HCT-8细胞miR-942防御微小隐孢子虫感染的调控机制

Cryptosporidium spp. can infect more than 280 species of animals, and cause the decreasing production performances or death. Cryptosporidium is the second most important diarrhoeal pathogen in humans, and cryptosporidiosis accounts for 8.4 million Disability-Adjusted Life Years (DALYs). Previous studies indicated that miRNAs of host cells play an important role in the innate immunity against Cryptosporidium infection. Apoptosis is one of the important mechanisms against Cryptosporidium infection, however, there is not study report about the regulation mechanism for cell apoptosis associated with Cryptosporidium. Applicant has analyzed the expression profiles of HCT-8 cells associated miRNAs following C. parvum infection, and deduced miR-942, miR-181d, and miR-122 are associated with the apoptosis/anti-apoptosis of cells, among which, miR-942 has the largest expression change following C. parvum infection. Therefore, in this project, the regulation mechanism against C. parvum infection associated the HCT-8 cell miR-942, including transcription regulation pathways, the target gene, anti-apoptosis role by the target gene coding protein, anti-apoptosis ways, and the effect to Cryptosporidium infection burden will be studied by using the Real-time PCR, transfection techniques, 5’-RACE PCR, Chromatin immunoprecipitation assays (ChIP), and Western blot, etc. The data obtained by this project should provide new strategies for design and development of drug targets against cryptosporidiosis.

隐孢子虫感染280多种动物，导致生产性能下降或死亡；是人体第二大致腹泻病原，该病发生导致840万伤残调整生命年（DALYs）。研究表明，miRNA在宿主防御隐孢子虫感染的先天性免疫中发挥重要作用。凋亡是宿主防御隐孢子虫感染重要机制之一，目前未见miRNA调控凋亡相关报道。申请人解析了微小隐孢子虫感染HCT-8细胞相关miRNA表达谱，推测miR-942、miR-181d、miR-122与细胞凋亡/抗凋亡有关，其中miR-942表达量变化最大。鉴于此，申请项目拟以miR-942为对象，采用Real-time PCR、转染技术、5’-RACE PCR、染色质免疫沉淀、Western Blot等技术阐明其在HCT-8细胞防御微小隐孢子虫感染中的调控机制，包括转录调控通路、互作靶基因、靶蛋白抗凋亡作用、抗凋亡途径以及对隐孢子虫感染负荷的影响，研究结果为设计开发抗隐孢子虫病药物靶标提供新的策略。

凋亡是宿主防御隐孢子虫感染的重要机制之一，本研究课题阐明了微小隐孢子虫感染诱导HCT-8细胞miR-942-5p调节细胞凋亡的机制。结果显示，HCT-8细胞表达所有已知的Toll样受体（TLRs）；微小隐孢子虫感染培养的HCT-8细胞可上调TLR2和TLR4，以及下游TLR效应分子，包括NF-κB和抑制性IκBα，miR-942-5p的表达在感染后4、8、12和24小时显著上调，尤其是8hpi。TLR4和TLR2特异性siRNA和NF-κB抑制的结果表明，p65亚基依赖的TLR2/TLR4-NF-κB信号通路促进miR-942-5p上调。. 双荧光素酶报告载体实验表明，在含有IFI27 mRNA 3‘UTR重组pmirGLO-wild质粒的HCT-8细胞中miR-942-5p mimics与 miR-942-5p mimics NC相对荧光差异显著（P<0.05）；qPCR和Western blotting检测显示miR-942-5p mimics组IFI27表达显著低于mimic NC组，而inhibitor组正好相反，证明了IFI27是miR-942的靶基因。. 流式细胞检测显示，IFI27过表达组细胞凋亡比例显著高于空载体组，而siRNA-IFI27组凋亡比例显著低于siRNA-NC组，证明miR-942-5p靶基因IFI27促进了HCT-8细胞凋亡。qPCR和Western blotting检测显示，Apaf-1、Casepase 3和Casepase 9表达变化不显著，而TRAIL和Casepase 3具有显著性变化，表明miR-942-5p靶基因编码蛋白IFI27抗凋通过外源性TRAIL途径。qPCR检测结果显示，miR-942-5p mimics组HCT-8细胞荷虫量显著高于mimic NC组，而inhibitor组正好相反，表明在早期miR-942可促进隐孢子虫感染HCT-8细胞。. 总之，微小隐孢子虫感染主要通过TLR4-NF-κB信号通路诱导miR-942-5p表达上调，从而下调靶基因IFI27的表达量起到抗凋亡作用，促进了隐孢子虫早期感染HCT-8细胞以及虫体发育。