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29 May 2015 : Laboratory Research  

XPD Functions as a Tumor Suppressor and Dysregulates Autophagy in Cultured HepG2 Cells

Jian-feng ZhengAE, Lin-lin LiBC, Juan LuDE, Kun YanEF, Wu-hua GuoBF, Ji-xiang ZhangAFG

DOI: 10.12659/MSM.894303

Med Sci Monit 2015; 21:1562-1568

Abstract

BACKGROUND: Recent clinical studies have linked polymorphisms in the xeroderma pigmentosum group D (XPD) gene, a key repair gene involved in nucleotide excision repair, to increased risk of hepatocellular carcinoma (HCC). However, the cellular effects of XPD expression in cultured HCC cells remain largely uncharacterized. Therefore, the aim of this study was to characterize the in vitro cellular effects of XPD expression on the HCC cell line HepG2.

MATERIAL AND METHODS: HepG2 cells were transfected as follows to create four experimental groups: pEGFP-N2/XPD plasmid (XPD) group, EGFP-N2 plasmid (N2) control group, lipofectamine™ 2000 (lipid) control group, and non-transfected (CON) control group. An MTT cell proliferation assay, Annexin V-APC apoptosis assay, colony formation assay, scratch wound migration assay, Transwell migration assay, and Western blotting of the autophagic proteins LC3 and p62 were conducted.

RESULTS: XPD expression significantly inhibited HepG2 cell proliferation (p<0.05), significantly promoted HepG2 cell apoptosis (p<0.05), significantly inhibited HepG2 colony formation (p<0.05), significantly decreased HepG2 cells’ migratory ability (p<0.05), and significantly lowered HepG2 cells’ invasive capacity (p<0.05). Western blotting showed that XPD expression significantly increased LC3 expression (p<0.05) and significantly reduced p62 expression (p<0.05).

CONCLUSIONS: XPD expression serves as a tumor suppressor and dysregulates autophagic protein degradation in HepG2 cells in vitro. Further in vivo pre-clinical studies and clinical trials are needed to validate XPD’s potential as a tumor-suppressive gene therapy.

Keywords: Apoptosis - physiology, Annexin A5, Autophagy - physiology, Blotting, Western, Cell Proliferation - physiology, Colony-Forming Units Assay, In Vitro Techniques, Tetrazolium Salts, Thiazoles, Tumor Suppressor Proteins - physiology, Xeroderma Pigmentosum Group D Protein - physiology

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750