Developing microRNA-509-3p as an inhibitor of metastatic progression and cisplatin sensitizer for osteosarcoma



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Osteosarcoma (OS) is the most common primary pediatric malignancy of the bone. Systemic chemotherapy, which benefits patients with localized OS to increase survival to 70%, has had minimal effects on metastatic OS with patient survival rates of < 30%. MicroRNAs (miRNAs) are non-coding RNAs that can target and silence hundreds of genes regulating the majority of biological processes. Here, we report, microRNA-509-3p as a strong tumor suppressor of OS, which inhibited migration of primary OS cell lines, HOS (p53mut/-) and SaOS2 (p53-/-); their metastatic derivatives 143B (p53mut/-) and LM7 (p53-/-), respectively; and the primary cell line U2OS (p53wt/wt). Overexpression of miR-509-3p inhibited proliferation and invasion of HOS and 143B and sensitized cells to cisplatin. miR-509-3p failed to sensitize U2OS (p53wt/wt) to cisplatin treatment. miR-509-3p inhibited migration by directly targeting ARHGAP1 only in HOS cell line while in 143B cell line knockdown of ARHGAP1 alone by siRNA had no effect on migration inhibition. These results are consistent with published findings that the action of microRNAs and their downstream targets are highly cell-type specific. Reverse Phase Protein Array (RPPA) analysis uncovered AXL, as significantly down-regulated upon miR-509-3p overexpression in HOS and 143B cells. Previous studies have shown that knockdown of AXL in 143B cells inhibits migration and invasion. RPPA data also showed significant down-regulation of AXL and ATM, both established to play a role in acquired cisplatin resistance, in miR-509-3p+cisplatin treated cells. We propose that AXL is a critical downstream effector of miR-509-3p-mediated inhibition of migration in 143B and cisplatin sensitization in 143B and HOS. In addition, to develop biocompatible microRNA delivery system, we optimized cysteamine-functionalized gold nanoparticle (cAuNP) based microRNA delivery system earlier developed in our lab. Here we, established optimal cAuNP:miR-509-3p ratio and incubation time for optimal delivery into LM7 OS cells. Lastly, as an out of box approach to treat OS we explored possibility of converting OS to fat by modulating peroxisome proliferator-activated receptor gamma (PPAR), a master regulator of adipogenesis through a combination of miR-130b inhibitor, diabetes drug, and unsaturated fatty acids. We, showed that linoleic acid can induce lipid droplet formation in HOS cells.



Osteosarcoma, MiR-509-3p, Cisplatin, ARHGAP1, AXL