[논문]Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway

Xie, Zhengyuan; Xiao, Zhihua; Wang, Fenfen

doi:10.14348/molcells.2017.2299

Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway 원문보기

Molecules and cells, v.40 no.3, 2017년, pp.202 - 210

Xie, Zhengyuan (Department of Gastroenterology, The Second Affiliated Hospital, Nanchang University) , Xiao, Zhihua (Department of Gastroenterology, The Second Affiliated Hospital, Nanchang University) , Wang, Fenfen (Department of Gastroenterology, The Second Affiliated Hospital, Nanchang University)

Abstract ▼ AI-Helper

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor $(TNF)-{\alpha}$. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of $NF-{\kappa}B$ and miR-503. We found that overexpression of NS5A inhibited $TNF-{\alpha}$-induced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the $TNF-{\alpha}$ induced Hep-mock cells was significantly less than the viability of the $TNF-{\alpha}$ induced Hep-NS5A cells, which demonstrates that NS5A inhibited $TNF-{\alpha}$-induced HepG2 cell apoptosis. Under $TNF-{\alpha}$ treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited $TNF{\alpha}$-induced $NF-{\kappa}B$ activation and $NF-{\kappa}B$ regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse $TNF-{\alpha}$-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits $NF-{\kappa}B$ activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.

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문제 정의

Thus, this present study aimed to investigate the mechanism of NS5A on TNF-α-induced hepatocyte apoptosis.

제안 방법

All experiments were repeated three times, and all data were presented as mean ± standard deviation.
1B, TNF-α reduced cell viability; However, as the concentrations of pcDNA-NS5A transfected into the cells increased, cell viability gradually increased. For the next experiment, two HepG2 cell lines were constructed, an Hep-NS5A cell line with stable NS5A expression and a Hep-mock cell line as the negative control. The NS5A protein expression in the two established cell lines is shown in Fig.

성능/효과

Consistent with the findings reported in those studies, our results showed that bcl-2 expression was significantly lower in the TNF-α induced Hep-mock cells, while c-caspase expression was higher in the Hep-mock cells without TNF-α treatment.
In conclusion, we observed decreased cell viability, upregulated miR-503 expression, reduced bcl-2 expression with TNF-α treatment in the Hep-mock cells, but all of these were reversed in the Hep-NS5A cells.
Our results suggest that NS5A plays a role in inhibiting TNF-α-mediated apoptosis in HepG2 cells via disturbed NF-kB activation.
Our results support those findings; we found that the expression of miR-503 and n65 were upregulated in the TNF-α-induced Hep-mock cells.
The result showed that TNF-α upregulated the expression of miR-503, Pri-miR-503 and miR-424 in the Hep-mock cells, but the expression of each of these factors was decreased in the Hep-NS5A cells (Figs. 4C and 4D).
To further explore the influence of NS5A on TNF-α-induced NF-kB activation, HepG2 cells were co-transfected with pNF-kB-luc plasmid and different concentrations of pcDNA-NS5A. The results showed an increase in the pcDNANS5A concentration accompanied by a gradual decreased in luciferase activity (Fig. 3C), and a decrease in np65 expression (Fig. 3D). In general, TNF-α induced NF-kB activation, which may have been inhibited by the high expression of NS5A in the HepG2 cells.

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Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway 원문보기

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Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway 원문보기

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