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. 2020 Feb 26;10:115.
doi: 10.3389/fonc.2020.00115. eCollection 2020.

METTL3 Promotes the Progression of Gastric Cancer via Targeting the MYC Pathway

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Free PMC article

METTL3 Promotes the Progression of Gastric Cancer via Targeting the MYC Pathway

Dong-Dong Yang et al. Front Oncol. .
Free PMC article

Abstract

Methyltransferase-like 3 (METTL3), a major component of the N6-methyladenosine (m6A) methyltransferase complex, has been suggested to function as an oncogene in several cancers. However, its biological mechanism and the involved pathways in gastric cancer (GC) remain unknown. Here, we reported that frequent upregulation of METTL3 was responsible for the aberrant m6A levels in gastric carcinoma. On the other hand, a high level of METTL3 was significantly associated with several clinicopathological features and poor survival in patients with GC. The knockdown of METTL3 effectively inhibited cell proliferation and migration and invasion capacity. Moreover, overexpression of METTL3 considerably augmented its oncogenic function. Integrated RNA-seq and m6A-seq analysis first indicated that several component molecules (e.g., MCM5, MCM6, etc.) of MYC target genes were mediated by METTL3 via altered m6A modification. Our work uncovers the oncogenic roles of METTL3 in GC and suggests a critical mechanism of GC progression.

Keywords: METTL3; MYC target genes; gastric cancer; minichromosome maintenance complex component 5; minichromosome maintenance complex component 6; prognostic factor.

Figures

Figure 1
Figure 1
The association between methyltransferase-like 3 (METTL3) upregulation and abnormal m6A levels in gastric cancer (GC). (A) Relative expression pf writers and erasers in the The Cancer Genome Atlas (TCGA) database (normal mucosa = 32, tumor tissue = 375). (B) Total RNA m6A levels in 13 paired gastric carcinoma tissues and adjacent tissues. (C) Relative messenger RNA (mRNA) expression of writers and erasers in the above 13 paired tissues. (D) The mRNA expression of METTL3 in 136 paired gastric carcinoma tissues and adjacent tissues. (E) METTL3 expression in GES-1, pGCC, and cancer cell lines at the protein level. (F) METTL3 protein expression in four paired tumor tissues and adjacent tissues. *p < 0.05, **p < 0.01, ***p < 0.001. ns, non-significant.
Figure 2
Figure 2
Immunohistochemistry (IHC) scores of methyltransferase-like 3 (METTL3) in different types of tissues and Kaplan–Meier survival curves in gastric cancer (GC) patients. (A) IHC scores of METTL3 in four different types of tissues from 196 patients with GC (paratumor, mean ± SD = 70.74 ± 46.29; primary tumor, mean ± SD = 105.60 ± 56.16; lymph node metastasis, mean ± SD = 130.80 ± 55.31; distant metastasis: mean ± SD = 162.6 ± 50.19). (B) Immunohistochemical staining of METTL3 in four different types of tissues from the same patient. (C) Overall survival analysis in all GC patients (n = 196). (D) Disease-free survival analysis in GC patients who were first diagnosed with tumor–node–metastasis (TNM) stage I, II, or III (n = 156). ***p < 0.001.
Figure 3
Figure 3
The effects of methyltransferase-like 3 (METTL3) knockdown on cell function in vitro and in vivo. (A) METTL3 knockdown efficiency and m6A levels of total RNAs in SGC7901 and AGS. (B) Cell proliferation assays after METTL3 knockdown in SGC7901 and AGS cells. (C) Colony formation assays of METTL3 knockdown in SGC7901 and AGS cells. (D) Transwell migration and invasion assays after METTL3 knockdown in SGC7901 and AGS. (E) The nude mice xenograft models (N = 5) of METTL3 knockdown SGC7901 cells. ***p < 0.001. ns, non-significant. All experiments were repeated three times. Scale bars = 50 μm.
Figure 4
Figure 4
Methyltransferase-like 3 (METTL3) overexpression enhances migration and invasion in vitro and promotes proliferation in vivo. (A) METTL3 overexpression efficiency and m6A levels of total RNA in SGC7901 and AGS. (B) Cell proliferation assays of METTL3-overexpressing SGC7901 and AGS cells. (C) Colony formation assays of METTL3 overexpressing SGC7901 and AGS cells. (D) Transwell migration and invasion assays of METTL3-overexpressing SGC7901 and AGS cells. (E) The nude mice xenograft models (N = 5) of METTL3 overexpressing SGC7901 cells. *p < 0.05, **p < 0.01, ***p < 0.001. ns, non-significant. All experiments were repeated three times. Scale bars = 50 μm.
Figure 5
Figure 5
Identification of the potential m6A-dependent targets of methyltransferase-like 3 (METTL3) in GC. (A) Identification of m6A peaks by two algorithms. The layers from outer to inner represent the m6A peaks identified by exomePeak, masc2, and the overlap from both algorithms. (B) The density distribution of m6A peaks in messenger RNA (mRNA) transcripts. (C) Distribution of the m6A peaks in exonic or intronic regions of protein-coding and non-coding genes and in other regions. The consensus motif from all identified m6A peaks. (D) Gene set enrichment analysis determined by both RNA-seq and m6A-seq selected MYC pathway as downstream of METTL3. (E) Relative m6A levels of MCM5 and MCM6 upon depletion of METTL3. (F) The protein levels of MCM5, MCM6, and MYC upon depletion of METTL3. *p < 0.05, **p < 0.01, ***p < 0.001. ns, non-significant.

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