Downregulation of miR-503 Promotes ESCC Cell Proliferation, Migration, and Invasion by Targeting Cyclin D1

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers in China, but the underlying molecular mechanism of ESCC is still unclear. Involvement of microRNAs has been demonstrated in cancer initiation and progression. Despite the reported function of miR-503 in several human cancers, its detailed anti-oncogenic role and clinical significance in ESCC remain undefined. In this study, we examined miR-503 expression by qPCR and found the downregulation of miR-503 expression in ESCC tissue relative to adjacent normal tissues. Further investigation in the effect of miR-503 on ESCC cell proliferation, migration, and invasion showed that enhanced expression of miR-503 inhibited ESCC aggressive phenotype and overexpression of CCND1 reversed the effect of miR-503-mediated ESCC cell aggressive phenotype. Our study further identified CCND1 as the target gene of miR-503. Thus, miR-503 functions as a tumor suppressor and has an important role in ESCC by targeting CCND1.

Keywords: Cyclin D1; Esophageal squamous cell carcinoma; Migration and invasion; Proliferation; miR-503.

Figure 1

miR-503 expression was down-regulated in ESCC tissues and cell lines A. miR-503 expression in NE2 and 10 ESCC cell lines was evaluated using qPCR and presented as fold change relative to the expression in NE2. B. Relative expression of miR-503 was evaluated using qPCR in 71 pairs of ESCC samples and the corresponding adjacent non-cancerous samples. C. Down-regulation of miR-503 expression was observed in 83% of ESCC tumor samples compared to the corresponding adjacent non-cancerous tissues. D. Overexpression of miR-503 mimic in KYSE30 and YES-2 cell lines was analyzed using qPCR. E. Silencing of miR-503 expression after transfecting the inhibitor in KYSE450 and KYSE510 cell lines was analyzed using qPCR. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, t-test. All the experiments were performed at least three times. U6 was used as the internal control for miR-503 expression.

Figure 2

Upregulation of miR-503 expression inhibits ESCC malignant phenotype After YES-2 and KYSE30 cells were transfected with miR-503 mimic or miR-NC 24 h, their proliferation ability was detected by RTCA-MP system (A), whereas migration and invasion assays were performed using Transwell (B). Original magnification, 100×. Each bar represents the mean of three independent experiments. Data were analyzed using two-sided t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 3

Silencing of miR-503 expression promotes ESCC malignant phenotype Cells were transfected with miR-503 inhibitor or miR-NC. A. The proliferation ability was detected by RTCA-MP system 24 h after transfection. B. Migration and invasion assays were performed with Transwell 24 h after transfection. Original magnification, 100×. Each bar represents the mean of three independent experiments and data were statistically analyzed by two-sided t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 4

Upregulation of miR-503 expression induces cell cycle G1/S arrest ESCC cell lines YES-2 A. and KYSE30 B. were transiently transfected with miR-NC or miR-503 mimic. Flow cytometry was performed 24 h after transfection. The histograms of PI staining are presented in the upper panels. Percentage of cells at G1, S, and G2 phases of the cell cycles representing the average of triplicate experiments is presented in the bottom panels for quantification. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 5

miR-503 regulates the expression of CCND1 by targeting its 3′UTR A. Venn diagram and the list of putative miR-503 target genes commonly predicted by TargetScan and miRDB. B. Predicted miR-503 target sequence in 3′UTR of CCND1 and the positions of mutated nucleotides (red). Relative luciferase activity of luciferase reporter plasmids containing the wild-type or mutant CCND1 3′UTR was determined in YES-2 and KYSE30 cells that were co-transfected with the miR-503 mimic or miR-NC. Renilla luciferase activity served as an internal control. C. qPCR analysis of CCND1 expression in YES-2 and KYSE30 cells 48 h after transfected with miR-503 mimic or negative control. D. Western blotting analysis of cyclin D1 in YES-2 and KYSE30 cells 48 h after transfected with miR-503 mimic or negative control. E. Western blotting analysis of endogenous cyclin D1 expression in NE2 and ten ESCC cell lines. F. qPCR analysis of CCND1 expression in 57 pairs of ESCC tissue samples. G. Correlation analysis of CCND1 mRNA expression and miR-503 expression in the paired ESCC tumor samples from 57 patients. Data shown in panels B, C, and F were analyzed using Student’s t-test. Pearson correlation was used to calculate r and P values in panel G. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 6

Overexpression of CCND1 could partly diminish the tumor suppressive effect of miR-503 on ESCC YES-2 and KYSE30 cells were cotransfected with miR-503 mimic, CCND1 siRNA or negative control and pcDNA3.1-vector or pcDNA3.1-CCND1 and incubated for 24 h. A. Western blotting analysis of cyclin D1, vimentin, and E-cadherin using β-actin as a loading control. B. The proliferation ability of YES-2 and KYSE30 cells after various transfections was evaluated using RTCA-MP system. C. Migration and invasion assays of YES-2 and KYSE30 cells after various transfections were performed using Transwell. Original magnification, 100×. Each bar represents the mean of three independent experiments. Data were analyzed using the two-sided t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Supplementary Figure 1

Expression of miR-15 and miR-19 transcripts in NE2 and 10 ESCC cell linesExpression of miR-15a (A), miR-15b (B), miR-19a (C), and miR-19b-2 (D) in NE2 and 10 ESCC cell lines was evaluated using qPCR and presented as fold change relative to the expression in NE2. All the experiments were performed at least three times.