Deciphering the porcine intestinal microRNA transcriptome

Background While more than 700 microRNAs (miRNAs) are known in human, a comparably low number has been identified in swine. Because of the close phylogenetic distance to humans, pigs serve as a suitable model for studying e.g. intestinal development or disease. Recent studies indicate that miRNAs are key regulators of intestinal development and their aberrant expression leads to intestinal malignancy. Results Here, we present the identification of hundreds of apparently novel miRNAs in the porcine intestine. MiRNAs were first identified by means of deep sequencing followed by miRNA precursor prediction using the miRDeep algorithm as well as searching for conserved miRNAs. Second, the porcine miRNAome along the entire intestine (duodenum, proximal and distal jejunum, ileum, ascending and transverse colon) was unraveled using customized miRNA microarrays based on the identified sequences as well as known porcine and human ones. In total, the expression of 332 intestinal miRNAs was discovered, of which 201 represented assumed novel porcine miRNAs. The identified hairpin forming precursors were in part organized in genomic clusters, and most of the precursors were located on chromosomes 3 and 1, respectively. Hierarchical clustering of the expression data revealed subsets of miRNAs that are specific to distinct parts of the intestine pointing to their impact on cellular signaling networks. Conclusions In this study, we have applied a straight forward approach to decipher the porcine intestinal miRNAome for the first time in mammals using a piglet model. The high number of identified novel miRNAs in the porcine intestine points out their crucial role in intestinal function as shown by pathway analysis. On the other hand, the reported miRNAs may share orthologs in other mammals such as human still to be discovered. RNA isolation from porcine intestinal tissue Intestinal samples (~ 2 cm circle segments) were taken from duodenum, proximal and distal jejunum, ileum, ascending and transverse colon of four 31 days old healthy piglets (EUROC x Pietrain). The piglets were weaned at the age of 28 days. Samples were quick-frozen in liquid nitrogen and stored at -80 °C. In order to obtain representative measurements in each intestinal locus, three cross sections of approximately 2 mm out of the 2 cm segment of frozen intestine were examined. These 3 sections were pooled and total RNA was isolated from samples using an automated homogenizer (FastPrep Instrument, MP Biomedicals, Heidelberg, Germany) and the mirVana miRNA Isolation Kit (Applied Biosystems, Darmstadt, Germany), according to the manufacturer’s protocol. The RNA quality and quantity of all samples were proven using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kits (Agilent, Waldbronn, Germany) and the Nanodrop 1000 Spectrophotometer (Thermo Scientific, MA, USA). Microfluidic miRNA microarrays Microfluidic microarray experiments were performed using customized Geniom biochips (febit). Customized microarrays were synthesized with the Geniom One device (febit) applying febit’s standard shortmer kit for oligonucleotide synthesis. The light-activated in situ oligonucleotide synthesis using a digital micromirror device was performed within the Geniom One instrument on an activated three-dimensional reaction carrier consisting of a glass-silicon-glass sandwich (biochip). Using standard DNA synthesis reagents and 3’-phosphoramidites carrying 5’-photolabile protective group, oligonucleotides were synthesized in parallel in eight individually accessible microchannels (referred to as microarrays) of one biochip. Prior to synthesis, the glass surface was activated by coating with spacer to facilitate probe-target interaction and to avoid probe-probe interface [37]. Two different microarray designs were used. The first one included only the 399 miRDeep predicted mature miRNAs and their star sequences in seven replicates allowing highly accurate validation of the miRDeep prediction plus 16 spike-in controls. The second design consisted of 1117 features in triplicate, including the identified conserved porcine mature miRNA sequences (18 mapping to the porcine genome and another 124, which did not map), 74 known porcine and 885 human miRNAs from miRBase (13.0) and 16 spike-in controls. For microarray experiments, intestinal total RNA samples from different loci of four subjects were isolated independently and respective pools of total RNA samples were prepared. All pools underwent a second quality control to determine the quality and quantity using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit according to the manufacturer’s instructions. For each microarray 250 ng of total RNA were suspended in 25 µl of febit’s proprietary miRNA hybridization buffer. The hybridization was performed automatically for 16h at 42°C using the Geniom RT-Analyzer (febit). After stringent washing a microfluidic-based extension assay was performed to label the miRNAs using biotinylated nucleotides. After washing, biotinylated nucleotides were detected by streptavidin-phycoerythrin and signal recognition and calculation were done automatically within milliseconds. The background of the chips was effectively corrected by global background subtraction and inter-array effects were corrected by variance stabilizing normalization.

背景 目前已在人类中发现超过700种微小核糖核酸（microRNAs, miRNAs），但在猪中鉴定出的同类RNA数量相对较少。由于与人类的系统发育距离较近，猪可作为研究肠道发育或疾病等问题的适宜模型。已有研究表明，miRNAs是肠道发育的关键调控因子，其表达异常会引发肠道恶性肿瘤。
结果 本文首次在猪肠道中鉴定出数百种疑似新型miRNAs。研究首先通过深度测序结合miRDeep算法预测miRNA前体，并搜索保守型miRNAs的方式实现miRNAs的鉴定；其次，基于已鉴定的序列、已知猪源和人源miRNAs，定制化制备miRNA芯片，以此解析整个肠道（十二指肠、空肠近端段、空肠远端段、回肠、升结肠及横结肠）的猪miRNA组。最终共检测到332种肠道miRNAs的表达，其中201种为假定的新型猪源miRNAs。鉴定出的形成发夹结构的前体部分在基因组中呈簇状分布，且多数前体分别位于3号和1号染色体上。对表达数据进行层次聚类后发现，存在特定的miRNA子集，它们在肠道不同区段特异性表达，提示其对细胞信号网络具有调控作用。
结论 本研究首次以仔猪为模型，采用简便直接的方法解析了哺乳动物的猪肠道miRNA组。在猪肠道中鉴定出大量新型miRNAs，结合通路分析结果，凸显了其在肠道功能中的关键作用。另一方面，本次报道的miRNAs可能在其他哺乳动物（如人类）中存在尚未被发现的同源基因。
猪肠道组织RNA提取 从4只31日龄健康仔猪（EUROC x 皮特兰）的十二指肠、空肠近端段、空肠远端段、回肠、升结肠及横结肠采集约2cm的环形肠段样本。仔猪于28日龄断奶。样本经液氮快速冷冻后保存于-80℃。为确保每个肠道位点的检测结果具有代表性，从2cm的冷冻肠段中选取3片约2mm的切片进行混合，随后使用全自动匀浆器（FastPrep仪器，MP生物医学公司，海德堡，德国）和mirVana miRNA分离试剂盒（Applied Biosystems，达姆施塔特，德国），按照制造商的操作流程从混合样本中提取总RNA。采用Agilent 2100生物分析仪及RNA 6000 Nano试剂盒（安捷伦科技公司，瓦尔德布龙，德国）、Nanodrop 1000分光光度计（赛默飞世尔科技公司，马萨诸塞州，美国）对所有样本的RNA质量和浓度进行验证。
微流控miRNA芯片 微流控芯片实验采用定制化Geniom生物芯片（febit公司）完成。使用Geniom One设备（febit公司），结合febit标准短链试剂盒进行寡核苷酸合成，制备定制化微阵列。在Geniom One仪器中，通过数字微镜器件实现光激活原位寡核苷酸合成，反应载体为经活化的玻璃-硅-玻璃夹层结构（生物芯片）。使用标准DNA合成试剂与带有5’光不稳定保护基团的3’-亚磷酰胺，在一块生物芯片的8个独立可访问微通道（即微阵列）中并行合成寡核苷酸。合成前，需通过间隔基涂层对玻璃表面进行活化，以促进探针与靶标的相互作用并避免探针间的非特异性结合[37]。本研究采用两种不同的微阵列设计：第一种设计仅包含399个经miRDeep预测的成熟miRNAs及其星型序列，设置7次重复，用于高精度验证miRDeep预测结果，同时包含16个spike-in对照；第二种设计包含1117个检测位点，设置3次重复，涵盖已鉴定的保守型猪源成熟miRNA序列（18个可比对至猪基因组，另有124个无法比对）、74个已知猪源miRNAs、885个来自miRBase（13.0版本）的人源miRNAs，以及16个spike-in对照。在芯片实验中，分别提取4只仔猪不同肠道位点的总RNA样本，并将样本混合制备总RNA混合池。所有混合池均再次通过Agilent 2100生物分析仪及RNA 6000 Nano试剂盒，按照制造商的操作说明进行质量和浓度检测。每个微阵列使用250ng总RNA，悬浮于25µl febit专有miRNA杂交缓冲液中。采用Geniom RT分析仪（febit公司）在42℃下自动杂交16小时。经过严格洗涤后，通过基于微流控的延伸实验，使用生物素标记的核苷酸对miRNAs进行标记。洗涤完成后，利用链霉亲和素-藻红蛋白检测生物素标记的核苷酸，信号识别与计算可在数毫秒内自动完成。通过全局背景减法有效校正芯片背景，并通过方差稳定归一化校正阵列间的差异。