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ISSN 1672-9145 Acta Biochim Biophys Sin 2005, 37(5): 310–316 CN 31-1940/Q

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Establishment of a Screening System for Selection of siRNA Target Sites Directed against Hepatitis B Virus Surface Gene

Xiu-Min ZHOU^1,3, Ju-Sheng LIN¹, Yi SHI³, De-An TIAN², Huan-Jun HUANG², He-Jun ZHOU¹, and You-Xin JIN³*

¹Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;

²Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;

³State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,

Chinese Academy of Sciences, Shanghai 200031, China

Abstract To study the inhibitory effects of plasmid-derived small interfering RNA (siRNA) and synthetic siRNA on the expression of the hepatitis B virus surface (HBs) gene, three plasmid-derived siRNAs and one synthetic siRNA that complement the coding region of the HBs gene were prepared. The HBs expression plasmid pHBs-EGFP was also constructed. HeLa cells were co-transfected with pHBs-EGFP and the above siRNAs. The HBs mRNA quantities were measured by reverse-transcription PCR, and the level of HBs-EGFP fusion protein was quantified by fluorescent microscope. The concentrations of the hepatitis B virus surface antigen (HBsAg) derived from the culture supernatant of transfected HepG2.2.15 cells were measured by an enzyme-linked immunosorbent assay (ELISA) kit. The results showed that the three plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the quantities of HBs mRNA and protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBs expression. It can inhibit HBs-EGFP expression by 63.3% and suppress HBs mRNA by 75.6%. To further substantiate the above observations, psiRNA1 was transfected into HepG2.2.15 cells (an HBV secreting cell line). The transfections resulted in almost complete blockage of HBsAg production, whereas control vector-transfected cells secreted high levels of HBsAg 7 days post-transfection. In conclusion, our data suggests that RNA interference (RNAi) is an efficient approach for reducing the level of HBs transcripts and proteins and for suppressing HBsAg production.

Key words RNA interference (RNAi); small interference RNA (siRNA); hepatitis B virus surface gene (HBs gene)

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Received: October 27, 2004 Accepted: March 3, 2005

This work was supported by the grants from the National Natural Science Foundation of China (No. 30270311, No. 30330680)

*Corresponding author: Tel, 86-21-54921222; Fax, 86-21-54921011; E-mail, [email protected]