miR-532-3p Inhibits the Progression of Tongue Squamous Cell Carcinoma by Targeting Podoplanin

Tongue squamous cell carcinoma (TSCC) is a prevalent malignancy within head and neck cancers, characterized by high rates of recurrence, metastasis, and poor survival outcomes. Despite advances in treatment modalities such as surgery, chemotherapy, and radiation therapy, the prognosis for TSCC patients remains suboptimal. Recent research has focused on identifying molecular mechanisms driving TSCC progression to uncover novel therapeutic targets. This study investigates the role of the miR-532-3p/podoplanin (PDPN) axis in TSCC development, revealing critical insights into its regulatory functions and potential clinical implications.

PDPN Is Upregulated in TSCC Tissues and Cell Lines

Bioinformatics analysis of the GEO dataset GSE34105 identified PDPN as a key gene overexpressed in TSCC. Among 414 upregulated genes associated with cell adhesion, proliferation, and migration, PDPN was prioritized due to its established oncogenic roles in other cancers. Validation experiments confirmed PDPN’s elevated expression in TSCC tissues (3.95 ± 1.94 vs. 1.00 ± 0.34 in adjacent normal tissues; P < 0.001) and cell lines (CAL-27, CTSC-3, and SCC-25) compared to normal oral epithelial cells (HOEC). Western blotting further demonstrated increased PDPN protein levels in TSCC cells (CAL-27: 1.86 ± 0.03; CTSC-3: 1.99 ± 0.05; SCC-25: 1.61 ± 0.03 vs. HOEC: 1.00 ± 0.01; P < 0.001).

Clinicopathological analysis revealed significant correlations between high PDPN expression and advanced TSCC features, including lymph node metastasis (P = 0.001), TNM stage (P = 0.010), and tumor grading (P = 0.010). These findings position PDPN as a biomarker of aggressive disease and a potential therapeutic target.

PDPN Knockdown Suppresses TSCC Cell Proliferation, Adhesion, and Migration

To assess PDPN’s functional role, siRNA-mediated knockdown was performed in CAL-27 and CTSC-3 cells. Transfection with si-PDPN reduced PDPN mRNA levels by 70% in CAL-27 (0.22 ± 0.02 vs. 1.00 ± 0.05; P < 0.001) and 60% in CTSC-3 cells (0.31 ± 0.01 vs. 1.01 ± 0.06; P < 0.001), with corresponding decreases in protein expression. Functional assays demonstrated that PDPN silencing significantly impaired TSCC cell viability (CAL-27: 0.85 ± 0.05 vs. 2.07 ± 0.05 at 72 hours; CTSC-3: 1.02 ± 0.07 vs. 2.13 ± 0.04; P < 0.001), adhesion (CAL-27: 60.60 ± 2.79% vs. 100.00 ± 3.97%; CTSC-3: 70.10 ± 4.54% vs. 100.00 ± 2.52%; P < 0.001), and migration (CAL-27: 46.50 ± 1.18% vs. 77.70 ± 4.24%; CTSC-3: 39.50 ± 2.14% vs. 72.20 ± 3.75%; P < 0.001). These results underscore PDPN’s critical role in promoting TSCC progression.

miR-532-3p Directly Targets PDPN in TSCC

Bioinformatic screening using TargetScan and starBase identified miR-532-3p as a potential regulator of PDPN, with complementary binding sites in PDPN’s 3′ untranslated region (UTR). Dual-luciferase reporter assays confirmed this interaction: co-transfection of miR-532-3p mimics with wild-type PDPN 3’UTR reduced luciferase activity by 63% in CAL-27 (0.37 ± 0.05 vs. 1.00 ± 0.02; P < 0.001) and 55% in CTSC-3 cells (0.45 ± 0.02 vs. 1.00 ± 0.02; P < 0.001), while mutant constructs showed no significant changes.

miR-532-3p expression was markedly downregulated in TSCC tissues (0.36 ± 0.13 vs. 1.00 ± 0.43 in normal tissues; P < 0.001) and cell lines (CAL-27: 0.28 ± 0.03; CTSC-3: 0.17 ± 0.02; SCC-25: 0.35 ± 0.04 vs. HOEC: 1.00 ± 0.12; P < 0.001). A strong inverse correlation between miR-532-3p and PDPN expression was observed in clinical samples (R² = 0.66; P < 0.001). Transfection with miR-532-3p mimics reduced PDPN protein levels by 79% in CAL-27 (0.21 ± 0.03 vs. 1.00 ± 0.02; P < 0.001) and 24% in CTSC-3 cells (0.76 ± 0.02 vs. 1.00 ± 0.06; P < 0.001), whereas miR-532-3p inhibitors increased PDPN expression.

miR-532-3p Inhibitor Reverses the Tumor-Suppressive Effects of PDPN Silencing

Rescue experiments evaluated the interplay between miR-532-3p and PDPN. Co-transfection of si-PDPN with miR-532-3p inhibitors restored TSCC cell viability (CAL-27: 1.78 ± 0.07 vs. si-PDPN alone: 1.02 ± 0.07; CTSC-3: 1.91 ± 0.08 vs. 1.13 ± 0.06; P < 0.001), adhesion (CAL-27: 81.30 ± 2.21% vs. 61.36 ± 3.33%; CTSC-3: 88.10 ± 3.80% vs. 72.23 ± 1.78%; P < 0.001), and migration (CAL-27: 70.60 ± 2.13% vs. 62.70 ± 3.29%; CTSC-3: 61.30 ± 4.29% vs. 53.40 ± 2.68%; P < 0.001). These findings confirm that miR-532-3p exerts tumor-suppressive effects by directly targeting PDPN.

Discussion

This study elucidates the miR-532-3p/PDPN axis as a critical regulator of TSCC progression. PDPN’s oncogenic role aligns with prior studies linking its overexpression to metastasis and poor prognosis in gastric, lung, and oral cancers. The inverse relationship between miR-532-3p and PDPN highlights miRNA-mediated post-transcriptional regulation as a key mechanism in TSCC. miR-532-3p’s tumor-suppressive function is consistent with its reported roles in ovarian and colorectal cancers, though contrasting roles in other malignancies underscore context-dependent miRNA activity.

The clinical relevance of these findings is underscored by correlations between PDPN/miR-532-3p expression and advanced TSCC features, suggesting their utility as prognostic biomarkers. However, limitations include the absence of in vivo validation and mechanistic insights into downstream signaling pathways. Future studies should explore PDPN’s interaction with pathways such as EMT or Wnt/β-catenin to fully delineate its oncogenic mechanisms.

Conclusion

This study demonstrates that miR-532-3p inhibits TSCC progression by targeting PDPN, thereby suppressing cell proliferation, adhesion, and migration. The miR-532-3p/PDPN axis represents a promising therapeutic target, offering potential strategies for improving TSCC prognosis and treatment efficacy.

doi:10.1097/CM9.0000000000001563

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