. In ischemia/reperfusion liver, knockdown of AK139328 increased survival signaling proteins including phosphorylated Akt (pAkt), glycogen synthase kinase 3 (pGSK3) and endothelial nitric oxide synthase (peNOS). Furthermore, knockdown of AK139328 also reduced macrophage infitration and inhibited NF-κB activity and inflammatory cytokines expression. In conclusion, these findings revealed that deregulated LncRNAs are involved in liver ischemia/reperfusion injury. Silencing of AK139328 ameliorated ischemia/reperfusion injury in the liver with the activation of Akt signaling pathway and inhibition of NF-κB activity. LncRNA AK139328 might be a novel target for diagnosis and treatment of liver surgery or transplantation.

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Hepatic knockdown of AK139328 on plasma activities of ALT and AST.3 days post tail vein injection of siRNA, the mice were subjected to ischemia/reperfusion treatment. A) siAK139328 treatment reduced AK139328 level in the livers. B) Knockdown of hepatic AK139328 on the expression levels of inflammatory cytokines in the livers. C) Knockdown of hepatic AK139328 reduced plasma activities of ALT and AST. D) Knockdown of AK139328 increased posphorylated Akt (pAkt), GSK-3 (pGSK3) and eNOS (peNOS) levels in the livers. E) Knockdown of AK139328 inhibited caspase 3 activation in the livers. F) Knockdown of AK139328 inhibited NF-κB activation in the livers. The distribution of IKBa and NF-κB in cytoplasm and nuclear extraction was analyzed byimmunoblotting assays, respectively. N=8-12, *P<0.05 versus sham group, #P<0.05 versus I/R-Scramble group. Sham, sham group; I/R-Scram or Scram, scrambled siRNA treated mice; I/R-siAK139 or siAK139, siAK139328 treated mice.
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pone-0080817-g004: Hepatic knockdown of AK139328 on plasma activities of ALT and AST.3 days post tail vein injection of siRNA, the mice were subjected to ischemia/reperfusion treatment. A) siAK139328 treatment reduced AK139328 level in the livers. B) Knockdown of hepatic AK139328 on the expression levels of inflammatory cytokines in the livers. C) Knockdown of hepatic AK139328 reduced plasma activities of ALT and AST. D) Knockdown of AK139328 increased posphorylated Akt (pAkt), GSK-3 (pGSK3) and eNOS (peNOS) levels in the livers. E) Knockdown of AK139328 inhibited caspase 3 activation in the livers. F) Knockdown of AK139328 inhibited NF-κB activation in the livers. The distribution of IKBa and NF-κB in cytoplasm and nuclear extraction was analyzed byimmunoblotting assays, respectively. N=8-12, *P<0.05 versus sham group, #P<0.05 versus I/R-Scramble group. Sham, sham group; I/R-Scram or Scram, scrambled siRNA treated mice; I/R-siAK139 or siAK139, siAK139328 treated mice.

Mentions: Because AK139328 is one of the most significantly upregulated LncRNAs with the highest expression level among the validated LncRNAs in the liver, its role in hepatic ischemia/reperfusion injury was further evaluated. The efficacy of siRNA against LncRNA AK139328 was firstly determined in primary cultured mouse hepatocytes. The results indicated that siAK139328 treatment reduced AK139328 level by about 60% (Figure S1). siRNA against AK139328 or scrambled siRNA was injected into normal mice via tail vein. 3 days post siRNA injection, the mice were performed for hepatic ischemia/reperfusion treatment. Real time PCR assay revealed that siAK139328 injection reduced the expression level of AK139328 in the livers by about 60% when compared with scrambled siRNA-treated livers (Figure 4A). Knockdown of AK139328 reduced the mRNA levels of IP-10 and MCP-1, whereas had little effect on that of TNF-α in ischemia/reperfusion-treated livers (Figure 4B). Notably, silencing of AK139328 significantly reduced plasma activities of ALT and AST by about 50% (Figure 4C). Moreover, phosphorylated Akt (pAkt), GSK-3 (pGSK3), eNOS (peNOS) levels were increased in siAK139328-treated mouse livers when compared with control livers (Figure 4D). Silencing of AK139328 also significantly reduced cleaved caspase 3 levels in the livers, suggesting the attenuation of apoptotic pathway(Figure 4E). In the cytoplasma, IKBa level was significantly increased after silencing AK139328. Basically, IKBa binds to NF-κB and inhibits its translocation to nucleus. Consistently, silencing of AK139328 repressed nuclear translocation of NF-κB in the livers (Figure 4F).


Silencing of long noncoding RNA AK139328 attenuates ischemia/reperfusion injury in mouse livers.

Chen Z, Jia S, Li D, Cai J, Tu J, Geng B, Guan Y, Cui Q, Yang J - PLoS ONE (2013)

Hepatic knockdown of AK139328 on plasma activities of ALT and AST.3 days post tail vein injection of siRNA, the mice were subjected to ischemia/reperfusion treatment. A) siAK139328 treatment reduced AK139328 level in the livers. B) Knockdown of hepatic AK139328 on the expression levels of inflammatory cytokines in the livers. C) Knockdown of hepatic AK139328 reduced plasma activities of ALT and AST. D) Knockdown of AK139328 increased posphorylated Akt (pAkt), GSK-3 (pGSK3) and eNOS (peNOS) levels in the livers. E) Knockdown of AK139328 inhibited caspase 3 activation in the livers. F) Knockdown of AK139328 inhibited NF-κB activation in the livers. The distribution of IKBa and NF-κB in cytoplasm and nuclear extraction was analyzed byimmunoblotting assays, respectively. N=8-12, *P<0.05 versus sham group, #P<0.05 versus I/R-Scramble group. Sham, sham group; I/R-Scram or Scram, scrambled siRNA treated mice; I/R-siAK139 or siAK139, siAK139328 treated mice.
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pone-0080817-g004: Hepatic knockdown of AK139328 on plasma activities of ALT and AST.3 days post tail vein injection of siRNA, the mice were subjected to ischemia/reperfusion treatment. A) siAK139328 treatment reduced AK139328 level in the livers. B) Knockdown of hepatic AK139328 on the expression levels of inflammatory cytokines in the livers. C) Knockdown of hepatic AK139328 reduced plasma activities of ALT and AST. D) Knockdown of AK139328 increased posphorylated Akt (pAkt), GSK-3 (pGSK3) and eNOS (peNOS) levels in the livers. E) Knockdown of AK139328 inhibited caspase 3 activation in the livers. F) Knockdown of AK139328 inhibited NF-κB activation in the livers. The distribution of IKBa and NF-κB in cytoplasm and nuclear extraction was analyzed byimmunoblotting assays, respectively. N=8-12, *P<0.05 versus sham group, #P<0.05 versus I/R-Scramble group. Sham, sham group; I/R-Scram or Scram, scrambled siRNA treated mice; I/R-siAK139 or siAK139, siAK139328 treated mice.
Mentions: Because AK139328 is one of the most significantly upregulated LncRNAs with the highest expression level among the validated LncRNAs in the liver, its role in hepatic ischemia/reperfusion injury was further evaluated. The efficacy of siRNA against LncRNA AK139328 was firstly determined in primary cultured mouse hepatocytes. The results indicated that siAK139328 treatment reduced AK139328 level by about 60% (Figure S1). siRNA against AK139328 or scrambled siRNA was injected into normal mice via tail vein. 3 days post siRNA injection, the mice were performed for hepatic ischemia/reperfusion treatment. Real time PCR assay revealed that siAK139328 injection reduced the expression level of AK139328 in the livers by about 60% when compared with scrambled siRNA-treated livers (Figure 4A). Knockdown of AK139328 reduced the mRNA levels of IP-10 and MCP-1, whereas had little effect on that of TNF-α in ischemia/reperfusion-treated livers (Figure 4B). Notably, silencing of AK139328 significantly reduced plasma activities of ALT and AST by about 50% (Figure 4C). Moreover, phosphorylated Akt (pAkt), GSK-3 (pGSK3), eNOS (peNOS) levels were increased in siAK139328-treated mouse livers when compared with control livers (Figure 4D). Silencing of AK139328 also significantly reduced cleaved caspase 3 levels in the livers, suggesting the attenuation of apoptotic pathway(Figure 4E). In the cytoplasma, IKBa level was significantly increased after silencing AK139328. Basically, IKBa binds to NF-κB and inhibits its translocation to nucleus. Consistently, silencing of AK139328 repressed nuclear translocation of NF-κB in the livers (Figure 4F).

Bottom Line: Eleven of the most significantly deregulated LncRNAs were further validated by quantitative PCR assays.Furthermore, knockdown of AK139328 also reduced macrophage infitration and inhibited NF-κB activity and inflammatory cytokines expression.In conclusion, these findings revealed that deregulated LncRNAs are involved in liver ischemia/reperfusion injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Peking University School of Basic Medical Sciences, Beijing, China ; MOE Key Laboratory of Molecular Cardiovascular Science, Peking University, Beijing, China.

ABSTRACT
Recently, increasing evidences had suggested that long noncoding RNAs (LncRNAs) are involved in a wide range of physiological and pathophysiological processes. Here we determined the LncRNA expression profile using microarray technology in mouse livers after ischemia/reperfusion treatment. Seventy one LncRNAs were upregulated, and 27 LncRNAs were downregulated in ischemia/reperfusion-treated mouse livers. Eleven of the most significantly deregulated LncRNAs were further validated by quantitative PCR assays. Among the upregulated LncRNAs confirmed by quantitative PCR assays, AK139328 exhibited the highest expression level in normal mouse livers. siRNA-mediated knockdown of hepatic AK139328 decreased plasma aminotransferase activities, and reduced necrosis area in the livers with a decrease in caspase-3 activation after ischemia/reperfusion treatment. In ischemia/reperfusion liver, knockdown of AK139328 increased survival signaling proteins including phosphorylated Akt (pAkt), glycogen synthase kinase 3 (pGSK3) and endothelial nitric oxide synthase (peNOS). Furthermore, knockdown of AK139328 also reduced macrophage infitration and inhibited NF-κB activity and inflammatory cytokines expression. In conclusion, these findings revealed that deregulated LncRNAs are involved in liver ischemia/reperfusion injury. Silencing of AK139328 ameliorated ischemia/reperfusion injury in the liver with the activation of Akt signaling pathway and inhibition of NF-κB activity. LncRNA AK139328 might be a novel target for diagnosis and treatment of liver surgery or transplantation.

Show MeSH
Related in: MedlinePlus