Construction of a recombinant pIRES2-EGFP-ARTS plasmid and its effect on LX-2 cells

• Published in: Molecular medicine reports Vol. 16; no. 4; pp. 4737 - 4743
• Main Authors: Xu, Feifan, Han, Yuanlong, Zhu, Dandan, Tian, Hua, Zhu, Huiming, Ren, Jingjing, Gu, Delin, Duan, Yinong
• Format: Journal Article
• Language: English
• Published: Greece Spandidos Publications 01.10.2017; Spandidos Publications UK Ltd; D.A. Spandidos
• Subjects: Apoptosis; Biological markers; Biotechnology; Care and treatment; Cell Line; Cell migration; Cell Movement; Cell Proliferation; Cellular signal transduction; Cloning; Cloning vectors; Deoxyribonucleic acid; Development and progression; DNA; DNA polymerase; E coli; Efficiency; Enzymes; Fibrosis; Gene amplification; Gene Expression; Genes, Reporter; Genetic aspects; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Health aspects; Hepatic Stellate Cells - metabolism; Humans; Immunoglobulins; Laboratories; Liver; Liver diseases; Plasmids; Plasmids - genetics; Polymerase chain reaction; Proteins; Recombinant molecules; Reverse transcription; Ribonucleic acid; RNA; Rodents; Signal Transduction; Stellate cells; Transfection; Transforming growth factor; Transforming Growth Factor beta - genetics; Transforming Growth Factor beta - metabolism; Transforming growth factor-b; Western blotting
• ISSN: 1791-2997; 1791-3004
• DOI: 10.3892/mmr.2017.7172

The inhibition of the activation of hepatic stellate cells (HSCs) and the induction of their apoptosis have been investigated as potential strategies to counteract the development and progression of liver fibrosis. Previous research has suggested that apoptosis‑related protein in the transforming growth factor‑β signaling pathway (ARTS) may serve a significant role in numerous cell types; however, little is known regarding its roles in HSCs. Total RNA was extracted from LX‑2 cells, and the human full‑length ARTS gene was obtained by reverse transcription‑polymerase chain reaction and inserted into the pIRES2‑EGFP cloning vector. Subsequently, the recombinant pIRES2‑EGFP‑ARTS plasmid was transfected into LX‑2 cells by FuGENE 6 transfection reagent, and the expression of ARTS was detected by western blotting and fluorescent microscopy. In addition, the effects of pIRES2‑EGFP‑ARTS on the activation, apoptosis, viability and migration of LX‑2 cells were assessed by western blot analysis, TUNEL staining, an MTT assay, and scratch and Transwell assays, respectively. The present results demonstrated that the pIRES2‑EGFP‑ARTS vector expressing human ARTS was successfully constructed, and the overexpression of ARTS contributed to enhance the apoptosis and inhibit the activation of human LX‑2 HSCs. The present findings suggested that ARTS overexpression may have potential as a novel therapeutic strategy to reverse hepatic fibrosis.