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. 2022 Nov 25;54(11):1619-1629.
doi: 10.3724/abbs.2022167.

LncRNA-Airn alleviates acute liver injury by inhibiting hepatocyte apoptosis via the NF-κB signaling pathway

Shuai Shao  1 , Yu Zhang  2 , Feng Zhou  2 , Xiaoxiang Meng  3 , Zhenjun Yu  2 , Guantong Li  2 , Lina Zheng  3 , Kun Zhang  3 , Yuhan Li  2 , Beichen Guo  2 , Qi Liu  3 , Mengxia Zhang  3 , Xiaoxiao Du  3 , Wei Hong  3 , Tao Han  1   2   4   5
Affiliations

LncRNA-Airn alleviates acute liver injury by inhibiting hepatocyte apoptosis via the NF-κB signaling pathway

Shuai Shao et al. Acta Biochim Biophys Sin (Shanghai). .

Abstract

Acute liver injury is a common and serious syndrome caused by multiple factors and unclear pathogenesis. If the injury persists, liver injury can lead to cirrhosis and liver failure and ultimately results in the development of liver cancer. Emerging evidence has indicated that long noncoding RNAs (lncRNAs) play an important role in the development of liver injury. However, the role of antisense Igf2r RNA (Airn) in acute liver injury and its underlying mechanism remain largely unclear. In this study, we show that Airn is upregulated in liver tissue and primary hepatocytes from an acute liver injury mouse model. Consistently, Airn is also overexpressed in serum samples of patients with acute-on-chronic liver failure and is negatively correlated with the Model for End-Stage Liver Disease (MELD) score. Moreover, gene knockout and rescue assays reveal that Airn alleviates CCl 4-induced liver injury by inhibiting hepatocyte apoptosis and oxidative stress in vivo. Further investigation reveals that Airn decreases H 2O 2-induced hepatocyte apoptosis in vitro. Mechanistically, we reveal that Airn represses CCl 4- and H 2O 2-induced enhancement of phosphorylation of p65 and IκBα, suggesting that Airn inhibits hepatocyte apoptosis by inactivating the NF-κB pathway. In conclusion, our results demonstrate that Airn can alleviate acute liver injury by inhibiting hepatocyte apoptosis via inactivating the NF-κB signaling pathway, and Airn could be a potential biomarker for acute liver injury.

Keywords: NF-κB signaling; acute liver injury; apoptosis; lncRNA; oxidative stress.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

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Figure 1
CCl 4-induced acute liver injury mouse model and the expression of Airn Mice were injected with CCl 4 (olive oil as a control) to induce acute liver injury and were sacrificed at different time points. Macroscopic appearance and H&E staining of liver tissues from CCl 4-treated mice (A). The areas of liver damage for H&E staining (B), serum ALT (C), AST (D), and LDH (E) were detected. qPCR analysis of the Airn transcript in injured liver tissues (F) and primary HCs (G). Primary HCs were isolated from the livers of Balb/c mice and treated with 3% H 2O 2 for 3 h. Then, the expressions of Airn, IL-6, Mcp-1, and TNF-α were detected by qPCR (H). qPCR analysis of the Airn transcript in serum samples of healthy people and patients with ACLF (I). The correlation between the Airn level and the MELD score of ACLF patients was assessed using Pearson’s correlation analysis, n=58 (J). Data are expressed as the mean±SD from at least three experiments, n=6. * P<0.05 vs control.
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Figure 2
Deletion of Airn aggravates CCl 4-induced acute liver injury C57BL/6N mice were divided into four groups. (A) qPCR analysis of the Airn transcript in liver tissues of the acute liver injury mouse model. (B) Macroscopic appearance and H&E staining of liver tissues from control and CCl 4- treated mice. (C‒E) Serum ALT, AST and LDH levels were detected. Data are expressed as the mean±SD from at least three experiments, n=6. P values were analyzed by Student’s t test. * P<0.05, WT+CCl 4 vs WT; # P<0.05, Airn-KO+CCl 4 vs WT+CCl 4. n.d., not detected.
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Figure 3
Rescue experiments confirming the role of Airn in CCl 4-induced acute liver injury Airn-KO mice were divided into four groups. (A) The expression of Airn in liver tissues was detected by qPCR. (B) Macroscopic examination and H&E staining of liver tissues from control and CCl 4- treated mice. (C‒E) The serum levels of ALT, AST and LDH were determined. Data are expressed as the mean±SD from at least three experiments, n=6. P values were analyzed by Student’s t test. * P<0.05, Airn-KO+AAV8-GFP+CCl 4 vs Airn-KO+AAV8-GFP; # P<0.05, Airn- KO+AAV8-Airn+CCl 4 vs Airn-KO+AAV8-GFP+CCl 4.
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Figure 4
Airn alleviates CCl 4-induced oxidative stress (A‒C) Mice were divided into four groups: WT, WT+CCl 4, Airn-KO and Airn+KO+CCl 4. The activities of SOD, CAT and GSH were detected. (D‒F) Airn-KO mice were divided into four groups: Airn-KO+AAV8-GFP, Airn-KO+AAV8- GFP+CCl 4, Airn-KO+AAV8-Airn, and Airn-KO+AAV8-Airn+CCl 4. The activities of SOD, CAT and GSH were detected. Data are expressed as the mean±SD from at least three experiments, n=6. P values were analyzed by Student’s t test. * P<0.05, WT+CCl 4 vs WT or Airn-KO+AAV8-GFP+CCl 4 vs Airn-KO+AAV8-GFP; # P<0.05, Airn-KO+CCl 4 vs WT+CCl 4 or Airn-KO+AAV8-Airn+CCl 4 vs Airn-KO+AAV8-GFP+CCl 4.
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Figure 5
Airn inhibits CCl 4-induced hepatocyte apoptosis in vivo (A,C) Mice were divided into four groups: WT, WT+CCl 4, Airn-KO and Airn- KO+CCl 4. (B,D) Airn-KO mice were divided into four groups: Airn-KO+AAV8-GFP, Airn-KO+AAV8-GFP+CCl 4, Airn-KO+AAV8-Airn and Airn- KO+AAV8-Airn+CCl 4. Western blot and TUNEL assays were used to detect the level of apoptosis in liver tissues and liver tissue sections, respectively. GAPDH was used as an internal control. The data are expressed in at least three experiments, n=3. P values were analysed by Student’s t test. * P<0.05, WT+CCl 4 vs WT or Airn-KO+AAV8-GFP+CCl 4 vs Airn-KO+AAV8-GFP; # P<0.05, Airn-KO+CCl 4 vs WT+CCl 4 or Airn-KO+AAV8- Airn+CCl 4 vs Airn-KO+AAV8-GFP+CCl 4.
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Figure 6
Airn inhibits H 2O 2-induced hepatocyte apoptosis in vitro Primary HCs were transfected with siRNA or lentivirus for 48 h, followed by treatment with 3% H 2O 2 for another 3 h. (A,B) The mRNA level of Airn was detected by qPCR. (C,D) The protein levels of apoptosis-related genes were detected by western blot analysis. GAPDH was used as an internal control. (E) Hoechst/PI double-staining was used to detect cell apoptosis. Scale bar: 100 μm. Data are expressed as the mean±SD from at least three experiments. P values were analyzed by Student’s t test. * P<0.05, siAirn vs siRNA-control or LV-Airn vs LV-control or NC+H 2O 2 vs NC or LV-con+H 2O 2 vs LV-con; # P<0.05, siAirn+H 2O 2 vs NC+H 2O 2 or LV-Airn+ H 2O 2 vs LV-con+H 2O 2.
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Figure 7
Airn inhibits hepatocyte apoptosis through NF-κB signaling (A) Mice were divided into four groups. The protein levels of p65, phos-p65, IκBα and phos-IκBα were detected by western blot analysis. GAPDH was used as an internal control. (B) Airn-KO mice were divided into four groups. The protein levels of p65, phos-p65, IκBα and phos-IκBα were detected by western blot analysis. GAPDH was used as an internal control. (C) Primary HCs were transfected with siRNA or lentivirus for 48 h, followed by treatment with 3% H 2O 2 for another 3 h. The protein levels of p65, phos-p65, IκBα and phos-IκBα were detected by western blot analysis. GAPDH was used as an internal control. (D,E) Primary hepatocytes were transfected with siRNA for 48 h, and then treated with 3% H 2O 2 and 10 μM of the NF-κB antagonist BAY11-7082 for another 3 h. The protein levels of PARP1, Bcl-2, BAX and cleaved caspase-3 were determined by western blot analysis. GAPDH was used as an internal control. Hoechst/PI double-staining was used to detect cell apoptosis. Scale bar: 100 μm. (F) pNF-κB-Luc and pRL-TK were cotransfected into primary hepatocytes using Lipofectamine 3000. Then, the cells were transfected with lentivirus-Airn and treated with 3% H 2O 2. NF-κB luciferase activity was detected using the Dual-luciferase reporter assay system, and the relative firefly luciferase activity was normalized to Renilla luciferase activity. Data are expressed as the mean±SD from at least three experiments, n=3. P values were analyzed by Student’s t test, * P<0.05, WT+CCl 4 vs WT or Airn-KO+AAV8-GFP+CCl 4 vs Airn-KO+AAV8-GFP or NC+H 2O 2 vs NC or LV-con+H 2O 2 vs LV-con or NC+H 2O 2+BAY vs NC+H 2O 2; # P<0.05, Airn-KO+CCl 4 vs WT+CCl 4 or Airn-KO+AAV8-Airn+CCl 4 vs Airn-KO+AAV8-GFP+CCl 4 or siAirn+H 2O 2 vs NC+H 2O 2 or LV-Airn+H 2O 2 vs LV-con+H 2O 2 or siAirn+H 2O 2+BAY vs NC+H 2O 2+BAY.
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Figure 8
The mechanism of Airn in hepatocyte apoptosis Airn represses CCl 4- and H 2O 2-induced enhancement of phosphorylation of p65 and IκBα, suggesting that Airn inhibits hepatocyte apoptosis by inactivating the NF-κB pathway.
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Grants and funding

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81870429, 82170630, 32171125, and 81971331) and the Natural Science Foundation of Tianjin (No. 19JCZDJC36700).
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