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Supplementary Figure 2 from Immunotherapy for Hepatoma Using a Dual-Function Vector with Both Immunostimulatory and Pim-3–Silencing Effects

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posted on 2023-04-03, 14:00 authored by Qie Guo, Peixiang Lan, Xin Yu, Qiuju Han, Jian Zhang, Zhigang Tian, Cai Zhang

PDF - 159K, Figure S2. shRNA and bi-functional vector suppresses the expression of anti-apoptotic genes in hepatoma cells. A, The expressions of apoptosis-related genes Pim-3, Bcl-XL, Bcl-2, and Bim in Hepa1-6 cells were detected by RT-PCR and Western blotting. B, The expressions of Pim-3, Bad, and phospho-Bad were detected by Western blotting. The relative expression of pim-3 was normalized to that of beta-actin, and that of phospho-Bad to that of total Bad. C, Concentration of caspase-8 in cell lysates of Hepa1-6 after transfection for 24 h with the indicated vectors, as measured by ELISA. Data are representative or means (plus or minus) SD of three independent experiments. **, P < 0.01, compared with the pSIREN transcription group.

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ARTICLE ABSTRACT

Tumorigenesis is an immortalization process in which the growth of normal cells is uncontrolled and programmed cell death is suppressed. Molecular biologic and immunologic studies have revealed that the aberrant expression of some proto-oncogenes boosts proliferation and inhibits apoptosis, which is vital for tumor development. The hypofunction of the host immune system also drives the development and metastasis of malignant tumors. Pim-3, a member of the Pim family, is aberrantly expressed in several cancers. Data suggest that Pim-3 inhibits apoptosis by phosphorylating the proapoptotic BH3-only protein Bad. Here, we constructed a dual-function small hairpin RNA (shRNA) vector containing an shRNA targeting Pim-3 and a TLR7-stimulating ssRNA. Stimulation with this bi-functional vector in vitro promoted significant apoptosis of Hepa1-6 cells by regulating the expression of apoptosis-related proteins and induced secretion of type I IFNs. Most importantly, this bi-functional vector more effectively inhibited subcutaneous Hepa1-6 cell growth than did single shRNA and ssRNA treatment in vivo. Natural killer (NK), CD4+ T, and CD8+ T cells and macrophages were required for effective tumor suppression, and CD4+ T cells were shown to play a helper role in the activation of NK cells, possibly by regulating the secretion of Th1 or Th2 cytokines. This ssRNA–shRNA bi-functional vector may represent a promising approach for tumor therapy. Mol Cancer Ther; 13(6); 1503–13. ©2014 AACR.