宿主ciRS-7作为ceRNA调控细胞凋亡影响隐孢子虫胞内生存的机制

隐孢子虫危害宿主的前提是其能在宿主细胞内生存，而对感染细胞凋亡的有效调控对其胞内生存至关重要，但目前对其调控机制知之甚少。课题组前期研究发现微小隐孢子虫感染的HCT-8细胞环状RNA ciRS-7上调表达，并与隐孢子虫胞内荷虫量呈正相关，而其结合的miR-7和miR-1270下调表达。有研究称ciRS-7作为ceRNA可靶向这些miRNA调控细胞凋亡。据此，我们推测ciRS-7可靶向miRNAs调控细胞凋亡影响隐孢子虫胞内生存。为验证此假设，本项目拟以微小隐孢子虫感染HCT-8细胞、乳鼠肠上皮细胞和乳鼠为模型，利用过表达、干扰、荧光素酶报告等技术确定ciRS-7/miRNAs/靶基因的靶向调控关系，利用抗体超迁移率试验等解析ciRS-7/miRNAs/靶基因轴对虫体感染细胞凋亡的调节机制，利用激光共聚焦等方法明确该轴对虫体胞内发育的调节机制，预期结果将为隐孢子虫病的防控提供新的靶点和策略。

Intracellular survival is the prerequisite of Cryptosporidium to endanger hosts, and effectively regulating apoptosis of infected cells is essential for intracellular survival of Cryptosporidium. However, the regulation mechanisms were poorly understood. Our previous study showed that the expression of host circRNA ciRS-7 was up-regulated in HCT-8 cells infected with C. parvum, positively correlating with intracellular burden of C. parvum, while the levels of ciRS-7 sponging miR-7 and miR-1270 were down-regulated. Previous studies indicated that ciRS-7 could regulate cell apoptosis as a ceRNA sponging these miRNAs. Based on these fact, we assume that ciRS-7 could regulate cell apoptosis to affect intracellular survival of Cryptosporidium through sponging these miRNAs. To address this hypothesis, using C. parvum infecting HCT-8 cells, suckling mouse intestinal epithelial cells and suckling mice as models, we will firstly test targeting regulation relationships of ciRS-7/miRNAs/targets using technologies of overexpression, RNAi and luciferase reporter assays, then investigate the mechanism of ciRS-7/miRNAs/targets axis to regulate apoptosis of cells infected with C. parvum using antibody supershift assays, and finally study the mechanism of this axis to regulate development of C. parvum in host cells using confocal laser scanning technology. The expected findings in this project will provide novel targets and strategies for controlling cryptosporidiosis.