开启细胞周期调控、基因转录等多功能的激酶被认为是肿瘤治疗的关键靶点。我们前期蛋白激酶文库筛查发现Mirk激酶在骨肉瘤生长、存活及预后方面起着关键作用，但其作用机制未明。新近研究发现Hedgehog（Hh）高表达能促进成骨细胞向骨肉瘤转化；而在与骨肉瘤发病机制类似的其他肿瘤中，Mirk被证实在开启Hh由细胞内到间质旁分泌这一过程中起着决定性作用；我们前期研究显示Mirk和Hh信号通路在骨肉瘤细胞系呈一致的持续高表达。因此我们假设：Mirk可能激活成骨细胞的Hh信号，并旁分泌到细胞间质，促进骨肉瘤形成及转移。本研究拟通过骨肉瘤细胞或成骨细胞CRIPSR基因编辑技术敲除或敲入Mirk基因，双向验证Mirk与Hh信号关联；并在骨肉瘤病理标本中验证它们与临床预后的关系，阐明Mirk-Hh通路介导骨肉瘤发生发展的机制。本研究从发病机制的初始关键环节解析骨肉瘤，将为骨肉瘤的防治开辟新的干预靶点和途径。

The protein kinase plays important roles in cellular function such as modulating cell-cycle、gene transcription、metabolism and may serve as a therapeutic target in tumor. The previous protein kinase library screening reveals that Mirk plays a key role in the growth, survival and prognosis of osteosarcoma. However, the mechanism of Mirk in osteosarcoma is not well understood. It has been demonstrated that High expression of Sonic Hedgehog mediated osteoblast cell conforming to osteosarcoma. Mirk functions as a switch on the Hedgehog intracellular and extracellular paracrine，which contribute to the same tumorigenesis pathway of osteosarcoma and other tumor. We find high-level expression of Mirk correlating well with expression of Hedgehog signaling pathway in osteosarcoma cell lines. Therefore, We hypothesis that Mirk actives the Hedgehog signaling pathway which secrete to extracellular matrix of osteoblast cell by paracrine mechanism, which plays important roles in promoting tumorigenesis and metastasis of osteosarcoma. This study aims to assess the relevance network between Mirk and Hedgehog signaling pathway by application of the CRISPR-Cas9 system to knockout/knock-in Mirk gene in osteosarcoma/osteoblast cell lines. The relationship between Mirk- Hedgehog pathway and the clinical characteristic of patients with osteosarcoma are illuminated. This research will elucidate critical initial factors of the signature in tumorigenesis of osteosarcoma and provide new insights into the targeted therapy for osteosarcoma.

我们在前期研究中发现蛋白激酶Mirk和Gli2在骨肉瘤生长及存活中发挥关键作用，进一步研究发现蛋白激酶Mirk通过旁分泌调控Hh信号，促进骨肉瘤形成、侵袭及转移。我们构建了Mirk/DYRK1B CRISPR激活系统(Mirk/pLV-Cas9Nick-Puro)，将其转染人成骨细胞，使成骨细胞稳定表达Mirk蛋白。Mirk蛋白在成骨细胞高表达后，促进成骨细胞增殖、克隆形成，提示Mirk蛋白可诱导成骨细胞向肿瘤细胞转化。对高表达Mirk蛋白的成骨细胞进行Co-IP实验探索Mirk激酶调控的蛋白，进行质谱分析为Gli2蛋白。我们构建了Gli2-GST融合蛋白载体，提取并纯化外源性Gli2-GST蛋白进行GST pull down实验，进一步证明Gli2蛋白可以结合Mirk蛋白。我们构建Gli2-shRNA慢病毒表达载体，载体转染骨肉瘤细胞，抑制Gli2的表达，我们发现Gli2表达被抑制，骨肉瘤细胞存活减少，调亡明显增加。进行ChIP实验发现，Gli2直接结合到基因 FOLR1启动子调控转录。我们将FOLR1/pLV-Cas9Nick-Puro表达载体转染骨肉瘤细胞U2-OS植入裸鼠皮下，建立骨肉瘤动物模型。应用四环素诱导肿瘤组织内表达咋载体活性，促进肿瘤内FOLR1的表达。我们发现用药4周后，肿瘤较对照组明显增加，说明通过促进肿瘤细胞内蛋白激酶Mirk的表达，促进肿瘤生长。我们的研究显示，FOLR1蛋白作为Gli2下游关键因子，在骨肉瘤的发生及生长中发挥重要作用， FOLR1蛋白在正常成骨细胞中高表达使成骨细胞有向肿瘤 细胞转化的倾向。而通过抑制骨肉瘤细胞内 FOLR1蛋白得表达，可以促进骨肉瘤细胞的凋亡，抑制肿瘤生长。因此转录因子Gli2和FOLR1蛋白可以成为骨肉瘤分子靶向治疗的潜在靶点。