Down-regulation of angiotensin II by shRNA reduces collagen synthesis in hepatic stellate cells.
The angiotensin-converting enzyme 2 (ACE-2), angiotensin II type I receptor (ATIR) antagonists and angiotensin-converting enzyme inhibitors (ACEI) were explored to block the renin-angiotensin-aldosterone system (RAAS). The experimental results were still not satisfactory, mainly due to excessive level of angiotensin II (AngII) in gene expression. RNA interference (RNAi) is a mature gene blocking technique, able to block target gene expression efficiently, specifically and continuously. In this study, we observed the effect of short hairpin RNA (shRNA) expression vectors targeting rat AngII on collagen synthesis in hepatic stellate cells (HSCs). According to rat AngII gene sequences, three AngII targeted shRNA expression vectors were designed and constructed. Using liposomes as transfection reagents, they were transfected into HSC-T6 cells. Enzyme digestion confirmed that the transfected shRNA target gene segment was successfully cloned to the vectors. Compared with the control group, AngII mRNA expression examined in shRNA1, shRNA2 and shRNA3 groups was inhibited by about 37, 30 and 61%, respectively. AngII protein expression in all three groups was also reduced by about 21, 24 and 59%, respectively. Furthermore, we revealed that the inhibitory effect exhibited a dose- and time-dependent relationship. In shRNA3 group, TGF-beta1 mRNA expression was reduced by about 51%. The levels of PIIIP, HA and LN were decreased by about 53, 47 and 58%, respectively. In conclusion, shRNA expression vectors targeting rat AngII can decrease collagen synthesis, which would hopefully serve as a foundation for RNAi study of liver fibrosis in vivo.
Yu
Fu Jun
FJ
Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, PR China. tjyufujun@163.com
Dong
Pei Hong
PH
Fan
Xu Fei
XF
Lin
Zhuo
Z
Chen
Yong Ping
YP
Li
Ji
J
D000804:Angiotensin II / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003094:Collagen / Q000096:biosynthesis*; D015536:Down-Regulation; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008103:Liver Cirrhosis / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism
D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D000804:Angiotensin II; D003094:Collagen