Restoration of miRNA-148a in pancreatic cancer reduces invasion and metastasis by inhibiting the Wnt/β-catenin signaling pathway via downregulating maternally expressed gene-3

Abstract

Various microRNAs (miRNA) have been recognized potential novel tumor markers and have a critical role in cancer development and progression. Recently, methylation of miRNA-148a was identified as a crucial biochemical process in the progression of cancer. However, its potential role and in pancreatic cancer as well as the underlying mechanisms have remained largely elusive. The present study investigated the potential antitumor effect of miR-148a as well as its impact on invasion and metastasis in pancreatic cancer. It was found that the expression of miRNA-148a and the potential predictive biomarker maternally expressed gene-3 (MEG-3) were obviously decreased in human pancreatic cancer tissues compared with those in adjacent non-tumorous tissues. Furthermore, miR-148a was found to be downregulated in pancreatic cancer cell lines compared with normal pancreatic cells through promoter methylation. An MTT assay and a clonogenic assay demonstrated that restoration of miRNA-148a inhibited the proliferation and colony formation of pancreatic cancer cells. In addition, miR-148a transduction led to the upregulation of MEG-3 expression and promoted apoptosis of pancreatic cancer cells. Western blot analysis revealed that transduction of miR-148a markedly decreased the expression levels of C-myc, cyclin D1 and β-catenin in pancreatic cancer cells. Methylation of miR-148a not only decreased the endogenous β-catenin levels but also inhibited the nuclear translocation of β-catenin to delay cell cycle progression. Furthermore, ectopic miR-148a methylation inhibited pancreatic cancer cell migration and invasion via causing an upregulation of MEG-3 expression. Most importantly, ectopic overexpression of miR-148a in pancreatic cancer cells inhibited tumor formation in an animal experiment. Taken together, miR-148a methylation is a crucial regulatory process to inhibit the proliferation and invasion of pancreatic cancer cells, and transduction of miR-148a suppressed the proliferation of pancreatic cancer cells through negative regulation of the Wnt/β-catenin signaling pathway. The findings of the present study suggested that miRNA-148a acts as a tumor suppressor in pancreatic cancer and may contribute to the development of novel treatments for pancreatic cancer.

Keywords: MEG-3; Wnt/β-catenin; metastasis; methylation; miR-148a; pancreatic cancer.

Figure 1.

Expression levels of miR-148a and MEG-3 in pancreatic cancer tissues. Tumors obtained from patients with pancreatic cancer showed (A) higher miR-148a levels and (B) MEG-3 levels than those from normal control tissues. Representative gels of polymerase chain reaction products and quantified expression levels are displayed. (C and D) Immunofluorescence analysis of (C) miR-148a and (D) MEG-3 expression in tumors obtained from patients with pancreatic cancer and normal control tissues. Representative immunostaining images and quantified expression levels are displayed (magnification, ×40). Values are expressed as the mean ± standard error of the mean. **P<0.01 vs. control. MEG-3, maternally expressed gene-3; miR, microRNA.

Figure 2.

Identification of differential methylation status/regions of miR-148a in pancreatic cancer. (A) Number of CpG islands of miR-148a in PANC-1 and Aspc-1 cells. (B) The extent of methylation in 5-aza-2′-deoxycytidine-treated PANC-1 and Aspc-1 cells. (C) Identification of differential methylation regions of miR-148a in PANC-1 and Aspc-1 cells. (D) Promoter activity of a luciferase reporter vector containing clones of CDP-CR for differential methylation following transfection of PANC-1 and Aspc-1. Relative promoter activity of miR-148 clones with differential methylation regions. (E) Relative reporter activity containing different methylation region clones of miR-148a after co-transfection of GATA1, GATA2 or USF1. (F) An electrophoretic mobility shift assay was performed to analyze the miR-148a promoter containing the USF1 binding sequence treated with a methylated probe. Purified bacteria-recombinant USF1 protein was used as a mock control. Values are expressed as the mean ± standard error of the mean. *P<0.05 and **P<0.01 vs. control. miR, microRNA; USF1, upstream stimulatory factor 1; CpG, C-phosphate-G; CDP-CR, catalytic domain protein-C region; GATA, G-A-T-A base.

Figure 3.

Inhibitory effects of miR-148a on growth and metastatic capacity of pancreatic cancer. (A) miR-148a exerted obvious inhibitory effects on growth of PANC-1 and Aspc-1 cells. (A-D) miR-148a exerted obvious inhibitory effects on (B) migration, (C) invasion and (D) colony formation by PANC-1 and Pasc-1 cells (magnification, ×40). (E and F) Transfection of miR-148a promoted (E) apoptosis and (F) MEG-3 expression of PANC-1 and Aspc-1 cells. Values are expressed as the mean ± standard error of the mean. **P<0.01 vs. control. miR, microRNA; MEG-3, maternally expressed gene-3; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 3.

Inhibitory effects of miR-148a on growth and metastatic capacity of pancreatic cancer. (A) miR-148a exerted obvious inhibitory effects on growth of PANC-1 and Aspc-1 cells. (A-D) miR-148a exerted obvious inhibitory effects on (B) migration, (C) invasion and (D) colony formation by PANC-1 and Pasc-1 cells (magnification, ×40). (E and F) Transfection of miR-148a promoted (E) apoptosis and (F) MEG-3 expression of PANC-1 and Aspc-1 cells. Values are expressed as the mean ± standard error of the mean. **P<0.01 vs. control. miR, microRNA; MEG-3, maternally expressed gene-3; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 4.

Restoration of miR-148a inhibits mesenchymal-epithelial transition through MEG-3 mediated via the Wnt/β-catenin signaling pathway in pancreatic cancer cells. Restoration of miR-148a (A) suppressed the expression of epithelial cell markers and (B) suppressed the expression of Wnt/β-catenin signaling molecules in PANC-1 and Aspc-1 cells. (C and D) Recombinant MEG-3 inhibited β-catenin, C-myc and Cyclin D1 expression in (C) PANC-1 and (D) Aspc-1 cells. (E) Immunofluorescence microscopy revealed that restoration of miR-148a decreased endogenous β-catenin levels and suppressed the activity of nuclear translocation (magnification, ×40). Nuclei were counterstained with DAPI. **P<0.01 miR, microRNA; MEG-3, maternally expressed gene-3; IR, inhibitor; C-myc, c-myelocytomatosis oncogene.

Figure 5.

Reconstruction of miR-148a leads to reduced pancreatic tumor growth and invasion in vivo. (A) Implantation of PANC-1-miR-148a inhibited formation of pancreatic tumor lesions (scale bar, 10 mm). (B) PANC-1-miR-148a showed higher miR-148a expression in vivo, as determined by histological analysis (magnification, ×40). (C) Quantification of pancreatic tumor nodules within animals after injection of PANC-1-miR-148a or PANC-1-control cells. (D) Reconstruction of miR-148a decreased Wnt/β-catenin expression levels by upregulating MEG-3 expression in vivo (magnification, ×40). Values are expressed as the mean ± standard error of the mean. **P<0.01 vs. control. miR, microRNA; MEG-3, maternally expressed gene-3.