长链非编码RNA DANCR作为ceRNA调控ROCK1介导骨肉瘤侵袭转移作用机制研究

ROCK1 mediates migration and invasion in osteosarcoma. Long non-coding RNA DANCR participates in regulation of osteoblast and precursor cell differentiation but the function and mechanism it plays in osteosarcoma migration and invasion mediated by ROCK1 remains unclear. The proposer found higher DANCR expression in osteosarcoma tissues and cells. The migration and invasion ability was down-regulated while ROCK1 expression was decreased when silence of DANCR in osteosarcoma cells. ROCK1 is one of target genes of miR-335. We theoretically predicted DANCR could bind to miR-335 and this may indicate us DANCR may regulate ROCK1 expression as ceRNA and effect migration and invasion in osteosarcoma. Our project intends to affirm the specific binding effect and the key binding-region between miR-335 and DANCR，investigate the correlation DANCR regulating migration/invasion and the expression of ROCK1 in osteosarcoma and to study the mechanism of DANCR regulating migration/invasion mediated by ROCK1 as ceRNA, while, to testify the effect in naked mice models. Our project aims at laying the further theoretical and experimental foundation of clarifying the developmental mechanism and finding out new target spot to reverse the formation of osteosarcoma.

ROCK1介导骨肉瘤侵袭转移。长链非编码RNA DANCR参与成骨细胞和前体细胞分化的调控，但在ROCK1介导骨肉瘤的侵袭转移中的作用与机制不清。申请者发现DANCR在骨肉瘤组织和细胞中高表达，沉默DANCR后，骨肉瘤细胞侵袭转移能力下降，同时ROCK1表达降低；骨肉瘤细胞中ROCK1是miR-335靶基因之一；理论预测DANCR存在与miR-335结合位点，提示DANCR可能作为ceRNA调控ROCK1表达，影响骨肉瘤的侵袭转移。本项目拟确认DANCR与miR-335特异结合作用及关键位点，考察 DANCR调控骨肉瘤细胞侵袭转移与ROCK1表达的相关性，研究DANCR作为ceRNA 竞争性调控 ROCK1介导的骨肉瘤侵袭和转移，并在裸鼠转移瘤模型上进行验证，为寻找骨肉瘤侵袭转移形成与逆转的新靶点、进一步阐明骨肉瘤发生发展机制奠定理论与实验基础。

越来越多的证据表明包括长链非编码RNA及微小RNA在内的非编码RNA可作为包括骨肉瘤在内的多种肿瘤的关键调控因子。我们前期研究发现ROCK1，一个侵袭转移相关因子，是miR-335-5p下游的靶基因并参与骨肉瘤的发生发展。目前有否长链非编码RNA参与调控miR-335/ROCK1轴未知。.我们通过qRT-PCR法及免疫原位杂交等方法检测了DANCR在骨肉瘤中的表达。我们通过细胞增殖实验及Transwell实验考察骨肉瘤细胞的增殖及侵袭转移能力变化。qRT-PCR及WB实验用于检测ROCK1的表达。我们通过Luciferase实验及RIP实验确认miR-335-5p/miR-1972与ROCK1和DANCR的靶向结合作用。最后我们通过裸鼠移植瘤模型在活体内考察不同DANCR表达水平对骨肉瘤肿瘤生长及肺转移发生的影响。.我们发现DANCR在骨肉瘤组织及细胞系中表达增高，且高表达的DANCR与骨肉瘤病人的不良预后及临床病理参数密切相关。功能学实验证实DANCR能够调控ROCK1的表达及其介导的骨肉瘤细胞侵袭转移。WB及qRT-PCR实验提示DANCR能够通过与miR-335-5p/miR-1972“交谈”的方式调控ROCK1的表达。Luciferase实验及RIP实验的结果证实ROCK1和DANCR共享了相似的miR-335-5p/miR-1972作用原件，miR-335-5p/miR-1972与ROCK1和DANCR的靶向结合。进一步的细胞行为学实验证实DANCR能够通过诱捕miR-335-5p/miR-1972的方式作为ceRNA促进ROCK1的表达并最终促进骨肉瘤增殖及侵袭转移。最后的动物学实验证实，DNACR能够明显促进骨肉瘤的肿瘤发生及肺转移。.我们的研究结果揭示了DANCR/miR-335-5p/miR-1972/ROCK1在骨肉瘤发生发展中的作用，为新的化疗药物的开发以及分子靶向治疗骨肉瘤的开展提供了新的治疗靶点。