[Selective knockdown of Angiotensin II receptor subtype 1a in rat vascular smooth muscle cells by RNA interference].
OBJECTIVE
To selectively knockdown the expression of Angiotensin II receptor subtype 1a (AT1aR) in rat vascular smooth muscle cells (VSMCs) by RNA interference and the sequential effects on cellular viability and proliferation.


METHODS
The primary cultured rat aortic VSMCs were transfected by plasmids pAT1a-shRNA1 and pAT1a-shRNA2, each carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence, or by a control plasmid pGenesil-Control (pCon) carrying a nonspecific shRNA-coding sequence. The mRNA and protein expressions of AT1a, AT2 were analyzed by semi-quantified RT-PCR and Western blot, respectively and normalized to the internal control gene beta-actin. Cellular viability and proliferation were determined with methylthiazoletetrazolium (MTT) assay.


RESULTS
AT1a mRNA and protein were reduced by 82% and 69% by pAT1a-shRNA1, 77% and 56% by pAT1a-shRNA2, respectively while no change was found in pCon treated VSMCs. AT2 receptor level in VSMCs remains unchanged after various treatments. The A(490nm) values obtained by MTT measurements were similar among groups in the absence of Ang II but decreased significantly in pAT1a-shRNA1 and pAT1a-shRNA2 treated VSMCs in the presence of Ang II.


CONCLUSIONS
RNA interference can selectively knockdown AT1a expression in cultured VSMCs and attenuate the Ang II induced cell proliferation. Future studies are warranted to explore the potential role of RNA interference on AT1 function and as a new gene therapy tool for cardiovascular diseases.
Zhang
Jing-qun
JQ
Department of Cardiology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China.
Ma
Ye-xin
YX
Wang
Dao-wen
DW
Xiao
Jian-min
JM
D000818:Animals; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D011945:Receptors, Angiotensin / Q000378:metabolism*; D014162:Transfection
D034741:RNA, Small Interfering; D011945:Receptors, Angiotensin