Deciphering the porcine intestinal microRNA transcriptome. Sus scrofa

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. Overall design: 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 目前已在人类中鉴定出超过700种微小RNA（microRNAs, miRNAs），但在猪中被发现的同类miRNA数量却相对较少。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课题的适宜模型。近期研究表明，miRNA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 本研究在猪肠道中鉴定出数百种疑似新型miRNA。首先，通过深度测序结合miRDeep算法进行miRNA前体预测，并搜索保守型miRNA，完成相关miRNA的鉴定；其次，基于鉴定得到的序列以及已知的猪和人类miRNA，定制miRNA微阵列，以此解析了整条肠道（十二指肠、近端空肠、远端空肠、回肠、升结肠和横结肠）的猪miRNA组（porcine miRNAome）。最终共检测到332种肠道miRNA的表达，其中201种为疑似新型猪miRNA。鉴定出的可形成发夹结构的前体部分在基因组中呈簇状分布，且大多数前体分别位于3号和1号染色体上。对表达数据进行层次聚类后发现，特定miRNA子集仅在肠道的不同区域特异性表达，这提示它们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。通过通路分析可知，猪肠道中鉴定出的大量新型miRNA，凸显了其在肠道功能中的关键作用。此外，本研究报道的miRNA可能在其他哺乳动物（如人类）中存在同源物，有待进一步发掘。

整体实验设计 猪肠道组织的RNA提取 从4只31日龄健康仔猪（EUROC×皮特兰）的十二指肠、近端空肠、远端空肠、回肠、升结肠和横结肠采集约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℃环境中。为确保每个肠道位点的检测结果具有代表性，对取自2cm肠段的3个约2mm的横截面进行检测。将这3个截面混合后，使用全自动均质器（FastPrep仪，MP Biomedicals，德国海德堡）和mirVana miRNA提取试剂盒（Applied Biosystems，德国达姆施塔特），按照制造商的标准操作流程提取总RNA。通过Agilent 2100生物分析仪及RNA 6000 Nano试剂盒（Agilent，德国瓦尔德布龙）、Nanodrop 1000分光光度计（Thermo Scientific，美国马萨诸塞州）对所有样本的RNA质量和浓度进行验证。

微流控miRNA微阵列 微阵列实验采用定制化Geniom生物芯片（febit公司）完成。使用Geniom One设备（febit公司）结合febit标准短链寡核苷酸合成试剂盒，合成定制化微阵列。在Geniom One仪器中，通过数字微镜器件进行光激活原位寡核苷酸合成，反应载体为玻璃-硅-玻璃夹层结构的活化三维载体（即生物芯片）。采用标准DNA合成试剂与带有5’光不稳定保护基团的3’-亚磷酰胺，在一个生物芯片的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，需对玻璃表面进行间隔基涂层活化，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]。本研究使用两种不同的微阵列设计：第一种仅包含399个经miRDeep预测的成熟miRNA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（spike-in control）；第二种设计包含1117个位点，设置3次重复，涵盖鉴定得到的保守型猪成熟miRNA序列（其中18个可比对至猪基因组，另外124个无法比对）、74个已知猪miRNA、885个来自miRBase数据库（版本13.0）的人类miRNA，以及16个spike-in对照。 微阵列实验中，独立提取4只仔猪不同肠道位点的总RNA样本，并制备相应的总RNA混合池。所有混合池均再次通过Agilent 2100生物分析仪和RNA 6000 Nano试剂盒，按照制造商的标准操作流程进行质量和浓度检测。每个微阵列实验使用250ng总RNA，悬浮于25μl febit专用miRNA杂交缓冲液中。使用Geniom RT分析仪（febit公司）在42℃下自动进行16小时杂交。经过严格洗涤后，采用微流控延伸实验对miRNA进行生物素化核苷酸标记。洗涤完成后，通过链霉亲和素-藻红蛋白检测生物素化核苷酸，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处理校正芯片间的差异。