DNA methyltransferase 3 beta regulates promoter methylation of microRNA-149 to augment esophageal squamous cell carcinoma development through the ring finger protein 2/Wnt/β-catenin axis

Abstract

Esophageal squamous cell carcinoma (ESCC) is an aggressive form of human squamous cell carcinomas with extremely aggressive pathological features. This study explores the functions of microRNA-149 (miR-149) and its interacted molecules in ESCC. The ESCC-related miRNA and messenger RNA (mRNA) datasets were applied to identify aberrantly expressed genes in ESCC. Forty-two patients with ESCC were included and their tissue samples were collected. miR-149 was poorly expressed whereas DNA methyltransferase 3 beta (DNMT3B) and ring finger protein 2 (RNF2) were abundantly expressed in ESCC tumor samples. Overexpression of miR-149 suppressed growth and invasiveness of ESCC cells in vitro and in vivo. DNMT3B bound to the promoter region of miR-149 to trigger its promoter methylation and downregulation. RNF2 mRNA was a target of miR-149. RNF2 overexpression blocked the inhibitory effect of miR-149 on ESCC cell growth. RNF2 activated the Wnt/β-catenin pathway to promote ESCC development. In conclusion, this study found that DNMT3B downregulates miR-149 level through methylation modification of the miR-149 promoter, while miR-149 suppresses RNF2 expression and inactivates the Wnt/β-catenin pathway to suppress growth of ESCC cells.

Keywords: DNMT3B; Esophageal squamous cell carcinoma; RNF2; methylation; miR-149; wnt/β-catenin.

Graphical abstract

Figure 1.

miR-149 is expressed at low levels in ESCC tissues and cells. A, DE miRNAs between ESCC tumor and normal tissues screened using a GEO GSE67268 dataset; B, miR-149 expression in ESCC and the paired adjacent tissue samples from 42 patients detected by RT-qPCR; C-E, association between miR-149 and the clinical staging (c), lymph node metastasis (d) and tumor differentiation (e); F, miR-149 expression in ESCC cell lines (TE-1, EC9706 and KYSE-150) and in HET-1A cells determined by RT-qPCR. In panels B-E, each plot indicates a single sample. **p < 0.01.

Figure 2.

miR-149 suppresses malignance of ESCC cells in vitro. a, expression of miR-149 in KYSE-150 and TE-1 cells after miR-149 mimic or mimic control transfection detected by RT-qPCR; b, colony formation ability of cells; c, Caspase-3 activity in cells detected by a Caspase-3 kit; d, apoptosis rate in cells determined by flow cytometry; e, mRNA levels of the EMT-related factors (E-cadherin, ZO-1, N-cadherin and Vimentin) in ESCC cells measured by RT-qPCR; f-g, migration (f) and invasiveness (g) of cells evaluated by Transwell assays. **p < 0.01.

Figure 3.

miR-149 suppresses growth of ESCC cells in vivo. a, weekly change in the volume of xenograft tumors; b, tumor weight on the 36^th day; c-d, expression of PCNA (c) and KI67 (d) in tumor examined by IHC; E, cell apoptosis rate in tumor tissues determined by TUNEL assay. N = 6 in each group. **p < 0.01.

Figure 4.

The promoter region of miR-149 is highly methylated in ESCC. a, location of miR-149 and GPC1 in genome measured using the Ensembl system; b, the CpG Island on the miR-139 promoter predicted using the UCSC browser; c, promoter methylation level of miR-149 in tumor and healthy tissues examined by MSP-qPCR; d, correlation between the promoter methylation level of miR-149 and its expression in tumor tissues; e, promoter methylation level of miR-149 in ESCC cell lines (TE-1, EC9706 and KYSE-150) and in HET-1A cells determined by MSP-qPCR; f, miR-149 expression in TE-1, EC9706 and KYSE-150 cells after 5-aZa-CDR treatment determined by RT-qPCR. In panels C and D, each spot indicates a single sample. **p < 0.01.

Figure 5.

DNMT3B regulates methylation of miR-149 promoter. a, DNMT1, DNMT3A, DNMT3B and DNMT3L mRNA levels in ESCC and normal tissues from 42 collected patients detected by RT-qPCR; b, DNMT1, DNMT3A, DNMT3B and DNMT3L expression predicted in TCGA-ESCA; c, an inverse correlation of miR-149 and DNMT3B in ESCC tissues; d, strong positive staining of DNMT3B in ESCC; E-F, binding between DNMT3B and miR-149 promoter verified through ChIP-qPCR (e) and luciferase (f) assays. In panels a and c, each spot indicates a sample. **p < 0.01.

Figure 6.

miR-149 directly targets RNF2 mRNA. a-b, DE genes between ESCC and normal tissues analyzed using the GEO GSE29001 and GSE20347 datasets; c, a Venn diagram for target mRNAs of miR-149 predicted via bioinformatics systems and DE genes screened from two datasets; d, RNF2 mRNA in ESCC and normal tissues detected by RT-qPCR; E-F, correlation of RNF2 expression with miR-149 (e) and DNMT3B (f) in ESCC tumor tissues; g, predicted RNF2 expression in TCGA-ESCA; H, staining intensity of RNF2 in ESCC and normal tissues examined by the IHC assay; i, mRNA expression of RNF2 in ESCC cells and in HET-1A cells determined by RT-qPCR; j, binding between miR-149 and RNF2 verified by luciferase assay; k, RNF2 mRNA expression in TE-1 and KYSE-150 cells after miR-149 mimic transfection determined by RT-qPCR. In panel D-F, each spot indicates a sample. **p < 0.01.

Figure 7.

Overexpression of RNF2 promotes ESCC cell growth and metastasis. a, RNF2 mRNA in KYSE-150 and TE-1 cells after oe-RNF-2 administration examined by RT-qPCR; b, colony formation ability of cells; c, Caspase-3 activity in cells determined using a Caspase-3 kit; d, apoptosis of cells detected by flow cytometry; E-F, migration (e), and invasiveness (f) of cells measured by Transwell assays. **p < 0.01.

Figure 8.

RNF2 activates the Wnt/β-catenin pathway. a, the RNF2-related signaling pathways predicted using GSEA; b, protein levels of Wnt1 and β-catenin in ESCC cells detected by Western blot analysis; c, sub-cellular localization of β-catenin in cells examined by immunofluorescence staining. **p < 0.01.

Figure 9.

A graphic abstract. DNMT3B induces CpG Island methylation at the promoter of miR-149 on the GPC1 transcript, which results in miR-149 downregulation and restoration of RNF2 mRNA, leading to Wnt/β-catenin pathway activation and augmented development of ESCC.