MiR-497 downregulation contributes to the malignancy of pancreatic cancer and associates with a poor prognosis

Abstract

Chemoresistance is one of the causes of poor prognosis in pancreatic cancer patients. However, the mechanisms of resistance remain unclear. Here we screened miRNAs associated with drug resistance in pancreatic cancer, and identified a panel of miRNAs dysregulated in gemcitabine-resistance pancreatic cancer cells, including 13 downregulated miRNAs and 20 upregulated miRNAs. Further studies focusing on miR-497 demonstrated that miR-497 suppressed cells proliferation, decreased the percentage of S phase cells, re-sensitized cells to gemcitabine and erlotinib, and attenuated migration and invasion capacities. Furthermore, fibroblast growth factor 2 and fibroblast growth factor receptor 1 were confirmed as miR-497 targets. Overexpression of miR-497 inhibited tumor growth in vivo. Additionally, miR-497 expression was significantly downregulated in pancreatic cancer tissues compared with tumor-adjacent samples (P=0.000). Low expression of miR-497 was an independent adverse prognostic factor of pancreatic cancer (P=0.01, hazard ratio=2.762, 95% confidence interval: 1.159-6.579). Together these results indicate that miR-497 could be a new therapeutic target and prognostic marker of pancreatic cancer.

Figure 1. MiRNA profile associated with drug resistance in pancreatic cancer cells

(A) The heat map shows 33 miRNAs whose expression is altered > 4- or < 0.25-fold in gemcitabine-resistant SW1990 cells (SW1990/GEM cells) compared with SW1990 cells. (B) Validation of miR-497 expression levels by qRT-PCR. U6 served as an internal control (*P < 0.05).

Figure 2. Effects of miR-497 on proliferation and chemosensitivity

(A) PDAC cells were transfected as indicated, and proliferation was analyzed by CCK8 assay. The optical density at the wavelength of 450 nm (OD450) was positively associated with the count of viable cells (*P < 0.05). (B) Transfected cells were treated with different doses of gemcitabine for 48 h. Cell viability was evaluated using the CCK8 assay, and the inhibition rate of each dose was calculated. (C) The sensitivity of PDAC cells to erlotinib was examined. Data are displayed as the mean ± SD (*P < 0.05).

Figure 3. MiR-497 induced cell cycle arrest

PDAC cells were transfected for 48 hours and collected for cell cycle analysis by fluorescence-activated cell sorting. Data are displayed as the mean ± SD (*P < 0.05).

Figure 4. MiR-497 attenuated migration and invasion capacities

Cell migration was analyzed by transwell membranes without Matrigel. Invasion was analyzed by transwell membranes with Matrigel. Cells that had migrated or invaded to the lower surface of the membrane were stained with hematoxylin and eosin and counted under a microscope at 100× magnification. (A) Upregulation of miR-497 by transfection with mimics reduced cell migration and invasion. (B) Downregulation of miR-497 by transfection of inhibitor promoted cell migration and invasion. Data are displayed as mean ± SD.

Figure 5. MiR-497 suppressed tumor growth in vivo

Mice were transplanted with SW1990 cells stably overexpressing miR-497 or control cells, and tumor growth was monitored. Tumor growth in vivo was analyzed by analysis of variance. Upper panel: tumors from experimental and control mice. Lower panel: tumor growth curves showing miR-497 inhibition of tumor growth.

Figure 6. MiR-497 directly inhibited the expression of FGF2 and FGFR1 by binding to the 3′-UTR

(A) Dual luciferase assay showed that FGF2 and FGFR1 were direct targets of miR-497. Cells were transfected with luciferase vectors containing either the wild-type or mutated binding site for miR-497 in FGF2 (left) or FGFR1 (right). The relative luciferase activity of cells co-transfected with vectors containing wild-type binding sequences along with miR-497 mimics was significantly decreased compared with that of cells co-transfected with mutated binding sequence vectors and mimics. Luciferase activities were also reduced compared with cells transfected with vectors containing wild-typed binding sequences and mimics control, as well as cells with mutant binding sequence vectors and mimics control. Data are displayed as the mean ± SD (*P < 0.05). (B) Protein expression levels of FGF2 and FGFR1 were detected by western blot.

Figure 7. Expression levels and prognostic values of miR-497 in pancreatic cancer tissues

(A) The expression levels of miR-497 in a panel of 90 pancreatic cancer and tumor-adjacent tissues were detected by ISH (200×). (B) The expression levels of miR-497 in 90 pancreatic cancer tissue samples and matched tumor-adjacent tissues were analyzed using Pearson χ² test. (C) Kaplan-Meier survival analysis in pancreatic cancer patients based on miR-497 expression in primary tumors.