Circ_0049447 Acts as a Tumor Suppressor in Gastric Cancer

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Circ_0049447 Acts as a Tumor Suppressor in Gastric Cancer Through Reducing Proliferation, Migration, Invasion, and Epithelial-Mesenchymal Transition

Gastric cancer (GC) remains a leading cause of cancer-related mortality globally, with late-stage diagnoses contributing to poor prognoses. Emerging studies highlight the role of circular RNAs (circRNAs) in cancer biology, yet their functions in GC remain underexplored. This study identifies circ_0049447 as a novel tumor suppressor in GC, demonstrating its ability to inhibit proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) through molecular and functional analyses.

Expression and Diagnostic Value of circ_0049447 in Gastric Cancer

The study analyzed 80 paired GC tissues and adjacent non-tumorous tissues to evaluate circ_0049447 expression. Reverse transcription-polymerase chain reaction (RT-PCR) revealed significant downregulation of circ_0049447 in GC tissues (P < 0.001), with median expression levels of −15.25 ± 1.05 in tumors compared to −13.50 ± 1.47 in normal tissues (Figure 1C). Notably, 83.75% (67/80) of GC samples exhibited low circ_0049447 expression (Figure 1B).

Diagnostic potential was assessed using receiver operating characteristic (ROC) curves, yielding an area under the curve (AUC) of 0.838, with 82.3% sensitivity and 77.2% specificity (Figure 1D). This positions circ_0049447 as a promising diagnostic biomarker. However, Kaplan-Meier survival analysis showed no significant correlation between circ_0049447 expression and overall survival (P = 0.125), suggesting its role may be more pronounced in early disease stages.

Clinicopathological analysis linked low circ_0049447 levels to advanced pN stage (lymph node metastasis; P = 0.029), though no associations were found with age, tumor size, histological grade, or TNM stage (Table 1).

Functional Role of circ_0049447 in Gastric Cancer Cells

To investigate circ_0049447’s biological effects, gain-of-function experiments were performed in GC cell lines (AGS, HGC-27, MGC-803) using pcDNA3.1-circ_0049447 overexpression vectors.

  1. Proliferation Assays

    • CCK-8 assays demonstrated that circ_0049447 overexpression suppressed cell proliferation at 24, 48, 72, and 96 hours post-transfection (P < 0.05 for all time points; Figure 2A).
    • Colony formation assays showed a 40–60% reduction in colony numbers across all cell lines (P < 0.05; Figure 2B, 2C).

  2. Migration and Invasion

    • Transwell migration assays revealed a 50–70% decrease in migrated cells in circ_0049447-overexpressing groups (P < 0.05; Figure 3A, 3B).
    • Matrigel invasion assays similarly showed a 45–65% reduction in invasive capacity (P < 0.05; Figure 3C, 3D).

  3. Epithelial-Mesenchymal Transition (EMT)
    Western blotting demonstrated that circ_0049447 upregulation:

    • Increased epithelial marker E-cadherin.
    • Decreased mesenchymal markers N-cadherin, Twist, Vimentin, and β-catenin (Figure 3E).

These findings confirm circ_0049447’s role in suppressing EMT, a critical driver of metastasis.

Molecular Mechanism: circ_0049447 as a miRNA Sponge

circRNAs often function as competing endogenous RNAs (ceRNAs) by binding miRNAs. Bioinformatics tools (Circinteractome, circBank, RegRNA) predicted six miRNAs with binding sites on circ_0049447, including miR-324-5p, which is implicated in cancer progression (Figure 4A, 4B).

  1. Validation of miR-324-5p Interaction

    • Luciferase reporter assays confirmed direct binding between circ_0049447 and miR-324-5p. Co-transfection of miR-324-5p mimics with wild-type (WT) circ_0049447 reduced luciferase activity by 60% (P < 0.01), while mutant (MUT) constructs showed no effect (Figure 4D).
    • miR-324-5p was upregulated in 30 GC tissues compared to normal controls (P < 0.001; Figure 4E).

  2. Downstream Targets
    circ_0049447 overexpression in MGC-803 cells:

    • Upregulated PTPRD (a miR-324-5p target), a tumor suppressor involved in migration inhibition.
    • No change in FBXO11, another miR-324-5p target (Figure 4F).

This suggests circ_0049447 inhibits GC progression partly by sequestering miR-324-5p, thereby restoring PTPRD expression (Figure 4G).

In Vivo Validation of circ_0049447’s Tumor-Suppressive Effects

A xenograft mouse model using MGC-803 cells stably overexpressing circ_0049447 was established:

  • Tumor volume was reduced by 50% in the circ_0049447 group compared to controls (P < 0.05; Figure 5C).
  • Tumor weight decreased by 45% (P < 0.05; Figure 5D).
  • Immunohistochemistry showed downregulation of β-catenin and Wnt1, key Wnt/β-catenin pathway components (Figure 5E).

These results validate circ_0049447’s ability to suppress tumor growth in vivo, likely via Wnt signaling modulation.

Clinical Implications and Future Directions

This study establishes circ_0049447 as a diagnostic biomarker and therapeutic target in GC. Its downregulation correlates with advanced lymph node metastasis, and functional assays confirm its role in inhibiting proliferation, migration, invasion, and EMT. The ceRNA mechanism involving miR-324-5p/PTPRD provides a molecular basis for its tumor-suppressive effects.

Limitations and Future Work:

  1. The lack of survival correlation suggests circ_0049447 may be more relevant in early-stage GC.
  2. Further studies should explore additional miRNA interactions and downstream pathways.
  3. Preclinical models testing circ_0049447 delivery (e.g., nanoparticle-based systems) could advance therapeutic applications.

Conclusion

circ_0049447 emerges as a critical regulator of GC progression, functioning through miRNA sponging and EMT inhibition. Its diagnostic potential and tumor-suppressive properties highlight its translational relevance, offering new avenues for GC management.

DOI: https://doi.org/10.1097/CM9.0000000000001494

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