Construction of shRNA Expressing Plasmid Targeted Rat NogoB and Observation of the Effection aboutNogoB on Hepatic Stellate Cells

WEN Mao-yao, MEN Ruo-ting, DAN Xue-lian.et al

Abstract

To construct shRNA expressing plasmid inhibiting rat NogoB and to observe its possible effect on rat primary hepatic stellate cells (HSCs) contraction. Methods Three pairs of shRNAs targeting different sequence of rat NogoB were designed and constructed into pSuper plasmid by DNA recombination technique. Culture-activated HSCs were transfected with NogoB-shRNA plasmids to scan the effective plasmid which could inhibit NogoB gene expression by Real-time PCR. And this depressant effect was also confirmed with Western blot. After NogoB was knocked-down effectively, ETA and ETB mRNA expression were assessed by Real-time PCR. Results Among the three pairs of recombinant plasmids, NogoB-shRNA2 plasmid could inhibit NogoB expression specifically. In HSCs, NogoB knockdown decreased the ratio of ETA and ETB. Conclusion We constructed specific NogoB-shRNA expression plasmid successfully which might be involved in contraction of HSCs.

 

Keywords: shRNA, NogoB, Hepatic stellate cells 

 

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References


Acevedo L. Yu J, Erdjument-Bromage H. et al. A new role for Nogo as a regulator of vascular remodeling. Nat Med, 2004; 10 (4): 382-388.

McKerracher L, Winton MJ. Nogo on the go. Neuron,2002;36 (3) :345-348.

Wang M, Han Y, Zhang XP, et al. Nogo, a star protein in reticulon family. Neurosci Bull,2006 ;22(3) ; 183-186.

Zhang D, Utsumi T, Huang HC, et al. Reticulon 4B (Nogo-B) is a novel regulator of hepatic fibrosis. Hepatology, 2011; 53 (4) :1306-1315.

Xu W, Hong W, Shao Y, et al. Nogo-B regulates migration and contraction of airway smooth muscle cells by decreasing ARPC 2/3 and increasing MYL-9 expression. Respir Res,2011; 12:14. doi;10. 1186/1465-9921-12-14.

Liu XJ. Yang L, Luo F, et al. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray. World J Gastroenterol, 2004;10(11);1600-1607.

Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference. Nat Biotechnol,2004 ;22(3) ;326-330.

Ui-Tei K, Naito Y, Takahashi F, et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res, 2004 ; 32 ( 3 ); 936-948.

Takashimizu S, Kojima S, Nishizaki Y, et al. Effect of endothelin A receptor antagonist on hepatic hemodynamics in cirrhotic rats. Implications for endothelin-1 in portal hypertension. Tokai J Exp Clin Med,2011;36(2);37-43.

Seo YS, Shah VH. Pathophysiology of portal hypertension and its clinical links. J Clin Exp Hepatol,2011; 1(2) ;87-93.

Hellerbrand С. Hepatic stellate cells the pericytes in the liver. Pflugers Arch ,2013 ;465( 6); 775-778.


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