Susceptibility of bovine to SARS-CoV-2 variants of concern: insights from ACE2, AXL, and NRP1 receptors.

The possibilities of cross-species transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) between humans and important livestock species are not yet known. Herein, we used the structural and genetic alignment and surface potential analysis of the amino acid (aa) in angiotensin-converting enzyme 2 (ACE2), tyrosine kinase receptor UFO (AXL), and neuropilin 1 (NRP1) in different species with substantial public health importance. The residues interfacing with the N-terminal domain (NTD) or receptor-binding domain (RBD) of S were aligned to screen the critical aa sites that determined the susceptibility of the SARS-CoV-2 to the host. We found that AXL and NRP1 proteins might be used as the receptors of SARS-CoV-2 in bovines. However, ACE2 protein may not be considered to be involved in the cross-species transmission of SARS-CoV-2 VOCs in cattle because the key residues of the ACE2-S-binding interface were different from those in known susceptible species. This study indicated that emerging SARS-CoV-2 variants potentially expand species tropism to bovines through AXL and NRP1 proteins.

Design of a novel multiepitope vaccine against Chlamydia pneumoniae using the extracellular protein as a target.

Chlamydia pneumoniae (C. pneumoniae) infection in humans is universal and causes various respiratory infectious diseases, making a safe and effective preventive vaccine essential. In this study, a multi-epitope vaccine with CTLA-4 extracellular structure was constructed by an immunoinformatics approach. Since MOMP protein is the major extracellular protein in C. pneumoniae and has good immunogenicity and high conservation, we selected the MOMP protein of C. pneumoniae as the antigen target, predicted the T and B cell epitopes of the MOMP protein and then connected the CTLA-4 extracellular structure with the predicted dominant epitopes by various linkers to construct a multi-epitope vaccine. The biochemical characterization of the multi-epitope vaccine showed its immunogenicity and anti-allergic properties. The tertiary structure of this vaccine, along with molecular docking, molecular dynamics simulation, and principal component analysis, showed that the multi-epitope vaccine structure interacted with B7 (B7-1, B7-2) and toll-like receptors (TLR-2, TLR-4). Ultimately, the vaccine was cloned and effectively expressed in silico on an insect baculovirus expression vector (pFastBac1). These analyses showed that the designed vaccine could potentially target antigen-presenting cells and was immune to C. pneumoniae, which provided novel strategies for developing the vaccine.

Emerging SARS-CoV-2 variants of concern potentially expand host range to chickens: insights from AXL, NRP1 and ACE2 receptors.

BACKGROUND: The possibilities of cross-species transmission of SARS-CoV-2 variants of concern (VOCs) between humans and poultry species are unknown. The analysis of the structure of receptor was used to investigate the potential of emerging SARS-CoV-2 VOCs to expand species tropism to chickens based on the interaction between Spike (S) protein and tyrosine kinase receptor UFO (AXL), angiotensin-converting enzyme 2 (ACE2), and neuropilin 1 (NRP1) with substantial public health importance. METHODS: The structural and genetic alignment and surface potential analysis of the amino acid (aa) in ACE2, AXL, and NRP1 in human, hamster, mouse, mink, ferret, rhesus monkey and chickens were performed by Swiss-Model and pymol software. The critical aa sites that determined the susceptibility of the SARS-CoV-2 to the host were screened by aligning the residues interfacing with the N-terminal domain (NTD) or receptor-binding domain (RBD) of Spike protein. RESULTS: The binding modes of chickens AXL and ACE2 to S protein are similar to that of the ferret. The spatial structure and electrostatic surface potential of NRP1 showed that SARS-CoV-2 VOCs could not invade chickens through NRP1 easily. CONCLUSION: These results suggested that emerging SARS-CoV-2 VOCs potentially expand the host range to chickens mainly through ACE2 and AXL receptors, while NRP1 receptor may rarely participate in the future epidemic of coronavirus disease 2019 in chickens.

Aryl hydrocarbon receptor is a proviral host factor and a candidate pan-SARS-CoV-2 therapeutic target.

The emergence of a series of SARS-CoV-2 variants has necessitated the search for broad-spectrum antiviral targets. The aryl hydrocarbon receptor (AhR) senses tryptophan metabolites and is an immune regulator. However, the role of AhR in SARS-CoV-2 infection and whether AhR can be used as the target of antiviral therapy against SARS-CoV-2 and its variants are yet unclear. Here, we show that infection with SARS-CoV-2 activates AhR signaling and facilitates viral replication by interfering with IFN-I-driven antiviral immunity and up-regulating ACE2 receptor expression. The pharmacological AhR blockade or AhR knockout reduces SARS-CoV-2 and its variants' replication in vitro. Drug targeting of AhR with AhR antagonists markedly reduced SARS-CoV-2 and its variants' replication in vivo and ameliorated lung inflammation caused by SARS-CoV-2 infection in hamsters. Overall, AhR was a SARS-CoV-2 proviral host factor and a candidate host-directed broad-spectrum target for antiviral therapy against SARS-CoV-2 and its variants, including Delta and Omicron, and potentially other variants in the future.

Global expression and functional analysis of human piRNAs during HSV-1 infection.

Piwi-interacting RNAs (piRNAs) are a class of non-coding RNAs that play a key role in spermatogenesis. However, little is known about their expression characterization and role in somatic cells infected with herpes simplex virus type 1 (HSV-1). In this study, we systematically investigated the cellular piRNA expression profiles of HSV-1-infected human lung fibroblasts. Compared with the control group, 69 differentially expressed piRNAs were identified in the infection group, among which 52 were up-regulated and 17 were down-regulated. The changes in the expression of 8 piRNAs were further verified by RT-qPCR with a similar expression trend. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the target genes of piRNAs were mainly involved in antiviral immunity and various human disease-related signaling pathways. Furthermore, we tested the effects of four up-regulated piRNAs on viral replication by transfecting piRNA mimics. The results showed that the virus titers of the group transfected with piRNA-hsa-28,382 (alias piR-36,233) mimic decreased significantly, and that of the group transfected piRNA-hsa-28,190 (alias piR-36,041) mimic significantly increased. Overall, our results revealed the expression characteristics of piRNAs in HSV-1-infected cells. We also screened two piRNAs that potentially regulate HSV-1 replication. These results may promote a better understanding of the regulatory mechanism of pathophysiological changes induced by HSV-1 infection.

Metabolomics Profiles Reveal New Insights of Herpes Simplex Virus Type 1 Infection.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen that can cause significant morbidity, primarily facial cold sores and herpes simplex encephalitis. Previous studies have shown that a variety of viruses can reprogram the metabolic profiles of host cells to facilitate self-replication. In order to further elucidate the metabolic interactions between the host cell and HSV-1, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze the metabolic profiles in human lung fibroblasts KMB17 infected with HSV-1. The results showed that 654 and 474 differential metabolites were identified in positive and negative ion modes, respectively, and 169 and 114 metabolic pathways that might be altered were screened. These altered metabolites are mainly involved in central carbon metabolism, choline metabolism, amino acid metabolism, purine and pyrimidine metabolism, cholesterol metabolism, bile secretion, and prolactin signaling pathway. Further, we confirmed that the addition of tryptophan metabolite kynurenine promotes HSV-1 replication, and the addition of 25-Hydroxycholesterol inhibits viral replication. Significantly, HSV-1 replication was obviously enhanced in the ChOKα (a choline metabolic rate-limiting enzyme) deficient mouse macrophages. These results indicated that HSV-1 induces the metabolic reprogramming of host cells to promote or resist viral replication. Taken together, these observations highlighted the significance of host cell metabolism in HSV-1 replication, which would help to clarify the pathogenesis of HSV-1 and identify new anti-HSV-1 therapeutic targets.

Inhibition of the MAPK/c-Jun-EGR1 Pathway Decreases Photoreceptor Cell Death in the rd1 Mouse Model for Inherited Retinal Degeneration.

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies that typically results in photoreceptor cell death and vision loss. Here, we explored the effect of early growth response-1 (EGR1) expression on photoreceptor cell death in Pde6brd1 (rd1) mice and its mechanism of action. To this end, single-cell RNA-seq (scRNA-seq) was used to identify differentially expressed genes in rd1 and congenic wild-type (WT) mice. Chromatin immunoprecipitation (ChIP), the dual-luciferase reporter gene assay, and western blotting were used to verify the relationship between EGR1 and poly (ADP-ribose) polymerase-1 (PARP1). Immunofluorescence staining was used to assess PARP1 expression after silencing or overexpression of EGR1. Photoreceptor cell death was assessed using the TUNEL assay following silencing/overexpression of EGR1 or administration of MAPK/c-Jun pathway inhibitors tanzisertib and PD98059. Our results showed differential expression of ERG1 in rd1 and WT mice via scRNA-seq analysis. The ChIP assay demonstrated EGR1 binding to the PARP1 promoter region. The dual-luciferase reporter gene assay and western blotting results revealed that EGR1 upregulated PARP1 expression. Additionally, the TUNEL assay showed that silencing EGR1 effectively reduced photoreceptor cell death. Similarly, the addition of tanzisertib and PD98059 reduced the expression of c-Jun and EGR1 and decreased photoreceptor cell death. Our study revealed that inhibition of the MAPK/c-Jun pathway reduced the expression of EGR1 and PARP1 and prevented photoreceptor cell death. These results highlight the importance of EGR1 for photoreceptor cell death and identify a new avenue for therapeutic interventions in RP.

Functional analysis and expression profile of human platelets infected by EBV in vitro.

Platelet activation is commonly detected after infection by multiple viruses such as human immunodeficiency virus (HIV), H1N1 influenza, Hepatitis C virus (HCV), Ebola virus (EBV), and Dengue virus (DENV). Non-coding RNAs (ncRNAs) constitute the majority of the human transcribed genome, but the biology of platelet ncRNAs is largely unexplored. In this study, we performed microarray profiling to characterize the expression profile of human platelets infected with EBV in vitro after 2 h. A total of 187 long non-coding RNAs (lncRNAs) displayed differences, of which 114 were upregulated and 73 were downregulated; 78 microRNAs (miRNAs) showed differences, including 73 upregulated and 5 downregulated; 808 mRNAs displayed differences, among which 367 were upregulated and 441 were downregulated. Gene ontology (GO) analysis mostly related to G protein-coupled receptor signaling pathway, detection of chemical stimulus involved in sensory perception of smell and regulation of transcription by RNA polymerase II. Pathway analysis showed that the differentially expressed genes were mainly enriched in cell metabolism and immune-related response. A ceRNA network was established based on predicting regulatory pairs in differentially expressed genes, in which hsa-miR-6877-3p had the highest regulatory capability (degree = 31), FAM230A was the lncRNA with the highest regulatory capability (degree = 28). According to the EBV related miRNA regulation network, it revealed that ebv-miR-BART19-3p had the most target genes and BRWD1, FAM126B, TFRC and JMY were the genes most regulated by EBV-related miRNAs. After overlapping the three networks, we found that the EIFAK2 gene was strongly correlated with autologous ncRNAs, including hsa-miR-1972, hsa-miR-504-3p and hsa-miR-6825-5p, as well as with EBV ncRNAs, including EBER1, EBER2, miR-BART7-3p and miR-BART16. The present study contributes to a better understanding of the expression profiling of ncRNAs and their functions in platelets activated by EBV in vitro, and paves the way to further study on platelet function.

Evolutionary analysis of a newly isolated Banna virus strain from Yunnan, China.

Banna virus (BAV) is a typical arbovirus whose infection is associated with fever and viral encephalitis. The whole genome of BAV is composed of 12 RNA segments. BAVs, which have been divided into three genotypes (A, B, and C) based on phylogenetic analysis of segment 12 or segment 9, are currently undergoing rapid evolution. Recent studies have shown that BAV variation can exceed intraspecific limits and generate novel viruses. In the current study, a new BAV strain, named 113c5, was isolated from Culex tritaeniorhynchus and found to be a member of genotype A2 based on phylogenetic analysis of segments 9 and 12. The complete genome sequence of the new BAV strain described in the current study might contribute to the surveillance of evolutionary characteristics of BAVs.

Whole genome analysis identifies intra-serotype recombinants and positive selection sites of dengue virus in mainland China from 2015 to 2020.

Immune pressure can select for escape mutants that can become epidemiologically relevant. Thus, surveillance of recombinants and positively selected mutants of the dengue virus (DENV) are vital for preventing and controlling the dengue fever outbreak. However, little is known about recombinants and positively selected mutants of circulating DENV strains in mainland China. In this study, those variants with recombination and adaptive evolutionary sites of circulating DENV strains were identified during 2015-2020. Phylogenetic analysis showed that the DENV-2 was the dominant epidemic serotype, and the dengue epidemic in China was closely related to the imported virus from Southeast Asian countries. Recombination analysis based on 291 complete genomes of naturally circulating DENV identified 10 new intra-serotype recombinant variants. Two or three recombination regions in a single dengue isolate were also observed. The breakpoints of recombinants were distributed in different regions of the genome. In particular, two recombinant strains (strain DENV-4/China/YN/15DGR394 (2015) and XLLM10666) with extremely large exchange fragments were detected. This large-scale gene fragment exchange (eight genomic regions) of strain DENV-4/China/YN/15DGR394 (2015) with substitutions at both the 5' and 3' ends of the genome, had never been described before. Moreover, selection pressure analyses revealed seven positive selection sites located in regions encoding the NS1, NS3 and NS5 proteins. Overall, this study is the first to report ten specific intra-serotype recombinants and seven positive selection sites of Chinese epidemic strains of DENV, which highlight their significance for DENV surveillance and effective control.

The Effect of Immunosuppressive Adjuvant Kynurenine on Type 1 Diabetes Vaccine.

Inducing antigen-specific tolerance is a promising treatment for preventing or reversing Type 1 diabetes (T1D). In contrast to a vaccine that induces immune responses against pathogens, a tolerogenic vaccine can suppress immunity against antigens causing diseases by administrating a mixture of self-antigens with an adjuvant that decreases the strength of antigen-specific response. Kynurenine (Kyn) is an endogenous substance that can inhibit the natural killer cell and T cell proliferation and promote the differentiation of naïve T cells into regulatory T cells (Tregs). In this study, we evaluated the efficacy of Kyn as a novel suppressive adjuvant. Kyn was co-immunized with GAD65 phage vaccine to induce Treg cells and tolerogenic responses for the prevention of T1D in NOD mouse model. Mice were subcutaneously immunized two times with 1011 Pfu (100µL,1012 Pfu/ml) GAD65 phage vaccine doses mixed with 200 µg of Kyn. Serum antibodies and cytokines were detected by ELISA and electrochemiluminescence, respectively. Flow cytometry assay was used to analyze DC and Treg. MTS was used for the analysis of spleen lymphocyte proliferation. RNA sequencing was used to investigate mRNA and miRNA expression profiles in spleen lymphocytes. Compared to GAD65 phage vaccine alone, co-immunization of Kyn and GAD65 phage vaccine resulted in the prevention of hyperglycemia in 60% of mice for at least one month. Further, Kyn enhances GAD65-specific Th2-mediated immune responses; regulates the Th1/Th2 imbalance and increases the secretion of Th2 cytokines and the number of CD4+CD25+Foxp3+T cells; suppresses DC maturation and GAD65-specific T lymphocyte proliferation. Moreover, we integrated Kyn related miRNA and mRNA expression profiles obtained from the spleen lymphocyte RNA-sequencing which was stimulated by Kyn in vitro. These data provide an important basis for understanding the mechanisms underlying Kyn as an immunosuppressive adjuvant which regulated the immune response. These findings suggest that Kyn can serve as an effective suppressive adjuvant candidate for Type 1 diabetes vaccines.

Differential host circRNA expression profiles in human lung epithelial cells infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging and highly pathogenic coronavirus that causes coronavirus disease (COVID-19), and might even lead to death. Circular RNAs (circRNAs), a new type of RNAs, are implicated in viral pathogenesis and host immune responses. However, their dynamic expression patterns and functions during SARS-CoV-2 infection remain to be unclear. We herein performed genome-wide dynamic analysis of circRNAs in human lung epithelial cells infected with SARS-CoV-2 at four time points. A total of 6118 circRNAs were identified at different genomic locations, including 5641 known and 477 novel circRNAs. Notably, a total of 42 circRNAs were significantly dysregulated, wherein 17 were up-regulated and 25 were down-regulated following infection at multiple phases. The gene ontology and KEGG enrichment analyses revealed that the parental genes of circRNAs were mainly involved in immune and inflammatory responses. Further, the RNA binding protein (RBP) prediction analysis indicated that the dysregulated circRNAs could regulate mRNA stability, immunity, cell death by binding specific proteins. Additionally, the circRNA-miRNA-gene network analysis showed that circRNAs indirectly regulated gene expression by absorbing their targeted miRNAs. Collectively, these results shed light on the roles of circRNAs in virus-host interactions, facilitating future studies on SARS-CoV-2 infection and pathogenesis.

Complete genome analysis identifies recombinant events and positive selection sites of hepatitis C virus from mainland China during 2010-2019.

Identification of new recombinant HCV strains and positive selection sites are crucially important for the formulation of virus intervention measures. However, little is known about the recombinant variant information and positive selection sites of circulating HCV strains in mainland China. In this study, we systematically identified recombinant variants and positive selection sites of HCV in mainland China during the 2010-2019. Phylogenetic analysis results indicated that HCV-6 was one of the dominant genotypes in mainland China during 2010-2019, whereas genotypes 7 and 8 were not detected. Recombinant analysis based on 102 full-length genome sequences of Chinese epidemic strains of HCV identified four intra-genotypic recombinants (strains WYHCV286, GB28, GZ2983, and HCV156) and one inter-genotypic recombinant (strain HH075). Specifically, two breakpoints in the 5' UTR of two recombinants, the strains HH075 and WYHCV286, are rather unusual and has not been described before. Further, selection pressure analyses revealed five positive selective sites, which were located in the core, E2, and NS5B protein. Notably, positive selective sites in NS5B and core protein may be partially responsible for the drug resistance and immune evasion. To the best of our knowledge, this study firstly reported five specific intertypic and intratypic recombinants of Chinese epidemic strains of HCV, which highlight their significance for anti-HCV treatment and vaccine development.

Natural intertypic and intratypic recombinants of enterovirus 71 from mainland China during 2009-2018: a complete genome analysis.

Surveillance of recombinant enterovirus 71 (EV71) and subgenotype replacement is vital for preventing and controlling hand, foot, and mouth disease (HFMD) outbreaks. Despite this, data on recombinant variants and phylogeny of circulating EV71 strains in mainland China are limited. In this study, recombinant variants of EV71 were identified in mainland China from 2009 to 2018. Phylogenetic analysis indicated that except for individual strains (CQ2014-86/CQ/CHN/2014 and EV71/Xiamen/2009 (B5)), almost all of the EV71 strains in mainland China belonged to the subgenotype C4a. Analysing complete genome sequences of 196 EV71 isolates, 3 intertypic recombination strains (VR1432, 30-2/2015/BJ, and Guangdong-2009) and 5 intratypic recombination strains (EV71/P1034/2013, VR1432, Henan-ZMD/CHN/2012, Hubei-WH/CHN/2012, and EV71/P868/2013/China) were identified among naturally circulating EV71. The breakpoints of these recombinant strains were located within the P1, P2, and P3 encoding regions. Notably, a double recombinant (VR1432) resulting from recombination between EV71 subgenotype C4a and C4b strain SHZH98 and a CA8 strain Donovan was identified. This study reports these specific intertypic and intratypic recombination events for the first time highlighting the importance of genetic recombination in the emergence of new enterovirus variants.

Hepatitis A virus-induced hsa-miR-146a-5p attenuates IFN-ß signaling by targeting adaptor protein TRAF6.

Hepatitis A virus (HAV), a unique hepatotropic human picornavirus, is the causative agent of acute hepatitis A in humans. Some studies have shown that HAV antagonizes the innate immune response by disrupting interferon-beta (IFN-ß) signaling by viral proteins. However, whether microRNAs (miRNAs), a class of non-coding RNAs, are involved in the antagonism of IFN-ß induction upon HAV infection is still unclear. In this study, we investigated the effects and mechanisms by which HAV-induced miRNAs antagonize IFN-ß signaling. A variety of analytical methods, including miRNA microarray, RT-qPCR, dual-luciferase reporter assay, and Western blotting, were performed using HAV-infected cells. The results indicated that HAV infection upregulates the expression of hsa-miR-146a-5p, which in turn partially suppresses the induction of IFN-ß synthesis, thereby promoting viral replication. Mechanistically, TRAF6 (TNF receptor-associated factor 6), a key adaptor protein in the RIG-I/MDA5-mediated IFN-I signaling pathway, is targeted and degraded by hsa-miR-146a-5p. As TRAF6 is necessary for IFN-ß induction, inhibition of this protein attenuates IFN-ß signaling. Taken together, the results from this study indicated that HAV disrupts RIG-I/MDA5-mediated IFN-I signaling partially through the cleavage of the essential adaptor molecule TRAF6 via hsa-miR-146a-5p.

Mannose-functionalized antigen nanoparticles for targeted dendritic cells, accelerated endosomal escape and enhanced MHC-I antigen presentation.

Cancer immunotherapy is an important tumor therapy option for prevention and treatment of tumors and has attracted tremendous interests. However, the therapeutic outcomes are limited by insufficient antigen uptake and presentation by antigen-presenting cells such as dendritic cells (DCs). In this study, mannose-functionalized antigen nanoparticles with endosome escape activity were designed for targeted DCs, accelerated endosomal escape and enhanced MHC-I antigen presentation for cancer immunotherapy. Mannose was selected as DCs targeting ligand to enhance antigen uptake. Model antigen ovalbumin (OVA) was directly conjugated with mannose to obtain DCs targeting antigen, which was then complexed with polyethylenimine (PEI) through electrostatic interaction to form mannose-functionalized antigen nanoparticles (MAN-OVA/PEI NPs). Flow cytometry analysis revealed that the MAN-OVA/PEI NPs greatly increased antigen uptake by DCs compared with OVA/PEI NPs. Confocal laser scanning microscopy further demonstrated that MAN-OVA/PEI NPs enhanced cytosolic antigen release. Moreover, MAN-OVA/PEI NPs significantly promoted cytokine production and DCs maturation in vitro. More importantly, MAN-OVA/PEI NPs treated DCs exhibited enhanced cross-presentation to B3Z T cell hybridoma in vitro. This work suggests that mannose-functionalized antigen nanoparticles provide a versatile delivery vehicle for targeted DCs, accelerated endosomal escape and enhanced MHC-I antigen presentation for cancer immunotherapy.

Comparison of nonhuman primates identified the suitable model for COVID-19.

Identification of a suitable nonhuman primate (NHP) model of COVID-19 remains challenging. Here, we characterized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in three NHP species: Old World monkeys Macaca mulatta (M. mulatta) and Macaca fascicularis (M. fascicularis) and New World monkey Callithrix jacchus (C. jacchus). Infected M. mulatta and M. fascicularis showed abnormal chest radiographs, an increased body temperature and a decreased body weight. Viral genomes were detected in swab and blood samples from all animals. Viral load was detected in the pulmonary tissues of M. mulatta and M. fascicularis but not C. jacchus. Furthermore, among the three animal species, M. mulatta showed the strongest response to SARS-CoV-2, including increased inflammatory cytokine expression and pathological changes in the pulmonary tissues. Collectively, these data revealed the different susceptibilities of Old World and New World monkeys to SARS-CoV-2 and identified M. mulatta as the most suitable for modeling COVID-19.

Phylogenetic and genetic analyses of the emerging Nipah virus from bats to humans.

Little is known about the genetic features of Nipah virus (NiV) associated with virulence and transmission. Herein, phylogenetic and genetic analyses for all available NiV strains revealed sequence variations between the two genetic lineages of NiV with pathogenic differences, as well as among different strains within Bangladesh lineage. A total of 143 conserved amino acid differences, distributed among viral nucleocapsid (N), phosphoprotein (P), matrix protein (M), fusion protein (F) and glycoprotein (G), were revealed. Structural modeling revealed one key substitution (S3554N) in the viral G protein that might mediate a 12-amino-acid structural change from a loop into a ß sheet. Multiple key amino acids substitutions in viral G protein were observed, which may alter viral fitness and transmissibility from bats to humans.

Comprehensive Analysis of the Expression Profiles of Long Non-Coding RNAs with Associated ceRNA Network Involved in the Colon Cancer Staging and Progression.

Long non-coding RNAs (lncRNAs) act as competing endogenous RNAs (ceRNAs) to compete with microRNAs (miRNAs) in cancer occurrence and development. However, the differential expression of RNAs and their ceRNA network during the development of colon cancer (CC) remains unclear. This study was aimed at comprehensive analysis of the lncRNAs and their ceRNA networks associated with CC. Whole transcriptome sequencing was performed on colorectal and adjacent normal tissues at different pathological stages. Forty-nine lncRNAs were differently expressed between the CC tissues and their adjacent normal tissues at all stages. Aberrant expression of lncRNA CDKN2B-AS1 and lncRNA MIR4435-2HG was confirmed by TCGA database. Moreover, 14 lncRNAs were differentially expressed between early and advance stages of the tumor tissues, and 117 miRNAs were specifically expressed in stage III & IV. Weighted gene co-expression network analysis of 17105 differently expressed mRNAs revealed that the mRNAs shown in module pink, midnight blue, black, and light cyan were related to TNM and pathological stage, and that these mRNAs were enriched in cancer related functions and pathways. As DElncRNA showed a trend of change similar to that of the DEmRNA and opposite to that of DEmiRNA, ceRNA network was constructed with 3 DEmiRNAs, 5 DElncRNAs, and 130 DEmRNAs. Real time PCR revealed that expression of MEG3 was decreased in the tumor tissues belonging to stage III and IV as compared to that in stage I. Moreover, hsa-miR-324-5p was upregulated, while FGFR3, PLCB4, and IKBKB were downregulated in the tumor tissues as compared to that in the adjacent normal tissues. Thus, this study revealed differentially expressed lncRNA between different stages of CC as well as suggested that lncRNA CDKN2B-AS1, MIR4435-2HG, and MEG3 may act as diagnostic biomarkers for the development of CC.