GNAI3 Promotes Aggressive Glioma Phenotypes and Is Associated with TGF-β-Related Mesenchymal Plasticity

Background/Objectives: Guanine nucleotide-binding protein G(i) subunit alpha 3 (GNAI3) participates in G-protein signaling and has been linked to glioma growth, but its contribution to invasive behavior and mesenchymal plasticity remains incompletely defined. This study examined the clinical relevance and functional effects of GNAI3 in glioma and evaluated its relationship with transforming growth factor-β (TGF-β)-associated mesenchymal changes. Methods:GNAI3 expression and clinical associations were analyzed using The Cancer Genome Atlas (TCGA)/Genotype-Tissue Expression (GTEx), Chinese Glioma Genome Atlas (CGGA), and Gene Expression Omnibus (GEO) datasets and were validated in five paired glioblastoma and peritumoral non-neoplastic tissues. Stable GNAI3 knockdown and overexpression models were established in U87-MG and U251-MG cells. Cell viability, migration, and invasion were evaluated using Cell Counting Kit-8 (CCK-8), Transwell, and wound-healing assays. Candidate GNAI3-associated proteins were screened by immunoprecipitation–mass spectrometry. The association between GNAI3 and TGF-β was examined by co-immunoprecipitation, and changes in protein abundance and phosphorylation of SMAD family members 2 and 3 (SMAD2/3) were assessed by Western blotting. The effect of GNAI3 depletion on tumor growth was evaluated in a U87-MG subcutaneous xenograft model. Results:GNAI3 expression was elevated in glioma and increased with World Health Organization (WHO) grade. Higher expression was associated with shorter overall survival in unadjusted analysis, whereas the association was attenuated after adjustment for clinical factors and molecular subtype. GNAI3 depletion reduced glioma-cell viability, migration, and invasion, whereas GNAI3 overexpression produced the opposite phenotype. GNAI3 expression correlated positively with cadherin 2 (CDH2) and vimentin (VIM), and these associations persisted after adjustment for histological grade. Co-immunoprecipitation showed co-enrichment of GNAI3 and TGF-β in protein complexes, and GNAI3 manipulation was accompanied by concordant changes in TGF-β, vimentin, N-cadherin, and zonula occludens-1 (ZO-1). In U251-MG cells, GNAI3 knockdown decreased the phosphorylated SMAD2 (p-SMAD2)/SMAD2/3 and phosphorylated SMAD3 (p-SMAD3)/SMAD2/3 ratios, whereas GNAI3 overexpression increased both ratios. In vivo, GNAI3 knockdown suppressed xenograft growth and reduced Ki67 and TGF-β expression. Conclusions: GNAI3 promotes aggressive glioma phenotypes in vitro and tumor growth in vivo. Its effects are accompanied by TGF-β-related mesenchymal changes, supporting further investigation of the molecular relationship between GNAI3 and TGF-β signaling without establishing pathway dependence from the present data.