miR-2944a-5p靶向调控TSL及相关MAPK信号通路促进大劣按蚊对疟原虫易感性的作用及机制研究

Malaria is one of the most serious vector-borne infectious diseases. Anopheles dirus is the important malaria vector in China. It is of great significance to unveil and clarify the interactions between the parasite and vector at the molecular level for the prevention and elimination of malaria. microRNAs have been proved to have critical physiological and pathological biology functions, serving as important regulatory factors at the post-transcription level to regulate gene expression. We have performed a genome-wide analysis of miRNA-mRNA interactions and discovered that the transcripts of some mosquito genes are differentially regulated by certain miRNAs in response to plasmodium berghei infection. Among them, miR-2944a-5p was the most down-regulated miRNA, and its predicted target gene TSL (Torso-like) was significantly up-regulated. Shanu Jain et al reported that miRNA2944 was expressed significantly in blood fed Anopheles stephensi (42 h), and it was also significantly down-regulated in iBF (infected blood fed) 5 d when compared with iBF 42 h. TSL is the initial member of MAPK (mitogen-activated protein kinase) signaling pathway which is critical to regulating both innate immunity and metabolism during infection. Activation of MAPK signaling pathway in the mosquito midgut could facilitate parasite infection. On the basis of comprehensive analysis of literatures and our previous work results, we proposed the hypothesis that miR-2944a-5p is an important susceptibility-related miRNA, which target TSL and related MAPK signaling pathways to promote the susceptibility of Anopheles dirus to Plasmodium parasite. We will study the function of miR-2944a-5p and its target genes TSL in Anopheles dirus physiology and immunity. We will also analyze the tissue specific expression and temporal expression pattern of them. We expect to clarify the relevant molecular mechanisms of Anopheles dirus miR-2944a-5p target TSL involved in the MAPK signaling pathway to regulate the susceptibility to parasite from the post-transcription level through the RNA interference, Luciferase reporter assay, western, and other experimental technologies in vivo and in vitro. Our proposed study will not only help interpreter pathogen-vector interactions, but also can provide new sight for the development of novel vector control strategies.

疟疾是重要的媒传疾病。已有研究显示蚊媒的miRNA可参与调控对病原体的抗感染防御，但目前尚缺乏关于其参与调控按蚊对疟原虫易感性的相关分子机制研究。本课题组前期研究发现，大劣按蚊感染疟原虫后其miRNA及mRNA表达谱均出现显著差异表达，其中miR-2944a-5p感染后表达水平下调最显著，其预测靶基因TSL是MAPK信号通路中的重要成员，呈显著上调。研究报导MAPK信号通路的活化可以促进按蚊对疟原虫易感性。据此，我们提出科学假设：miR-2944a-5p是重要的易感性相关miRNA，可通过靶向TSL基因调控MAPK信号通路促进按蚊对疟原虫易感性。本研究拟通过对miR-2944a-5p及其靶基因TSL进行表达及功能分析，并利用RNA干扰、报告基因检测、western等实验技术手段在体内外进行验证。本研究不仅有助于阐释媒介-病原体的相互作用机制，同时也能够为新的媒介控制策略的开发提供新的思路。

miRNA是转录后基因沉默的特异性和敏感性的关键因素。从基因组中鉴定miRNA，预测其靶基因并进一步推断其功能和调节机制对于理解生物体的生物学过程至关重要，并有助于阐明其在疾病的病理生理学中的作用。既往研究表明，miRNA可作为各种生物过程而成为发育和细胞稳态的关键调节剂，调节靶mRNA并对蛋白质输出进行精细调整。因此，miRNA功能鉴定是miRNA研究的重要部分，也是阐明其参与机体多种具体分子调控机制的基础。. 本研究探讨按蚊miR-2944a-3p及其预测靶基因torso-like（tsl）对疟原虫感染按蚊的调控作用。按蚊体内的miR-2944a-3p在疟原虫感染后显著下调，而tsl基因在控制果蝇细胞免疫系统发育中起重要作用。本研究从基因组水平鉴定了大劣按蚊tsl基因，分析预测了其蛋白结构，并获得了其组织特异性表达特征。采用双荧光素报告酶系统验证miR-2944a-3p是对大劣按蚊tsl基因（ADIR006646-RA）产生靶向调节作用。进一步验证miRNA及其靶基因功能，并分别通过miR-2944a-3p 过表达、体外构建表达载体对按蚊进行活体RNAi（敲低tsl基因）进行疟原虫感染按蚊实验显示，然而，二者均对疟原虫感染没有显著影响。. 通过实验验证按蚊miR-2944a-3p及其预测靶基因tsl在按蚊组织的分布及二者的靶向关系，并进一步探索其对疟原虫感染按蚊过程中的作用，了解两者之间互相作用的分子机制，开发新的疟疾控制策略，为推进我国疟疾消除计划及全球无疟疾愿景提供理论研究基础。