METTL1-modulated LSM14A facilitates proliferation and migration in glioblastoma via the stabilization of DDX5

Abstract

Glioblastoma (GBM) is characterized by aggressive growth, invasiveness, and poor prognosis. Elucidating the molecular mechanisms underlying GBM is crucial. This study explores the role of Sm-like protein 14 homolog A (LSM14A) in GBM. Bioinformatics and clinical tissue samples analysis demonstrated that overexpression of LSM14A in GBM correlates with poorer prognosis. CCK8, EdU, colony formation, and transwell assays revealed that LSM14A promotes proliferation, migration, and invasion in GBM in vitro. In vivo mouse xenograft models confirmed the results of the in vitro experiments. The mechanism of LSM14A modulating GBM cell proliferation was investigated using mass spectrometry, co-immunoprecipitation (coIP), protein half-life, and methylated RNA immunoprecipitation (MeRIP) analyses. The findings indicate that during the G1/S phase, LSM14A stabilizes DDX5 in the cytoplasm, regulating CDK4 and P21 levels. Furthermore, METTL1 modulates LSM14A expression via mRNA m⁷G methylation. Altogether, our work highlights the METTL1-LSM14A-DDX5 pathway as a potential therapeutic target in GBM.

Keywords: Bioinformatics; Cancer; Molecular biology.

Graphical abstract

Figure 1

LSM14A expression in gliomas (A–E) mRNA levels of LSM14A from TCGA, CGGA, and GSE16011 datasets categorized by disease, WHO classification, tissue type, IDH mutation, and 1p/19q codeletion. (F–H) Kaplan-Meier survival curves for glioma patients from the TCGA, CGGA, and GSE16011 datasets stratified by LSM14A expression levels. (I) LSM14A protein levels variation between the normal and GBM groups in the PDC000204 dataset. (J) LSM14A mRNA levels in glioma tissues, analyzed via RT-qPCR. (K) Protein levels of LSM14A in glioma samples, ascertained by immunoblotting (“A”: astrocytoma, “O”: oligodendroglioma, “OA”: oligoastrocytoma). The data are represented as the mean ± S.D. of three independent experiments. ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 2

Inhibition of GBM cell proliferation and migration by LSM14A reduction (A) Efficiency of LSM14A silencing at the protein level in U87 and U251 cells, utilize shLSM14A #1 and shLSM14A #3 to construct LSM14A-KD1 and LSM14A-KD2, respectively. (B–D) Proliferation in LSM14A-knockdown U87 and U251 cells, as evaluated by CCK-8 (B), EdU (C), and colony formation assays (D). (E) Flow cytometric analysis of cell cycle distribution in LSM14A-knockdown U87 cells. (F) Transwell migration assay for LSM14A-NC, LSM14A-KD1, and LSM14A-KD2 groups in U87 and U251 cells. (G and H) Proliferation assays (CCK-8 [G] and EdU [H]) for LSM14A-KO U87 and U251 cells. (I) Transwell assay evaluating migration in LSM14A-KO U87 and U251 cells. The data are represented as the mean ± S.D. of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

LSM14A overexpression augments GBM cell proliferation and migration (A) Efficiency of LSM14A overexpression at the protein level in LN229 and U118 cells. (B–D) Cell proliferation in LN229 and U118 cells overexpressing LSM14A was assessed via CCK-8 (B), EdU (C), and colony formation (D) assays. Differences between LSM14A-EV and LSM14A-OE were quantified. (E) The migration capability of LN229 and U118 cells overexpressing LSM14A was evaluated using a Transwell assay. (F) Left: Images showcase tumors from each mice group, right: statistical analysis of the tumor weights from each group revealed a significant difference. The data are represented as the mean ± S.D. of three independent experiments. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 4

LSM14A and DDX5 interaction enhances DDX5 stability (A) Venn diagram displaying LSM14A-interacting proteins identified through mass spectrometry and correlation analyses. (B and C) Correlation analysis of LSM14A and DDX5 protein levels in the PDC000180 (B) and PDC000204 (C) datasets. (D) RT-qPCR analysis of DDX5 mRNA levels in LSM14A-KD1 U87 cells. (E) Immunoblotting analysis highlighting differences in DDX5 protein levels in U87 and U251 cells with LSM14A knockdown and LN229 cells with LSM14A overexpression compared to controls. (F) Immunoblot assessment of LSM14A, P21, and CDK4 protein expression in LSM14A knockdown or knockout U87 and U251 cells and in LSM14A-overexpressing LN229 and U118 cells. (G) Immunofluorescence images depicting LSM14A (green), DDX5 (red), and DAPI-stained nuclei (blue) in cells at different phases of the cell cycle after 20h of starvation (starv.) or regular (CC) culture conditions. (H) CoIP assay to examine endogenous LSM14A and DDX5 interaction in U87, U251, LN229, and U118 cells. Left panel: LSM14A binding to DDX5 detected using an anti-LSM14A antibody; Right panel: DDX5 binding to LSM14A identified using an anti-DDX5 antibody. (I) Immunoblotting analyses of DDX5 protein half-life in LSM14A knockdown U251 cells and LSM14A overexpressing LN229 cells, right: grayscale value statistics.

Figure 5

LSM14A-mediated proliferation and migration through DDX5 (A–C) Proliferation (A and B) and migration (C) in U87 and U251 cells overexpressing DDX5 with LSM14A knockdown. (D) Immunoblot analysis of CDK4 and P21 expression upon DDX5 overexpression following LSM14A knockdown. (E–G) Proliferation (E and F) and migration (G) in U87 and U251 cells overexpressing DDX5 after LSM14A knockdown and quantitatively analyzed. (H) Immunoblot analysis of CDK4 and P21 levels after DDX5 overexpression in LSM14A-knockdown cells. (I–K) Proliferation (I and J) and migration (K) in LN229 and U118 cells with DDX5 knockdown overexpressing LSM14A and quantitatively analyzed. (L) Immunoblot analysis of CDK4 and P21 levels in LN229 cells after DDX5 knockdown and LSM14A overexpression. The data are represented as the mean ± S.D. of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 6

METTL1 regulates LSM14A-induced proliferation and migration, acting upstream of LSM14A (A) RT-qPCR detected LSM14A mRNA levels in U87 cells with METTL1 knockdown. (B) Levels of LSM14A and DDX5 proteins in U87 cells with METTL1 knockdown (left panel) and LN229 cells overexpressing METTL1 were analyzed using immunoblotting. (C) The MeRIP assay detected m⁷G modifications in LSM14A mRNA. RT-qPCR was used to assess the enrichment efficiency of LSM14A mRNA (right panel: DNA gel electrophoresis results). (D–I) Effects of LSM14A expression regulation on cell proliferation (D–G) and migration (H and I) based on METTL1 modulation. (J) The effect of LSM14A expression regulation on DDX5 protein levels in U87 and LN229 cells with METTL1 modulation was determined by immunoblotting. The data are represented as the mean ± S.D. of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 7

Schematic representation of the METTL1-LSM14A-DDX5 axis in mediating GBM cell proliferation and migration