miRNA-296-5p Functions as a Potential Tumor Suppressor in Human Osteosarcoma by Targeting SND1

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miRNA-296-5p Functions as a Potential Tumor Suppressor in Human Osteosarcoma by Targeting SND1

Osteosarcoma (OS) is the most prevalent primary malignant bone tumor, predominantly affecting children and adolescents. Despite advancements in neoadjuvant chemotherapy, the 5-year survival rate for patients with metastatic or locally invasive OS remains alarmingly low at 20%. The high mortality and treatment resistance associated with OS underscore the urgent need to identify novel therapeutic targets and molecular mechanisms driving tumor progression. MicroRNAs (miRNAs) have emerged as critical regulators of tumorigenesis, with growing evidence linking their dysregulation to cancer development. Among these, miRNA-296-5p has been implicated in various cancers, but its role in OS remained unexplored. This study systematically investigates the tumor-suppressive function of miRNA-296-5p in OS and elucidates its mechanism of action through targeting Staphylococcal Nuclease and Tudor Domain Containing 1 (SND1).

Downregulation of miRNA-296-5p in Osteosarcoma

The study first evaluated miRNA-296-5p expression in 45 pairs of human OS tissues and adjacent normal bone tissues using quantitative reverse transcription PCR (qRT-PCR). A significant reduction in miRNA-296-5p levels was observed in tumor tissues compared to controls (control: 1.802 ± 0.313 vs. OS: 0.618 ± 0.235; t = 6.402, P < 0.01). This downregulation was further validated in four OS cell lines (HOS, MG63, U-2OS, Saos-2) relative to normal human osteoblasts (HOB). Among these, Saos-2 and HOS exhibited the lowest miRNA-296-5p expression, making them ideal models for functional studies.

Tumor-Suppressive Effects of miRNA-296-5p Overexpression

To investigate the functional consequences of miRNA-296-5p loss, Saos-2 and HOS cells were transfected with miRNA-296-5p mimics or a negative control (NC). qRT-PCR confirmed successful overexpression, with miRNA-296-5p levels increasing 7.9-fold in HOS (P < 0.01) and 6.2-fold in Saos-2 (P < 0.01). Functional assays revealed profound anti-tumor effects:

  • Proliferation: MTT assays demonstrated that miRNA-296-5p overexpression reduced HOS cell viability by 34.6% (NC: 0.897 ± 0.032 vs. mimic: 0.587 ± 0.369; P < 0.01) and Saos-2 viability by 41.3% (NC: 1.000 ± 0 vs. mimic: 0.587 ± 0.023; P < 0.01) over 72 hours.
  • Migration and Invasion: Wound healing assays showed that miRNA-296-5p mimics reduced the migratory capacity of HOS and Saos-2 cells by approximately 60% compared to controls. Transwell invasion assays further revealed a 55% decrease in invasive potential (P < 0.01).

These findings establish miRNA-296-5p as a potent inhibitor of OS cell proliferation, migration, and invasion.

SND1 as a Direct Target of miRNA-296-5p

Bioinformatic analysis using TargetScan predicted SND1, an oncogenic protein involved in RNA interference and tumor progression, as a potential target of miRNA-296-5p. Experimental validation confirmed this interaction:

  • Inverse Correlation: In 12 OS tissue samples, miRNA-296-5p levels negatively correlated with SND1 mRNA (r = -0.72, P < 0.01).
  • Dual-Luciferase Reporter Assay: Co-transfection of miRNA-296-5p mimics with a luciferase reporter vector containing the wild-type SND1 3′-UTR reduced luciferase activity by 71.7% in HOS (P < 0.01) and 61% in Saos-2 (P < 0.01). Mutations in the miRNA-296-5p binding sites abolished this suppression.
  • Expression Validation: Western blot and qRT-PCR analyses confirmed that miRNA-296-5p mimics reduced SND1 protein and mRNA levels by 65% and 58%, respectively, in transfected cells (P < 0.01).

Oncogenic Role of SND1 in Osteosarcoma

The study further characterized SND1’s role in OS pathogenesis:

  • Elevated Expression: SND1 protein and mRNA levels were significantly higher in OS tissues and cell lines compared to normal controls. Immunohistochemistry (IHC) of clinical samples revealed intense SND1 staining in tumor cells, contrasting with minimal expression in adjacent normal bone.
  • Functional Knockdown: Short hairpin RNA (shRNA)-mediated SND1 silencing in HOS and Saos-2 cells reduced proliferation by 50% (P < 0.01), migration by 55%, and invasion by 60% (P < 0.01), mirroring the effects of miRNA-296-5p overexpression.

These results position SND1 as a critical driver of OS malignancy.

Rescue Experiments Confirm miRNA-296-5p/SND1 Axis

To confirm that miRNA-296-5p exerts its effects via SND1, rescue experiments were performed. HOS cells co-transfected with miRNA-296-5p mimics and a SND1-overexpressing plasmid (lacking the 3′-UTR) showed:

  • Restored SND1 Levels: qRT-PCR and Western blot confirmed that exogenous SND1 expression bypassed miRNA-296-5p-mediated repression (miRNA-296-5p: 0.294 ± 0.159 vs. miRNA-296-5p + SND1: 2.300 ± 0.277; P = 0.003).
  • Reversal of Anti-Tumor Effects: SND1 overexpression nullified the growth inhibition (MTT assay: 1.8-fold increase; P < 0.01) and restored migratory/invasive capacities to near baseline levels.

This mechanistic link underscores SND1 as the primary mediator of miRNA-296-5p’s tumor-suppressive activity.

Clinical Implications and Future Directions

The study highlights miRNA-296-5p as a promising therapeutic candidate for OS. Its downregulation in tumors correlates with aggressive phenotypes, while restoration of miRNA-296-5p levels suppresses malignancy via SND1 inhibition. Clinically, miRNA-296-5p-based therapies could complement existing treatments to reduce metastasis and chemoresistance. Future studies should validate these findings in vivo and explore delivery mechanisms for miRNA-296-5p mimics in preclinical models. Additionally, investigating SND1’s downstream effectors could uncover novel pathways for intervention.

doi.org/10.1097/CM9.0000000000001400

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