Raw Data for the article: Potential Anti-Metastatic Role of the Novel miR-CT3 in Tumor Angiogenesis and Osteosarcoma Invasion

Osteosarcoma (OS) is the most common primary bone tumor mainly occurring in young adults and derived from primitive bone-forming mesenchyme. OS develops in an intricate tumor microenvironment (TME) where cellular function regulated by microRNAs (miRNAs) may affect communication between OS cells and the surrounding TME. Therefore, miRNAs are considered potential therapeutic targets in cancer and one of the goals of research is to accurately define a specific signature of a miRNAs, which could reflect the phenotype of a particular tumor, such as OS. Through NGS approach, we previously found a specific molecular profile of miRNAs in OS and discovered 8 novel miRNAs. Among these, we deepen our knowledge on the fifth candidate renamed now miR-CT3. MiR-CT3 expression was low in OS cells when compared with human primary osteoblasts and healthy bone. Through TargetScan, VEGF-A was predicted as a potential biological target of miR-CT3 and luciferase assay confirmed it. We showed that enforced expression of miR-CT3 in two OS cell lines, SAOS-2 and MG-63, reduced expression of VEGF-A mRNA and protein, inhibiting tumor angiogenesis. Enforced expression of miR-CT3 also reduced OS cell migration and invasion as confirmed by soft agar colony formation assay. Interestingly, we found that miR-CT3 behaves inducing the activation of p38 MAP kinase pathway and modulating the epithelial-mesenchymal transition (EMT) proteins, in particular reducing Vimentin expression. Overall, our study highlights the novel role of miR-CT3 in regulating tumor angiogenesis and progression in OS cells, linking also to the modulation of EMT proteins.骨肉瘤（Osteosarcoma, OS）是最常见的原发性骨肿瘤，主要发病人群为青年群体，起源于原始成骨间充质。OS所处的肿瘤微环境（tumor microenvironment, TME）结构复杂，其中微小RNA（microRNAs, miRNAs）对细胞功能的调控可影响OS细胞与周围肿瘤微环境之间的信号交流。因此，miRNAs被视为癌症的潜在治疗靶点，当前研究的核心目标之一便是精准界定一类特异性miRNA特征，该特征可反映骨肉瘤等特定肿瘤的表型。本研究团队此前通过下一代测序（next-generation sequencing, NGS）技术，在OS样本中鉴定出特异性miRNA分子谱，并发现了8种新型miRNA。本研究针对其中第五个候选miRNA展开深入探究，该分子现已被命名为miR-CT3。相较于人原代成骨细胞与健康骨组织，miR-CT3在OS细胞中的表达水平显著降低。通过TargetScan软件预测，血管内皮生长因子A（vascular endothelial growth factor A, VEGF-A）被确定为miR-CT3的潜在生物学靶点，该结果经荧光素酶实验得到验证。本研究证实，在SAOS-2与MG-63这两种OS细胞系中过表达miR-CT3，可下调VEGF-A的mRNA与蛋白表达水平，进而抑制肿瘤血管生成。过表达miR-CT3还可降低OS细胞的迁移与侵袭能力，该结论经软琼脂集落形成实验得到确认。值得注意的是，本研究发现miR-CT3可通过激活p38丝裂原活化蛋白激酶通路（p38 MAP kinase pathway），并调控上皮间质转化（epithelial-mesenchymal transition, EMT）相关蛋白的表达，具体表现为下调波形蛋白（Vimentin）的表达水平。综上，本研究揭示了miR-CT3在调控OS细胞的肿瘤血管生成与进展中的全新作用，并将该过程与EMT蛋白的调控建立了关联。