Characteristics of circular RNA expression of pulmonary macrophages in mice with sepsis‐induced acute lung injury

Circular RNAs (circRNAs) make up a large class of non‐coding RNAs and play important roles in the pathology of a variety of diseases. However, their roles in pulmonary macrophage polarization after sepsisinduced lung injury is unknown. In this study, mice were divided into two groups: Sham control group and cecal ligation and puncture (CLP)‐induced ALI group. Macrophages were isolated from lung homogenates 24 hours after SCLP/CLP. We started with RNA‐seq of circRNA changes in macrophages and validated by RT‐PCR in the following experiments. A total of 4318 circRNAs were detected in the two groups. Of these, 11 and 126 circRNAs were found to be significantly upregulated and downregulated, respectively, compared to the control (p≤0.05, Fold Change ≥2). Differentially expressed circRNAs with a high foldchange (fold‐change >4, P<0.05) were selected for validation by qRT‐PCR, 10 of which were verified. Furthermore, the most differentially expressed circRNAs within all the comparisons were annotated in detail with circRNA/miRNA interaction information using miRNA target prediction software. The network of circRNA‐miRNA‐mRNA was illustrated by cytoscape software. Gene ontology analyses indicated the upregulated circRNAs were involved in the multiple biological functions such as regulation of mitochondrion distribution and Notch binding, while the down‐regulated circRNAs mainly involved in the biological process as histone H3K27 methylation. KEGG pathway analysis revealed TGF‐beta signaling pathway was related to the upregulated circRNAs. The present study provides a novel insight into the roles of circRNAs in pulmonary macrophage differentiation and polarization post septic lung injury.     

Has_circ_0055625 from circRNA profile increases colon cancer cell growth by sponging miR-106b-5p

Circular RNAs (circRNA) are endogenous noncoding RNAs and play important roles in cancer; however, the roles of circRNAs in colon cancer are far from clear. The circRNA expression profile in colon cancer tissues was analyzed by microarray. The data from microarray showed that there were 198 upregulated and 136 downregulated circRNAs in colon cancer tissues. Among the top 10 upregulated circRNAs, hsa_circ_0055625 (circ_0055625) was confirmed to be significantly upregulated in colon cancer tissues. Further analysis demonstrated that circ_0055625 might get involved in the pathogenesis of colon cancer by functioning as miRNA sponges and performed bioinformatics analysis of the predicted circ_0055625/miR-106b-5p (miR-106b)/ITGB8 network. Moreover, we found that circ_0055625 expression was associated with pathological TNM stage and metastasis. These data indicated that circ_0055625/miR-106b/ITGB8 played a role in promoting tumor growth and metastasis, which suggested that circ_0055625 was a potential biomarker of colon cancer.     

意大利蜜蜂工蜂中肠的环状RNA及其调控网络分析

【目的】环状RNA(circRNA)在可变剪接、转录调控和来源基因的表达调控等方面具有重要功能。本研究旨在分析意大利蜜蜂Apis mellifera ligustica工蜂中肠circRNA的数量、种类、结构特征和作用,并通过构建和分析circRNA的调控网络探索circRNA的调控功能。【方法】在实验室条件下人工饲养意大利蜜蜂工蜂,利用circRNA-seq技术对意大利蜜蜂7和10日龄成年工蜂中肠样品进行深度测序。利用find_circ软件从质控后的数据中预测circRNA。通过BLAST比对GO和KEGG数据库,对circRNA的来源基因进行功能和代谢通路注释。利用TargetFinder软件预测circRNA靶向结合的miRNA及miRNA靶向结合的mRNA,通过Cytoscape v.3.2.1软件对circRNA-miRNA和circRNA-miRNA-mRNA调控网络进行构建及可视化。通过设计背靠背引物和线性扩增引物RT-PCR对预测出的circRNA进行验证。【结果】意大利蜜蜂工蜂中肠样品的测序平均得到136 463 071条clean reads,去除rRNA后各样品的anchor reads均在136 779 122条及以上。共预测出10 833个circRNA,长度主要介于15~1 000 nt;上述circRNA的类型丰富,其中已注释的外显子circRNA数量最多,分布在西方蜜蜂1号染色体的circRNA数量最多,其次为8号染色体。CircRNA的来源基因可注释到包括结合、细胞进程和细胞在内的45个GO条目,以及包括内吞作用、内质网蛋白加工及核糖体在内的121条KEGG代谢通路,表明circRNA在意大利蜜蜂工蜂中肠的生长、发育、新陈代谢和细胞生命活动等生物学过程中发挥重要作用。进一步构建circRNA-miRNA和circRNA-miRNA-mRNA调控网络,分析结果显示部分circRNA可能作为竞争性内源RNA吸附结合microRNA,从而调控基因的表达水平。最后,对随机选择的3个circRNA的RT-PCR结果验证了其真实存在。【结论】本研究对意大利蜜蜂工蜂中肠中的circRNA进行预测、分析及鉴定。研究结果提供了中肠circRNA的数量、种类、结构特征、作用和调控网络的信息,揭示了circRNA可能通过作用于来源基因和作为竞争性内源RNA在意大利蜜蜂工蜂中肠的生长发育和免疫防御中发挥作用,为深入研究circRNA在意大利蜜蜂中肠发育及胁迫响应过程中的功能奠定了基础。     

Circulating microRNA signature of steroid‐induced osteonecrosis of the femoral head

Objectives

Steroid‐induced osteonecrosis of the femoral head (ONFH) is a common orthopaedic disease of which early detection remains clinically challenging. Accumulating evidences indicated that circulating microRNAs (miRNAs) plays vital roles in the development of several bone diseases. However, the association between circulating miRNAs and steroid‐induced ONFH remains elusive.

Materials and methods

miRNA microarray was performed to identify the differentially abundant miRNAs in the serums of systemic lupus erythematosus (SLE) patients with steroid‐induced ONFH as compared with SLE control and healthy control group. We predicted the potential functions of these differentially abundant miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and reconstructed the regulatory networks of miRNA‐mRNA interactions.

Results

Our data indicated that there were 11 differentially abundant miRNAs (2 upregulated and 9 downregulated) between SLE‐ONFH group and healthy control group and 42 differentially abundant miRNAs (14 upregulated and 28 downregulated) between SLE‐ONFH group and SLE control group. We also predicted the potential functions of these differentially abundant miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and reconstructed the regulatory networks of miRNA‐mRNA interactions.

Conclusions

These findings corroborated the idea that circulating miRNAs play significant roles in the development of ONFH and may serve as diagnostic markers and therapeutic targets.     

Dysregulated circRNAs in plasma from active tuberculosis patients

Endogenous circular RNAs (circRNAs) have been reported in various diseases. However, their role in active TB remains unknown. The study was aimed to determine plasma circRNA expression profile to characterize potential biomarker and improve our understanding of active TB pathogenesis. CircRNA expression profiles were screened by circRNA microarrays in active TB plasma samples. Dysregulated circRNAs were then verified by qRT‐PCR. CircRNA targets were predicted based on analysis of circRNA‐miRNA‐mRNA interaction. GO and KEGG pathway analyses were used to predict the function of circRNA. ROC curve was calculated to evaluate diagnostic value for active TB. A total of 75 circRNAs were significantly dysregulated in active TB plasma. By further validation, hsa_circRNA_103571 exhibited significant decrease in active TB patients and showed potential interaction with active TB‐related miRNAs such as miR‐29a and miR‐16. Bioinformatics analysis revealed that hsa_circRNA_103571 was primarily involved in ras signalling pathway, regulation of actin cytoskeleton, T‐ and B‐cell receptor signalling pathway. ROC curve analysis suggested that hsa_circRNA_103571 had significant value for active TB diagnosis. Circulating circRNA dysregulation may play a role in active TB pathogenesis. Hsa_circRNA_103571 may be served as a potential biomarker for active TB diagnosis, and hsa_circRNA_103571‐miRNA‐mRNA interaction may provide some novel mechanism for active TB.     

结节性甲状腺肿circRNA-miRNA-mRNA调控网络的构建

基于生物信息分析筛选结节性甲状腺肿中差异表达的环状RNA(circRNA),并揭示circRNA-miRNA-mRNA调控网络在结节性甲状腺肿中的作用。从GEO数据库中检索结节性甲状腺肿组织基因芯片数据,利用R软件筛选出差异表达的circRNA。联合多个生物信息数据库预测差异表达circRNA下游的miRNA及mRNA, 并对靶mRNA进行GO及KEGG富集分析。利用STRING在线数据库及Cytoscape软件筛选核心基因。确定了2个circRNA,42个miRNA及546个mRNA。GO及KEGG富集分析表明靶mRNA主要涉及细胞生长及基因表达调控过程。基于Cytoscape软件筛选出了14个核心基因(SP1、IGF1R、RPS6KB1、SMAD2、SMAD3、SMAD4、VEGFA、CCND1、CDK2、HSPA4、HIF1A、CREB1,NR3C1和STAT5A)。最终基于2个circRNA、11个miRNA和14个核心mRNA构建了circRNA-miRNA-mRNA调控网络。结节性甲状腺肿组织中异常表达的circRNA及相关的circRNA-miRNA-mRNA调控网络可能成为结节性甲状腺肿诊断与治疗的新靶点。     

Cover Image,Volume 120, Number 8, August 2019

Circular RNAs (circRNAs) are novel noncoding RNAs and play crucial roles in various biological processes. However, little is known about the functions of circRNAs in osteogenic differentiation. The current study aimed to investigate the differential expression of circRNAs in rat dental follicle cells (rDFCs) during osteogenic differentiation, identified by RNA high-throughput sequencing and quantitative real-time polymerase chain reaction. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to further explore the biofunctions of circRNA biofunctions. Two hundred sixty-six differentially-expressed circRNAs that are involved in several important signaling pathways, including mitogen-activated protein kinases (MAPK) and transforming growth factor-β (TGF-β) signaling pathways were revealed. Among these, circFgfr2 and its predicted downstream targets, miR-133 and BMP6 (bone morphogenetic protein-6), were identified both in vivo and in vitro. For further validation, circFgfr2 was overexpressed in rDFCs, the results showed that the expression of miR-133 was downregulated and the expression of BMP6 was upregulated. Taken together, the results revealed the circRNA expression profiles and indicated the importance of circRNAs of rDFCs. In addition, circFgfr2 might promote osteogenesis by controlling miR-133/BMP6, which is a potential new target for the manipulation of tooth regeneration and bone formation.     

Profiling and integrated analysis of differentially expressed circRNAs as novel biomarkers for breast cancer

Breast cancer (BC) is a globally common cancer with the highest and increasing morbidity and mortality among females. Novel biomarkers are warranted to be discovered for the early detection, treatment, and prognosis of BC. In this study, we investigated the profiles of differentially expressed (DE) circular RNAs (circRNAs) by competing endogenous RNAs (ceRNA) microarray to construct a genome-wide circRNA profile. Then, we performed Gene Ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis of the host genes (HGs) of circRNAs. A total of 4,370 DE circRNAs were detected and GO and KEGG analysis showed that they were significantly associated with cell cycle, DNA replication, BC, and familial BC. We validated the differential circRNAs and relevant HGs through quantitative real-time polymerase chain reaction and constructed a putative circRNA–microRNA–messenger RNA regulatory network. Eight circRNAs, including hsa_circ_0069094, hsa_circ_0062558, hsa_circ_0074026, hsa_circ_0079876, hsa_circ_0017536, hsa_circ_0023302, hsa_circ_0017650, and hsa_circ_0017545, were validated significantly DE in BC tissue and associated with TNM staging, lymph node infiltration, and Ki67. Hsa_circ_0069094, hsa_circ_0079876, hsa_circ_0017650, and hsa_circ_0017526 were upregulated in plasma. This study revealed the general expression characteristics of specific DE circRNAs in BC and hsa_circ_0069094, hsa_circ_0079876, hsa_circ_0017650, and hsa_circ_0017526 might be promising candidate targets.     

CircRNA expression profiles and function prediction in peripheral blood mononuclear cells of patients with acute ischemic stroke

Most circular RNAs (circRNAs) belong to a novel class of noncoding RNAs that are produced by back-splicing reactions, and they regulate physiological and pathophysiological processes in human disease. Although circRNA expression has been shown to be altered in the ischemic cerebral tissue in animal studies, the expression profile of circRNA in the patients with acute ischemic stroke (AIS) has not been investigated to date. In this investigation, high-throughput sequencing was carried out to compare the circRNA expression of peripheral blood mononuclear cells (PBMCs) from five patients with AIS and five healthy subjects. A total of 521 circRNAs were expressed differentially between the patients with AIS and healthy controls (p < .05, fold difference ≥2) including 373 upregulated circRNAs and 148 downregulated circRNAs in patients with AIS compared to controls. Thirteen candidate circRNAs were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics analyses showed that these differentially expressed circRNAs were highly conserved, as well as eight circRNAs that were confirmed by qRT-PCR containing binding sites to multiple microRNAs. Kyoto Encyclopedia of Genes and Genomes pathway enrichment and gene ontology analyses indicated that the aberrantly expressed circRNAs participated in many pathophysiological processes of AIS, especially regarding inflammation and immunity. In conclusion, patients with AIS differentially express certain circRNAs in PBMCs, which may be diagnostic biomarkers or potential therapeutic targets.     

A novel strategy of identifying circRNA biomarkers in cardiovascular disease by meta-analysis

Circular RNAs (circRNAs) are stable and abundantly expressed in vivo but are abnormally expressed in several diseases. This study aimed to identify circRNAs acting as potential biomarkers for cardiovascular disease (CVD). Research were retrieved from the articles published by September 2018 in eight databases to compare circRNA expression profiles between CVD and non-CVD in human and animal models. Meta-analysis under a random effects model was conducted. Subgroup analysis of tissue, species, and disease-specific circRNAs was examined. Sensitivity analysis was performed to explain the uncertainty among all studies. Diagnostic accuracy of circRNAs in CVD was analyzed to testify the discriminative ability. Bioinformatics analysis including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted. Among 6,284 differentially expressed circRNAs from 32 original studies, only 322 circRNAs were reported in three or more studies. The meta-analysis identified 63 significantly dysregulated circRNAs, 44 upregulated and 19 downregulated. Among the tissue-specific or disease-specific circRNAs identified in the subgroup analysis, two circRNAs (circCDKN2BAS and circMACF1) showed the potential to be circulating biomarkers for CVD. Sensitivity analysis demonstrated 69% of circRNAs were in conformity with the overall analysis. The pooled diagnostic odds ratio was 2.94 (95% confidence interval [CI], 2.35–3.58), and the overall area under the curve value was 0.86 (95% CI, 0.83–0.89). GO and KEGG enrichment analyses indicated that the target genes of circRNAs participate in cardiogenesis-related processes and pathways. This study demonstrates circRNAs have a high diagnostic value as potential biomarkers for CVD, and two candidate circRNAs, circCDKN2BAS and circMACF1, are potential circulating biomarkers for CVD diagnosis and treatment.     

Altered plasma exosome miRNAs and novel potential biomarkers in pediatric fulminant myocarditis

Previous studies have indicated that exosome-mediated intercellular microRNAs (miRNA) can influence fulminant myocarditis (FM) pathogenesis between immune and cardiac cells. This study explored plasma exosome miRNA profile in pediatric FM using a small RNA microarray. As per our analysis, we observed the differential expression of 266 miRNAs, including 197 upregulated and 69 downregulated candidate genes. Differentially expressed mRNAs in pediatric FM patients' peripheral blood mononuclear cells (PBMCs) were intersected with miRNA target genes predicting tools to screen for FM-specific target genes. The hub genes and their biological and mechanistic pathways related to inflammation and/or the immune system were identified. CeRNA networks of lncRNAs, circRNAs, miRNAs, and mRNAs between cardiomyocytes and PBMCs were finally established. Furthermore, we verified that hsa-miR-146a-5p, hsa-miR-23a-3p, and hsa-miR-27a-3p had higher expression levels in exosomes of pediatric FM patients by qRT-PCR, and hsa-miR-146a-5p shown high sensitivities and specificities for FM diagnosis. Overall, the results demonstrate that the exosome miRNAs play a regulatory role between immune and cardiac cells and provide research targets.