HBx gene transfection affects the cycle of primary renal tubular epithelial cells through regulating cyclin expression

Han,Wenlun; Luo,Meiliang; He,Mengying; Zhu,Yunyun; Zhong,Yu; Ding,Huideng; Hu,Gang; Liu,Liansheng; Chen,Qin; Lu,Ying

doi:10.3892/mmr.2018.9197

HBx gene transfection affects the cycle of primary renal tubular epithelial cells through regulating cyclin expression

Authors:
- Wenlun Han
- Meiliang Luo
- Mengying He
- Yunyun Zhu
- Yu Zhong
- Huideng Ding
- Gang Hu
- Liansheng Liu
- Qin Chen
- Ying Lu
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Affiliations: Department of Nephrology, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang 310012, P.R. China
Published online on: June 20, 2018 https://doi.org/10.3892/mmr.2018.9197
Pages: 1947-1954
Copyright: © Han et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Hepatitis B virus X protein (HBx) has been previously demonstrated to be associated with the regulation of cell proliferation; however, the exact mechanisms underlying this effect remain unclear. The present study aimed to investigate the regulatory mechanism of HBx on the cycle progression of primary renal tubular epithelial cells. Primary renal tubular epithelial cells of Sprague Dawley (SD) rats were separated and cultured. The morphology of cultured cells was characterized by immunohistochemical analysis and the results demonstrated that primary renal tubular epithelial cells with the expected morphology and distribution were successfully separated and cultured from SD rats. HBx gene pcDNA3.1/myc vector and empty vector were constructed and transfected into cells as HBx and empty groups, respectively. Following transfection, the mRNA and protein levels of HBx, cyclin A, cyclin D1 and cyclin E in cells were determined by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. The results demonstrated that following HBx gene transfection, the mRNA and protein levels of HBx, cyclin A, cyclin D1 and cyclin E in cells were significantly upregulated, compared with the empty control group (P<0.05). Furthermore, cell apoptosis and the cell cycle were evaluated by Annexin V‑fluorescein isothiocyanate/propidium iodide staining and flow cytometry. HBx gene transfection significantly inhibited the cell apoptosis (P<0.05), promoted cell cycle progression from the G1 to S phase and arrested the cell cycle in the S phase. Therefore, the results of the present study indicated that HBx gene transfection may regulate the apoptosis and cell cycle of primary renal tubular epithelial cells by affecting the expression of cyclins. The results of the present study may improve the understanding of pathogenesis associated with HBV‑associated glomerulonephritis, and may also provide insight and theoretical support for the future design and development of drugs for the treatment of hepatitis B virus.

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1	Zhang Q and Xue C: Research progress in pathogenesis of hepatitis B virus associated nephritis. Med Recap. 2543–2545. 2013.
2	Yi Z, Jie YW and Nan Z: The efficacy of anti-viral therapy on hepatitis B virus-associated glomerulonephritis: A systematic review and meta-analysis. Ann Hepatol. 10:165–173. 2011.PubMed/NCBI
3	Seeger C and Mason WS: Hepatitis B virus biology. Microb Mol Biol Rev. 64:51–68. 2000. View Article : Google Scholar
4	Zhai LL, Liu J and Xie YH: Dose-dependent modulation of cell apoptosis by hepatitis B virus X protein. J Microb Infect. 72–77. 2011.
5	Xia LM, Huang WJ, Wu JG, Yang YB, Zhang Q, Zhou ZZ, Zhu HF, Lei P, Shen GX and Tian DA: HBx protein induces expression of MIG and increases migration of leukocytes through activation of NF-kappa B. Virology. 385:335–342. 2009. View Article : Google Scholar : PubMed/NCBI
6	Clippinger AJ, Gearhart TL and Bouchard MJ: Hepatitis B virus X protein modulates apoptosis in primary rat hepatocytes by regulating both NF-kappa B and the mitochondrial permeability transition pore. J Virology. 83:4718–4731. 2009. View Article : Google Scholar : PubMed/NCBI
7	Tan C, Guo H, Zheng M, Chen Y and Huang W: Involvement of mitochondrial permeability transition in hepatitis B virus replication. Virus Res. 145:307–311. 2009. View Article : Google Scholar : PubMed/NCBI
8	He P, Shang H, Li D and Feng G: Effects of HBx gene on proliferation and apoptosis of human renal tubular epithelial cells. Chin J Nephrol. 29:380–381. 2013.
9	Chen HY, Tang NH, Lin N, Chen ZX and Wang XZ: Hepatitis B virus X protein induces apoptosis and cell cycle deregulation through interfering with DNA repair and checkpoint responses. Hepatol Res. 38:174–182. 2008.PubMed/NCBI
10	Huang YQ, Wang LW, Yan SN and Gong ZJ: Effects of cell cycle on telomerase activity and on hepatitis B virus replication in HepG22.2.15 cells. Hepatobiliary Pancreat Dis Int. 3:543–547. 2006.
11	General Administration of Quality Supervision, Inspection and Quarantine of China: Laboratory animal-Guideline of welfare ethical review (Draft for approval). Mar 18–2017.
12	Ono Y, Onda H, Sasada R, Igarashi K, Sugino Y and Nishioka K: The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw. Nucleic Acids Res. 11:1747–1757. 1983. View Article : Google Scholar : PubMed/NCBI
13	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
14	He P, Li H, Li D and Feng G: HBx gene promotes apoptosis in HK-2 cells through regulating apoptosis-relatedprotein ratio of Bax/Bcl-2. J Chin Med Univer. 43:38–44. 2014.
15	An S, Zhang R, Yang W, Zhou H, Zhang Z, Yang Y and Guo X: Research advances in hepatitis B virus-associated glomerulonephritis. J Clin Hepatol. 32:366–369. 2016.
16	Jiang W, Xu Y, Guan G, Ma R and Dong H: Renal amyloidosis and hepatitis B virus-associated glomerulonephritis in a patient with nephrotic syndrome. Chin Med J (Engl). 127:11992014.PubMed/NCBI
17	Deng CL, Song XW, Liang HJ, Feng C, Sheng YJ and Wang MY: Chronic hepatitis B serum promotes apoptotic damage in human renal tubular cells. World J Gastroenterol. 12:1752–1756. 2006. View Article : Google Scholar : PubMed/NCBI
18	Zhu N, Zhou Y, Yuan WJ, Liu J, Shang MH, Wang L and Gu LJ: Toll-like receptor 4 deposition and its significance in hepatitis B virus associated nephropathy. Zhonghua Nei Ke Za Zhi. 50:1008–1012. 2011.(In Chinese). PubMed/NCBI
19	Han WL and Huang ZX: Experimental study of direct pathogenic effects of hepatitis B virus on human tubular kidney cell. J Wenzhou Med College. 38:144–147. 2008.
20	Han WL and Huang ZX: Infection of hepatitis B virus on human tubular kidney cells in vitro. J Zhejiang Med Univer. 32:1148–1150. 2010.
21	Hong L, Zhang J, Li Q, Min J, Lu J, Li F, Li H and Guo S: Role of NF-κB activiation by MHBst167/HBx in human renal tubular cells. Chin J Nephrol Dial Transplant. 19:135–141. 2010.
22	Hong L, Zhang J, Min J, Lu J, Li F, Li H, Guo S and Li Q: A role for MHBst167/HBx in hepatitis B virus-induced renal tubular cell apoptosis. Nephrol Dial Transplant. 25:2125–2133. 2010. View Article : Google Scholar : PubMed/NCBI
23	Zhou Y, Wang X, Yuan W and Zhu N: Effect of hepatitis B virus X gene on transdifferentiation of human proximal tubular epithelial cells. Chin J Nephrol. 28:956–960. 2012.
24	Zhou Y, Zhu N, Wang X, Wang L, Gu LJ and Yuan WJ: The role of the toll-like receptor TLR4 in hepatitis B virus-associated glomerulonephritis. Arch Virol. 158:425–433. 2013. View Article : Google Scholar : PubMed/NCBI
25	Marshall CB and Shankland SJ: Cell cycle regulatory proteins in podocyte health and disease. Nephron Exp Nephrol. 106:e51–e59. 2007. View Article : Google Scholar : PubMed/NCBI
26	Madden CR and Slagle BL: Stimulation of cellular proliferation by hepatitis B virus X protein. Dis Markers. 17:153–157. 2001. View Article : Google Scholar : PubMed/NCBI
27	Zhan SD and Huang JX: Progression of Cyclin A, PTEN, Survivin and p27 expression in esophageal cancer. J Modern Oncol. 2245–2248. 2014.
28	Li YJ and Li YY: Advances in the research of cyclin E and p27 in gynecological. Foreign Med Sci (Obstet Gynecol Fascicle). 177–180. 2007.
29	Lin T: Expression and significance of Cell cycle regulatory proteins in the hepatitis B virus associated nephritsFujian Med Univer. Fuzhou: pp. 522011
30	Wu RS: Cell cycle regulation of p16, cdk4 and its role in the formation of middle ear cholesteatoma. J Basic Med Shandong Univer. 53–55. 2004.
31	Lu HZ, Liu D and Zhou JH: Effects of HBV X protein on glomerular mesangial cell proliferation and proinflammation factor expression. Chongqing Med. 40:2227–2230. 2011.
32	Matsuda Y and Ichida T: Impact of hepatitis B virus X protein on the DNA damage response during hepatocarcinogenesis. J Med Mol Morphol. 42:138–142. 2009. View Article : Google Scholar
33	Park SG, Min JY, Chung C, Hsieh A and Jung G: Tumor suppressor protein p53 induces degradation of the oncogenic protein HBx. Cancer Lett. 282:229–237. 2009. View Article : Google Scholar : PubMed/NCBI
34	Bouehard MJ and Sehneider RJ: The enigmatic X gene of hepatitis B virus. J Virol. 78:12725–12734. 2004. View Article : Google Scholar : PubMed/NCBI