High throughput sequencing of whole transcriptome and construct of ceRNA regulatory network in RD cells infected with enterovirus D68.

With the advancement of sequencing technologies, a plethora of noncoding RNA (ncRNA) species have been widely discovered, including microRNAs (miRNAs), circular RNAs (circRNAs), and long ncRNAs (lncRNAs). However, the mechanism of these non-coding RNAs in diseases caused by enterovirus d68 (EV-D68) remains unclear. The goal of this research was to identify significantly altered circRNAs, lncRNAs, miRNAs, and mRNAs pathways in RD cells infected with EV-D68, analyze their target relationships, demonstrate the competing endogenous RNA (ceRNA) regulatory network, and evaluate their biological functions. The total RNAs were sequenced by high-throughput sequencing technology, and differentially expressed genes between control and infection groups were screened using bioinformatics method. We discovered the targeting relationship between three ncRNAs and mRNA using bioinformatics methods, and then built a ceRNA regulatory network centered on miRNA. The biological functions of differentially expressed mRNAs (DEmRNAs) were discovered through GO and KEGG enrichment analysis. Create a protein interaction network (PPI) to seek for hub mRNAs and learn more about protein-protein interactions. The relative expression was verified using RT-qPCR. The effects of Fos and ARRDC3 on virus replication were confirmed using RT-qPCR, virus titer (TCID50/ml), Western blotting. 375 lncRNAs (154 upregulated and 221 downregulated), 33 circRNAs (32 upregulated and 1 downregulated), 96 miRNAs (49 upregulated and 47 downregulated), and 239 mRNAs (135 upregulated and 104 downregulated) were identified as differently in infected group compare to no-infected group. A single lncRNA or circRNA can be connected with numerous miRNAs, which subsequently coregulate additional mRNAs, according to the ceRNA regulatory network. The majority of DEmRNAs were shown to be connected to DNA binding, transcription regulation by RNA polymerase II, transcription factor, MAPK signaling pathways, Hippo signal pathway, and apoptosis pathway, according to GO and KEGG pathway enrichment analysis. The hub mRNAs with EGR1, Fos and Jun as the core were screened through PPI interaction network. We preliminarily demonstrated that the Fos and ARRDC3 genes can suppress EV-D68 viral replication in order to further verify the results of full transcriptome sequencing. The results of whole transcriptome analysis after EV-D68 infection of RD cells were first reported in this study, and for the first time, a ceRNA regulation network containing miRNA at its center was established for the first time. The Fos and ARRDC3 genes were found to hinder viral in RD cells. This study establishes a novel insight host response during EV-D68 infection and further investigated potential drug targets.

Si J ，Tang X ，Xu L ，Fu H ，Li H ，He Y ，Bao J ，Tang J ，Li A ，Lu N ，Yang C ... -  《Virology Journal》

Construction and functional enrichment analysis of the competitive endogenous RNA regulatory network for nonarteritic anterior ischemic optic neuropathy based on high-throughput sequencing.

Based on the transcriptome high-throughput sequencing of nonarteritic anterior ischemic optic neuropathy (NAION), this study constructed a competitive endogenous RNA (ceRNA) network, enriched and analyzed it, screened long noncoding (lnc)RNAs and circular (circ)RNAs that may participate in the competitive endogenous mechanism in NAION, and inferred its function. Four milliliters of peripheral blood from NAION patients and the control group was extracted from clinical samples, transcriptome high-throughput sequencing was performed, and the sequencing data were visualized. Based on the principle of the ceRNA network, the lncRNA-micro (mi)RNA-messenger (m)RNA interaction axis and circRNA-miRNA‒mRNA interaction axes were constructed. The differentially expressed genes (DEGs) in the interaction axis were enriched and analyzed by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the functions and signalling pathways of lncRNAs and circRNAs in the interaction network were speculated. Fifty-one circRNAs were differentially expressed in the sequencing data: 25 were upregulated, and 26 were downregulated. For 996 differentially expressed lncRNAs, 317 were upregulated and 679 were downregulated, and for 1161 differentially expressed mRNAs, 698 were upregulated and 463 were downregulated. Thirty-three differentially expressed miRNAs, upregulated miRNA 18 and downregulated miRNA 15 were identified. After screening, 13 coexpressed mRNAs, 15 lncRNAs, and 3 miRNAs were finally constructed in the lncRNA-miRNA-mRNA network, and the circRNA-miRNA-mRNA network was constructed by 159 mRNAs, 26 miRNAs, and 34 circRNAs. In the lncRNA network, GO enrichment analysis obtained 182 biological processes, 12 cell components and 38 molecular functions, which are related mainly to the regulation of the activity of proteins and enzymes such as cyclic nucleotide-dependent protein kinase activity and magnesium ion-dependent protein serine/threonine phosphatase activity. KEGG analysis involved mainly the forkhead box O (FoxO) signalling pathway, apelin signalling pathway, etc. In the circRNA enrichment results, 353 biological processes, 52 cell components, and 45 molecular functions were obtained, involving mainly calcium channel regulation, neutrophil activation, mRNA transport, and metabolism. KEGG mainly involved the Wnt signalling pathway, apelin signalling pathway, Hippo signalling pathway, oxytocin signalling pathway, etc. This paper provides a new idea for lncRNAs and circRNAs to mediate the occurrence and development of NAION through the ceRNA mechanism. This study lays a foundation for the in-depth study of the pathogenesis of NAION.

Zhang M ，Wang X 《-》

Whole transcriptome analysis of HCT-8 cells infected by Cryptosporidium parvum.

Cryptosporidium species are zoonotic protozoans that are important causes of diarrhoeal disease in both humans and animals. Non-coding RNAs (ncRNAs) play an important role in the innate immune defense against Cryptosporidium infection, but the underlying molecular mechanisms in the interaction between human ileocecal adenocarcinoma (HCT-8) cells and Cryptosporidium species have not been entirely revealed. The expression profiles of messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) in the early phase of infection of HCT-8 cells with Cryptosporidium parvum and at 3 and 12 h post infection were analyzed using the RNA-sequencing technique. The biological functions of differentially expressed RNAs (dif-RNAs) were discovered through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The targeting relationships between three ncRNAs and mRNAs were analyzed using bioinformatics methods, followed by building a competing endogenous RNA (ceRNA) regulatory network centered on miRNAs. After strictly filtering the raw data, our analysis revealed 393 dif-lncRNAs, 69 dif-miRNAs and 115 dif-mRNAs at 3 hpi, and 450 dif-lncRNAs, 129 dif-miRNAs, 117 dif-mRNAs and one dif-circRNA at 12 hpi. Of these, 94 dif-lncRNAs, 24 dif-miRNAs and 22 dif-mRNAs were detected at both post-infection time points. Eleven dif-lncRNAs, seven dif-miRNAs, eight dif-mRNAs and one circRNA were randomly selected and confirmed using the quantitative real-time PCR. Bioinformatics analyses showed that the dif-mRNAs were significantly enriched in nutritional absorption, metabolic processes and metabolism-related pathways, while the dif-lncRNAs were mainly involved in the pathways related to the infection and pathogenicity of C. parvum (e.g. tight junction protein) and immune-related pathways (e.g. cell adhesion molecules). In contrast, dif-miRNAs and dif-circRNA were significantly enriched in apoptosis and apoptosis-related pathways. Among the downregulated RNAs, the miRNAs has-miR-324-3p and hsa-miR-3127-5p appear to be crucial miRNAs which could negatively regulate circRNA, lncRNA and mRNA. The whole transcriptome profiles of HCT-8 cells infected with C. parvum were obtained in this study. The results of the GO and KEGG pathway analyses suggest significant roles for these dif-RNAs during the course of C. parvum infection. A ceRNA regulation network containing miRNA at its center was constructed for the first time, with hsa-miR-324-3p and hsa-miR-3127-5p being the crucial miRNAs. These findings provide novel insights into the responses of human intestinal epithelial cells to C. parvum infection.

Sun L ，Li J ，Xie F ，Wu S ，Shao T ，Li X ，Li J ，Jian F ，Zhang S ，Ning C ，Zhang L ，Wang R ... -  《Parasites & Vectors》

High-Throughput Sequencing and Exploration of the lncRNA-circRNA-miRNA-mRNA Network in Type 2 Diabetes Mellitus.

Long noncoding RNA (lncRNA) and circular RNA (circRNA) are receiving increasing attention in diabetes research. However, there are still many unknown lncRNAs and circRNAs that need further study. The aim of this study is to identify new lncRNAs and circRNAs and their potential biological functions in type 2 diabetes mellitus (T2DM). RNA sequencing and differential expression analysis were used to identify the noncoding RNAs (ncRNAs) and mRNAs that were expressed abnormally between the T2DM and control groups. The competitive endogenous RNA (ceRNA) regulatory network revealed the mechanism of lncRNA and circRNA coregulating gene expression. The biological functions of lncRNA and circRNA were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The candidate hub mRNAs were selected by the protein-protein interaction (PPI) network and validated by using the Gene Expression Omnibus (GEO) database. Differential expression analysis results showed that 441 lncRNAs (366 upregulated and 75 downregulated), 683 circRNAs (354 upregulated and 329 downregulated), 93 miRNAs (63 upregulated and 30 downregulated), and 2923 mRNAs (1156 upregulated and 1779 downregulated) were identified as remarkably differentially expressed in the T2DM group. The ceRNA regulatory network showed that a single lncRNA and circRNA can be associated with multiple miRNAs, and then, they coregulate more mRNAs. Functional analysis showed that differentially expressed lncRNA (DElncRNA) and differentially expressed circRNA (DEcircRNA) may play important roles in the mTOR signaling pathway, lysosomal pathway, apoptosis pathway, and tuberculosis pathway. In addition, PIK3R5, AKT2, and CLTA were hub mRNAs screened out that were enriched in an important pathway by establishing the PPI network. This study is the first study to explore the molecular mechanisms of lncRNA and circRNA in T2DM through the ceRNA network cofounded by lncRNA and circRNA. Our study provides a novel insight into the T2DM from the ceRNA regulatory network.

Yang F ，Chen Y ，Xue Z ，Lv Y ，Shen L ，Li K ，Zheng P ，Pan P ，Feng T ，Jin L ，Yao Y ... -  《-》

Identification of a Plasma Exosomal lncRNA- and circRNA-Based ceRNA Regulatory Network in Patients With Lung Adenocarcinoma.

Exosomes have been established to be enriched with various long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) that exert various biological effects. However, the lncRNA- and circRNA-mediated coexpression competing endogenous RNA (ceRNA) regulatory network in exosomes derived from the plasma of patients with lung adenocarcinoma (LUAD) remains elusive. This study enrolled nine patients with lung adenocarcinoma and three healthy individuals, and the differential expression of messenger RNAs (mRNAs), lncRNAs, and circRNAs was detected using microarray analysis, while microRNAs (miRNAs) were detected through RNA sequencing. Additionally, bioinformatics algorithms were applied to evaluate the lncRNA-miRNA-mRNAs/circRNA-miRNA-mRNA network. Differentially expressed cicRNAs were identified via quantitative reverse transcription polymerase chain reaction (RT-qPCR). A total of 1016 lncRNAs, 1396 circRNAs, 45 miRNAs, and 699 mRNAs were differentially expressed in the plasma exosomes of patients with LUAD compared with healthy controls. Among them, 881 lncRNAs were upregulated and 135 were downregulated, 916 circRNAs were upregulated while 480 were downregulated, 45 miRNAs were upregulated while none were downregulated, and 591 mRNAs were upregulated while 108 were downregulated (p ≤ 0.05, and fold change ≥ 2). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed the biological functions of differentially expressed RNAs. Meanwhile, the RNA networks displayed the regulatory relationship between dysregulated RNAs. Finally, RT-qPCR validated that the expression of circ-0033861, circ-0043273, and circ-0011959 was upregulated in the plasma exosome of patients with LUAD compared to healthy controls (p = 0.0327, p = 0.0002, p = 0.0437, respectively). This study proposed a newly discovered ncRNA-miRNA-mRNA/circRNA-miRNA-mRNA ceRNA network and identified that the expression of circulating circ-0033861, circ-0043273, and circ-0011959 was up-regulated in the plasma exosomes of patients with LUAD, offering valuable insights for exploring the potential function of exosomal noncoding RNA and identifying potential biomarkers for LUAD.

Zhu W ，Zhang H ，Tang L ，Fang K ，Lin N ，Huang Y ，Zhang Y ，Le H ... -  《-》