In human osteosarcoma 3AB-OS cancer stem cells let-7d seems to have the dual function of oncogene/tumor-suppressor miRNA

Introduction: Osteosarcoma (OS), an aggressive tumor a!ecting adolescents, shows therapy resistance and recurrence which can depend on cancer stem cells (CSCs), the source for tissue renewal and hold malignant potential; thus, OS treatment requires their eradication. Here, using 3AB-OS CSCs previously obtained from the OS-MG63 cells, we focused on the role of let-7d in managing stemness properties of 3AB-OS CSCs. Methods: 3AB-OS CSCs were stably transfected with pCDomH-plasmid, containing mir-let7-d, or empty vector as a control. Cell proliferation and viability were evaluated by EdU and Trypan blue assays, respectively. Sarcosphere and colony assays and wound healing and transwell invasion assays were performed to evaluate let-7d e!ects on self-renewal and migratory/invasive abilities of 3AB-OS cells, respectively. Publicly available databases were used to identify let-7d predicted target genes. RT-PCR and western blot analyses were employed to validate the targets of let-7d. Results: We have found that let-7d-overexpression reduces cell proliferation, lowers CCND2 and E2F2, increases p21 and p27. Let-7d also decreases sarcosphere- and colony-forming ability and decreases Oct3/4, Sox2, Nanog, Lin28B and HMGA2. Moreover, let-7d induces mesenchymal-to-epithelium-transition, as shown by both N-Cadherin-E-cadherin-switch and vimentin decrease. "is switch is accompanied by enhanced migratory/invasive capacities and by MMP9, CXCR4 and VersicanV1 increases. Resistance to serum starvation and chemotherapy, decrease in caspase-3 and increase in Bcl-2 were also observed. Conclusions: "is study shows that let-7d displays both suppressor and oncogenic functions, suggesting that, before prospecting new therapeutic strategies by let-7d use, it is urgent to better understand its functions.