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  • br Immunohistochemistry and western blot analyses were

    2020-08-18


    Immunohistochemistry and western blot analyses were performed
    on transformed versus wild-type organoids on day 10 after infection (Fig. 2F). Expression of KrasG12D and deletion of P53 in transformed
    organoids were associated with BIRC5 upregulation (Fig. 2H, upper panel), whereas absence of KrasG12D and expression of wild type P53 in wild-type organoids was associated with complete absence of BIRC5 expression (Fig. 2H, bottom panel). These data demonstrate that BIRC5
    Fig. 2. BIRC5 was selected as the prototype for PDAC overexpressed gene. Initiation of expression of BIRC5 was triggered by overexpression of KrasG12D or P53(1−320) (expression of 1–320 amino LY500307 of P53) in benign human primary pancreatic epithelial cells (HPPE) and significantly increased by overexpression of KrasG12D and P53(1−320 as shown in immunostaining (A) and western blot analysis (B). The pattern of expression of BIRC5 was further confirmed by CRISPR/Cas9 engineered HPPE cells and mouse pancreatic ductal organoids (ORGs). Driver mutation KrasG12D/p53Del engineered HPPE cells were obtained by co-transfection with both gRNA (green) and Cas9 (red) (C) and enriched by flowtometry (D). The expression profiling of BIRC5 in driver mutation-engineered HPPE cells was determined by western blot (E). Successful infection of ORGs with lentiviral Cas9 (red) and gRNA (green) (F) resulted in transformation of organoids (G upper 2 panels) compared to wild-type organoids morphologically (G bottom panel). Absence of expression of p53 and over-expression of KrasG12D in transformed organoids resulted in over expression of BIRC5, as shown in the immunofluorescence staining (H upper panel); wild-type organoids expressing p53 and an absence of KrasG12D had no expression of BIRC5 (H bottom panel) (scale bar = 50 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    upregulation in PDAC reflects activation of common PDAC driver mu-tations and support our selection of BIRC5 as a prototype PDAC-upre-gulated gene.
    3.3. BIRC5 super-promoter enhances gene expression in PDAC cells
    Utilizing our technique of engineering synthetic promoters of overexpressed genes to target PDAC, a BIRC5 super-promoter (BIRC5-SP) was generated based on the analysis of key cis- and trans-regulatory elements located in the endogenous BIRC5 promoter between −327 and −1 (ATP), which were randomly repeated [13]. The elements of the promoter included the binding sites for EGR-1 (Early growth re-sponse protein 1), NF-kB, KLF5, STAT3, P53, E2F/RB, SP1, the CDE (Cell Cycle-dependent Elements) and CHR (Cell Cycle Genes Homology Region) (Fig. 3A) [14]. Fourteen BIRC5 synthetic promoter sequences were cloned into firefly luciferase reporter vector (S2-4) and screened in PDAC cells versus benign human pancreatic ductal cells (HPDE) and HPPE cells using a bioluminescent reporter assay (S5). The one with greatest promoter activity in PDAC cells and lowest activity in benign cells was defined as BIRC5-SP for further studies (S5); BIRC5-SP_S2; red arrows). BIRC5-SP drove GLuc expression with 15, 9, 13, and 6-fold higher activity compared to BIRC5-endogenous promoter (BIRC5-EP) in PANC-1, Mia PaCa2, Capan2, and AsPC-1 cells, respectively (p < 0.05) (Fig. 3B); no activity was observed in benign HPPE and HPDE cells due to absence of BIRC5 expression in these benign cells (p > 0.05). Fur-thermore, BIRC5-SP's activity was further enhanced by a two-step
    transcriptional amplification (TSTA) system (S6) [15]. TSTA sig-nificantly enhanced BIRC5-SP's activity up to 10-fold in BIRC5-SPTSTA transfected PDAC and PDAC patient-derived cell lines, which express BIRC5, with no significant activity in benign HPDE cells, which do not express BIRC5 (Fig. 3C). Enhanced BIRC5-SPTSTA activity was further 
    validated using a GFP reporter (S7). The brightest signals were ob-
    served in the BIRC5-SPTSTA-GFP transfected PDAC cells, whereas no signals were observed in benign HPPE cells. BIRC5-SPTSTA-GLuc was
    further tested in transformed HPPE cells using BIRC5-SP-GLuc and CMV-GLuc as controls. BIRC5-SPTSTA-GLuc resulted in significantly greater secreted GLuc levels compared to BIRC5-SP-GLuc and CMV-GLuc in both HPPEKrasG12D and HPPEKrasG12D/P53Del cells (Fig. 3D). Both BIRC5-SPTSTA-GLuc and BIRC5-SP-GLuc resulted in no expression of
    GLuc in benign HPPE cells, whereas CMV-GLuc resulted in high ex-pression of GLuc in wild-type HPPE cells (Fig. 3D). Lentiviral vector BIRC5-SPTSTA-GLuc was selected to infect transformed versus wild-type mouse pancreatic ductal organoids (ORGs), resulting in secreted GLuc