miR-281是媒介蚊虫白纹伊蚊中一种丰富的中肠特异性微小RNA，它能增强登革病毒的复制。
miR-281, an abundant midgut-specific miRNA of the vector mosquito Aedes albopictus enhances dengue virus replication.
作者信息
Zhou Yanhe, Liu Yanxia, Yan Hui, Li Yiji, Zhang Hao, Xu Jiabao, Puthiyakunnon Santhosh, Chen Xiaoguang
机构信息
Key Laboratory of Prevention and Control for Emerging Infectious Diseases of Guangdong, Department of Pathogen Biology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China.
出版信息
Parasit Vectors. 2014 Oct 22;7:488. doi: 10.1186/s13071-014-0488-4.
BACKGROUND
Emerging evidence indicates that microRNAs (miRNAs) are involved in host-virus interaction. We previously reported that some miRNAs were differentially expressed in sugar-fed and blood-fed females of Aedes albopictus (Ae. albopictus). Here, we analysis the role in the host-virus system of an abundant midgut-specific miRNA in the mosquito Ae. albopictus.
METHODS
The expression profiles of miR-281 in different body parts of Ae. albopictus and following dengue virus infection were determined using RT-qPCR and Northern blot. miR-281 mimics, antagomiRs and corresponding negative controls were designed and their overexpression and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting female mosquitoes. Dengue virus serotype-2 (DENV-2) viral genomic RNA abundance was determined by RT-qPCR. The levels of DENV-2 E protein were detected using Western blot. Virus titers were tested using TCID50. RNAhybrid was used to predict targets of miR-281 in the DENV-2 genome. The EGFP plasmid-based reporter system was used to investigate the interaction between miR-281 and the predicted binding site in the C6/36 cell line.
RESULTS
miR-281 is specifically expressed in the female midgut where dengue virus first invades. After DENV-2 infection, this miRNA is up-regulated in response to viral infection. Functional intervention analyses in vitro with specifically designed miR-281 mimics and corresponding antagomiRs indicated that miR-281 enhances DENV-2 viral replication. Further depletion of miR-281 in female mosquitoes by injection of its specific antagomiRs led to a significant reduction in DENV-2 abundance. The interaction between miR-281 and its predicted target sequence, the DENV-2 genomic 5'-untranslated region (UTR), is confirmed in the context of a plasmid-based reporter system.
CONCLUSION
These findings confirm that miR-281, an abundant midgut-specific miRNA, facilitates DENV-2 replication.
背景
新出现的证据表明，微小RNA（miRNA）参与宿主与病毒的相互作用。我们之前报道过，一些miRNA在白纹伊蚊（Ae. albopictus）吸食糖水和吸食血液的雌蚊中差异表达。在此，我们分析了白纹伊蚊中一种丰富的中肠特异性miRNA在宿主-病毒系统中的作用。
方法
使用逆转录定量聚合酶链反应（RT-qPCR）和Northern印迹法测定白纹伊蚊不同身体部位以及登革病毒感染后miR-281的表达谱。设计了miR-281模拟物、反义寡核苷酸（antagomiR）及其相应的阴性对照，并在转染蚊细胞系C6/36后通过qRT-PCR分析它们的过表达和敲低效率，也通过注射雌蚊进行分析。通过RT-qPCR测定登革病毒2型（DENV-2）病毒基因组RNA丰度。使用蛋白质免疫印迹法检测DENV-2 E蛋白水平。使用半数组织培养感染剂量（TCID50）检测病毒滴度。使用RNAhybrid预测DENV-2基因组中miR-281的靶标。基于增强绿色荧光蛋白（EGFP）质粒的报告系统用于研究miR-281与C6/36细胞系中预测的结合位点之间的相互作用。
结果
miR-281在登革病毒首先侵入的雌蚊中肠中特异性表达。DENV-2感染后，这种miRNA会因病毒感染而上调。用专门设计的miR-281模拟物和相应的反义寡核苷酸进行的体外功能干预分析表明，miR-281增强DENV-2病毒复制。通过向雌蚊注射其特异性反义寡核苷酸进一步降低miR-281水平，导致DENV-2丰度显著降低。在基于质粒的报告系统中证实了miR-281与其预测的靶序列即DENV-2基因组5'-非翻译区（UTR）之间的相互作用。
结论
这些发现证实，miR-281是一种丰富的中肠特异性miRNA，它促进DENV-2复制。
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