Background: G protein subunit alpha 13 (GNA13) plays a role in the progression of many malignant tumors, but its expression in esophageal carcinoma (ESCA) cells and impact on their biological behavior are unknown. This research aims to clarify the functions and mechanisms of GNA13 on the malignant phenotype of ESCA cells. Methods: GNA13 expression in ESCA tissues and cells was assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot. Sh-GNA13 was transfected into ESCA cells (TE-1 and KYSE-150) to assess the efficiency of GNA13 silencing. The effects of GNA13 silencing on cell viability, clone formation rate, invasion, and apoptosis of ESCA cells were investigated via Cell Counting Kit-8, clone formation, Transwell, and flow cytometry. The expression of epithelial-mesenchymal transition (EMT) marker proteins, apoptotic proteins, and beta-catenin pathway proteins was evaluated by Western blot. Results: GNA13 expression in ESCA tissues and cells was significantly higher than in paracancerous tissues and normal human esophageal cells, HET1A (p < 0.05). Silencing GNA13 suppressed the malignant phenotypes of ESCA cells: cell viability at 48 h was decreased by 25.8%-42.2% (p < 0.01), clone formation rate was reduced by 70%-89% (p < 0.05), and invasion capacity was attenuated by 51%-56% (p < 0.05). Meanwhile, GNA13 knockdown inhibited the EMT process and increased the apoptotic rate by 3.0-3.2-fold (p < 0.05). Silencing GNA13 reduced the expression of beta-catenin pathway proteins (p < 0.05), while the beta-catenin pathway activator SKL2001 reversed the tumor-suppressive effects induced by GNA13 silencing (p < 0.05). Conclusion: GNA13 increased ESCA cell invasion and prevented apoptosis by stimulating the beta-catenin pathway. The findings may lead to new approaches for diagnosing and treating ESCA.
Background:The inhibitor of MyoD family (Mdfi) has been characterized as a myogenic repressor that regulates transcription factor activity through cytoplasmic retention; however, its specific function in myoblast proliferation remains poorly understood. This study aimed to elucidate the precise role ofMdfiin regulating myoblast cell cycle progression and proliferation using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing.Methods:We employed the CRISPR/Cas9 system to construct Mdfi-knockout (Mdfi(-/-)) C2C12 cell lines. Cell cycle distribution was analyzed by flow cytometry, proliferation was assessed by EdU incorporation assays, and molecular mechanisms were investigated through quantitative RT-PCR, Western blotting, and RNA sequencing (RNA-Seq). Differentially expressed genes (DEGs) between wild-type (WT) andMdfi-overexpressing (Mdfi-OE) were identified and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.Results:Mdfi overexpression significantly increased the proportion of cells in G1 (p< 0.001) phase while reducing S and G2 phase populations (p< 0.001), accompanied by decreased EdU-positive cells. Conversely,Mdfiknockout promoted cell cycle progression into S phase (p< 0.05) and enhanced proliferation. Mechanistically, Mdfi overexpression downregulated cyclin B1 (Ccnb1) (p< 0.01), cyclin D1 (Ccnd1) (p< 0.05), and proliferating cell nuclear antigen (Pcna) (p< 0.05), while upregulating the cyclin-dependent kinase inhibitor P21 (p< 0.01). RNA-Seq analysis identified 889 DEGs (FDR < 0.05 and |log(2)FC| > 1), with enrichment in cell cycle and calcium signaling pathways.Conclusions:Mdfi functions as a negative regulator of myoblast proliferation by inducing G1 phase arrest, potentially through modulation of cyclin D within the cyclin-CDK-P21 pathway. These findings advance our understanding of the molecular mechanisms governing muscle cell proliferation and identify Mdfi as a potential therapeutic target for muscle regeneration and repair.
