Knockdown of apolipoprotein B, an atherogenic apolipoprotein, in HepG2 cells by lentivirus-mediated siRNA.
ApoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target for lowering atherogenic lipoproteins. In the present study, we used a lentiviral vector to express short hairpin RNAs for inhibition of apoB production in HepG2 cells. We first demonstrated that lentivirus could efficiently deliver transgene into HepG2 cells by using GFP lentivirus. We then made three lentiviral siApoB constructs, two of which were highly efficient for silencing apoB expression in HepG2 cells. We showed that siApoB lentivirus specifically knocked down apoB but had no effects on other proteins such as apoAI and albumin. Consequently, the secretion of apoB was reduced markedly. The silencing effect of siApoB lentivirus appeared to be permanent. Knocking down apoB did not alter the expression of cytoplasmic stress proteins (HSP70 and HSP90) and their ER homologues (GRP78 and GRP94). Furthermore, neither IKKalpha and JNK nor phosphorylated IKK and JNK were increased in long-term apoB-deficient hepatocytes as compared to the control cells. Consistent with these findings, apoB-deficient hepatocytes responded to insulin to a similar extent as the control cells as determined by measuring insulin-induced phosphorylation of IRS and ERK. Our studies indicate that lentiviral siRNAs provide an excellent approach for delivering siRNA into HepG2 cells and may be used for gene therapy for hyperlipidemia.
Liao
Wei
W
Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, CA 92093, USA. wliao@ucsd.edu
Ning
Guang
G
D001055:Apolipoproteins B / Q000235:genetics* / Q000378:metabolism*; D050197:Atherosclerosis / Q000378:metabolism; D002460:Cell Line; D000091342:Endoplasmic Reticulum Chaperone BiP; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods; D015316:Genetic Therapy / Q000379:methods; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics*; D014162:Transfection / Q000379:methods
D001055:Apolipoproteins B; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D034741:RNA, Small Interfering