title: Hepatitis B virus X protein activates human hepatic stellate cells through upregulating TGFβ1.
abstract: We investigated the effects of the hepatitis B virus X gene (HBV X) on the activation of human hepatic stellate cells (HSCs) and the possible mechanisms underlying the pathway. Recombinant plasmid pHBV-X-IRES2-EGFP was constructed and transfected into HL-7702 cells using a lipid-mediated method. Transfected cells were screened by G418, which detected stable expression of the X gene by reverse transcription (RT)-PCR and Western blot analysis, and named L02/x. Cells not subjected to G418-selection were analyzed to confirm the transient expression of the X gene and named L02/48x. Subsequently, L02/x and L02/48x, together with non-HBx-expressing cells, were co-cultured with HSCs in a non-contact transwell system. After 36 h of co-culture, the proliferation and migration of HSCs was detected using different cell counting methods. Finally, the mRNA and protein levels of α-SMA, Col I, and TGFβ1 in HSCs were detected by real-time PCR and western blot analysis. RT-PCR and Western blot analysis showed that L02/x and L02/48x cells can express HBV X gene mRNA and protein. Additionally, HSCs co-cultured with L02/x or L02/48x cells showed significantly higher proliferation and migration levels than control groups. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of α-SMA, Col I, and TGFβ1 in HSCs co-cultured with HBx-expressing liver cells were higher than those in control groups. HBx protein activated HSCs in vitro, leading to increased proliferation and migration of HSCs and upregulation of α-SMA and Col I. The TGFβ1 gene may be involved in this pathway.
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: H-Y
authors.initials: HY
authors.lastname: Chen
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Z-X
authors.initials: ZX
authors.lastname: Chen
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: R-F
authors.initials: RF
authors.lastname: Huang
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: N
authors.initials: N
authors.lastname: Lin
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, China drwangxz@163.com.
authors.forename: X-Z
authors.initials: XZ
authors.lastname: Wang
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D000199:Actins; D024042:Collagen Type I; D012333:RNA, Messenger; D015534:Trans-Activators; D053773:Transforming Growth Factor beta1; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein