miR-186 and 326 predict the prognosis of pancreatic ductal adenocarcinoma and affect the proliferation and migration of cancer cells

Abstract

MicroRNAs can function as key tumor suppressors or oncogenes and act as biomarkers for cancer diagnosis or prognosis. Although high-throughput assays have revealed many miRNA biomarkers for pancreatic ductal adenocarcinoma (PDAC), only a few have been validated in independent populations or investigated for functional significance in PDAC pathogenesis. In this study, we correlated the expression of 36 potentially prognostic miRNAs within PDAC tissue with clinico-pathological features and survival in 151 Chinese patients. We then analyzed the functional roles and target genes of two miRNAs in PDAC development. We found that high expression of miR-186 and miR-326 predict poor and improved survival, respectively. miR-186 was over-expressed in PDAC patients compared with controls, especially in patients with large tumors (>2 cm), lymph node metastasis, or short-term survival (< 24 months). In contrast, miR-326 was down-regulated in patients compared with controls and displayed relatively increased expression in the patients with long-term survival or without venous invasion. Functional experiments revealed that PDAC cell proliferation and migration was decreased following inhibition and enhanced following over-expression of miR-186. In contrast, it was enhanced following inhibition and decreased after over-expression of miR-326. A luciferase assay indicated that miR-186 can bind directly to the 3'-UTR of NR5A2 to repress gene expression. These findings suggest that miR-186 over-expression contributes to the invasive potential of PDAC, likely via suppression of NR5A2, thereby leading to a poor prognosis; high miR-326 expression prolongs survival likely via the decreasing invasive potential of PDAC cells. These two miRNAs can be used as markers for clinical diagnosis and prognosis, and they represent therapeutic targets for PDAC.

Fig 1. Kaplan-Meier curve for overall survival in patients with PDAC based on venous invasion (A), resection margin status (B), tumor differentiation (C), and adjuvant chemotherapy (D).

Survival curves were compared by log-rank analysis for 11 demographic and clinico-pathological variables, and those with P < 0.05 are shown in this figure. FDR for each test result was calculated by the Benjamini and Hochberg method.

Fig 2. Kaplan-Meier curve for overall survival in patients with PDAC based on the binomial variable of high or low expression relative to the mean expression of miR-186 (A), miR-326 (B), miR-224 (C), miR-219 (D), miR-30a (E) using RT-PCR.

Survival curves were compared by log-rank analysis for 36 miRNAs, and the ones with P < 0.05 are shown in this figure. FDR for each test result was calculated by the Benjamini and Hochberg method.

Fig 3. Functional analyses of miR-186 or miR-326 regulation in PDAC cell lines.

Analysis of miR-186 (A) and miR-326 (B) expression in three PDAC cell lines, HPDE cells, PDAC tissues and adjacent normal pancreatic tissues. (C) Analysis of miR-186 expression in three PDAC cell lines after the transfection of the miR-186 mimic, Anti-miR-186, and respective negative controls. (D) Analysis of miR-326 expression in three PDAC cell lines after the transfection of the miR-326 mimic, Anti-miR-326, and respective negative controls. Cell proliferation was assessed in the PDAC cell lines transfected with miR-186 mimics, Anti-miR-186 or the corresponding negative control (E) and in the PDAC cell lines transfected with miR-326 mimics, Anti-miR-326 or the corresponding negative control (F) using the WST-1 assay. Relative cell growth was normalized to its respective control-treated cells. The graphs show relative cell migration after either inhibition or over-expression experiments for miR-186 (G) and miR-326 (H), as evaluated by the Transwell migration assay. Relative cell migration was normalized to its respective control-treated cells. Data presented are the mean of three independent experiments. Error bars represent standard deviations from the mean. *, P < 0.05, **, P < 0.01 and **, P < 0.001.

Fig 4. MiR-186 directly targets the NR5A2 gene.

(A) qRT-PCR analyses of NR5A2 expression levels following the treatment of MiaPaca-2, BxPC3, Panc-1 cells with miR-186 inhibitors or mimics. (B) Human NR5A2 3′-UTR fragment containing a wild-type (WT) or mutant (Mut) miR-186–binding sequence was cloned downstream of the luciferase reporter gene. (C) The intensity of EGFP fluorescence was decreased in cancer cells transfected with both miR-186 mimics and NR5A2 WT 3′-UTR, but was unaltered in those transfected with both miR-186 mimics and the 3′-UTR mutant vector. (D) Relatively lower expression of NR5A2 was found in cancer samples compared with their normal counterparts using western blot assay. (E) There is an inverse correlation between the expression level of miR-186 and NR5A2. The expression relationship was evaluated by Pearson’s correlation analysis. P < 0.05 was considered statistically significant. *, P < 0.05, **, P < 0.01 and **, P < 0.001. Hsa, Homo sapiens; Ptr, Pan troglodytes; Mml Macaca mulatta; Oga, Otolemur garnettii; Mmu, mouse.