mRNA profiling of hypoxia+SU5416-induced pulmonary hypertension (PH) mouse model treated with an inhibitor of miR-130/301 family

Purpose: Guided by an in silico combination of microRNA (miRNA) target prediction, analysis of transcriptomic changes in 137 human diseases, and advanced gene network modeling, we predicted the miR-130/301 family of miRNAs as a shared regulator of a fibrotic gene network across human diseases, thus orchestrating broad control over disease manifestation. The goals of this study are to compare the lung mRNA profile of mouse model of Pulmonary hypertension, one of the most fibrotic pathology uncovered by our in silico prediction, treated with an inhibitor of miR-130/301 (Short-130) to mice treated with a control inhibitor (Short-NC). Methods: Eight-week-old mice (C57BL/6) were injected with SU5416 (20 mg/kg/dose; Sigma-Aldrich), followed by exposure to normobaric hypoxia (10% O2; OxyCycler chamber, Biospherix Ltd.) for 2 weeks. After 2 weeks and confirmation of PH development in 5 mice (right heart catheterization), mice were further treated with 3 intrapharyngeal injections (every 4 days) of control or miR-130/301 shortmer oligonucleotides, designed as fully modified antisense oligonucleotides complementary to the seed sequence of the miR-130/301 miRNA family (10 mg/kg/dose; Regulus). Specifically, the control and miR-130/301 shortmer oligonucleotides were nontoxic, lipid-permeable, high-affinity oligonucleotides. The miR-130/301 shortmer carried a sequence complementary to the active site of the miR-130/301 miRNA family, containing a phosphorothioate backbone and modifications (fluoro-, methoxyethyl, and bicyclic sugar) at the sugar 2' position. Three days after the last injection, right heart catheterization was performed followed by harvesting of lung tissue for RNA extraction. Lung mRNA profiles of those mice or control mice (Normoxia+SU5416) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the gene-level count. The gene level counts were then normalized with the R/Bioconductor package limma using the voom /variance stabilization method. The data were quality controlled for outliers using principal component analysis (PCA). Differential expression analysis between transcriptome profiles of experimental groups was performed using the R / Bioconductor package limma. Results: Transcriptomic analyses of whole lung from mice with hypoxia+SU5416-induced PH revealed a generalized de-repression of miR-130/301 targets by Short-130 treatment. Importantly, although whole lung transcriptomics likely captured only a subset of the miR-130/301 targets affecting the diseased pulmonary vasculature, pathway enrichment nonetheless revealed pronounced representation of several pathways known to be involved in fibrosis. Thus, the miR-130/301 family indeed induces a programmatic shift at the molecular level toward the fibrotic pathophenotype in vivo Overall design: Whole lung mRNA profiles of Normoxia (Control) and hypoxia+SU5416-induced PH mice treated with Short-NC or Short-130 were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.

研究目的：本研究依托虚拟的微小RNA（microRNA，miRNA）靶基因预测、137种人类疾病的转录组变化分析以及先进的基因网络建模方法，预测miR-130/301家族微小RNA是跨人类疾病的纤维化基因网络的共同调控因子，进而对疾病表型发挥广谱调控作用。本研究的核心目标是对比两组小鼠的肺脏mRNA表达谱：一组为经miR-130/301抑制剂（Short-130）处理的肺动脉高压（Pulmonary hypertension，PH）小鼠模型——该模型是本研究虚拟预测发现的纤维化程度最高的病理模型之一；另一组为经阴性对照抑制剂（Short-NC）处理的同模型小鼠。
实验方法：选取8周龄C57BL/6小鼠，按20 mg/kg/剂的剂量注射SU5416（Sigma-Aldrich），随后置于常压低氧环境（10% O₂；OxyCycler饲养箱，Biospherix Ltd.）饲养2周。2周后，通过右心导管术确认5只小鼠成功构建肺动脉高压模型，后续每4天经咽内注射1次，共3次给予对照或miR-130/301短寡核苷酸抑制剂（剂量为10 mg/kg/剂，Regulus）。该抑制剂为完全修饰的反义寡核苷酸，序列与miR-130/301家族微小RNA的种子序列互补。其中，阴性对照与miR-130/301短寡核苷酸均为无毒、脂质通透、高亲和力的寡核苷酸；miR-130/301短寡核苷酸的序列与该家族微小RNA的活性位点互补，其骨架为硫代磷酸酯修饰，且在核糖2'位进行了氟代、甲氧基乙基及双环糖修饰。末次注射3天后，对小鼠实施右心导管术，随后摘取肺组织用于RNA提取。采用Illumina HiSeq 2000平台对各组小鼠（包括常氧+SU5416对照组小鼠）的肺脏mRNA进行深度测序，每组设置3次生物学重复。对通过质量过滤的测序读段进行基因水平计数分析，随后使用R/Bioconductor的limma包结合voom/方差稳定化方法对基因计数数据进行标准化处理；通过主成分分析（Principal Component Analysis，PCA）对数据进行异常值质控。最后采用limma包对各组转录组表达谱进行差异表达分析。
实验结果：对低氧+SU5416诱导的肺动脉高压小鼠的全肺组织进行转录组分析发现，经Short-130处理后，miR-130/301的靶基因普遍出现去抑制现象。值得注意的是，尽管全肺转录组分析仅能捕获到部分影响病变肺血管的miR-130/301靶基因，但通路富集分析仍显示多个已知与纤维化相关的通路显著富集。由此证实，miR-130/301家族确实可在体内通过分子层面的程序化调控，驱动向纤维化病理表型的转变。
实验设计：采用Illumina HiSeq 2000平台，对常氧对照组、低氧+SU5416诱导的肺动脉高压且经Short-NC处理组、低氧+SU5416诱导的肺动脉高压且经Short-130处理组的小鼠全肺mRNA进行深度测序，每组设置3次生物学重复。