miRNA-376a/c-TGF-α通路调控骨肉瘤干细胞增殖和转移的实验研究

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. They are associated with poor prognosis and the overall survival rate for patients with advanced disease remains low. A better understanding of osteosarcoma biology and pathogenesis isneeded to advance the development of targeted therapies for osteosarcoma.We found that the expression of miR-376a/c are down-regulated in CD133 positive osteosarcoma cells through miRNA microarray and validation. It has been proved that miR-376a/c can inhibit the proliferation and metastasis of CD133 positive cells, namely, the osteosarcoma stem cells. The targeted inhibit of TGFA may be involved within this procedure. We firstly harvest CD133 positive osteosarcoma cells by magnetic sorting. The cell sources include human osteosarcoma cell lines (Saos-2, MG-63, U2OS) and cells obtained from primary culture of tumor tissue. Constructing the CD133+ cells differentially expressed the miRNA using the miR-376a/c lentivirus and miR-376a/c Sponge lentivirus. The ability of cell growth, tumor formation and invasiveness will be evaluated. The related genes and proteins are being examined by PCR or WB. This study is being planned to demonstrate that miR-376a/c can regulate TGFA in direct way to suppress the proliferation and metastasis ability of osteosarcoma stem cells, in vitro and in vivo. This study will provide theoretical and experimental basis for the treatment of osteosarcoma using miRNAs or cancer stem cells as targets.

骨肉瘤是最常见的原发恶性骨肿瘤，治疗效果欠佳，预后差。深入研究其成瘤机制并鉴定新治疗靶点非常必要。前期试验发现CD133+骨肉瘤细胞具有肿瘤干细胞的生物学特性，miR-376a/c在CD133+骨肉瘤细胞中低表达，过表达miR-376a/c可抑制CD133+细胞增殖和转移。该过程与miR-376a/c直接调控TGFA相关。本项目拟从多个骨肉瘤细胞系及原代培养的骨肉瘤细胞中分离CD133+细胞，构建pri-miR-376a/c EGFP慢病毒及miR-376a/c Sponge EGFP慢病毒，感染并筛选稳转的CD133+细胞。从离体和在体两个层面比较miR-376a/c对CD133+骨肉瘤干细胞增殖和转移能力的影响，并通过TGFA的回复实验，探讨miR-376a/c靶向抑制TGFA调控骨肉瘤干细胞的分子机制。项目从肿瘤干细胞这全新视角认识miRNA的生物学功能，为骨肉瘤靶向治疗提供理论基础

骨肉瘤是最常见的原发恶性骨肿瘤，治疗效果欠佳，预后差，深入研究其成瘤机制并鉴定新治疗靶点非常必要。经磁珠分选和鉴定CD133+和CD133-骨肉瘤细胞，探讨miR-376a/c在的差异表达并进行功能研究。本项目通过芯片技术、慢病毒包装与感染、裸鼠成瘤等手段，MTT、BrdU、克隆形成、Transwell等功能检测，证实了miR-376a/c通过同时靶向TGFA参与CD133+骨肉瘤细胞的增殖和侵袭能力，明确MiR-376a/c可作为骨肉瘤新的潜在诊断和防止靶点。