Functional Characterization of miR-216a-5p and miR-125a-5p on Pancreatic Cancer Stem Cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death. Its poor prognosis is closely related to late-stage diagnosis, which results from both nonspecific symptoms and the absence of biomarkers for early diagnosis. MicroRNAs (miRNAs) exert a regulatory role in numerous biological processes and their aberrant expression has been found in a broad spectrum of diseases, including cancer. Cancer stem cells (CSCs) represent a driving force for PDAC initiation, progression, and metastatic spread. Our previous research highlighted the interesting behavior of miR-216a-5p and miR-125a-5p related to PDAC progression and the CSC phenotype. The present study aimed to evaluate the effect of miR-216a-5p and miR-125a-5p on the acquisition or suppression of pancreatic CSC traits. BxPC-3, AsPC-1 cell lines, and their CSC-like models were transfected with miR-216a-5p and miR-125a-5p mimics and inhibitors. Following transfection, we evaluated their impact on the expression of CSC surface markers (CD44/CD24/CxCR4), ALDH1 activity, pluripotency- and EMT-related gene expression, and clonogenic potential. Our results show that miR-216a-5p enhances the expression of CD44/CD24/CxCR4 while negatively affecting the activity of ALDH1 and the expression of EMT genes. MiR-216a-5p positively influenced the clonogenic property. MiR-125a-5p promoted the expression of CD44/CD24/CxCR4 while inhibiting ALDH1 activity. It enhanced the expression of Snail, Oct-4, and Sox-2, while the clonogenic potential appeared to be affected. Comprehensively, our results provide further knowledge on the role of miRNAs in pancreatic CSCs. Moreover, they corroborate our previous findings about miR-216a-5p's potential dual role and miR-125a-5p's promotive function in PDAC.

Keywords: CSC; PDAC; functional study; miR-125a-5p; miR-216a-5p; miRNA; miRNA regulation.

Figure 1

Effect of miR-216a-5p on CD44/CD24/CxCR4 expression in (a) adherent BxPC-3 and their CSC-like models; and (b) adherent AsPC-1 and their CSC-like models. Effect of miR-125a-5p on CD44/CD24/CxCR4 expression in (c) adherent BxPC-3 and their CSC-like models; and (d) adherent AsPC-1 and their CSC-like models. Values were normalized with the control cells in monolayer and in CSC enrichment. We attributed different p-values to the asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

Effect of miR-216a-5p on ALDH1 activity and representative flow cytometry cytogram plot of (a) adherent BxPC-3 and their CSC-like models; and (b) adherent AsPC-1 and their CSC-like models. Effect of miR-125a-5p on ALDH1 activity and representative flow cytometry cytogram plot in (c) adherent BxPC-3 and their CSC-like models; and (d) adherent AsPC-1 and their CSC-like models. x-axis: FSC-A/ALDH1, y-axis: SSC-A/side scatter. Red dots refer to the entire population while green dots are representatives of ALDH1+ population. Values were normalized with the control cells in monolayer and in CSC enrichment. We attributed different p-values to the asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Pluripotency-related gene relative expression following miR-216a-5p mimic and inhibitor treatments on monolayer-grown BxPC-3 and their CSC-like models (a) and on monolayer-grown AsPC-1 and their CSC-like models (b). We attributed different p-values to the asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Pluripotency-related gene relative expression following miR-125a-5p mimic and inhibitor treatments on monolayer-grown BxPC-3 and their CSC-like models (a) and on monolayer-grown AsPC-1 and their CSC-like models (b). We attributed different p-values to the asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

EMT-related gene expression following miR-216a-5p mimic and inhibitor transfection in (a) adherent BxPC-3 and CSC-like models and (b) in adherent AsPC-1 and their CSC-like models. We attributed different p-values to the asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

EMT-related gene expression following miR-125a-5p mimic and inhibitor transfection in (a) adherent BxPC-3 and CSC-like models and (b) AsPC-1 and CSC-like models. We attributed different p-values to the asterisks: * p < 0.05; ** p < 0.01.

Figure 7

(a) Effect of miR-216a-5p on the number and size of BxPC-3 colonies; (b) representative images of untreated BxPC-3 and miR-216a-5p mimic-treated and miR-216a-5p inhibitor-treated BxPC-3 in the soft-agar colony formation assay; (c) effect of miR-216a-5p on the number and size of AsPC-1 colonies; (d) representative imagines of untreated AsPC-1 and miR-216a-5p mimic-treated and miR-216a-5p inhibitor-treated AsPC-1 in the soft-agar colony formation assay. We attributed different p-values to the asterisks: * p < 0.05; ** p < 0.01.

Figure 8

(a) Effect of miR-125a-5p on the number and size of BxPC-3 colonies; (b) representative images of untreated BxPC-3 and miR-125a-5p mimic-treated and miR-125a-5p inhibitor-treated BxPC-3 in the soft-agar colony formation assay; (c) effect of miR-125a-5p on the number and size of AsPC-1 colonies; (d) representative images of untreated AsPC-1 and miR-125a-5p mimic-treated and miR-125a-5p inhibitor-treated AsPC-1 in the soft-agar colony formation assay. We attributed different p-values to the asterisks: *** p < 0.001.