METTL14-mediated N6-methyladenosine modification of SOX4 mRNA inhibits tumor metastasis in colorectal cancer

Abstract

Background: Colorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide, and its main cause of death is distant metastasis. Methyltransferase-like 14(METTL14), a major RNA N6-adenosine methyltransferase, is involved in tumor progression via regulating RNA function. The goal of the study is to uncover the biological function and molecular mechanism of METTL14 in CRC.

Methods: Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) assays were employed to detect METTL14 and SOX4 in CRC cell lines and tissues. The biological functions of METTL14 were demonstrated using in vitro and in vivo experiments. Chromatin immunoprecipitation (ChIP), Transcrptomic RNA sequencing (RNA-Seq), m6A-RNA immunoprecipitation sequencing (MeRIP-Seq), RNA immunoprecipitation and luciferase reporter assays were used to explore the mechanism of METTL14 action.

Results: METTL14 expression was significantly downregulated in CRC and decreased METTL14 was associated with poor overall survival (OS). Both the univariate and multivariate Cox regression analysis indicated that METTL14 was an independent prognostic factor in CRC. Moreover, lysine-specific histone demethylase 5C(KDM5C)-mediated demethylation of histone H3 lysine 4 tri-methylation(H3K4me3) in the promoter of METTL14 inhibited METTL14 transcription. Functionally, we verified that METTL14 inhibited CRC cells migration, invasion and metastasis through in vitro and in vivo assays, respectively. Furthermore, we identified SRY-related high-mobility-group box 4(SOX4) as a target of METTL14-mediated m6A modification. Knockdown of METTL14 markedly abolished SOX4 mRNA m6A modification and elevated SOX4 mRNA expression. We also revealed that METTL14-mediated SOX4 mRNA degradation relied on the YTHDF2-dependent pathway. Lastly, we demonstrated that METTL14 might inhibit CRC malignant process partly through SOX4-mediated EMT process and PI3K/Akt signals.

Conclusions: Decreased METTL14 facilitates tumor metastasis in CRC, suggesting that METTL14 might be a potential prognostic biomarker and effective therapeutic target for CRC.

Keywords: Colorectal cancer (CRC); METTL14; N6-methyladenosine(m6A); SOX4; YTHDF2.

Fig. 1

METTL14 expression is downregulated and is associated with prognosis in CRC. a. Expression of METTL14 in the TCGA CRC cohort. b. Expression of METTL14 in the GSE9348, GSE44076 and GSE41657 CRC cohorts. c. The METTL14 mRNA expression levels in 136 CRC tissues and matched normal tissues were detected using qRT-PCR. d. Representive images of IHC staining for METTL14 protein on a tissue microarray constructed from 136 CRC tissues and matched normal tissues. e.Kaplan-Meier OS analysis of METTL14 expression in CRC patients. f. Univariate and multivariable analyses were performed in the CRC cohort. All bars correspond to 95% CIs. **P < 0.01, ***P < 0.001

Fig. 2

KDM5C-mediated demethylation of H3K4me3 inhibited METTL14 transcription. a. Analysis of H3K4me3 ChIP-Seq data of CRC cells and colorectal tissues in the METTL14 locus. b. The mRNA levels of METTL14 in KDM5A-IN-1(5 μM)-treated HCT116 and HCT8 cells were measured using qRT-PCR. c. The METTL14 and H3K4me3 protein levels in HCT116 and HCT8 cells were detected by western blot after KDM5A-IN-1(5 μM) treatment. d. The KDM5C knockdown efficiency was determined at the protein levels in HCT116 and HCT8 cells using western blot. e. The METTL14 and H3K4me3 protein levels in HCT116 and HCT8 cells were detected using western blot. f. ChIP assays were employed to measure the levels of KDM5C binding f and the enrichment of H3K4me3 g at the promoter of METTL14 in KDM5C deficiency or control HCT116 and HCT8 cells. ***P < 0.001

Fig. 3

METTL14 inhibits CRC cell migration, invasion and metastasis. a. The expression of METTL14 in CRC cell lines (HCT116, HCT8, HT29, SW620, SW480 and DLD-1) compared with NCM460 detected using qRT-PCR. b. The protein levels of METTL14 in HCT116 and HCT8 cells with METTL14 knockdown or METTL14 overexpression were measured by western blot. c. Transwell migration assays were used to determine the migratoy abilities of HCT116 and HCT8 cells with METTL14 knockdown or overexpression. d. Transwell invasion assays were employed to detect the invasive abilities of HCT116 and HCT8 cells with METTL14 knockdown or overexpression. e. Left panel, representative images of metastatic nodes in the lungs from LV-shNC and LV-shME-1 groups. Middle panel, quantification of the metastatic nodes from LV-shNC and LV-shME-1 groups. Right panel, HE-stained lung sections from LV-shNC and LV-shME-1 groups. f. Left panel, representative images of metastatic nodes in the lungs from LV-NC and LV-METTL14 groups..Middle panel, quantification of the metastatic nodes from LV-NC and LV-METTL14 groups. Right panel, HE-stained lung sections from LV-NC and LV-METTL14 groups.**P < 0.01, ***P < 0.001

Fig. 4

SOX4 is a downstream target of METTL14. a. MeRIP-Seq and RNA-Seq identidied differentially expressed genes in METTL14 stable knockdown cells when compared with their corresponding controls. b, c. The mRNA b and protein c levels of SOX4 in METTL14-deficient HCT116 and HCT8 cells were detected using qRT-PCR and western blot, respectively. d. Expression of SOX4 mRNA in the TCGA cohort. e. The METTL14 mRNA expression levels in 136 CRC tissues and matched normal tissues were detected using qRT-PCR. f. Representive images of IHC staining for SOX4 protein on a tissue microarray constructed from 136 CRC tissues and matched normal tissues. g. The correlation between METTL14 and SOX4 in CRC. ***P < 0.001

Fig. 5

METTL14 knockdown enhances SOX4 mRNA stability via an m6A-YTHDF2-dependent pathway. a. The m6A contents of total RNAs in METTL14-knockdown HCT116 and HCT8 cells (left panel) were detected using dot blot with m6A antibody. Methylene blue staining were served as the loading control (right panel). b. MeRIP-qPCR analysis was used to demonstrate METTL14-mediated SOX4 m6A modification in HCT116 and HCT8 cells. m6A modification of SOX4 was depleted upon METTL14 knockdown. c. Wild-type or m6A consensus sequence mutant SOX4 cDNA was fused with firely luciferse reporter. d. Mutation of m6A consensus sequences or knockdown of METTL14 relieved the posttranscriptional repression of SOX4 in HCT116 and HCT8 cells. e. The mRNA levels of YTHDF2 and SOX4 in YTHDF2 knockdown CRC cells were detected by qRT-PCR. f. The protein levels of YTHDF2 and SOX4 in YTHDF2 knockdown CRC cells were detected by western blot. GAPDH was used as control. g. Precursor and mature mRNA of SOX4 in METTL14 stable knockdown and control HCT116 cells. h. i. The precursor h and mature i SOX4 mRNA expression were detected at indicated times. j. RIP-qPCR assay using YTHDF2-specifc antibody and IgG control antibody to measure the enrichment of YTHDF2 binding to SOX4 m6A modification sites. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

SOX4 served as an oncogene and reversed the effects of METTL14 in CRC. a. The SOX4 knockdown efficiency was proved at the protein levels in HCT116 and HCT8 cells by western blot assay. b. Quantified results of the cell migration abilities of HCT116 and HCT8 cells with SOX4 deficiency. c. Quantified results of the cell invasion abilities of HCT116 and HCT8 cells with SOX4 deficiency. d. The protein levels of METTL14 and SOX4 in HCT116 and HCT8 cells transfected with METTL14 and SOX4 expression plasmid or blank vector (vector) were measured using western blot. e, f. Quantified results of the cell migration e and invasion f abilities of cell migration abilities of METTL14-overexpressing HCT116 and HCT8 cells added with SOX4 expression plasmid or blank vector. g. Left panel, representative images of the gross lesion in the lung tissues from sh-NC and shSOX4 group. Middle panel, quantification of the metastatic nodes from indicated groups. Right panel, representative microscopic views of pulmonary metastatic foci from indicated groups using HE staining. h. Left panel, representative images of the gross lesion in the lung tissues from negative control (NC), LV-METTL14 + vector and LV-METTL14 + SOX4 group. Middle panel, quantification of the metastatic nodes from indicated groups. Right panel, representative microscopic views of pulmonary metastatic foci from indicated groups using HE staining. **P < 0.01, ***P < 0.001

Fig. 7

METTL14 inhibits CRC malignant process through SOX4-mediated EMT process and PI3K/Akt signals. a. Protein levels of METTL14, SOX4, E-cadherin, Vimentin and N-cadherin were detected by western blot in HCT116 and HCT8 cells with indicated treatment. b. Protein levels of PI3K, p-PI3K, Akt and p-Akt were detected by western blot in HCT116 and HCT8 cells with indicated treatment. GAPDH was used as a control