N6-Methyladenosine Reader YTHDF2 Enhances Non-Small-Cell Lung Cancer Cell Proliferation and Metastasis through Mediating circ_SFMBT2 Degradation

Abstract

Objective: circ_SFMBT2 was reported to facilitate malignant progression in various cancers, but its function in non-small-cell lung cancer (NSCLC) has not been fully uncovered. This study aimed to investigate the effects of N6-methyladenosine (m6A) methylation of circ_SFMBT2 (circ_0017628) on non-small-cell lung cancer (NSCLC) and its underlying mechanisms.

Methods: Paired tumor and noncancerous tissues from NSCLC patients were surgically collected from January 2020 to March 2021 in our hospital. The levels of circ_SFMBT2 and LATS2 in NSCLC and human bronchial epithelial cells were assayed with qRT-PCR. Overexpression or silencing of circ_SFMBT2, LATS2, or YTHDF2 was performed in the NSCLC cells. CCK-8, colony-forming, and transwell assays were performed to analyze cell proliferation, viability, and migration, respectively. Meanwhile, the expression of MMP-9, E-cadherin, vimentin, and the Hippo/YAP pathway components was examined by western blotting. The m6A enrichment in circ_SFMBT2 was verified using methylated RNA immunoprecipitation, and interaction between circ_SFMBT2 and YTHDF2 was assessed by RNA pull-down and immunoprecipitation assays.

Results: Both circ_SFMBT2 and LATS2 were lowly expressed in NSCLC cells and tissues. A positive correlation of circ_SFMBT2 with LATS2 was identified, and circ_SFMBT2 was localized predominantly in the cytoplasm. circ_SFMBT2 overexpression negatively regulated cell proliferation, viability, migration, and epithelial-mesenchymal transition while promoting the Hippo/YAP pathway activation. Notably, knockdown of LATS2 effectively abrogated the inhibitory effects of circ_SFMBT2 overexpression on NSCLC cell malignancies. Besides, m6A was specifically enriched in circ_SFMBT2, and circ_SFMBT2 could bind to YTHDF2. Silencing of YTHDF2 led to an increase in circ_SFMBT2 expression while inhibiting the malignancy of cancer cells.

Conclusion: Our results showed that YTHDF2 could facilitate NSCLC cell proliferation and metastasis via the Hippo/YAP pathway activation by mediating circ_SFMBT2 degradation.

Figure 1

circ_SFMBT2 was lowly expressed in NSCLC cells and tissues A and B. Detection of the circ_SFMBT2 level in the paired tumor and normal tissue samples (a) or various NSCLC cell lines as well as HBE cells (b) via qRT-PCR analysis. ^∗∗p < 0.01 versus the normal group; ^∗∗p < 0.05 versus the HBE group. Nuclear or cytoplasmic expression of circ_SFMBT2 in A549 (c) or H1299 cells (d) was analyzed with qRT-PCR. ^∗p < 0.05 versus the nucleus group; ^∗∗p < 0.05 versus the nucleus group. (e) Subcellular localization of circ_SFMBT2 was observed by immunofluorescence (scale = 20 μm).

Figure 2

Regulatory effects of circ_SFMBT2 and LATS2 on the NSCLC cell malignancies A and B. Analysis of the expression level of LATS2 in paired tumor and noncancerous tissues (a) or various NSCLC cells as well as HBE cells (b) via qRT-PCR. ^∗∗p < 0.01 versus the normal group; ^∗∗p < 0.05 versus the HBE group. Determination of circ_SFMBT2 (c) or the LATS2 (d) expression level in the cir_cSFMBT2-overexpressing cells via qRT-PCR. ^∗∗p < 0.01 versus the vector group. (e) Pearson's correlation analysis identified a correlation between circ_SFMBT2 and LATS2 expression in the tissues. (f–i) The cell proliferative activity (f), colony-forming ability (g), and migratory ability (h, i) measured using CCK-8 assay, colony formation assay, and transwell assay, respectively. (j) Western blot analysis of MMP-9, E-cadherin, and vimentin expression in the cells. ^∗∗p < 0.01 versus the vector group, and ^##p < 0.01 versus the circ_SFMBT2 group.

Figure 3

Effects of circ_SFMBT2 in NSCLC cells on the Hippo/YAP pathway. ^∗∗p < 0.01 vs the vector group, and ^##p < 0.01 vs the circ_SFMBT2 group.

Figure 4

YTHDF2 facilitates the degradation of m6A-modified circ_SFMBT2. (a) The m6A sites in circ_SFMBT2 were predicted using the m6A modification site predictor. (b) The m6A methylation in circ_SFMBT2 was verified by MeRIP. ^∗p < 0.01 versus the IgG group. (c) Verification of YTHDF2 knockdown in the cells. (d) Expression analysis of circ_SFMBT2 in YTHDF2 knockdown cells. (e) The binding of circ_SFMBT2 to YTHDF2 was assayed using RNA pull-down experiments. (f) Measurement of circ_SFMBT2 stability in YTHDF2 knockdown cells with qRT-PCR. ^∗p < 0.01 versus the siNC group.

Figure 5

Effects of circ_SFMBT2 and YTHDF2 on NSCLC progression (a–d). The cell proliferation (a), viability (b), and migratory ability (c, d) were determined using CCK-8 assay, colony-forming assay, and transwell assay, respectively. Western blot analysis of the expression levels of MMP-9, E-cadherin, and vimentin (e) or Hippo/YAP pathway components (f) in the cells. ^∗∗p < 0.01 versus the siNC group, and ^##p < 0.01 versus the si-YTHDF2 group.