Anti-Proliferative and Apoptosis-Promoting Effect of microRNA-125b on Pancreatic Cancer by Targeting NEDD9 via PI3K/AKT Signaling

Abstract

Purpose: The expression of microRNA-125b (miR-125b) is low in a variety of cancers, including gastric, lung, bladder, thyroid, and esophageal cancers. However, its specific mechanism in pancreatic ductal adenocarcinoma (PDAC) remains unclear. This study is aimed to explore the role of miR-125b in PDAC.

Methods: PDAC tissues and adjacent tissues were collected for miR-125b analysis by qRT-PCR. Different PDAC cell lines were cultured for miR-125b detection by qRT-PCR, and CAPAN1 cells were selected for the downstream experiments. Cell proliferation was characterized by methyl thiazolyl tetrazolium (MTT) and 5-bromo-2-deoxyUridine (BrdU) staining. Flow cytometry was utilized for apoptosis and cell cycle changes. Cell invasion was determined by the Transwell assay and the dual-luciferase assay was utilized for validating the target gene. Western blotting was used to detect apoptosis related and PI3K/AKT signaling proteins.

Results: miR-125b was significantly down-regulated in human PDAC tissues and cell lines (P < 0.05). miR-125b inhibited the growth and invasion of CAPAN1 cells, facilitated apoptosis, and blocked the cell cycle at the G0/G1 phase. Furthermore, miR-125 directly targeted NEDD9. The high expression of NEDD9 impaired the anti-proliferative and anti-apoptotic activity of miR-125b. miR-125b also inhibited apoptosis-related proteins and PI3K/AKT signaling pathways via NEDD9.

Conclusion: miR-125b decreased cell growth and invasion, and facilitated apoptosis in CAPAN1 cells through PI3K/AKT inhibition via targeting NEDD9.

Keywords: NEDD9; PI3K/AKT; apoptosis; miR-125b; pancreatic ductal adenocarcinoma.

Figure 1

Low expression of miR-125b in PDAC tissues and PDAC cell lines. Notes: (A) miR-125b expression in paired PDAC tissues and adjacent tissues; (B) miR-125b expression in PDAC cell lines by qRT-PCR analysis. **p< 0.01, ***p< 0.001 vs the respective controls.

Figure 2

MiR-125b inhibits CAPAN1 cell proliferation. Notes: (A) miR-125b levels in CAPAN1 cells with different plasmids; (B) proliferation rates of CAPAN1 cells determined by MTT transfected with different plasmids; (C) BrdU incorporation assay for cell proliferation. The values are presented as mean ± SD (n = 3), *p<0.05, **p< 0.01 vs the respective controls.

Figure 3

MiR-125b promotes apoptosis, regulates cell cycle and inhibits invasion in CAPAN1 cells. Notes: (A) Apoptosis of cells with different plasmids by flow cytometry; (B) flow cytometric analysis of cell cycles; (C) representative images of cell invasion across the membrane. The mean values ± SD (n = 3) are shown. *p<0.05, **p<0.01, ***p<0.001 vs the respective control.

Figure 4

MiR-125b regulates apoptosis-related proteins and PI3K/AKT signaling pathways in CAPAN1 cells. Notes: (A) The mRNA levels of BAX, caspase 3, PI3K and AKT; (B) the protein levels of BAX, caspase 3, cleaved caspase 3, PI3K and AKT. The mean values ± SD (n = 3) are shown. *p<0.05, **p<0.01, ***p<0.001 vs the respective control.

Figure 5

NEDD9 is a binding target of miR-125b. Notes: (A) Binding region between miR-125b and NEDD9 and the luciferase reporter constructs containing the NEDD9/WT or NEDD9/MUT sequence. (B) Relative luciferase activity of miR-125b co-transfected into CAPAN1 cells. (C) Relative mRNA expression of NEDD9 in CAPAN1 cells with different transfections. (D) Western blot analysis and band intensity analysis of NEDD9 in CAPAN1 cells with different transfections. Data presented as mean ± SD of three independent experiments. *p<0.05, **p<0.01 vs the respective control.

Figure 6

NEDD9 reverses the effect of miR-125b on proliferation and invasion of CAPAN1 cells. Notes: (A) Relative mRNA expression of NEDD9 in CAPAN1 cells with different transfections; (B) Western blot analysis and band intensity analysis of NEDD9 in CAPAN1 cells with different transfections; (C) proliferation of CAPAN1 cells determined by MTT after different transfections; (D) BrdU incorporation assay for cell proliferation; (E) representative images of cell invasion across the membrane. Data presented as mean ± SD (n = 3), **p<0.01 vs the miR-125b mimics group.

Figure 7

NEDD9 reverses the effect of miR-125b on apoptosis-related proteins and PI3K/AKT signaling pathways in CAPAN1 cells. Notes: (A) The mRNA levels of BAX, caspase 3, PI3K and AKT; (B) the protein levels of BAX, caspase 3, cleaved caspase 3, PI3K and AKT. The mean values ± SD (n = 3) are shown. *p<0.05, **p<0.01 vs the miR-125b mimic group.