The miRNA-targeted transcriptome of porcine alveolar macrophages upon infection with Porcine Reproductive and Respiratory Syndrome Virus (RISC bound RNA). Sus scrofa

Porcine alveolar macrophages were challenged in vitro with a low virulent strain of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) to identify and characterize the population of host genes subjected to miRNA post-transcriptional regulation durign early viral infection. We employed a high-throughput biochemical assay, i.e. the immunoprecipitation of RISCs followed by microarray analysis of the RISC-bound miRNA targets (RIP-Chip). qPCR analyses were carried out to validate the resolution of the microarray and to determine levels of expression of a panel of eight miRNAs (ssc-miR-142-3p, ssc-miR-142-5p, ssc-let-7f, miR-146a-5p, miR-155-5p, ssc-miR-181a, ssc-miR-21 and ssc-miR-335). Overall design: Lung alveolar macrophages were obtained by lung lavage from 4 animals. Cells were infected in vitro with the strain Finistère at multiplicity of infection of 2, or mock infected. Infected cells were harvested at 7 and 10 hours post-infection; control (mock infected) cells were collected at the same times. Total RNAs were extracted from total cell contents and from RISC (RNA-induced Silencing Complex) immunoprecipitates. RNAs were hybridized on microarrays and transcripts analyzed for relative enrichment in RISC (RIP-Chip technology).

本研究以低毒力猪繁殖与呼吸综合征病毒（Porcine Reproductive and Respiratory Syndrome Virus, PRRSV）毒株体外攻毒猪肺泡巨噬细胞，旨在鉴定并解析病毒感染早期受微小RNA（microRNA, miRNA）转录后调控的宿主基因群体。本研究采用高通量生化检测手段，即RNA诱导沉默复合体（RNA-induced Silencing Complex, RISC）免疫沉淀技术，结合靶向结合RISC的miRNA靶基因芯片分析（RIP-Chip）。通过实时定量聚合酶链反应（quantitative PCR, qPCR）分析，验证芯片检测的准确性，并定量检测8种miRNA（ssc-miR-142-3p、ssc-miR-142-5p、ssc-let-7f、miR-146a-5p、miR-155-5p、ssc-miR-181a、ssc-miR-21及ssc-miR-335）的表达水平。整体实验设计：通过肺灌洗法从4头实验动物体内分离得到猪肺泡巨噬细胞；将细胞以感染复数（multiplicity of infection, MOI）为2的Finistère毒株进行体外感染，同时设置空白感染（mock感染）对照组。分别于感染后7小时和10小时收取感染组细胞，同期收取空白感染对照组细胞。从全细胞裂解物及RISC免疫沉淀产物中提取总RNA，将提取的RNA进行芯片杂交，通过RIP-Chip技术分析转录本在RISC中的相对富集情况。