Role of transgelin-2 in diabetes-associated pancreatic ductal adenocarcinoma

Figure 1. Transgelin-2 expression in PDAC and adjacent normal tissues (A) Representative pictures of transgelin-2 immunostaining in PDAC (left) and adjacent normal (right) tissues. Location of the high magnification regions (upper panels) is indicated by the rectangles in the upper panels. Magnified images of cells counterstained with transgelin-2 antibody are shown in lower panel. Scale bar, 100 μm. (B) Mean staining score of transgelin-2 in PDAC tissues compared with and adjacent normal tissues. (C) Overall survival as a function of transgelin-2 expression. High levels of transgelin-2 was labeled as blue, while low levels of transgelin-2 was labeled as black. Analysis of statistical significance was performed using the long-rank test. (D) Transgelin-2 level in pancreatic cancer tissues and corresponding normal tissues in Pei's dataset (p<0.01), Logsdon's dataset (p<0.01) and Badea's dataset (p<0.01).

Figure 2. Impact of insulin on transgelin-2 expression PANC-1 (A), BxPC-3 (B), AsPC-1 (C) and SW-1990 (D) cells were treated with insulin at dose 0, 0.2, 2, 20 and 200 nM for 24 h, respectively. Transgelin-2 was detected by quantitative PCR. The relative transgelin-2 level was quantified by 2−ΔΔCT method, with GAPDH as a reference gene. (E) PANC-1 cells were treated with 200 nM insulin for 24 h. Transgelin-2 level, AKT level and AKT S473 phosphorylation were analyzed by western blotting. Quantitative PCR analysis of transgelin-2 mRNA in pancreatic tissues from ob/ob (F) or db/db (G) mice demonstrating increased transgelin-2 in type 2 diabetes models. (H) Quantitative PCR analysis displaying increased transgelin-2 mRNA by IGF-1 in a dose-dependent manner. In all cases, data are represented as mean±SD of three independent experiments.

Figure 3. Impact of transgelin-2 depletion on proliferation of PDAC cells (A) Three specific siRNAs against transgelin-2 were transfected into PANC-1 cells for 48 h. The knockdown efficiency was analyzed by quantitative PCR. (B) Western blot analysis of transgelin-2 or transgelin protein level in PANC-1 cells after transfection for 48 h. PDAC cell lines (PANC-1, AsPC-1, BxPC-3 and SW-1990) were transfected by specific siRNAs against transgelin-2 for 48 h. Transgelin-2 (C) or transgelin (D) mRNA level was analyzed by quantitative PCR. Cell proliferation was analyzed in PANC-1 (E), AsPC-1 (F), BxPC-3 (G) and SW-1990 (H) cell lines treated with non-silencing siRNA (siRNA-NC) or specific siRNA against transgelin-2. Vehicle group indicates cells treated with transfection reagent alone. (I) Relative cell viability was analyzed in PANC-1 cells treated with siRNA against transgelin-2 in the presence or absence of insulin. (J) Colony formation was performed in PANC-1 cells with siRNA against transgelin-2 in the presence or absence of insulin. In all cases, data are represented as mean±SD of three independent experiments.

Figure 4. Transgelin-2 is a target gene of SREBP-1 (A) Schematic diagram of promoter region of the human TALGN2 gene. A putative SRE site is located at −2562/−2552 bp. (B) Validation of SREBP-1 knockdown efficiency using quantitative PCR. (C) Luciferase activity assay of transgelin-2 reporter containing different fragments spanning the promoter region, indicating that the SRE sequence was located between 1 and 5 kb. (D) HEK293T cells were co-transfected by non-silencing siRNA (siRNA-NC) or siRNA against SREBP-1 (siRNA-SREBP-1) with wild-type (WT) or mutant (MT) transgelin-2 firefly luciferase reporter for 24 h. Luciferase activity was measured. The fold of luciferase activity was calculated by normalization of the firefly luciferase activity by the transfection containing reporter plasmids and the siRNA-NC. (E) HEK293T cells were co-transfected by vector or SREBP-1 with wild-type (WT) or mutant (MT) transgelin-2 firefly luciferase reporter for 24 h. The fold of luciferase activity was calculated by normalization of the firefly luciferase activity by the transfection containing reporter plasmids and the siRNA-NC. (F) ChIP analysis using SREBP-1 antibody demonstrated SREBP-1 association with…

Figure 5. Correlation between transgelin-2 and SREBP-1 (A) Scatter plot between transgelin-2 and SREBP-1 staining scores in a sample of PDAC patients. The correlation analysis was calculated using Spearman statistical analysis (Spearman's correlation coefficient: 0.617, p<0.001). R2 is 0.3807. (B) Representative photomicrographs of transgelin-2 (left) and SREBP-1 (right) staining in PDAC tissues from the same patient.