circRNA-associated ceRNA network construction reveals the circRNAs involved in the progression and prognosis of breast cancer

Recently, increasing evidences show that circular RNAs (circRNAs) are important regulators of various diseases, especially cancer. However, the regulatory role and the potential mechanism of action of circRNAs in breast cancer remain largely unknown. In this study, weighted gene co-expression network analysis was conducted with the differentially expressed miRNAs and mRNAs in breast cancer from The Cancer Genome Atlas database to identify the key modules associated with the carcinogenesis of breast cancer. In the significant turquoise and brown modules, 22 miRNAs and 1877 mRNAs were identified, respectively. Then, We compared and predicted the target genes and performed survival analysis to identify the miRNAs and mRNAs related to the prognosis of breast cancer. A circRNA-related competitive endogenous RNA network was identified by database co-screening, and deleted in liver cancer 1 (DLC1) was identified as a key gene. Finally, to assess how genes in key modules and key genes contribute to the development of breast cancer, relevant pathway information was obtained through DAVID and Gene Set Enrichment Analysis. These data demonstrated that three circRNAs (hsa-circ-0083373, hsa-circ-0083374, and hsa-circ-0083375) that regulate DLC1 expression via hsa-mir-511 and are involved in the pathogenesis and development of breast cancer.     

PGC-Enriched miRNAs Control Germ Cell Development

Non-coding microRNAs (miRNAs) regulate the translation of target messenger RNAs (mRNAs) involved in the growth and development of a variety of cells, including primordial germ cells (PGCs) which play an essential role in germ cell development. However, the target mRNAs and the regulatory networks influenced by miRNAs in PGCs remain unclear. Here, we demonstrate a novel miRNAs control PGC development through targeting mRNAs involved in various cellular pathways. We reveal the PGC-enriched expression patterns of nine miRNAs, including miR-10b, -18a, -93, -106b, -126-3p, -127, -181a, -181b, and -301, using miRNA expression analysis along with mRNA microarray analysis in PGCs, embryonic gonads, and postnatal testes. These miRNAs are highly expressed in PGCs, as demonstrated by Northern blotting, miRNA in situ hybridization assay, and miRNA qPCR analysis. This integrative study utilizing mRNA microarray analysis and miRNA target prediction demonstrates the regulatory networks through which these miRNAs regulate their potential target genes during PGC development. The elucidated networks of miRNAs disclose a coordinated molecular mechanism by which these miRNAs regulate distinct cellular pathways in PGCs that determine germ cell development.     

Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.     

Investigation of circular RNAs and related genes in pulmonary fibrosis based on bioinformatics analysis

Pulmonary fibrosis is a lethal inflammatory disease. In this study, we aimed to explore the potential-related circular RNAs (circRNAs) and genes that are associated with pulmonary fibrosis. Pulmonary fibrosis rat models were constructed and the fibrosis deposition was detected using hematoxylin and eosin and Masson staining. The differentially expressed circRNAs were obtained through RNA sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were further performed to uncover the key function and pathways in pulmonary fibrosis. The interaction networks between circRNAs and their downstream micro RNAs (miRNAs) and genes were constructed by Cytoscape Software. The quantitative polymerase chain reaction was performed to validate the expression of 10 candidate circRNAs and five of them were performed ringwise sequencing in pulmonary fibrosis rats. We further selected five candidate circRNAs target miRNAs and messenger RNAs and validated by real-time polymerase chain reaction. The pulmonary fibrosis models were successfully constructed according to the pathological examination. circRNAs were differentially expressed between the pulmonary fibrosis and normal pulmonary tissues. GO analysis verified that the differentially expressed circRNAs were significantly clustered in the cellular component, molecular function, and biological process. In the KEGG analysis, circRNAs were enriched in the following pathways: antigen processing and presentation, phagosome, PI3K-AKt signaling pathway, HTLV-I infection, and Herpes simplex infection. After validation in pulmonary fibrosis rat models, it was found that five of those circRNAs (chr9:113534327|113546234 [down], chr1:200648164|200672411 [down], chr5:150850432|150865550 [up], chr20:14319170|14326640 [down], and chr10:57634023|57634588 [down]) showed a relatively consistent trend with predictions. Validation of these circRNAs target miRNAs and genes showed that chr9:113534327|113546234, chr20:14319170|14326640, and chr10:57634023|57634588 were implicated in Notch1 activated transforming growth factor-β (TGF-β) signaling pathway. The study demonstrated that a series of circRNAs are differentially expressed in pulmonary fibrosis rats. These circRNAs, especially TGF-β- and Notch1-related circRNAs might play an important role in regulating pulmonary fibrogenesis.     

An evaluation of gastric adenocarcinoma-associated CircRNAs based on microarray meta-analysis and ceRNA networks

Gastric cancer is the fourth leading cause of cancer-related mortality and one of the most commonly diagnosed malignancies worldwide. Gastric adenocarcinoma (GAC) accounts for the majority of gastric cancer cases. Circular RNAs (circRNAs) have been shown to be associated with carcinogenesis and cancer progression. This research aims to investigate GAC-associated circRNAs and the underlying mechanisms of circRNA-miRNA-mRNA networks in the development and progression of GAC. Differentially expressed miRNAs and mRNAs (DEMs and DEGs) were identified in Gene Expression Omnibus (GEO) microarray datasets using the R package Limma. A microarray meta-analysis was performed to identify potential GAC-associated circRNAs with high statistical power, resulting in 13 up-regulated and 19 down-regulated circRNAs. CircRNA-miRNA-mRNA networks were constructed by combining predicted and experimentally validated databases and considering differentially expressed miRNAs and mRNAs. The constructed ceRNA networks revealed the potential regulatory effect of hsa_circ_0002019 and hsa_circ_0074736 on key survival-related genes. The expression levels of these two circRNAs were measured in plasma samples from GAC patients and healthy controls using SYBR Green-based real-time PCR. Axon guidance, cellular senescence, AGE-RAGE signaling pathway in diabetic complications, and AMPK signaling pathway were among the major significant (P-value <0.05) enriched pathways of "main mRNAs" in the constructed ceRNA networks. In conclusion, we identified strongly correlated circRNAs and their likely mechanisms of action in GAC, which may improve the knowledge of regulatory networks underlying GAC formation and contribute to developing better strategies for early diagnosis, prognosis, and treatment.     

Cigarette smoke extract alters genome‐wide profiles of circular RNAs and mRNAs in primary human small airway epithelial cells

As a novel kind of non‐coding RNA, circular RNAs (circRNAs) were involved in various biological processes. However, the role of circRNAs in the developmental process of chronic obstructive pulmonary disease (COPD) is still unclear. In the present study, by using a cell model of COPD in primary human small airway epithelial cells (HSAECs) treated with or without cigarette smoke extract (CSE), we uncovered 4,379 previously unknown circRNAs in human cells and 903 smoke‐specific circRNAs, with the help of RNA‐sequencing and bioinformatic analysis. Moreover, 3,872 up‐ and 4,425 down‐regulated mRNAs were also identified under CSE stimulation. Furthermore, a putative circRNA‐microRNA‐mRNA network was constructed for in‐depth mechanism exploration, which indicated that differentially expressed circRNAs could influence expression of some key genes that participate in response to pentose phosphate pathway, ATP‐binding cassette (ABC) transporters, glycosaminoglycan biosynthesis pathway and cancer‐related pathways. Our research indicated that cigarette smoke had an influence on the biogenesis of circRNAs and mRNAs. CircRNAs might be involved in the response to CSE in COPD through the circRNA‐mediated ceRNA networks.     

小鼠正常黑素细胞和小鼠B16黑素瘤细胞的环状RNAs表达谱

黑素瘤是一种多发于皮肤的恶性肿瘤,因其侵袭性强,预后差等特点一直是科研人员关注的热点。环状RNAs(circRNAs)是一种新型内源性非编码RNA,广泛参与动物生长发育、细胞分化和信号转导等生理过程,但circRNAs在黑素瘤细胞内的分子机制尚未被充分解析。本研究以小鼠(C57BL/6J)正常黑素细胞及B16黑素瘤细胞为研究对象,采用二代测序技术分析两种细胞间circRNAs表达特性。测序结果显示,小鼠正常黑素细胞和黑素瘤细胞中共有851个circRNAs,其中195个差异表达circRNAs(DECs)。GO及KEGG数据库注释发现,DECs的来源基因主要参与细胞周期(cell cycle)、紧密结合(tight junction)、Rap1 信号通路(Rap1 signaling pathway)、TGF-beta 信号通路(TGF-beta signaling pathway)等与细胞增殖、迁移相关的信号通路;探究发现,黑素瘤细胞中显著性高表达的circE2F5(circ-3:14578602|14606309)通过上调E2F5的表达促进黑素瘤细胞增殖。circRNA靶基因预测发现,73个DECs 存在miRNA的结合位点,多个潜在靶向miRNAs参与黑素细胞增殖和迁移等过程。本研究通过对DECs来源基因功能注释、DECs靶向miRNAs预测,获得多个与黑素瘤细胞增殖和迁移相关的DECs,期望为黑素瘤研究提供新的见解。     

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。     

Changing expression profiles of mRNA,lncRNA, circRNA,and miRNA in lung tissue reveal the pathophysiological of bronchopulmonary dysplasia (BPD) in mouse model

New perinatal care technologies have improved the survival rate of preterm neonates, but the prevalence of bronchopulmonary dysplasia (BPD), one of the most intractable problems in neonatal intensive care unit (NICU), remains unchanged. In present study, high-throughput sequencing (HTS) was performed to detect the expression profiles of long noncoding RNAs (lncRNAs), messenger RNAs (mRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs) in hyperoxia-induced BPD mouse model. Significant differentially expressed RNAs were selected and clustered between the BPD group and the control group. The results revealed that expressions of 1778 lncRNAs, 1240 mRNAs, 97 circRNAs, and 201 miRNAs were significantly altered in the BPD group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to predict the potential functions of differentially expressed RNAs. lncRNA-mRNA and circRNA-miRNA coexpression networks were constructed to detect their association with the pathogenesis of BPD. Our study provides a systematic perspective on the potential function of RNAs during BPD.