RNA interfering effects of CCL20 shRNA expressing vectors on human embryonic kidney cells

HE Shengdong; PENG Daizhi; HE Bin; LIU Jing; ZHOU Xin; WANG Yong; WANG Lihua; ZHENG Bixiang; ZUO Haibin

Abstract：Objective To investigate the inhibiting effects of short hairpin RNA on the expression of CCL20 mRNA and protein in human embryonic kidney cells(293FY cells)by using CCL20 shRNA expressing vectors(pSCS-EGFP).Methods The three previously constructed pSCS-EGFP vectors were used in this study(pSCS-EGFP-1 is CCL20 gene mismatch,while pSCS-EGFP-2 and pSCS-EGFP-3 are CCL20 gene specific).The vectors were transfected into 293FT cells by using liposome,and their transfection rates were observed by inverted fluorescence microscope and flow cytometry 24 hours after transfection.These cells were cultured for 48 hours with the three vectors of pSCS-EGFP.respectively.and then treated with TNF-α and IL-1β for 24 hours.The expression of CCL20 mRNA and protein was determined by Real-time fluorescent-quantitative PCR and ELISA,respectively.Results The transfection rates of pSCS-EGFP-1,2,3 were(73.8±5.3)%,(50.0±4.8)%,and(56.8±2.9)%,respectively.The inhibition rates of pSCS-EGFP-1,2,3 on293FT cells stimulated with cytokines were(18.53±34.44)%,(90.40 4-3.94)%,and(92.50 4-6.15)%fnr CCL20 mRNA expre-sion,and were(14.88±17.39)%,(71.76±8.27)%,and(88.56±1.74)%for CCL20 protein secretion,respectively.Without cytokine stimulation,the corresponding inhibition rates of the three vectors were(61.37±11.72)%,(84.33±12.78)%,and(80.27±15.84)%for CCL20 mRNA,and were(19.91±48.12)%,(-27.87±50.24)%,and(87.21±8.36)%for CCL20 protein secretion,respectively.Conclusion Short hairpin RNAs specifically against CCL20 gene have significant down-regulation effect on the expression of CCL20 mRNA and protein in human embryonic kidney cells stimulated with inflammatory cytokines.These results could provide useful experimental evidences for the prevention and treatment of CCL20 associated diseases,such as kidney allograft rejection.