METTL14-mediated m6A modification of CXCL9 mRNA regulates macrophage CS polarization to suppress OSCC growth and stemness.

چکیده اصلی

Oral squamous cell carcinoma (OSCC), the most prevalent histological subtype of oral cancer, is characterized by an immunosuppressive tumor microenvironment (TME) predominantly modulated by tumor-associated macrophages (TAMs). The CXCL9/SPP1 ratio (CS ratio) has been validated as a more reliable prognostic biomarker than the conventional M1/M2 classification. Although methyltransferase-like 14 (METTL14) participates in TAM polarization, its specific role in regulating the CS balance in OSCC remains elusive. This investigation elucidates the mechanism by which METTL14 modulates TAM CS polarization and affects OSCC growth and stemness. We utilized single-cell RNA sequencing (scRNA-seq) data for OSCC and normal samples from Gene Expression Omnibus (GEO) to characterize TAM CS polarization and screen critical regulatory molecules. To independently validate the expression of key candidate genes, we additionally analyzed three independent bulk transcriptomics cohorts from GEO, comprising 330 patient tissue samples. A METTL14-overexpressing TAM model was constructed to explore the effects of METTL14 on CS polarization and OSCC growth and stemness. The target gene of METTL14-mediated m6A modification was identified using the RM2Target database and validated by cellular experiments. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), an m6A reader protein, mediates the downstream biological effects of this epigenetic modification. Finally, the impact of METTL14-driven TAM CS polarization on OSCC tumor growth and stemness was verified in vitro and in vivo in a macrophage-specific Mettl14 knockout mouse model. scRNA-seq re-analysis uncovered mutually exclusive CXCL9⁺ and SPP1⁺ TAM populations in OSCC. METTL14 was markedly enriched and co-expressed in CXCL9⁺ TAMs, where it showed a negative correlation with cancer stemness pathways. Independent validation in three bulk transcriptomics cohorts (n = 330) confirmed significant differential expression of CXCL9 and METTL14 between OSCC tumor and normal tissues. In vitro, METTL14 overexpression in TAMs promoted CXCL9⁺ TAM generation and suppressed OSCC cell growth and stemness, an effect attributed to METTL14-mediated m6A modification of CXCL9 mRNA, which was recognized and stabilized by the m6A reader protein IGF2BP3. Consistently, in vivo assays using macrophage-specific METTL14-knockout mice revealed that METTL14 knockdown reduced m6A modification levels of CXCL9 mRNA, promoted the polarization of TAMs toward the SPP1⁺ phenotype, and ultimately accelerated OSCC tumor growth and enhanced cancer stemness. Collectively, this study identifies a novel mechanism wherein METTL14 regulates TAM CS polarization by promoting m6A modification of CXCL9 mRNA, thereby driving TAMs towards a CXCL9⁺ phenotype and suppressing OSCC growth and stemness.

ترجمه فارسی چکیده

سرطان اسکوما سلولی دهانی (OSCC)، شایع‌ترین زیرنوع هیستولوژیک سرطان دهان، با محیط تومور ایمونوساپرسیو مشخص می‌شود که عمدتاً توسط ماکروفاژهای مرتبط با تومور (TAMs) تنظیم می‌شود. نسبت CXCL9/SPP1 (CS ratio) به عنوان یک نشانگر پرونوستیک قابل اعتمادتر از طبقه‌بندی سنتی M1/M2 تأیید شده است. این مطالعه مکانیسمی را که در آن METTL14 پولاریزاسیون TAM CS را تنظیم می‌کند و بر رشد و استخوان‌زایی OSCC تأثیر می‌گذارد، elucidates. از داده‌های ریزساختار RNA sequencing (scRNA-seq) برای OSCC و نمونه‌های عادی از Gene Expression Omnibus (GEO) برای توصیف پولاریزاسیون TAM CS و غربالگری مولکول‌های تنظیم‌کننده کلیدی استفاده شد. یک مدل TAM با ابربروز METTL14 برای بررسی اثرات METTL14 بر پولاریزاسیون CS و رشد و استخوان‌زایی OSCC ساخته شد. هدف mRNA CXCL9 توسط METTL14 mediated m6A modification با استفاده از پایگاه داده RM2Target شناسایی و با آزمایش‌های سلولی تأیید شد. IGF2BP3، یک پروتئین خواننده m6A، اثرات زیستی پایین‌دستی این اصلاح اپی‌ژنتیک را mediates. در نهایت، اثر پولاریزاسیون TAM CS driven توسط METTL14 بر رشد تومور و استخوان‌زایی OSCC در vitro و در vivo در یک مدل موش knockout متل14 ماکروفاژ-خاص تأیید شد.

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