MiR-532-3p suppresses colorectal cancer progression by disrupting the ETS1/TGM2 axis-mediated Wnt/β-catenin signaling

Abstract

The expression panel of plasma microRNA defined miR-532-3p as a valuable biomarker for colorectal adenoma (CRA). However, its expression pattern and function in colorectal cancer (CRC) have remained unclear. The present study investigated the expression levels of miR-532-3p and found that it was in situ downregulated both in CRA and CRC. Moreover, it functioned as a sensitizer for chemotherapy in CRC by inducing cell cycle arrest and early apoptosis via its activating effects on p53 and apoptotic signaling pathways. In addition, miR-532-3p was found to restrain cell growth, metastasis, and epithelial-mesenchymal transition (EMT) phenotype of CRC. A study on the mechanism behind these effects revealed that miR-532-3p directly binds to 3'UTR regions of ETS1 and TGM2, ultimately repressing the canonical Wnt/β-catenin signaling. Further investigation showed that TGM2 was transcriptionally regulated by ETS1 and ETS1/TGM2 axis served as a vital functional target of miR-532-3p in suppressing CRC progression. To conclude, miR-532-3p mimics could act as potential candidate for molecular therapy in CRC through inactivation of the canonical Wnt/β-catenin signaling and enhancement of chemosensitivity.

Fig. 1. The downregulation of MiR-532-3p in colorectal cancer.

a The expression patterns of eight miRNAs in normal colorectal mucosa (N), colorectal adenoma (CRA), and colorectal adenocarcinoma (CRC) tissues with a public expression profiling dataset GSE41655. b The normalized miRNAs expression levels of miR-532-3p and miR-532-5p in GSE41655 dataset. c Expression of miR-532-3p was verified in 54 pairs of CRC tissues from a clinical cohort, which was normalized against an endogenous U6 RNA control. d Relative miR-532-3p expression in CRC compared with that in the paired normal colorectal mucosa, the fold change was normalized with log₂(CRC/N). e Expression of miR-532-3p in seven colorectal cancer cell lines and a colorectal mucosal cell line FHC. f Detection of miR-532-3p in HT29 cells transfected with miR-532-3p mimics and RKO cells with miR-532-3p inhibitors by qPCR. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. non-significant

Fig. 2. MiR-532-3p enhances p53-induced cell cycle arrest and apoptosis of CRC.

a Panther Pathway enrichment of significant altered genes in HT29-miR-532-3p cells and top ten pathways have been shown. b MiR-532-3p-induced activation of cell cycle arrest and apoptosis signaling in CRC cells. c Cell cycle analysis showed that miR-532-3p-induced cell cycle arrest in HT29 cells and miR-532-3p inhibitors could accelerate the transition in RKO cells. d Transfection of miR-532-3p mimics could induce apoptosis in HT29 after treatment with 5-Fluorouracil (5-Fu) or cisplatin. Further, miR-532-3p inhibitors could enhance apoptosis resistance in RKO cells. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. non-significant

Fig. 3. MiR-532-3p suppresses cell proliferation and migration of CRC.

a MiR-532-3p mimics decreased the cell growth and the inhibitors restored the proliferation in HT29 and RKO cells by CCK-8. b Clone formation assay showed a decrease or increase in the proliferation of HT29 and RKO cells after transfection with miR-532-3p mimics and inhibitors as indicated. c Subcutaneous tumor growth curve of HT29 cells and RKO cells infected with NC, miR-532-3p, i-NC, and i-miR-532-3p lentivirus as indicated. d The transwell assay for HT29-NC, HT29-miR-532-3p, RKO-i-NC, RKO-i-miR-532-3p groups. e The wound-healing assay in HT29 and RKO cells transfected with miR-532-3p mimics or inhibitors as indicated. f Western blot analysis of EMT markers and Smad, MAPK proteins in HT29 and RKO cells. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4. ETS1 and TGM2 as the direct targets of miR-532-3p.

a The comparison among different miRNA target prediction engines (MiRanda, miR-walk, and rna22) combined with microarray data. b QPCR verification of the potential target genes of miR-532-3p in HT29 cells transfected with NC, miR-532-3p mimics. ETS1 and TGM2 were both significantly downregulated in HT29-miR-532-3p group. c. Western blot analysis and qPCR analysis of ETS1 and TGM2 in HT29 or RKO cells transfected with miR-532-3p mimics or inhibitors. d Bioinformatics analysis revealed the 3′UTR region of ETS1 and TGM2 contained binding sites of the seed sequence of miR-532-3p. e Dual-luciferase reporter assay illustrating the binding of miR-532-3p and ETS1 or TGM2 in 293FT cells. *p < 0.05, **p < 0.01, n.s. non-significant

Fig. 5. ETS1 activates Wnt/β-catenin pathway by directly regulating TGM2 transcription and induces CRC progression.

a Western blot analysis of ETS1, TGM2, β-catenin in RKO cells transfected with TGM2, ETS1 plasmids, and P-Enter used as control. b Spearman’s correlation analysis showing a positive correlation between ETS1 and TGM2 mRNA levels according to TCGA datasets. c Chip-qPCR analysis of ETS1-binding regions in TGM2 promoter showing the important sites including −186/−13 and −358/−252. d Western blot analysis of β-catenin and downstream molecules of Wnt/β-catenin signaling in HT29-miR-532-3p cells and RKO-i-miR-532-3p cells. e Apoptosis analysis of RKO-P-Enter, RKO-ETS1, RKO-TGM2 cells after treatment with 5-Fu, cisplatin. f The CCK-8 assay was performed to detect the effect of ETS1 and TGM2 in the proliferation ability of RKO cells. g Wound-healing assay evaluating the motility of RKO-P-Enter, RKO-ETS1, RKO-TGM2 cells. *p < 0.05, **p < 0.01, n.s. non-significant

Fig. 6. MiR-532-3p restrains Wnt/β-catenin signaling via suppression of ETS1/TGM2 axis.

a MiR-532-3p-induced G0/G1 cell cycle arrest and ETS1 and TGM2 rescued the transition in HT29 cells. b ETS1 or TGM2 could rescue HT29-miR-532-3p cells from early apoptosis induced by 5-Fu and cisplatin. c Proliferation ability was determined with CCK-8 assay in HT29 cells; and ETS1 and TGM2 could elevate the cell growth by the suppression of miR-532-3p. d Western blot analysis of ETS1, TGM2, β-catenin, downstream molecules of β-catenin, and caspase proteins in HT29 cells after co-transfection with miR-532-3p mimics, ETS1, and TGM2 plasmids. *p < 0.05, n.s. non-significant

Fig. 7. A hypothetical model illustrating that miR-532-3p directly targeted ETS1/TGM2 axis and activated p53 signaling and apoptosis signaling via the suppression of β-catenin and Wnt/β-catenin pathway downstream molecules.

Meanwhile, it supposed a transcript regulation function of ETS1 in TGM2, indicating the indirect inhibition effect of miR-532-3p on TGM2