Effects of silencing CTGF on apoptosis and expression of extracelluar matrix proteins in rat hepatic stellate T6 cells

Abstract

Objective To investigate the effects of silencing connective tissue growth factor (CTGF) gene on growth, apoptosis, and expression of proliferating cell nuclear antigen (PCNA), CTGF, integrin alpha 5 (Itgα5) and collagen Ⅰ (ColⅠ) in rat hepatic stellate cell line T6 (HSC_T6). Methods The CTGF shRNA lentiviral vectors with high infectivity were transfected into HSC_T6 cells, and the cells transfected with empty virus or not transfected served as control cells. Fluorescence inverted microscopy was used to observe the cell growth and the expression of green fluorescent protein (GFP) in the transfected HSC_T6 cells. CCK-8 assay and flow cytometry were employed respectively to detect the cell viability and apoptosis. The expression changes in PCNA, CTGF, Itgα5 and ColⅠ were measured by real-time PCR, Western blotting and immunofluorescence assay. Results Microscopic observation displayed that transfection with CTGF shRNA lentiviral vectors resulted in high expression of GFP in HSC_T6 cells. CCK-8 assay showed that silencing CTGF induced inhibited growth in the transfected cells, with significant difference since 72 h after transfection (P < 0.05). Flow cytometry indicated that transfection of CTGF shRNA lentivirus led to the apoptosis of HSC_T6 cells. The results of real-time PCR, Western blotting and immunofluorescence assay confirmed that CTGF shRNA lentivirus effectively down-regulated the expression of PCNA, CTGF, Itgα5 and ColⅠ, with notable differences when compared the expression in control cells (P < 0.05). Conclusion Silencing CTGF can down-regulate the expression of CTGF, Itgα5 and ColⅠ, inhibit the growth and induce the apoptosis of HSC_T6 cells, which might be closely associated with its interference for the CTGF/Itgα5 signaling pathway.

Keywords

• connective tissue growth factor
• hepatic stellate cells
• integrin alpha 5
• collagen ⅰ
• apoptosis