Soft Substrate Promotes Osteosarcoma Cell Self-Renewal, Differentiation, and Drug Resistance Through miR-29b and Its Target Protein Spin 1

Abstract

Stiffening of the extracellular matrix (ECM) is considered a typical remolding of the microenvironment in multistep tumor progression. However, the molecular mechanisms by which the tumor cell responds to the ECM mechanical cues remain elusive. Here, we demonstrated that microRNA-29b (miR-29b) and its downstream signaling play critical regulatory roles that osteosarcoma cells sense the ECM stiffness to maintain the cancer stem cell-like ability. Polyacrylamide gels with a stiffness of 7, 20, and 55 kPa were used to mimic the rigidity of connective tissue, muscle tissue, and bone tissue. It was found that the stemness properties including self-renewal ability, differentiation potential, and drug resistance of osteosarcoma cells were strongly enhanced with reducing substrate stiffness, whereas spreading area, proliferation, and migration were inhibited. Moreover, miR-29 was obviously downregulated in soft substrate-cultured osteosarcoma cells, and the expression of stemness-related transcription factors (Sox2, Nanog, and Oct4) and the sphere formation ability were significantly inhibited by ectopic expression of miR-29b-5p. The soft substrate-induced miR-29 downregulation could increase Spin 1 expression and activate phosphatidylinositol 3-kinase (PI3K)/Akt and Stat3 signaling, which were suppressed by the increase in miR-29b-5p. Taken together, our results elucidated that miR-29 could be a novel mechanical sensor which manipulates osteosarcoma cell stemness. This finding uncovers the fact that the mechanical cue of the cancer niche could take part in the regulation of cancer progression through operating microRNAs and their downstream signaling.