旋毛虫感染宿主肠上皮细胞过程中microRNA的差异表达及其作用机制的研究

Trichinellosis is a serious food-borne parasitic zoonosis. Following ingestion, Trichinella muscle larvae are activated by bile in small intestine and developed to the intestinal infective larvae. The further development of the infective larvae invading into intestinal epithelium is the key steps for establishing Trichinella infection. Our previous studies showed that the expression of many genes in larvae were activated or obviously up-regulated after co-cultured with intestinal epithelial cells (IECs), and some of them could significantly bond to normal mouse IECs. However, what kinds of miRNAs regulate these up-regulated genes? And whether they play important roles in the invasion of host IECs by the intestinal infective larvae was not clear at the present. Based on the previous study, the differently expressed miRNAs from intestinal infective larvae were screened by using high-throughput sequencing. The target gene and transcription factor of the chosen miRNA were predicted by bioinformatics tools. Then, dual-luciferase reporter assay and chromatin immunoprecipitation assay were used to identify the expression of the target gene regulated by the miRNA, and the expression of miRNA regulated by the transcription factor. The effect on invasion, molt and maturation of the infective larvae was observed in vitro and animal experiments by over-expressing or suppressing the miRNA. The project will lay the foundation for illustrating the molecular mechanism of regulation of invasion by Trichinella infective larvae, searching the effective vaccines for trichinellosis, and screening the new drugs against Trichinella.

旋毛虫病是一种严重的食源性人兽共患寄生虫病，肌幼虫在小肠被胆汁激活为肠道感染性幼虫后侵入肠粘膜中蜕皮发育是建立感染的关键。我们研究发现，在侵入肠上皮细胞（IECs）的过程中肠道感染性幼虫多个基因被激活或表达量显著上调，然而这些基因受哪些miRNA的调控，侵袭前后幼虫差异表达的miRNA在其侵入宿主IECs的过程中发挥何种作用，目前尚不清楚。本项目将在前期研究的基础上，建立旋毛虫-正常小鼠IECs体外侵袭模型，高通量测序筛选肠道感染性幼虫侵袭IECs前后差异表达的miRNA；预测表达下调最显著miRNA的靶基因，应用双荧光素酶报告基因实验及染色质免疫共沉淀技术验证该miRNA与上游转录因子、下游靶基因的调控关系；体内外实验观察miRNA过表达与抑制对肠道感染性幼虫侵入肠粘膜及其发育的影响。该项目为阐明旋毛虫侵入肠粘膜的分子调控机制、寻找旋毛虫病的强保护性抗原疫苗及筛选抗旋毛虫新药等奠定基础。

旋毛虫病是一种危害严重的食源性人兽共患寄生虫病，旋毛虫肠道感染性幼虫侵入宿主肠粘膜中蜕皮发育是其成功建立感染的关键。课题组前期研究发现，在侵入宿主肠上皮细胞（IECs）的过程中旋毛虫的多个基因被激活或表达显著上调，然而这些基因受哪些miRNA的调控，差异表达的miRNA在旋毛虫侵袭IECs的过程中发挥着何种作用，目前仍不清楚。据此，本研究通过高通量基因测序对旋毛虫侵袭宿主IECs前后的miRNA表达谱进行鉴定并筛选差异miRNA；对表达显著下调的miRNA进行靶基因的预测及相互作用的验证；体外实验观察目标miRNA的过表达和抑制对旋毛虫体外侵入IECs的影响。结果表明，在旋毛虫侵入小鼠IECs前后共有3431个miRNA发生差异表达，其中1466个表达上调，1965个表达下调。从中选取明显下调、且感染前后差异度达29.9倍的Tsp-let-7为目标miRNA，双荧光素酶报告基因技术证实Tsp-let-7的靶基因为Tsp_07700（羧肽酶E）。体外实验发现Tsp-let-7的下调明显促进旋毛虫的侵入及蜕皮发育，而Tsp-let-7的上调则抑制其侵入及其蜕皮发育。本研究为阐述旋毛虫与宿主肠上皮细胞间的相互关系、寻找潜在的旋毛虫病新型保护性疫苗及治疗靶标等奠定了基础。