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. 2021 Mar 15:9:647702.
doi: 10.3389/fcell.2021.647702. eCollection 2021.

The m6A Reader YTHDF1 Facilitates the Tumorigenesis and Metastasis of Gastric Cancer via USP14 Translation in an m6A-Dependent Manner

Xiao-Yu Chen  1 , Rui Liang  1 , You-Cai Yi  1 , Hui-Ning Fan  1 , Ming Chen  1 , Jing Zhang  1 , Jin-Shui Zhu  1
Affiliations

The m6A Reader YTHDF1 Facilitates the Tumorigenesis and Metastasis of Gastric Cancer via USP14 Translation in an m6A-Dependent Manner

Xiao-Yu Chen et al. Front Cell Dev Biol. .

Abstract

Objectives: N6-methyladenosine (m6A) RNA methylation is implicated in the progression of multiple cancers via influencing mRNA modification. YTHDF1 can act as an oncogene in gastric cancer (GC), while the biological mechanisms via which YTHDF1 regulates gastric tumorigenesis through m6A modification remain largely unknown.

Methods: GEO and TCGA cohorts were analyzed for differentially expressed m6A modification components in GC clinical specimens and their association with clinical prognosis. Transwell and flow cytometry assays as well as subcutaneous xenograft and lung metastasis models were used to evaluate the phenotype of YTHDF1 in GC. Intersection of RNA/MeRIP-seq, luciferase assay, RIP-PCR, RNA pull-down and MeRIP-PCR was used to identify YTHDF1- modified USP14 and its m6A levels in GC cells.

Results: High-expressed YTHDF1 was found in GC tissues and was related to poor prognosis, acting as an independent prognostic factor of poor survival in GC patients. YTHDF1 deficiency inhibited cell proliferation and invasion (in vitro), and gastric tumorigenesis and lung metastasis (in vivo) and also induced cell apoptosis. Intersection assays revealed that YTHDF1 promoted USP14 protein translation in an m6A-dependent manner. USP14 upregulation was positively correlated with YTHDF1 expression and indicated a poor prognosis in GC.

Conclusion: Our data suggested that m6A reader YTHDF1 facilitated tumorigenesis and metastasis of GC by promoting USP14 protein translation in an m6A-dependent manner and might provide a potential target for GC treatment.

Keywords: N6-methyladenosine; USP14; YTHDF1; gastric cancer; metastasis; tumorigenesis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
High expression of YTHDF1 predicts a poor prognosis in patients with GC. (A) Gene expression of m6A-related compositions in patients with GC according to TCGA dataset (unpaired or paired). (B) Gene expression of YTHDF1 in patients with GC according to GSE29272 dataset (n = 126). (C) Correlation analysis between gene expression and the copy number status of YTHDF1 in TCGA-GC dataset. (D,E) Kaplan–Meier analysis of GC patients in GSEs dataset for the correlations between YTHDF1 expression and overall survival. as well as tumor recurrence. (F) Kaplan–Meier analysis of GC patients with Stage III and Stage IV in GSEs database. ADJ, adjacent normal; GC, gastric cancer. Data are shown as means ± S.D. *P < 0.05, **P < 0.01.
FIGURE 2
FIGURE 2
Inhibition of YTHDF1 suppresses cell growth, migration and invasion in vitro. (A) Western blot analysis for YTHDF1 expression in GES-1 and GC cell lines (AGS, BGC-823, SGC-7901, MKN-28, and HGC-27). (B) Western blot analysis for YTHDF1 expression in AGS and BGC-823 cells infected with two independent shRNAs targeting YTHDF1 (sh-YTHDF1#1/2) or a control shRNA (sh-NC). (C) Colony formation assays of AGS and BGC-823 cells described in (B). (D) CCK8 assays were performed to determine cell growth in YTHDF1 deficient AGS and BGC-823 cells as described in (B). (E,F) Knockdown of YTHDF1 decreased the abilities of migration and invasion of AGS and BGC-823 cells. Scale bar, 50 μm. (G) Cell apoptosis analysis was used to compare down-expression of YTHDF1 (sh-YTHDF1#1/2) with the sh-NC group in BGC-823 and AGS cells. *P < 0.05, **P < 0.01, ***P < 0.001; ns, no significance.
FIGURE 3
FIGURE 3
YTHDF1 deficiency inhibits tumor growth and metastasis in vivo. (A) Different systems were applied including GC cell lines and cell line-derived xenograft. (B) Images of the sacrificed nude mice injected with YTHDF1 deficient BGC-823 cells and its control group in mice subcutaneous tumor model (n = 5). (C) (Left) Tumors were dissected from the nude mice of each group and photographed at 36 days after transplantation. (Right) Representative histology images in mice subcutaneous tumor model (n = 5, 200×). Scale bar, 50 μm. (D,E) Tumor weight and the tumor growth curve were measured in YTHDF1 down-expressed cells and its control group. (F) (Left) Images of pulmonary metastases in mice lung metastasis model (n = 4). Black arrows: metastatic nodules. (Right) Representative histology images in mice lung metastasis tumor model (n = 4, 100×). Scale bar, 200 μm. (G) The numbers of lung metastatic nodules in YTHDF1 down-expressed cells and its control group. GC, gastric cancer. Data are shown as means ± S.D. *P < 0.05, **P < 0.01.
FIGURE 4
FIGURE 4
Identification of the YTHDF1 targets in GC cells. (A) Heatmap of DEGs identified by RNA-seq. (B) GO enrichment analysis of DEGs. (C–G) GSEA plots showing the pathways of DEGs enriched by YTHDF1 were involved in GC cells. (H) Metagene profiles of m6A enrichment across mRNA transcriptome in BGC-823 cells. (I) The m6A motif detected by the DREME motif analysis with m6A-seq results. (J) The distribution of m6A peaks on different chromosomes. DEGs, differentially expressed genes; GC, gastric cancer; GO, Gene ontology; GSEA, gene set enrichment analysis. ***P < 0.001.
FIGURE 5
FIGURE 5
USP14 as the m6A modification target of YTHDF1. (A) Overlapping analysis of genes identified by MeRIP-seq, RIP-seq, and RNA-seq. (B) GO analysis of genes described in (A). (C) The expression of genes related to proteasomal protein catabolic process in patients with GC according to TCGA dataset. (D–F) Correlation analysis between YTHDF1 expression and USP14, ANK1B1 and SOCS4 expression in TCGA-GC dataset. (G–J) Kaplan-Meier analysis of GC patients in TCGA dataset for the correlations between USP14/SIRT2/SOCS4/GABARAPL2 expression and overall survival. (K) Predicted m6A sites in USP14 mRNA by SRAMP program, especially in 3’UTR. (L) The m6A abundance on USP14 mRNA transcripts in sh-YTHDF1 and sh-NC infected AGS cells as examined by MeRIP-seq. Data represented the mean ± SD. GC, gastric cancer; GO, Gene ontology; TCGA, The Cancer Genome Atlas; MeRIP, Methylated RNA immune-precipitation. Data are shown as means. *P < 0.05, **P < 0.01, ****P < 0.0001; ns, no significance.
FIGURE 6
FIGURE 6
USP14 rescues the tumor suppressive effect caused by YTHDF1 deficiency. (A) RT-qPCR analysis of the relative RNA level of USP14 in sh-YTHDF1 and sh-NC infected AGS and BGC-823 cells. (B) Western blot analysis of the protein level of USP14 in sh-YTHDF1 and sh-NC infected AGS and BGC-823 cells. (C) YTHDF1-RIP-qPCR confirmed the interrelationship between YTHDF1 and USP14 mRNA in BGC-823 cells. (D) MeRIP-qPCR validation of m6A levels of USP14 in AGS cells. Primers to m6A negative region of MAP2K4 as the negative control and primers to m6A positive region of MAP2K4 as the positive control. (E) RNA pull-down with a biotin-labeled USP14 probe was implemented in BGC-823 cells, followed by western blot to test the enrichment of YTHDF1. (F) (up) Diagrammatic sketch showed the construction of YTHDF1-WT and YTHDF1-MUT. (down-left) Diagrammatic sketch showed that the fragment of wild-type USP14 CDS (USP14) containing predicted YTHDF1 target sites was cloned into pGL3 vector with Firefly luciferase reporter genes. (down-right) The luciferase activity analysis in AGS cells. (G) (Left) Western blot analysis of the protein levels of YTHDF1 and USP14 in YTHDF1-deficient AGS and BGC-823 cells under overexpression of USP14 (USP14-WT) or potential m6A binding site mutated USP14 (USP14-MUT). (Right) Diagrammatic sketch showed the construction of USP14-WT and USP14-MUT. (H,I) Cell growth was measured by colony formation and CCK8 assays in AGS and BGC-823 cells described in (G). (J) Different dosages of IU1 (0, 1, 5, 25, 50, 75, and 100 μM) were used in MKN-28, BGC-823 and AGS GC cell lines. (K) Western blot analysis and gray value analysis of the protein level of USP14 and YTHDF1 in YTHDF1-overexpressed and normal control MKN-28 cells. (L) Cell growth was measured by CCK8 assays in MKN-28 cells described in (K). GC, gastric cancer; MeRIP, Methylated RNA immune-precipitation. Data represented the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, no significance.
FIGURE 7
FIGURE 7
The correlation between YTHDF1 and USP14 expression in GC patients. (A) Representative immunohistochemical images of YTHDF1 expression in primary gastric tumor tissues and normal gastric gland. Scale bar, 50 μm. (B) Relative USP14 protein expression in normal gastric gland and GC specimens assessed by immunohistochemistry in TMA2 (n = 28). Scale bar, 50 μm. (C) Comparison between protein expression of YTHDF1 in patients with GC and their adjacent normal tissues according to TMA1 dataset (n = 80). (D) Comparison between protein expression of USP14 in patients with GC and their adjacent normal tissues according to TMA2 dataset (n = 28). (E) The correlation analysis of the protein expressions of YTHDF1 and USP14 in GC samples and their adjacent normal tissues (n = 28; Pearson’s and spearman’s correlation test). (F) Kaplan-Meier analysis of GC patients in TMA1 dataset (n = 80) for the correlations between YTHDF1 expression and overall survival. (G) Proposed model underlying the roles of YTHDF1-mediated USP14 translation in GC. GC, gastric cancer; TMA, tissue microarray; Data are shown as means ± S.D.
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