Dual interference with novel genes mfgl2 and mTNFR1 ameliorates murine hepatitis virus type 3-induced fulminant hepatitis in BALB/cJ mice.
Our studies and those of many others have implicated hepatocyte necrosis and apoptosis mediated by fibrinogen-like protein-2 (fgl2) prothrombinase and tumor necrosis factor receptor (TNFR) in the development of fulminant viral hepatitis, a disease with a mortality rate greater than 80% in cases lacking immediate organ transplantation. This study was designed to explore the efficacy of dual short hairpin RNA (shRNA) interference with fgl2 and TNFR1 in the treatment of murine hepatitis virus strain 3 (MHV-3)-induced fulminant hepatitis in mice. Plasmids p-mfgl2shRNA and p-mTNFR1shRNA, complementary to the sequences for mfgl2 and mTNFR1, were constructed. Plasmids pEGFP-mfgl2 and pEGFP-mTNFR1 expressing mfgl2-EGFP (enhanced green fluorescent protein) and mTNFR1-EGFP fusion proteins were also constructed to screen the inhibitory effect of p-mfgl2shRNA and p-mTNFR1shRNA on mfgl2 and mTNFR1 expression. Cotransfection of individual shRNA plasmids and pcDNA3.0-mfgl2 and pcDNA3.0-mTNFR1 expression constructs into Chinese hamster ovary (CHO) cells significantly inhibited mfgl2 and mTNFR1 gene expression, as evidenced by fluorescence microscopy, reverse transcription-polymerase chain reaction, and Western blotting. In vivo hydrodynamic delivery of dual-interference shRNA plasmids for mfgl2 and mTNFR1 significantly decreased mfgl2 and mTNFR1 expression; markedly ameliorated fibrin deposition, hepatocyte necrosis, and apoptosis; and prolonged survival against fulminant viral hepatitis induced by MHV-3 in BALB/cJ mice compared with mfgl2 or TNFR1 single-gene interference. These results indicate that in vivo interference with genes for more than one key target provides superior treatment efficacy compared with single-gene interference.
Gao
Sui
S
Department and Institute of Infectious Disease, Huazhong University of Science and Technology, 430074 Wuhan, People's Republic of China.
Wang
Ming
M
Ye
Huali
H
Guo
Jianwen
J
Xi
Dong
D
Wang
Zhimo
Z
Zhu
Chuanglong
C
Yan
Weiming
W
Luo
Xiaoping
X
Ning
Qin
Q
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004195:Disease Models, Animal; D005260:Female; D005340:Fibrinogen / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D057446:Hydrodynamics; D017114:Liver Failure, Acute / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D006517:Murine hepatitis virus / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
C490637:Fgl2 protein, mouse; D034741:RNA, Small Interfering; D047888:Receptors, Tumor Necrosis Factor, Type I; C506236:Tnfrsf1a protein, mouse; D005340:Fibrinogen; D013925:Thromboplastin