The Small Non-coding RNA miR-16-1-3p Hampers Cancer Stem Cell Self-renewal and Invasiveness, Boosting Chemosensitivity by Adjusting TGF-β1 Signaling via MDM2/p53 Axis in Human Osteosarcoma

The TGF-β signaling pathway has both tumor-suppressing and metastasis-promoting effects in cancer. However, the molecular determinants governing this switch remain unclear. Here, we explored the miR-16-1-3p/MDM2/p53 axis as a critical conductor of the TGF-β-Smad pathway in osteosarcoma.

Although miR-16-1-3p overexpression by itself markedly reduces proliferative and clonogenic potential of U2OS cells, when paired with TGF-β treatment, it significantly increase arrest cells in G1 phase and nearly extinguishing the growth capability of these cells. MiR-16-1-3p overexpression inhibited TGF-induced actin remodeling and EMT featuring, significantly decreasing vimentin levels. TGF-β enhances both 2D and 3D migration, but miR-16-1-3p overexpression, alone or with TGF-β, strongly counteracts its pro-migratory effects. MiR-16-1-3p restored p53 stability by targeting MDM2, redirecting TGF-β-Smad signaling toward p21 activation and proliferation inhibition while attenuating its EMT-promoting capacity. Administration of TGF-β together with miR-16-1-3p dramatically increases the sensitivity of wild-type U2OS cells to cisplatin, exceeding that of TGF-β therapy alone by more than an order of magnitude. Administering TGF-β and miR-16-1-3p together significantly reduces the tumor nodule volume and Ki67 expression, while effectively eradicates metastases in the chicken chorioallantoic membrane (CAM) in vivo model. Analysis of the TARGET-OS cohort revealed that high TGFB1 and low MDM2 expression correlated with improved survival.

For the first time, our research demonstrates that miR-16-1-3p shifts TGF-β1 signaling from a facilitator of metastasis to a promoter of anti-growth effects through MDM2 inhibition and p53 stabilization, effectively reducing the self-renewal and invasiveness of cancer stem cells in human osteosarcoma model. This process preserves TGF-β’s tumor-suppressive role while limiting its associated cancer risks.