Expression profile analysis of circular RNAs during embryonic lung development of C57BL/6 mice

Objectives: The contribution of circular RNAs (circRNAs) in the regulation of embryonic lung development is not yet clear. This study aims to uncover the differentially expressed circRNAs in mouse embryonic lung tissue. Methods: High-throughput sequencing (HTS) was used to determine the cirRNA expression profiles at four time points of mouse embryonic lung tissue [embryonic (E) Day 14.5 (E14.5/S1), (E16.5/S2), (E18.5/S3), and newborn (N) Day 7.5 (N7.5/S4)]. CircRNAs that were significantly differential were subjected to bioinformatic analysis and functional prediction for their host genes. Finally, quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was conducted to examine their levels. Results: Dynamically expressed circRNAs in the mouse embryonic lung tissue were identified. After that, a total of 231 circRNAs differentially expressed between the three groups (S1 vs. S2, S1 vs. S3 and S1 vs. S4) in embryonic lung tissue (fold change = 2.0; p < 0.05). These circRNAs were subjected to bioinformatic analysis to predict their potential biological functions. Finally, six circRNAs (circRNA3_Ttn, circRNA56_Galnt18, circRNA38_Alg12, circRNA42_Filip1l, circRNA43_Ptprm, and circRNA57_Ncoa3) that were further validated using qRT-PCR. Conclusions: These findings illustrate the vital functions of circRNAs in mouse embryonic lung development. Combined with our results and previous researches, circRNAs may serve as potential miRNA sponges in the embryonic lung development. Our research is conducive to further explore the development of embryonic lungs, and the dynamic expressed circRNAs may be novel molecules that regulate mouse lung development. Overall design: High-throughput sequencing was conducted assess the differentially expressed circRNAs in embryonic lung tissues divided into four different groups: pseudo glandular phase (S1), canalicular phase (S2), terminal sac phase (S3), and alveolar phase (S4) .

研究目的：环状RNA（circular RNAs, circRNAs）在胚胎肺发育调控中的作用尚未明确。本研究旨在揭示小鼠胚胎肺组织中差异表达的环状RNA。
研究方法：采用高通量测序（High-throughput sequencing, HTS）检测小鼠胚胎肺组织四个时间点的circRNA表达谱，分别为胚胎第14.5天（E14.5/S1）、胚胎第16.5天（E16.5/S2）、胚胎第18.5天（E18.5/S3）以及新生第7.5天（N7.5/S4）。对筛选得到的差异表达circRNA进行宿主基因的生物信息学分析与功能预测。最后通过实时定量逆转录聚合酶链反应（quantitative real-time reverse transcriptase polymerase chain reaction, qRT-PCR）验证其表达水平。
研究结果：本研究鉴定出小鼠胚胎肺组织中动态表达的circRNA。经筛选，胚胎肺组织中三组对照（S1 vs. S2、S1 vs. S3及S1 vs. S4）共获得231个差异表达circRNA（差异倍数=2.0；p < 0.05）。对上述circRNA进行生物信息学分析以预测其潜在生物学功能。最终选取6个circRNA（circRNA3_Ttn、circRNA56_Galnt18、circRNA38_Alg12、circRNA42_Filip1l、circRNA43_Ptprm及circRNA57_Ncoa3）通过qRT-PCR进行了进一步验证。
研究结论：本研究结果阐明了circRNA在小鼠胚胎肺发育中的关键功能。结合本研究结果与既往研究，circRNA可能作为胚胎肺发育过程中的潜在miRNA海绵（miRNA sponge）。本研究有助于进一步探究胚胎肺发育的机制，且动态表达的circRNA或可成为调控小鼠肺发育的新型分子靶点。
整体实验设计：本研究通过高通量测序评估分为四个不同发育阶段的胚胎肺组织中的差异表达circRNA，四个阶段分别为假腺期（pseudo glandular phase, S1）、小管期（canalicular phase, S2）、终末囊泡期（terminal sac phase, S3）以及肺泡期（alveolar phase, S4）。