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Lentivirus‐mediated RNA Interference Knockdown of Inositol Requiring 1 Enhances Sorafenib Lethality in Hepatoma Hep3B Cells Involving Autophagy Inhibition
Author(s) -
Hu Ye,
Ye HuiLan,
Zhang Guo,
He LiJun,
Liu ZeFeng,
Chen Li
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.938.1
Subject(s) - small hairpin rna , gene knockdown , rna interference , multiplicity of infection , gene silencing , transfection , microbiology and biotechnology , sorafenib , cell culture , green fluorescent protein , viral vector , biology , chemistry , cancer research , rna , recombinant dna , hepatocellular carcinoma , gene , biochemistry , genetics
Inositol requiring 1 (IRE1), a component of the unfolded protein response signaling pathway, has been found to be correlated with sorafenib resistance in hepatocellular carcinoma (HCC). The present study aimed to investigate the effects of lentivirus vector mediated short hairpin RNA interference knockdown of IRE1 on hepatoma Hep3B cells and explore the possibility of IRE1 silencing as a therapeutic target for HCC. Firstly, three pairs of human IRE1 targeted specific interference sequences tagged with green fluorescent protein were designed, synthesized and subcloned into lentiviral vectors(pL‐shRNA‐IRE1‐GFP) successfully, which were further identified by PCR and sequencing. Then, recombinant and lentiviral packaging plasmids were co‐transfected into 293T cell lines effectively in order to produce lentiviral particles using fluorescence determined the titer. After stable infected Hep3B cell lines established with pL‐shRNA‐IRE1‐GFP and pL‐GFP‐Negative control at the optimum multiplicity of infection, flow cytometry was used to sort green fluorescent protein‐positive cells which showed a very high degree of positive rate. QPCR and western blot analysis were employed to identify the interference silencing sequence with the most efficient silencing profile. Results showed that the expression profiles of IRE1 mRNA and protein were significantly lower (P<0.05) in the specific interference group (LV142‐pL‐shRNA‐IRE1‐GFP) than that of the control group with a maximum of 90% inhibition. Although there was no significant difference of proliferation, growth as well as apoptosis in basal condition, sorafenib (5μM) was found to cause a substantial increment about 50% on cell death in IRE1 silencing LV142 cells vs control. Interestingly, mechanistic investigations showed that such a stronger sorafenib lethality was coupled with decreasing expression of autophagic molecule light chain 3 (LC3)‐II as well as accumulation of p62 in these cells. Collectively, these data suggests that RNA interference mediated IRE1 knockdown might enhance sorafenib lethality in hepatoma Hep3B cells involving autophagy inhibition, which brings insight into a novel and potential HCC therapeutic strategy in the future. Support or Funding Information Supported by NSFC Grants 881172260, 30630145, 81360077 and GXNSFC Grants 2015GXNSFDA139022 to Guo Zhang