ABSTRACT
Objective To investigate the effect of alteration of CRELD1 gene expression on the related genes in the endocardial cushion development.Methods Over-expression and silence of CRELD1 gene were realized by the construction of lentiviral vector.Afterwards,the lentiviral vectors were used to infect the human fetal lung fibroblasts (HFL)-I.All of the cells were divided into the following 5 groups:the blank control group,the negative control group of interference,the interference group,the negative control group of over-expression,and the over-expression group.Western blot and real-time fluorescent quantitative polymerase chain reaction were applied to examine the mRNA and protein expression of CRELD1,Sox9,Aggrecan,Scleraxis and Tenascin-C.Results The DNA sequences of 2 recombinant plasmids pLV3-shRNA-CRELD1 and pLV4-CRELD1 matched very well with those which were designed according to the DNA sequence analysis.HFL-I was successfully infected with lentiviral vectors and displayed fluorescent green light under inverted fluorescence microscope.The results of real-time PCR detection and Western blot test were consistent:expressions of Sox9 and Aggrecan in the interference group were significantly higher than those in the negative control group of interference,while the expressions of the 2 genes in the over-expression group were significantly lower than those in the negative control group of over-expression.Expressions of Scleraxis increased in both the interference group and the over-expression group when compared with the negative control groups respectively.Compared to the corresponding negative control groups,Tenascin-C expression decreased markedly in the interference group,whereas it increased significantly in over-expression group.Conclusions CRELD1 gene has negative effect on the expression of the related genes Sox9 and Aggrecan in the endocardial cushion development,whereas it has positive effect on the Tenascin-C expression.It serves as a theoretical framework to illustrate the effect of CRELD1 gene on the atrioventricular septal defect.