Effect of knocking down EPCR in hUC-MSCs on differentiation to fibroblasts

Abstract:

Objective：To construct a lentiviral vector that is capable of knocking down EPCR in hUC-MSCs and to explore the effect of knocking down EPCR on differentiative capacity of hUC-MSCs into fibroblasts. Methods：Three siRNAs were synthesized according to the mRNA sequence of human EPCR and were transferred into hUC-MSCs. Western blot was conducted to analyze the suppression efficiency of three siRNAs to screen for an optimum siRNA. Then the shRNA targeting the optimum siRNA sequence was synthesized，cloned into lentiviral vector and packaged by lentivirus. HUC-MSCs were infected with the recombinant lentivirus. The effici-ency of knocking down was detected both at mRNA and protein levels. HUC-MSCs were stimulated with TGF-β1. The expression levels of α-SMA and Col Ⅰ were detected by Real-time PCR and Western blot for evaluation of differentiative capacity of hUC-MSCs into fibroblasts. Results：Lentiviral vector that targeting to the optimal siRNA sequence of EPCR and can down-regulate the mRNA（t=28.86，P=0.000） and protein（t=88.60，P=0.000） expression of EPCR was constructed successfully. And knocking down of EPCR in hUC-MSCs decreased the mRNA（P=0.044，P=0.000） and protein（P=0.000，P=0.000） expression of COL1A1 and COL1A2. TGF-β1 could induce up-expression of α-SMA（F=369.713，P=0.000；F=451.060，P=0.000），COL1A1（F=42.051，P=0.000；F=759.041，P=0.000） and COL1A2（F=359.205，P=0.000；F=764.348，P=0.000） in mRNA and protein level and transform hUC-MSCs into fibroblasts. However，knocking down of EPCR in hUC-MSCs significantly decreased the expression of α-SMA（P=0.002，P=0.002） and COL1A1（P=0.002，P=0.000） and COL1A2（P=0.000，P=0.000） induced by TGF-β1 compared with that of control group. Conclusion：Knocking down of EPCR in hUC-MSCs are successfully constructed and could decrease the expression of Col Ⅰ as well as suppress the differentiative capacity of hUC-MSCs into fibroblasts.