miR-484 suppresses proliferation and epithelial-mesenchymal transition by targeting ZEB1 and SMAD2 in cervical cancer cells

Abstract

Background: MicroRNAs (miRNAs) play important roles in cancer initiation and development. Epithelial-mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and metastasis. The purpose of the study was to determine the function and mechanism of miR-484 in initiation and development of cervical cancer (CC).

Methods: We determined the expression levels of miR-484 in cervical cancer tissues and cell lines with RT-qPCR. Prediction algorithms and EGFP reporter assay were performed to evaluate the targets for miR-484. MTT assay, colony formation assay, flow cytometric analysis, transwell cell migration and invasion assays, and detection of EMT markers were employed to investigate the roles of miR-484 and the targets in regulation of cell proliferation and EMT process. We also used rescue experiments to confirm the effect of miR-484 on CC cells through directly regulating the expression of its targets.

Results: Firstly we found miR-484 was down-regulated in cervical cancer tissues and cell lines compared with their matched non-cancerous tissues or normal cervical keratinocytes cells. Further studies revealed that overexpression of miR-484 suppressed the cell proliferation, while exacerbates apoptosis. Besides, miR-484 suppressed cellular migration, invasion and EMT process of CC cells. EGFP reporter assay showed that miR-484 binds to ZEB1 and SMAD2 3'UTR region and reduced their expression. The expression of miR-484 had reverse correlation with SMAD2/ZEB1, and SMAD2/ZEB1 had positive correlation with each other in cervical cancer tissues and cell lines. Furthermore, the ectopic expression of ZEB1 or SMAD2 could rescue the malignancies suppressed by miR-484, suggesting that miR-484 down-regulates ZEB1 and SMAD2 to repress tumorigenic activities.

Conclusion: We found miR-484 inhibits cell proliferation and the EMT process by targeting both ZEB1 and SMAD2 genes and functions as a tumor suppressor, which may served as potential biomarkers for cervical cancer.

Keywords: Cervical cancer; EMT; SMAD2; ZEB1; miR-484.

Fig. 1

miR-484 suppresses the proliferation of CC cells. a RT-qPCR showed the expression of miR-484 in 15 pairs of human cervical cancer tissues and the adjacent noncancerous tissues. U6 snRNA was used as the internal control. b After transfection 48 h, RT-qPCR was performed to detect miR-484 levels in HeLa or C33A cells transfected with pcDNA3/pri-miR-484 or ASO-miR-484. c After transfection 24, 48, 72 h, MTT assay was performed to measure cell viability in HeLa or C33A cells transfected with pcDNA3/pri-miR-484 or ASO-miR-484. d Colony formation assay was performed to determine the effect of miR-484 on the long-term proliferative activity of HeLa or C33A cells. The control was normalized to 1

Fig. 2

miR-484 suppresses cell cycle of CC cells and exacerbates apoptosis. a, b The cell cycle progression of HeLa cells was analyzed by flow cytometry. The chart shows the population of cells in G1-, S- and G2-phase (a) as well as the proliferation index (b). Proliferation index = (S + G2)/G1 (S, G2 and G1 are the percentages of cells in S-phase, G2-phase and G1-phase, respectively). c After transfection 24 h, apoptosis of HeLa cells was monitored by Annexin V-PI double staining and flow cytometry assay. Paclitaxel was used to induce cell apoptosis 6 h before collecting cells. The right lower quadrant of each plot contains early apoptotic cells, whereas the right upper quadrant contains late apoptotic cells. All data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

miR-484 suppresses the migration and invasion of CC cells and down-regulates the EMT process. a, b After transfection 48 h, cell migration (a) and invasion (b) were evaluated using a transwell system with 8 μm pores in polycarbonate membranes. Representative views of migratory or invasive cells on the membrane were presented below. All pictures were photographed at ×20 magnification. c Protein levels of EMT-associated markers were assessed by western blotting after transfection 48 h. d RT-qPCR analysis for the expression of EMT transcription factors ZEB1, Snail, Slug and Twist2 in HeLa cells transfected with miR-484 or the control vector. The control was normalized to 1. All data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

miR-484 directly targets SMAD2/ZEB1 and down-regulates their expressions. a The venn diagram shows the potential target genes (overlapping fraction) that were shared by all three databases. b The predicted binding sites for miR-484 in the 3′UTR of SMAD2 and ZEB1 and the mutations in the binding sites are shown. c The EGFP reporter assay was performed in HeLa or C33A cells co-transfected with pcDNA3/EGFP-SMAD2/ZEB1 3′UTR wild type or pcDNA3/EGFP-SMAD2/ZEB1 3′UTRmut with pri-miR-484 or ASO-miR-484. The fluorescence intensities were measured at 48 h after transfection. d Western blot assays were used to detect the SMAD2 and ZEB1 protein levels in HeLa or C33A cells transfected with pcDNA3/pri-miR-484, ASO-miR-484 or their corresponding controls. e Western blot assays tested the effect of SMAD2 on the expression of ZEB1. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance. All the bars indicate the means ± SDs. All experiments were performed at least triplicates

Fig. 5

The ectopic expression of SMAD2 and ZEB1 counteracts the inhibition of cell proliferation induced by miR-484. a CC cells were co-transfected with pcDNA3/pri-miR-484 and pFlag-SMAD2/ZEB1 without its 3′UTR or the control vector and western bolt assay was used to determine the restoration of SMAD2 or ZEB1 protein levels by pFlag-SMAD2/ZEB1 in the presence of miR-484. b After transfection 48 h, MTT assay was performed to detect cell viability in CC cells. c The transfected cells were submitted to colony formation assays to test the proliferation of CC cells. All data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

The ectopic expression of SMAD2 and ZEB1 counteracts the inhibition of cell cycle and the promotion of apoptosis induced by miR-484. a The cell cycle progression of HeLa cells was analyzed by flow cytometry. b After transfection 24 h, apoptosis of HeLa cells was monitored by Annexin V-PI double staining and flow cytometry analysis. All data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

The ectopic expression of SMAD2 and ZEB1 counteracts the inhibition of migration, invasion and EMT progression induced by miR-484. a, b Transwell migration/invasion assays and three-dimensional Matrigel culture to test the cells’ abilities to migration (a) and invasion (b). c Protein levels of EMT-associated markers were assessed by western blotting after transfection for 48 h. *p < 0.05, **p < 0.01, ***p < 0.001. All the bars indicate the means ± SDs. All experiments were repeated at least three times

Fig. 8

Knockdown of SMAD2/ZEB1 abrogates the effects induced by ASO-miR-484. a After co-transfection with ASO-miR-484 and pshR-SMAD2/ZEB1 or the control vector for 48 h, MTT assay was performed to detect cell viability in CC cells. b The transfected cells were submitted to colony formation assays to test the proliferation of CC cells. c The expression of EMT markers (E-cadherin and vimentin) was detected by western blot analysis. All data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 9

The inverse correlation between the expression of miR-484 and ZEB1/SMAD2 in cervical cancer tissues and cell lines. a RT-qPCR showing the expression of SMAD2 and ZEB1 in 15 pairs of human cervical cancer tissues and the adjacent noncancerous tissues. U6 snRNA was used as the internal control. b Pearson’s correlation analysis indicated the negative correlation between the expression of miR-484 and ZEB1 (r = −0.55*) and SMAD2 (r = −0.65***). c RT-qPCR showing the expression of miR-484, SMAD2 and ZEB1 in cervical cancer cell lines compared with NCx. U6 snRNA and β-actin were used as the internal controls. d Western blot were used to detect the ZEB1 and SMAD2 protein levels in cervical cancer cell lines compared with NCx. All data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. e Representative IHC images showing the expression level of SMAD2 and ZEB1 in cervical cancer and normal cervix (×20)

Fig. 10

Schematic that describes the role of miR-484 in CC cells. miR-484 regulated proliferation and EMT process through both directly and indirectly targeting ZEB1 in CC cells