Construction of COX-2 short hairpin RNA expression vector and its inhibitory effect on hepatic fibrosis

• Main Authors: Ni Xie, Hong Wu Liao, Wen Sheng Ou, Xu Zhou, Yang Hu, Nian Fu, Xue Feng Yang, Duan-Fang Liao
• Format: Article
• Language: English
• Published: Taylor & Francis Group 2018-05-01
• Series: Biotechnology & Biotechnological Equipment
• Subjects: Cyclooxygenase-2; recombinant adenovirus vector; liver fibrosis; short hairpin RNA
• Online Access: http://dx.doi.org/10.1080/13102818.2018.1431569

The aim of this study was to construct recombinant adenovirus vector carrying a short hairpin RNA (shRNA) that can exclusively target cyclooxygenase-2 (COX-2) gene expression and observe its inhibitory effect on hepatic fibrosis. We designed and synthesized three oligonucleotide sequences, and cloned those into a shuttle vector, pYr-1.1-hU6-EGFP, after annealing. The restriction-enzyme digestion and sequencing analyses confirmed that the constructed recombinant eukaryotic expression vector was correct. A recombination reaction using LR Clonase was performed for the pYr-1.1-hU6-EGFP COX-2shRNA and the adenovirus vector pAd/BL-DEST to form Ad-COX-2shRNA. The adenoviruses containing the recombinant plasmids were transfected into hepatic stellate cells (HSCs). The transfection efficiency of the three COX-2 shRNAs exceeded 70%. Reverse transcription PCR (RT-PCR) and western blot confirmed that the target gene expression was decreased at the level of mRNA and protein, and the interference effect of COX-2 shRNA-1 was better than that of the other two COX-2 shRNAs. COX-2 shRNA-1 recombinant adenovirus vectors (1 × 109 PFU/mL) were injected via the tail vein into rats fed a high-fat diet with a 40% carbon tetrachloride peanut oil lavage, which induced liver fibrosis. Rats were euthanized at the end of the 12th week, and their liver was removed. Liver expression of COX-2 mRNA and protein was detected by RT-PCR and immunohistochemistry, respectively. RT-PCR and immunohistochemistry showed that COX-2 shRNA-1 inhibited COX-2 expression in liver tissue. Hematoxylin/eosin and Masson staining showed that COX-2 shRNA-1 ameliorated the severity of liver fibrosis. The COX-2 shRNA eukaryotic expression vectors were constructed successfully and COX-2 shRNA-1 ameliorated liver fibrosis.