## 4 Discussion  
miR-320a was described as a tumor suppressor for osteosarcoma [9; 11] and involved in osteoporotic bone [13], but its effect has never been compared between both cellular contexts. Hence, we overexpressed or inhibited miR-320a in two osteosarcoma cell lines, and the miRNA effects on these two cells lines were compared to that on primary hOBs. We observed an alteration in osteoblast function in all cells tested, involving reduced mineralization and increased oxidative stress. However, while viability and proliferation were impaired in osteosarcoma, no effect was detected in hOB viability and an increased proliferation was even observed. Interestingly, this miRNA is significantly downregulated in osteosarcoma tissues compared to in noncancerous tissues [9], and its overexpression significantly inhibited cell proliferation in U2 OS [16]. In cervical cancer, miR-320a has also been found to be decreased and, similar to our study, to inhibit cell proliferation and to induce cell apoptosis [18]. In our study, this miRNA, at the tested doses, appeared to reduce the viability of osteosarcoma cells without affecting primary osteoblastic cells, suggesting that tumorigenic cells may be more sensitive to miR-320a toxicity. On the contrary, the overexpression of miR-320a in hOBs led to increased proliferation but a decreased mineralization capability and ALP activity, consistent with a loss of osteoblastic differentiation. It is in accordance with the increased levels found in fragility fracture [2], where high miR-320a levels may be involved in the osteoporotic phenotype. All these evidences suggested a different effect of miR-320a on tumorigenic cells compared to that on primary cells, where on one hand the viability and proliferation is reduced and on the other hand cells undergo reduced functionality. It is noteworthy that these osteosarcoma cells, which survived after miR-320a transfection, also showed an altered functioning in terms of osteoblastic performance. All these results strongly fit with an involvement of the TGF-beta signaling pathway predicted by DIANA-miRPath using miR-320a target genes with experimental support. The crucial role of TGF-beta signaling pathway in osteoblast proliferation, differentiation, and function is well-known [19]. In fact, TGF-beta/BMP signaling was proposed as an altered pathway in osteosarcoma cells, according to [PERSON] et al. (2016) [20]. In that study, they analyzed transcriptional profiles of osteosarcoma, including two osteosarcoma biopsy specimens, two cell lines, and two xenografts derived from patient specimens-one from normal osteoblasts and one from mesenchymal stem cells. They concluded that alterations in the signaling axes of IGF-1 and TGF-beta, in concert with cell cycle regulators, could be involved in the pathogenic basis of osteosarcoma. However, of the 13 miR-320a target genes predicted in the TGF-beta pathway, only SMAD5 was validated in an RNA microarray analysis [13] after miR-320a mimic or inhibitor transfection (Supplementary Table S2). On the other hand, several genes predicted in cancer pathways have been validated in this microarray (Supplementary Table S3). Since cellular oxidative stress can affect cell viability and function, we measured ROS in hOBs and osteosarcoma cells. The results showed that the overexpression of miR-320a produced increased ROS levels whereas miR-320a inhibition reduced ROS levels. In primary cells, with a normal functioning, minimal ROS levels were observed, since they were processed by the cellular antioxidant defense system. The increased ROS levels due to miR-320a overexpression could be involved in part in the altered functionality [21] provoking cell proliferation increase [22]. The elevated ROS levels of osteosarcoma cells, compared to those of untransformed cells, may reflect mitochondrial dysfunction and an accelerated metabolism [23]. However, cancer cells are more sensitive than untransformed cells to an acute increase in ROS levels.