RNA interference inhibits hepatitis B virus gene expression and replication in HepG2-N10 cells.
OBJECTIVE
RNA interference (RNAi) refers to the phenomenon of sequence-specific degradation of homologous mRNA induced by double-stranded RNA. It has been successfully utilized to down-regulate endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, the effect of vector-based small interfering RNA (siRNA) promoted by pSilencer2.0-U6 inhibit hepatitis B virus (HBV) replication in cell culture was evaluated.


METHODS
Three fragments of short nucleic acids, respectively, targeting on S, X and C region of HBV genome were inserted into pSilencer vectors after they were annealed with their partly antisense strands. The recombination plasmids were pS, pX and pC. These expression plasmids were transfected into HepG2-N10 cells, a cell line which stably expresses hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg) and adw2 subtype Dane particles. The effect of RNAi was evaluated from the changes of DNA, RNA and protein levels. Viral antigens were measured by ELISA. Viral mRNA was analyzed by RT-PCR. The covalent closed circular DNA and genome DNA of HBV secreted into the culture media were measured by quantitative real-time PCR. Analysis of variance was performed for the results.


RESULTS
Vector-based RNA interference could potently reduce HBsAg (pS vs pN: 47%, pX vs pN: 30%, and pC vs pN: 25%, P < 0.001) and HBeAg (pX vs pN: 57% and pC vs pN: 66%, P < 0.001) expression in cell culture. Furthermore, RT-PCR analysis showed that viral mRNAs were effectively degraded, thus eliminating the messengers for protein expression as well as templates for reverse transcription (pS and pC vs pN, P < 0.001; pX vs pN, P = 0.003). Quantitative real-time PCR analysis of HBV DNA revealed that vector-based RNA interference can inhibit HBV replication efficiently (pS, pX and pC vs pN, P < 0.001).


CONCLUSIONS
Our results indicate that RNAi can inhibit HBV gene expression and replication, and it might have the potential to revolutionize the treatment of HBV.
Ren
Jian Lin
JL
Department of Gastroenterology, Zhongshan Hospital, Xiamen University, Xiamen, Fujian Province, China. jianlinr@msn.com
Pan
Jin Shui
JS
Cheng
Tong
T
Dong
Jing
J
Lu
Ya Pi
YP
Huang
Song Jie
SJ
Shi
Hua Xiu
HX
Wang
Lin
L
Lian
Ya Mei
YM
D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000187:drug effects; D006513:Hepatitis B e Antigens / Q000187:drug effects; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D008055:Lipids / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D012367:RNA, Viral / Q000378:metabolism; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D034741:RNA, Small Interfering; D012367:RNA, Viral