miR-125在青蒿琥酯预防治疗日本血吸虫病中的作用机制研究

Human schistosomiasis is a parasitic disease caused by trematode flukes of the genus Schistosoma, at least 230 million people worldwide are infected with Schistosoma spp. Artesunate has high prophylactic efficacy against Schistosoma japonicum infections, however, the molecular mechanism of artesunate effects on Schistosoma japonicum remains unclear. Our previous research demonstrated that miR-125b significantly differentially expressed after artesunate treatment in schistosomula. The potential targets of miR-125b were involved in drug metabolic enzymes and the genes related to the metabolism of hemoglobin. miR-125b could play a significant role in the molecular mechanism of artesunate effects on Schistosoma japonicum by down-regulating the potential target genes that are important for drug function. In the present study, we will further explore the association between the expression level of miR-125b and artesunate action on Schistosoma japonicum infection in vitro by real-time quantitative PCR. We will also construct a lentivirus-based transduction system to silence or over-express miR-125b in schistosomula and then detect the expression of the potential targets of miR-125b. To confirm the targets of miR-125b further, we will employ dual luciferase reporter assay experiment. This study will elucidate the regulational mechanism of miRNA-125b in the preventive treatment for schistosomiasis by artesunate and establish the basis for further studies in potential targets for drug development for schistosomiasis.

血吸虫病是一种裂体吸虫引起的感染性寄生虫病，严重危害世界上2.3亿感染者的健康。青蒿琥酯能够有效杀灭日本血吸虫童虫，但是青蒿琥酯抗日本血吸虫的分子机制不明确。项目组前期研究发现，miR-125b在青蒿琥酯作用日本血吸虫后表达水平呈显著性差异，其候选作用靶点涉及药物代谢酶与血红蛋白代谢相关基因，因此提出miR-125b可能通过靶向调控青蒿琥酯代谢相关酶或血红蛋白代谢相关基因发挥药物抗日本血吸虫作用的假说。本研究拟通过日本血吸虫童虫体外培养模型的构建，结合实时荧光定量 PCR技术，进一步分析miR-125b表达谱与青蒿琥酯作用日本血吸虫的相关性；利用慢病毒转染过表达、抑制日本血吸虫体内的miR-125b，分析候选靶基因的表达水平；同时利用双荧光素酶报告系统验证miR-125b作用的确切靶点，阐明miR-125b参与青蒿琥酯抗日本血吸虫的调控机制，有望为日本血吸虫病有效治疗提供新靶标。

血吸虫病，一种由裂体吸虫引起的感染性寄生虫病，影响着全世界2.3亿感染患者的身心健康。青蒿琥酯能够有效杀灭人体内的日本血吸虫童虫，具有较好的预防治疗日本血吸虫病的效果。目前，青蒿琥酯抗日本血吸虫的机制尚未明确。本研究首次分析了青蒿琥酯作用日本血吸虫童虫后的miRNA差异表达谱，发现miR-125b在内的7个miRNAs在青蒿琥酯作用日本血吸虫后表达水平呈显著性差异变化，miR-125b候选的靶点涉及药物代谢酶与血红蛋白代谢相关基因。本研究构建了日本血吸虫体外培养模型，结合实时荧光定量 PCR技术，分析了miR-125b表达谱与青蒿琥酯作用日本血吸虫的相关性；相比对照组，随着给药剂量的增加，青蒿琥酯处理后的虫体miR-125b的表达量呈显著性降低。同时，还发现PC-5P-108表达量呈显著性上调。本研究构建了miR-125b过表达慢病毒载体pHRS-1cla-miR-125b-CMV-EGFP，结果显示转染阳性载体的293FT 细胞miR-125b的表达水平比转染空质粒的对照组高约750倍（p<0.01）。miR-125b候选靶基因的表达水平检测分析发现Heme-binding protein 2，AMPK gamma 2，CLPP，SJCHGC03376，SJCHGC05717和SJCHGC03211的转录表达水平与对照组相比无显著性差异变化。使用双萤光素酶报告系统等技术验证的miR-125b的靶基因近60个，涉及细胞的分化、增殖、凋亡、免疫和代谢等生物学功能。针对日本血吸虫miR-125b在转录水平上的调控未能引起相关候选基因表达水平的显著性差异，推测候选靶基因可能存在其它代偿机制。