METTL14 inhibits malignant progression of oral squamous cell carcinoma by targeting the autophagy-related gene RB1CC1 in an m6A-IGF2BP2-dependent manner

Abstract

N6-methyladenosine (m6A) plays crucial roles in tumorigenesis and autophagy. However, the underlying mechanisms mediated by m6A and autophagy in the malignant progression of oral squamous cell carcinoma (OSCC) remain unclear. In the present study, we revealed that down-regulated expression of METTL14 was correlated with advanced clinicopathological characteristics and poor prognosis in OSCC. METTL14 knockdown significantly inhibited autophagy and facilitated malignant progression in vitro, and promoted tumor growth and metastasis in vivo. A cell model of rapamycin-induced autophagy was established to identify RB1CC1 as a potential target gene involved in m6A-regulated autophagy in OSCC, through RNA sequencing and methylated RNA immunoprecipitation sequencing (meRIP-seq) analysis. Mechanistically, we confirmed that METTL14 posttranscriptionally enhanced RB1CC1 expression in an m6A-IGF2BP2-dependent manner, thereby affecting autophagy and progression in OSCC, through methylated RNA immunoprecipitation qRT-PCR (meRIP-qPCR), RNA stability assays, mutagenesis assays and dual-luciferase reporter. Collectively, our findings demonstrated that METTL14 serves as an OSCC suppressor by regulating the autophagy-related gene RB1CC1 through m6A modification, which may provide a new insight for the diagnosis and therapy of OSCC.

Keywords: autophagy; epigenetics; methyltransferase-like 14; oncogenesis; oral squamous cell carcinoma.

Figure 1. Decreased METTL14 expression was associated with poor prognosis in OSCC patients

(A) Representative image of IHC staining in OSCC tissues and ANCT, and statistical analysis of METTL14 IHC staining scores (bar = 100 µm). (B) Statistical analysis of METTL14 IHC staining scores in OSCC tissues stratified by advanced tumor stage, differentiation, and lymph node metastasis. (C) Kaplan–Meier OS analysis based on METTL14 expression in OSCC patients. P-values are indicated in the figure.

Figure 2. METTL14 expression affected autophagic flux and migration, invasion, and proliferation of OSCC cells

(A) CAL33 and HSC3 cells were transfected with mRFP-GFP-LC3 adenovirus vector and treated with shMETTL14, oeMETTL14. The quantification of red dots (autolysosomes) and yellow dots (autophagosomes) per cell was calculated (bars = 40 μm). (B) TEM detected the change of autophagosome (yellow arrow) and autolysosomes (red arrow) in CAL33 and HSC3 cells with METTL14 knockdown or overexpression, respectively (bar = 1 μm). (C,D) CCK-8 (C) and colony formation assay (D) were performed to measure the proliferation of cells transfected with shMETTL14. (E,F) Transwell invasion assay (E) and migration assay (F) were performed to detect the invasive and migratory abilities of cells transfected with shMETTL14 (bar = 100 μm). P-values are indicated in the figure, ns non-significant.

Figure 3. METTL14 recognized m6A residues on the RB1CC1 mRNA and enhanced its stability and affected autophagy in CAL33 and HSC3 cells

(A) m6A peaks were enriched in the exon 15 of RB1CC1 mRNA. Squares showed collective increases in m6A peaks in autophagy-induced OSCC cells. (B) METTL14 knockdown decreased the level of RB1CC1 mRNA. (C) Western blot analysis of RB1CC1 and LC3 in control, shMETTL4 CAL33 and HSC3 cells. (D) RNA stability assay showed the RB1CC1 mRNA half-life (t_1/2) in shMETTL14 and control cells. (E) MeRIP-qPCR analysis confirmed that METTL14 knockdown depleted the m6A modification of RB1CC1 mRNA. (F) Relative luciferase activities of CAL33 and HSC3 cell co-transfected wild-type or mutant RB1CC1 luciferase reporter and shMETTL14. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. P-values are indicated in the figure, ns non-significant.

Figure 4. METTL14 modulated RB1CC1 expression in IGF2BP2-mediated manner

(A) Heatmap based to RNA-seq data revealed the IGF2BP2 up-regulation in both autophagy-induced OSCC cells. (B) IGF2BP2 knockdown down-regulated the mRNA level of RB1CC1. (C) Relative luciferase activities of cells co-transfected with wild-type or mutant RB1CC1 luciferase reporter and siIGF2BP2. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (D) METTL14 overexpression resulted in the RB1CC1 up-regulation, while the up-regulation could be partially reversed in IGF2BP2 silencing. P-values are indicated in the figure, ns non-significant.

Figure 5. RB1CC1 regulation had impact on autophagic flux and proliferation, migration, invasion in OSCC cells.

(A) RB1CC1 silencing decreased the LC3B-II/I ratio in CAL33 and HSC3 cells. (B) RB1CC1 silencing resulted in decreased LC3-II/I ratio, whereas METTL14 overexpression partially rescued the down-regulation of RB1CC1 and the reduction of LC3B-II/I ratio. (C) HSC3 and CAL33 cells were transfected with mRFR-GFP-LC3 vector and treated with oeMETTL14, siRB1CC1#3 for 48 h. The changes were observed using a confocal microscope (bars = 40 μm). (D) TEM analysis of autophagosomes (yellow arrow) and autolysosomes (red arrow) in CAL33 and HSC3 cells with RB1CC1 overexpression or knockdown, respectively (bars = 1 μm). (E,F) CCK-8 (E) and colony formation assays (F) revealed that RB1CC1 silencing significantly promoted the proliferation of OSCC cells. (G,H) Transwell assays showed the accelerating invasion (G) and migration (H) in HSC3 and CAL33 cells, followed RB1CC1 silencing (bar = 100 μm). P-values are indicated in the figure.

Figure 6. METTL14 knockdown promoted OSCC growth and metastasis in vivo

(A) Xenograft tumor formed by shMETTL14 or shNC OSCC cells in nude mice. (B) Quantitative analysis of xenografts tumors volume. (C) Representative images of IHC-stained cervical lymph node and statistical results of lymph node metastasis in METTL14 knockdown (bar = 300 μm). (D) Representative images of IHC staining in xenograft tumors and statistical results of RB1CC1 protein abundance in shMETTL14 and shNC groups (bar = 200 μm). P-values are indicated in the figure, ns non-significant.