Effects of connective tissue growth factor gene-silencing on cell growth and cell cycle of hepatic stellate cells / 上海交通大学学报（医学版）

ABSTRACT

Objective • To investigate the effects of silencing connective tissue growth factor (Ctgf) gene on the growth, cell cycle and the expression of TGF-β1, Smad3 and Smad7 of rat hepatic stellate cell line HSCT6. Methods • The recombinant lentivirus vector pCDH/Ctgf-shRNA of Ctgf gene was constructed by RNA interference. The recombinant vector was packaged to obtain highly infectious pCDH/Ctgf-shRNA lentivirus particles for HSCT6 infection. The expression of green fluorescent protein (GFP) in the transfected HSCT6 cells was observed under fluorescence microscope. The effects of Ctgf-shRNA lentivirus on the growth of HSCT6 cells were tested by CCK-8. The effects of Ctgf-shRNA lentivirus on the cell cycle of HSCT6 cells were analyzed by flow cytometry (FCM). The effects of Ctgf-shRNA lentivirus on the expression of mRNA of Ctgf, Tgf-β1, Smad3 and Smad7, and their proteins in HSCT6 cells were detected by real-time PCR and Western blotting, respectively. Results • The lentiviral vector pCDH/Ctgf-shRNA has been constructed successfully. The HSCT6 cells transfected by Ctgf-shRNA lentivirus significantly expressed GFP under fluorescence microscope. The results of CCK-8 confirmed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was slower than that of controls and the differences were statistically significant after being cultured for 72 h (P<0.05). The results of FCM revealed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was blocked in the S phase of cell cycle. The results of real-time PCR and Western blotting showed that the Ctgf-shRNA lentivirus effectively silenced Ctgf gene, down-regulated the expression of genes and encoding proteins of TGF-β1, and Smad3 of HSCT6 and up-regulated the expression of genes and encoding proteins of Smad7 of HSCT6 cells. The differences between transfected cells and controls were statistically significant (P<0.05). Conclusion • Silencing Ctgf gene can effectively inhibit the growth of HSCT6 cells, down-regulate the expression of TGF-β1 and Smad3 and up-regulate the expression of Smad7. The inhibition of the growth of HSCT6 cells may be closely related to interference of the TGF-β1/Smads (Smad3 and Smad7) signaling pathway.