Fig 2: miR-320a modulated the oncogenic MACC1/MET signaling pathway through SP1. Previous study identified SP1-binding elements within the MACC1 promoter region. We performed a luciferase reporter assay to confirm whether SP1 modulates MACC1 transcriptionally. a A schematic diagram showing the previously identified SP1 binding site (- 172 to - 166) and a predicted one (- 445 to - 436) within 500 bp upstream of the MACC1 TSS. (B) SP1 enhanced luciferase activity by recognizing the wild-type MACC1 promoter, and the enhancement was abrogated by its mutated counterpart. (P < 0.0001). c Immunoblotting assays showed that ectopic miR-320a expression apparently inhibited SP1 expression followed by MACC1 inhibition. As downstream effectors of MACC1, c-MET, p-ERK1/2, and p-AKT were repressed along with ectopic miR-320a expression. Normalized intensity of each immunoblot was analyzed and is annotated below the stripes. d Decreased mRNA expression of SP1 and MACC1 induced by miR-320a was also observed in xenograft mouse tumor tissues. SP1: SP1 expressing vector; Ctrl: control vector; pMACC1-wt: wild-type promoter sequence of MACC1 (- 500 to + 1 bp) subcloned into a pGL4.10 luciferase reporter vector; pMACC1-mut: mutant counterpart of MACC1 promoter sequence created by deletion mutation of the two mentioned SP1 binding elements

Fig 3: MACC1 promotes PC cell migration and invasion by upregulating FN1.A Heatmap of the upregulated gene expression clusters in control (Ctrl) and MACC1-overexpressing (OE) SUIT-2 cells. B, C Quantitative real-time PCR validation of selected differentially regulated genes after overexpression of MACC1 in PANC-1 and SUIT-2 cells. **p < 0.01, ***p < 0.001, ****p < 0.0001. D Quantitative real-time PCR validation of selected differentially regulated genes after knockdown of MACC1 in BxPC-3 cells. *p < 0.05, **p < 0.01, ****p < 0.0001. E–G Western blot for FN1, PTGS2 and MMP10 after overexpression of MACC1 in PANC-1 and SUIT-2 cells or knockdown of MACC1 in BxPC-3 cells. H, I GO analyses (H) and KEGG pathway analyses (I) of the upregulated genes in MACC1 overexpressing cells. J, K MACC1 and FN1 mRNA levels in primary tumor tissues and matched adjacent normal tissues from 10 PC patients. *p < 0.05, **p < 0.01, ****p < 0.0001. L MACC1 and FN1 protein levels in primary tumor tissues and matched adjacent normal tissues from 10 PC patients. M Correlation between MACC1 and FN1 mRNA levels in PC tissues from the TCGA datasets. R, Pearson correlation coefficient. N–P Immunofluorescence staining was performed in PANC-1, SUIT-2 and BxPC-3 cells using an anti-FN1 antibody. Representative images of immunofluorescent staining in PANC-1 and SUIT-2 cells stably expressing empty vector (lv-ctrl) or MACC1 (lv-MACC1) or in BxPC-3 cells stably expressing shNT, shMACC1#1 or shMACC1#2. Data are representative of at least three independent experiments. Scale bars, 20 µm. D–F, J, K, Data represent the mean ± SD. of three biologically independent experiments (two-tailed t test). G–I, L, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments.

Fig 4: MACC1 expression is upregulated in PC tissues and correlates with poor outcomes in PC patients.A Representative images of immunohistochemistry for MACC1 expression levels in PC TMAs. Scale bar, left, 200 µm; right, 20 µm. B Comparison of MACC1 staining score in paired PC (tumor, n = 71) and the corresponding adjacent noncancerous tissues (Adj NT, n = 71). Two-tailed t test. ****p < 0.0001. C Comparison of MACC1 staining scores in unpaired PC (tumor, n = 98) and adjacent noncancerous tissues (Adj NT, n = 71). Two-tailed t test. ****p < 0.0001. D MACC1 expression in TCGA PC and normal tissues. Two-tailed t test. ***p < 0.001. E MACC1 protein expression levels in PC samples grouped by clinical stage. Two-tailed t test. *p < 0.05. F MACC1 mRNA expression levels in PC samples grouped by pathological stage (data retrieved from the TCGA database). Mann–Whitney test. **p < 0.01. G ROC analysis to evaluate the predictive value of MACC1 levels for patient survival time. H Kaplan–Meier curve for overall survival by MACC1 protein levels. I Survival plot in TCGA PC patients according to MACC1 mRNA expression levels.

Fig 5: MACC1 expression level is associated with FN1 expression level in PC.A Representative IHC staining of FN1 in PC TMAs. Scale bar, left, 200 µm; right, 20 µm. B Comparison of FN1 staining scores in paired PC (tumor, n = 71) and the corresponding adjacent noncancerous tissues (Adj NT, n = 71). Two-tailed t test. ****p < 0.0001. C Comparison of FN1 staining scores in unpaired PC (tumor, n = 96) and adjacent noncancerous tissues (Adj NT, n = 71). Two-tailed t test. ****p < 0.0001. D FN1 protein expression levels in PC samples grouped by clinical stage. Two-tailed t test. *p < 0.05. E ROC analysis to evaluate the predictive value of FN1 for patient survival time. F Kaplan–Meier curve for overall survival by FN1 expression levels. G Correlations between MACC1 and FN1. R, Pearson correlation coefficient. H Combined analysis of MACC1 and FN1 expression levels in the prognostic value of patients with PC by Kaplan–Meier survival curves. I Survival plot in TCGA PC patients according to the combination of MACC1 and FN1 mRNA expression.