miR-23a-5p inhibits cell proliferation and invasion in pancreatic ductal adenocarcinoma by suppressing ECM1 expression

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a genetic disease and a leading cause of cancer-related mortality. However, the molecular mechanism underlying PDAC progression remains unclear. In this study, we first confirmed that ECM1 is significantly upregulated in PDAC tissues and that its high levels of expression are closely associated with an advanced histologic grade and a poor prognosis using The Cancer Genome Atlas (TCGA) dataset and the Gene Expression Omnibus (GEO) database. We then found that miR-23a-5p binds directly to the ECM1 3'-untranslated region (3'-UTR), thereby inhibiting ECM1 expression. Functional studies revealed that the induced expression of ECM1 promoted oncogenic abilities and reversed the suppressive effects induced by miR-23a-5p. Collectively, our findings indicate that ECM1 is a proto-oncogene and show that targeting the miR-23a-5p/ECM1 axis may represent a promising therapeutic strategy for PDAC.

Keywords: ECM1; invasion; miR-23a-5p; pancreatic ductal adenocarcinoma (PDAC); proliferation.

Figure 1

ECM1 is overexpressed in PDAC and ECM1 overexpression is associated with poorer overall survival of PDAC patients. A. GEO dataset (GSE28735) analyses revealed relative ECM1 expression in 45 matched PDAC tissues and adjacent non-tumor tissues by microarray analysis. Paired Student’s t-test was applied, the data are represented as the mean ± SD. ***, P < 0.001. B. The cluster heat map showed the aberrantly upregulated genes in PDAC tissues with P < 0.05. Red color indicates high expression level and green color indicates low expression level. C. Relative expression of ECM1 was detected by RT-qPCR in 14 matched PDAC tissues and adjacent non-tumor tissues. β-actin was used as an endogenous control. Paired Student’s t-test was applied, the data are represented as the mean ± SD. *, P < 0.05. D. Kaplan-Meier analysis revealed that high ECM1 expression was associated with reduced overall survival time of PDAC patients. The clinical data of 176 PDAC patients was downloaded from the TCGA database. E. IHC staining confirmed that ECM1 was upregulated in pancreatic tissues compared with normal pancreatic tissues. Unpaired Student’s t-test was applied, the data are represented as the mean ± SD. ***, P < 0.001. F. The overall survival of PDAC patients was evaluated using Kaplan-Meier analysis according to high and low ECM1 expression. The log-rank test was applied, ***P < 0.001.

Figure 2

ECM1 overexpression promotes cell proliferation and invasion and accelerated cell cycle in vitro. ECM1 Overexpression vector (OV) was transfected to upregulate ECM1 and empty vector (EV) was transfected as Negative control. A. The effect of ECM1 on pancreatic cancer cell growth was evaluated via CCK-8 assay. B. The effect of ECM1 on colony formation ability of pancreatic cancer cells was evaluated. C. The effect of ECM1 on cell cycle was evaluated via flow cytometric analysis. D. The effect of ECM1 on pancreatic cancer cell invasion ability was measured via Transwell^® assay. Data are presented as mean ± SD of three independent experiments; statistical significance was analyzed by unpaired t-test. *, P < 0.05, **, P < 0.01, ***, P < 0.001. E. Western blot analysis was used to analyze the protein level of E-cadhein, Vimentin, ECM1, Cyclin D1 and CDK4.

Figure 3

ECM1 is directly targeted and negatively regulated by miR-23a-5p. A. TargetScan identified the potential binding site between miR-23a-5p and the 3’-UTR of ECM1. B. Western blot analysis revealed that overexpression of miR-23a-5p by mimics inhibited ECM1 expression. C. For the dual-luciferase reporter assay, mutations were generated in the ECM1 3’-UTR sequence at the complementary sites for the seed regions in miR-23a-5p. D. Dual-luciferase reporter assay revealed that miR-23a-5p reduced the luciferase activity of the wild type ECM1 3’-UTR compared to the mutant type. The data are represented as the mean ± SD of three independent experiments. Student’s t-test was used. ***, P < 0.001.

Figure 4

MiR-23a-5p is downregulated in PDAC tissues and closely associated with poorer overall survival of PDAC patients. A. The expression of miR-23a-5p was detected in 14 paired PDAC and adjacent noncancerous tissues. Data are presented as mean ± SD. Paired t-test was applied. **, P < 0.01. B. Kaplan-Meier analysis showed that PDAC patients with low miR-23a-5p expression had lower overall survival rate. Log-rank test was applied, P = 0.0203. C. Biological process classification of target genes by GO annotation. D. Significantly enriched pathways of target genes.

Figure 5

Overexpression of miR-23a-5p abrogated ECM1 carcinogenic effects in vitro. In this section, miR-23a-5p mimics and ECM1 overexpression vector (mimics + OV) were transfected to investigate the rescue potential of ECM1. Cells transfected with ECM1 mimics NC and empty vector (NC + EV) were used as negative control. A. CCK-8 assay was applied to investigate proliferation ability; B. Colony formation assay was applied to investigate colony formation ability; C. Flow cytometric analysis was used to detect changes in cell cycle; D. Transwell^® assay was applied to investigate invasion ability. All experiments were performed in triplicate in two pancreatic cancer cell lines, AsPC-1 and CFPAC-1. E. Western blot analysis was used to analyze the protein levels of E-cadhein, Vimentin, ECM1, Cyclin D1 and CDK4. Data are presented as mean ± SD of three independent experiments. Statistical significance was assessed by unpaired t-test. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Figure 6

Proposed model for miR-142-3p inhibiting ECM1 expression in PDAC.