弓形虫棒状体蛋白38通过宿主细胞周期素依赖蛋白激酶5抑制人肝肿瘤细胞生长作用的机制研究

Toxoplasma gondii is a specialized intracellular parasite. Toxoplasma gondii can cause opportunistic pathologies in cancer patients. Recent studies have found that Toxoplasma gondii infection can inhibit the growth of host tumors. After preliminary studies, we found that Toxoplasma gondii rhoptry protein 38 (ROP38) can act on the host cell cyclin dependent protein kinase 5 (CDK5) protein, thereby inhibiting the growth and proliferation of human liver tumor cells. This topic intends to explore the interaction between ROP38 protein and CDK5 protein，to find the key amino acids using the techniques of point mutation, RT-PCR, Western blot, siRNA, stable cell transfection and CRISPR / Cas9 knockout. This study will explore the downstream molecules of CDK5, elucidate the relationship between the molecules, clarify the mechanism of ROP38 inhibition of tumor cell growth and proliferation. This topic will provide a theoretical basis for the new strategy of human tumor prevention and treatment, be significant for prevention and treatment of human oncological diseases.

弓形虫是一种专性细胞内寄生原虫。在肿瘤患者中，弓形虫会引起机会性致病。近期研究发现，弓形虫感染会抑制宿主肿瘤的生长。经过前期研究，我们发现弓形虫棒状体蛋白38（ROP38）能够作用于宿主细胞周期素依赖蛋白激酶5（CDK5）蛋白，从而抑制人肝肿瘤细胞的生长增殖。本课题拟利用基因点突变、RT-PCR、Western blot、siRNA、细胞稳定转染、CRISPR/Cas9基因敲除等实验技术探究ROP38蛋白与CDK5蛋白的相互作用区域及关键氨基酸。本研究将要探寻CDK5的下游作用分子，阐明各分子之间的联系，理清ROP38抑制肿瘤细胞生长增殖作用的机制。本课题将为人类肿瘤防治的新策略提供理论基础，对人类肿瘤疾病的防治意义重大。

项目通过大量筛选实验找到了CDK5与ROP38相互作用的关键氨基酸位点，揭示了两个分子更密切的联系。并对ROP38影响肝肿瘤生长的可能下游分子做了体外和体内验证。一方面，通过上调和下调肝肿瘤细胞内ROP38和CDK5的表达量验证了两者作用的下游分子变化，CDK5的下调和上调分别降低和增加了下游分子PD-L1的表达量，同样ROP38的上调和下调也分别降低和增加了PD-L1表达量。ROP38和CDK5表达量的变化对ROS和DLC1表达量没有显著性的影响。动物实验显示ROP38高表达组降低了小鼠肿瘤CDK5和PD-L1的表达量，同时一定程度上减小了小鼠肿瘤的重量并延长了小鼠生存时间。本项目理清了一条ROP38作用于CDK5而调控PD-L1表达量进而影响肝肿瘤的分子通路。本项目为人类肿瘤防治的新路径提供理论依据。