Small hairpin RNA targeting rat C5aR can inhibit the cell apoptosis induced by LPS in kidney epithelial cells / 中国免疫学杂志

ABSTRACT

Objective:To investigate RNA interference and apoptosis induced by LPS in kidney epithelial cells through silencing C5aR gene with small hairpin RNA (shRNA).Methods:We construct the eukaryotic expression vector of small hairpin RNA targeting rat C5aR gene,and transfected RK3E cell by electroporation,after G418 selection,so we got the stable cell line expressing C5aR shRNA.The experiment was designed into 3 groups①normal control group,RK3E cells without transfection;②negative control group,RK3E cells transfected with blank vector;③ experimental group,RK3E cells transfected with C5aR shRNA.After incubation with LPS for 12 h,the ratio of apoptosis was tested by flow cytometry,the level of mRNA was tested by RT-PCR,and binding of ~(125)I-rrC5a to RK3E cells stimulated with LPS were performed to examine the expression of C5aR in RK3E cells.Results:Compared with the normal control group and negative control group,in the experimental group the ratio of apoptosis was significantly decreased(P＜0.01),and the expression of C5aR mRNA was significantly inhibited(P＜0.01),and binding of ~(125)I-rrC5a to RK3E cells was significantly decreased also.Conclusion:Hairpin shRNA targeting C5aR gene can lead to obvious gene silence in vitro and inhibit the cell apoptosis induced by LPS in kidney epithelial cell.