靶向沉默蛋白激酶CDK11治疗耐药骨肉瘤的机制研究

Osteosarcoma is the most common malignancy of bone in adolescents. After the treatment of chemotherapeutic drugs, 1/3 of patients with osteosarcoma eventually have local recurrence or distant metastasis. Our preliminary data demonstrated that many protein kinases were aberrantly activated in osteosarcoma and knockdown of specific kinases could inhibit cell proliferation and induce apoptosis. Our group first reported that the protein kinase CDK11 played an important role in cell growth and survival in osteosarcoma cell lines. Nevertheless, that targeting protein kinases CDK11 reverse multidrug resistance in osteosarcoma via specific signaling pathway was not investigated. In this study, after transfection of multidrug resistant osteosarcoma cell lines KHOS R2 and U2OS MR with CDK11 siRNA, we examined cellular biological characteristics (cell growth, cell proliferation and cell apoptosis), the expression levels of Hh-GLI signaling pathway and apoptosis related genes to identify that inhibition of CDK11 lead to impaired cell proliferation and apoptosis due to deactivation of Hh-SMO-GLI signaling pathway. Furthermore, In Vivo Ready CDK11 siRNA was injected into mouse xenografts with multidrug resistant osteosarcoma to evaluate the effects of antitumor and reversing multidrug resistance. We expected that targeting protein kinase CDK11 would serve as a valuable addition and important tool for the treatment and reversal of multidrug resistance in osteosarcoma.

骨肉瘤是青少年最常见的恶性肿瘤，化疗后约1/3患者因为对化疗药物产生耐药而复发或转移。本课题组前期研究发现许多蛋白激酶在骨肉瘤中表达水平异常增高，抑制其表达可以阻碍骨肉瘤细胞增殖并促进其凋亡。本课题组首次发现蛋白激酶CDK11在骨肉瘤生长及存活中发挥关键作用。靶向沉默CDK11是否能通过影响特定信号通路逆转骨肉瘤耐药，至今尚无研究证实。本研究拟通过转染CDK11 siRNA抑制耐药骨肉瘤细胞CDK11的表达，检测细胞生物学特性，检验转染后Hh-SMO-GLI信号通路靶基因及细胞凋亡相关蛋白的表达水平，明确CDK11能否通过引起Hh-GLI通路抑制导致耐药骨肉瘤细胞增殖受限和细胞凋亡。使用In Vivo Ready CDK11 siRNA治疗裸鼠耐药骨肉瘤动物模型，观察其在裸鼠体内抑制耐药骨肉瘤成瘤的作用，进一步证实其逆转骨肉瘤耐药的能力，为临床治疗骨肉瘤、逆转骨肉瘤耐药奠定理论和实验基础。

本课题发现蛋白激酶CDK11在骨肉瘤组织和细胞中高表达，而在人成骨细胞中低表达；进一步通过转染 CDK11 特异性siRNA发现，靶向沉默CDK11 能够抑制骨肉瘤细胞的增殖，并促进骨肉瘤细胞凋亡及凋亡相关基因的表达。通过检测Hh-GLI 信号通路关键靶基因发现，靶向沉默CDK11 能抑制骨肉瘤细胞中Hedgehog信号通路关键因子和靶基因的表达。进一步使用 In Vivo Ready CDK11 siRNA治疗裸鼠骨肉瘤动物模型，发现其能抑制裸鼠体内肿瘤的生长。通过对骨肉瘤患者肿瘤组织中蛋白激酶CDK11 表达水平的测定，我们发现肿瘤组织中CDK11 表达水平高的患者预后较中度表达或低度表达的患者预后差。我们也研究了蛋白激酶CDK11在卵巢癌发生中的作用，发现靶向沉默CDK11的表达可导致卵巢癌细胞增殖受阻并发生凋亡，增加卵巢癌细胞对化疗药物的敏感性，抑制卵巢癌肿瘤在体内的生长；此外，我们也发现卵巢癌组织中CDK11 表达水平高的患者预后较中度表达或低度表达的患者预后差。. 此外我们也发现长链非编码RNA MALAT1在不同肉瘤细胞系和骨肉瘤肿瘤组织高表达，而在正常成骨细胞中呈现低表达。进一步应用RNA干扰阻断长链非编码RNA MALAT1的表达可导致骨肉瘤细胞增殖受阻、诱导细胞发生凋亡和周期抑制。靶向沉默MALAT1能降低骨肉瘤细胞的迁移，并抑制血管生成拟态的形成和导致细胞骨架的破坏。体内实验进一步证实沉默MALAT1能抑制骨肉瘤体内成瘤。Western Blot结果显示MALAT1的促瘤作用可能与RhoA/ROCK信号通路有关。我们也首次报道了碳纳米材料G/SWCNT能诱导骨肉瘤细胞凋亡，且骨肉瘤细胞的凋亡伴随着ROS的增加、线粒体膜电位的降低以及线粒体凋亡途径相关基因的改变，且G/SWCNT对体内主要的器官没有导致损伤或感染的作用。我们通过对骨肉瘤组织的分离培养，成功构建出转移型成软骨分化骨肉瘤细胞株—CHOS。