Changes In circRNA Expression Research Articles

Genome-wide perturbations of A-to-I RNA editing dysregulated circular RNAs promoting the development of cervical cancer

Cervical cancer, the second most common female malignant tumor, seriously threatens women's health and lives. Despite the availability of the HPV vaccine, effective treatment options for cervical cancer are still lacking. New research perspectives now clarify that RNA editing dysregulation and changes in circRNA expression are jointly involved in disease pathogenesis, so molecular changes associated with circRNA and RNA editing may provide clues for the development of new therapeutic strategies for cervical cancer. In this study, we designed a series of pipelines to identify and analyze dysregulated RNA editing events in circRNAs. Our findings indicate a decrease in A-to-I RNA editing levels in cervical cancer compared to normal tissues, and editing may influence the back-splicing process of circRNAs through structural modifications of Alu elements. Moreover, our research reveals that RNA editing could modulate circRNA biogenesis by influencing RNA binding protein (RBP) binding on a transcriptome-wide scale, as well as influence the expression and coding potential of circRNAs. Importantly, we identified three RNA editing sites that could serve as potential biomarkers. In summary, our study presents a comprehensive landscape of RNA editing perturbations in circRNAs, providing new insights into the complex relationship between RNA editing and circRNA dysregulation in cervical cancer.

Circular RNA hsa_circ_0098181 inhibits metastasis in hepatocellular carcinoma by activating the Hippo signaling pathway via interaction with eEF2

Introduction and ObjectivesThe development of hepatocellular carcinoma (HCC) is a multi-step process that accumulates genetic and epigenetic alterations, including changes in circular RNA (circRNA). This study aimed to understand the alterations in circRNA expression in HCC development and metastasis and to explore the biological functions of circRNA. Materials and MethodsTen pairs of adjacent chronic hepatitis tissues and HCC tissues from patients without venous metastases, and ten HCC tissues from patients with venous metastases were analyzed using human circRNA microarrays. Differentially expressed circRNAs were then validated by quantitative real-time PCR. In vitro and in vivo assays were performed to assess the roles of the circRNA in HCC progression. RNA pull-down assay, mass spectrometry analysis, and RNA-binding protein immunoprecipitation were conducted to explore the protein partners of the circRNA. ResultsCircRNA microarrays revealed that the expression patterns of circRNAs across the three groups were significantly different. Among these, hsa_circ_0098181 was validated to be lowly expressed and associated with poor prognosis in HCC patients. Ectopic expression of hsa_circ_0098181 delayed HCC metastasis in vitro and in vivo. Mechanistically, hsa_circ_0098181 sequestered eukaryotic translation elongation factor 2 (eEF2) and dissociated eEF2 from filamentous actin (F-actin) to prevent F-actin formation, which blocked activation of the Hippo signaling pathway. In addition, the RNA binding protein Quaking-5 bound directly to hsa_circ_0098181 and induced its biogenesis. ConclusionsOur study reveals changes in circRNA expression from chronic hepatitis, primary HCC, to metastatic HCC. Further, the QKI5-hsa_circ_0098181-eEF2-Hippo signaling pathway exerts a regulatory role in HCC.

Roles of Aging, Circular RNAs, and RNA Editing in the Pathogenesis of Amyotrophic Lateral Sclerosis: Potential Biomarkers and Therapeutic Targets

Amyotrophic lateral sclerosis (ALS) is an incurable motor neuron disease caused by upper and lower motor neuron death. Despite advances in our understanding of ALS pathogenesis, effective treatment for this fatal disease remains elusive. As aging is a major risk factor for ALS, age-related molecular changes may provide clues for the development of new therapeutic strategies. Dysregulation of age-dependent RNA metabolism plays a pivotal role in the pathogenesis of ALS. In addition, failure of RNA editing at the glutamine/arginine (Q/R) site of GluA2 mRNA causes excitotoxicity due to excessive Ca2+ influx through Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, which is recognized as an underlying mechanism of motor neuron death in ALS. Circular RNAs (circRNAs), a circular form of cognate RNA generated by back-splicing, are abundant in the brain and accumulate with age. Hence, they are assumed to play a role in neurodegeneration. Emerging evidence has demonstrated that age-related dysregulation of RNA editing and changes in circRNA expression are involved in ALS pathogenesis. Herein, we review the potential associations between age-dependent changes in circRNAs and RNA editing, and discuss the possibility of developing new therapies and biomarkers for ALS based on age-related changes in circRNAs and dysregulation of RNA editing.

Global circRNA expression changes predate clinical and histological improvements of psoriasis patients upon secukinumab treatment.

Psoriasis is a common chronic inflammatory skin disease accompanied by heterogenous clinical and histological features, including a characteristic keratinocyte hyperproliferation and dermal immunogenic profile. In addition, psoriasis is associated with widespread transcriptomic alterations including changes in microRNA (miRNA) and circular RNA (circRNA) abundance, which constitute non-coding RNA (ncRNA) classes with specific regulatory capacities in diverse physiological and pathological processes. However, the knowledge about the expression dynamics of ncRNA during psoriasis treatment is sparse. To elucidate the dynamics of miRNA and circRNA abundance during secukinumab (anti-IL-17A) treatment, we studied their expression patterns in skin biopsies from 14 patients with severe plaque-type psoriasis before and during an 84-day secukinumab therapy at day 0, 4, 14, 42, and 84 using NanoString nCounter technology. We found a comprehensive downregulation of the majority of investigated circRNAs and specific alterations in the miRNA profile, including an upregulation of miR-203a-3p, miR-93-5p, and miR-378i in lesional compared to non-lesional skin before treatment. During treatment, the circRNAs progressively returned to the expression levels observed in non-lesional skin and already four days after treatment initiation most circRNAs were significantly upregulated. In comparison, for miRNAs, the normalization to baseline during treatment was delayed and limited to a subset of miRNAs. Moreover, we observed a strong correlation between multiple circRNAs, including ciRS-7 and circPTPRA, and the psoriasis area and severity index (PASI). Similar pronounced correlations could, however, not be found for miRNAs. Finally, we did not observe any significant changes in circRNA expression in peripheral blood mononuclear cells during treatment. In conclusion, we uncovered a rapid shift in global circRNA abundance upon anti-IL-17A treatment, which predated clinical and histological improvements, and a strong correlation with PASI, indicating a biomarker potential of individual circRNAs.

Profiling and Bioinformatics Analysis Revealing Differential Circular RNA Expression about Storage Lesion Regulatory in Stored Red Blood Cells

Introduction: Circular RNA (circRNA) plays an important role in regulating metabolism of red blood cells (RBCs) and their storage lesions, but the study of how circRNA expression changes in stored RBCs has rarely been conducted. Methods: The expression change of circRNA was systemically evaluated via high-throughput sequencing on healthy RBCs on day 0, 20, and 40. And then we confirmed the reliability of the high-throughput sequencing analysis by RT-qPCR characterization on selected circRNAs. A higher parental gene enrichment was used to explore circRNA function in pathways. In addition, we deciphered a dysregulated circRNA-related ceRNAs network, and identified three circRNA-miRNA-mRNA regulatory axes related to storage lesion. Results: We identified 2,586 known and 6,216 putative novel circRNAs, more than 100 circRNAs expression levels were shifted, and the number of downregulated circRNAs was greater with longer storage time. Furthermore, a higher parental gene enrichment related to circRNA was found in pathways, including cAMP signaling pathway, ubiquitin-mediated proteolysis, apoptosis, adhesion, MAPK signaling pathway, cystine methionine metabolism, RNA degradation, RNA transport, TGF-β, and actin regulatory pathway. hsa_circ_0007127-miR-513a-5p-SMAD4, hsa_circ_0000033-miR-19a-3p-VAMP3, and hsa_circ_0005546-miR-4720-CCND3 regulatory axes related to storage lesion was found. Conclusions: Through investigation in circRNAs profile and circRNA-miRNA-mRNA interactions, this study provides insights on stored RBC circRNA expression changes, which closely relate to the storage lesion of RBCs and their physiological functions.

Exosomal hsa_circRNA_104484 and hsa_circRNA_104670 may serve as potential novel biomarkers and therapeutic targets for sepsis

In order to explore the role of exosomal circRNAs in the occurrence and development of sepsis, we looked for potential diagnostic markers to accurately identify sepsis and to lay a molecular basis for precise treatment. Ultracentrifugation was used to extract exosomes from the serum of patients with sepsis and healthy individuals. Then, changes in circRNA expression in exosomes were studied by circRNA microarray analysis. Gene ontology (GO) analysis and Kyoto City Encyclopaedia of Genes and Genomes (KEGG) pathway analysis were used to annotate the biological functions and pathways of genes, and a circRNA-miRNA-mRNA regulatory network was constructed. In the microarray analysis, 132 circRNAs were significantly differentially expressed, including 80 and 52 that were upregulated and downregulated, respectively. RT-qPCR verified the results of microarray analysis: hsa_circRNA_104484 and hsa_circRNA_104670 were upregulated in sepsis serum exosomes. ROC analysis showed that hsa_circRNA_104484 and hsa_circRNA_104670 in serum exosomes have the potential to be used as diagnostic markers for sepsis. The circRNA-miRNA-mRNA network predicted the potential regulatory pathways of differentially expressed circRNAs. There are differences in the expression of circRNA in serum exosomes between patients with sepsis and healthy individuals, which may be involved in the occurrence and development of the disease. Among them, elevations in hsa_circRNA_104484 and hsa_circRNA_104670 could be used as novel diagnostic biomarkers and molecular therapeutic targets.

CircIMPACT: An R Package to Explore Circular RNA Impact on Gene Expression and Pathways.

Circular RNAs (circRNAs) are transcripts generated by back-splicing. CircRNAs might regulate cellular processes by different mechanisms, including interaction with miRNAs and RNA-binding proteins. CircRNAs are pleiotropic molecules whose dysregulation has been linked to human diseases and can drive cancer by impacting gene expression and signaling pathways. The detection of circRNAs aberrantly expressed in disease conditions calls for the investigation of their functions. Here, we propose CircIMPACT, a bioinformatics tool for the integrative analysis of circRNA and gene expression data to facilitate the identification and visualization of the genes whose expression varies according to circRNA expression changes. This tool can highlight regulatory axes potentially governed by circRNAs, which can be prioritized for further experimental study. The usefulness of CircIMPACT is exemplified by a case study analysis of bladder cancer RNA-seq data. The link between circHIPK3 and heparanase (HPSE) expression, due to the circHIPK3-miR558-HPSE regulatory axis previously determined by experimental studies on cell lines, was successfully detected. CircIMPACT is freely available at GitHub.

Deregulated expression of circular RNAs in acute myeloid leukemia

Circular RNAs (circRNAs) are dynamically regulated during differentiation and show cell type–specific expression, which is altered in cancer and can have a direct impact on its various hallmarks. We hypothesized that circRNA expression is deregulated in acute myeloid leukemia (AML) and that circRNA candidates might contribute to the pathogenesis of the disease. To identify leukemia-associated and differentiation-independent changes in circRNA expression, we determined the circular RNAome of 61 AML patients and 16 healthy hematopoietic stem and progenitor cell (HSPC) samples using ribosomal RNA–depleted RNA sequencing. We found hundreds of circRNAs that were differentially expressed between AML and healthy HSPCs. Gene set analysis found that many of these circRNAs were transcribed from genes implicated in leukemia biology. We discovered a circRNA derived from the T-cell transcription factor gene B cell CLL/lymphoma 11B, circBCL11B, which was exclusively expressed in AML patients, but not detected in healthy HSPCs, and associated with a T-cell–like gene expression signature. We were able to validate this finding in an independent cohort of 332 AML patients. Knockdown of circBCL11B had a negative effect on leukemic cell proliferation and resulted in increased cell death of leukemic cells, thereby suggesting circBCL11B as a novel functionally relevant candidate in AML pathogenesis. In summary, our study enables comprehensive insights into circRNA expression changes upon leukemic transformation and provides valuable information on the biology of leukemic cells and potential novel pathway dependencies that are relevant for AML therapy.

Expression and function of circular RNAs in the mammalian brain.

Mammalian brain presents extraordinary complexity reflected in the structure, function, and dynamic changes in the biological and physiological processes of development, maturity, and aging. Recent transcriptomic profiles from the brain tissues of distinct species have described a novel class of transcripts with a covalently closed-loop structure, called circular RNAs (circRNAs), which are produced by alternative back-splicing and derived from genes associated with synaptogenesis and neural activities. Brain is a tightly regulated and largely unexplored organ where circRNAs are highly enriched and expressed in the cell type-, spatiotemporal-specific, sex-biased, and age-related manner. Although the biological functions of most of the circRNAs in the brain remain elusive, increased evidence suggests that dynamic changes in circRNA expression are critical for brain function and the maintenance of physiological homeostasis in the brain. Here, we review the latest immense progresses in the understanding of circRNA expression and function in the mammalian brain. We also discuss possibly biological functions of circRNAs in the brain, which may provide new sights of understanding brain development and aging, as well as the pathogenesis of mental diseases.

Characterization of circular RNA transcriptomes in psoriasis and atopic dermatitis reveals disease-specific expression profiles.

Atopic dermatitis (AD) and psoriasis are two common chronic inflammatory skin diseases that are associated with various comorbidities. Circular RNA (circRNA) constitutes a major class of non-coding RNAs that have been implicated in many human diseases, although their potential involvement in inflammatory skin diseases remains elusive. Here, we compare and contrast the circRNA expression landscapes in paired lesional and non-lesional skin from psoriasis and AD patients relative to skin from unaffected individuals using high-depth RNA-seq data. CircRNAs and their cognate linear transcripts were quantified using the circRNA detection algorithm, CIRI2, and in situ hybridization and Sanger sequencing was used for validation purposes. We identified 39,286 circRNAs among all samples and found that psoriasis and AD lesional skin could be distinguished from non-lesional and healthy skin based on circRNA expression landscapes. In general, circRNAs were less abundant in lesional relative to non-lesional and healthy skin. Differential expression analyses revealed many significantly downregulated circRNAs, mainly in psoriasis lesional skin, and a strong correlation between psoriasis and AD-related circRNA expression changes was observed. Two individual circRNAs, ciRS-7 (also known as CDR1as) and circZRANB1, were specifically dysregulated in psoriasis and show promise as biomarkers for discriminating AD from psoriasis. In conclusion, the circRNA transcriptomes of psoriasis and AD share expression features, including a global downregulation relative to healthy skin, but this is most pronounced in psoriasis, and only psoriasis is characterized by several circRNAs being dysregulated independently of their cognate linear transcripts. Finally, specific circRNAs could potentially be used to distinguish AD from psoriasis.

Prenatal Alcohol Exposure Results in Sex-Specific Alterations in Circular RNA Expression in the Developing Mouse Brain.

Fetal alcohol spectrum disorders (FASD) are heterogeneous disorders associated with alcohol exposure to the developing fetus that are characterized by a range of adverse neurodevelopmental deficits. Despite the numerous genomics and genetic studies on FASD models, the comprehensive molecular understanding of the mechanisms that underlie FASD-related neurodevelopmental deficits remains elusive. Circular RNAs (circRNAs) are a subtype of long non-coding RNAs that are derived from back-splicing and covalent joining of exons and/or introns of protein-coding genes. Recent studies have shown that circRNAs are highly enriched in the brain, where they are developmentally regulated. However, the role of the majority of brain-enriched circRNAs in normal and pathological brain development and function has not been explored yet. Here we carried out the first systematic profiling of circRNA expression in response to prenatal alcohol exposure (PAE) in male and female embryonic day 18 (E18) whole brains. We observed that the changes in circRNA expression in response to PAE were notably sex-specific and that PAE tended to erase most of the sex-specificity in circRNA expression present in control (saccharin-treated) mice. On the other hand, RNA sequencing (RNA-seq) in the same samples showed that changes in protein-coding gene expression were not predominantly sex-specific. Using circRNA quantitative real-time PCR (qRT-PCR), we validated that circSatb2, which is generated from the special AT-rich sequence-binding protein 2 (Satb2) gene, is significantly upregulated in the brain of E18 male PAE mice. We also show that circPtchd2, a circRNA synthesized from dispatched RND transporter family member 3 (Disp3, also known as Ptchd2), exhibits significantly higher expression in E18 control but not PAE female mouse brain relative to males. Taken together, our results demonstrate that PAE differentially alters circRNA expression in the developing brain in a sex-specific manner.

Identification of atrial fibrillation-related circular RNAs and constructing the integrative regulatory network of circular RNAs, microRNAs and mRNAs by bioinformatics analysis

Atrial fibrillation (AF) is the most common cardiac arrhythmia with a high incidence of stroke. Many circular RNAs (circRNAs) have been demonstrated they are elated to various heart diseases and may play important roles in diagnostics or many pathophysiological processes. Nevertheless, there is Few studies on circRNAs functions in persistent AF. To identify AF-related circRNAs and construct the integrative regulatory network of circular RNAs, miRNAs, and mRNAs, we collected human right atrial appendage tissues from 5 patients suffering persistent AF (AF group) and 5 patients with normal sinus rhythm (NSR group) and characterized the global changes in circRNA expression with high-throughput sequencing technology. The differential expression of circRNAs and the interactions between circRNAs and microRNAs were analyzed. The microRNA expression file GSE68475 dataset was downloaded from the Gene Expression Omnibus (GEO) database to explore the differentially expressed microRNAs. The target genes of overlapped miRNAs were predicted by using DIANA-TarBase v8. We constructed the circRNA- miRNA-mRNA network using Cytoscape (version 3.4.0) and the network topology was analyzed by utilizing CentiScaPe app. Results showed that all of 600 differentially expressed circRNAs related to AF were screened, including 340 up-regulated and 260 down-regulated circRNAs. An integrative regulatory network was constructed, which included 30 circRNAs, 9 miRNAs and 130 target mRNAs of these miRNAs. It was concluded that that 30 circRNAs, including 8 upregulated circRNAs and 22 downregulated circRNAs, were predicted to highly possibly function as sponges of 9 miRNAs to regulate gene expression by using bioinformatics analysis. Moreover, the interactions of hsa-miR-339-5p with its related circRNAs and target mRNAs constructed the hub regulatory network in persistent AF by utilizing topology analysis. Our proposed regulatory network of circRNAs-miRNAs-mRNAs may provide new insight into the potential mechanism underlying persistent AF. Additionally, these important molecular may become novel biomarkers providing a new strategy in diagnosis and therapy of AF.

Circular RNAs as important players in human gastric cancer.

As one of the most prevalent gastrointestinal diseases, gastric cancer (GC) is the second leading cause of cancer-related deaths worldwide. Since GC has no clinical manifestations in the early stage of the disease, most patients are detected in the later phases of disease and have an unfortunately lower chance of recovery. Circular RNAs (circRNAs), a novel category of non-coding RNAs (ncRNAs), are mainly engaged in the regulation of gene expression at the transcriptional and post-transcriptional levels. Numerous evidences have revealed that circRNAs play key roles in GC as they are involved in cell proliferation, growth, and apoptosis via modulating the expression of some target genes, miRNAs, and proteins. Many studies have addressed the impact of circRNA dysregulation on GC initiation, progression, and invasion via binding to miRNAs or RNA binding proteins. Moreover, changes in circRNA expression are associated with pathological and clinical features of GC highlighting their potentials as diagnostic or prognostic biomarkers in GC. In the current study, the recent findings on the significance of circRNAs in the development and progression of GC are reviewed. We focus on the implications of circRNAs as potential diagnostic or prognostic biomarkers in this malignancy.

Commentary on Some Recent Theses Relevant to Combating Aging: December 2019.

Theses reviewed in this issue include "Atrophied Thymus, a Tumor Reservoir for Harboring Melanoma Cells," "Evolutionary Adaptations in Developmental Signaling Pathways Underlie Regenerative Scar-Free Wound Repair in African Spiny Mouse (Genus Acomys)," "Integrated Immunoassays on Paper/Polymer Hybrid Microfluidic Devices for Low-Cost Detection of Disease Biomarkers," "RNA Regulation in the Nervous System: CircRNA Expression Changes During Aging, and Function of the Calm1 Extended 3' UTR Isoform," "The Role of Amylin in Alzheimer's Disease," and "Therapeutic Monoclonal Antibodies to Detect and Halt ATTR Cardiac Amyloidosis and Neuropathy."

An atlas of cortical circular RNA expression in Alzheimer disease brains demonstrates clinical and pathological associations.

We generated parietal cortex RNA-seq data from individuals with and without Alzheimer disease (AD; ncontrol = 13; nAD = 83) from the Knight-ADRC. Using this and an independent (MSBB) AD RNA-seq dataset, we quantified cortical circular RNA (circRNA) expression in the context of AD. We identified significant associations between circRNA expression and AD diagnosis, clinical dementia severity, and neuropathological severity. We demonstrated that a majority of circRNA AD-associations are independent from changes in cognate linear mRNA expression or brain cell-type proportions. We provided evidence for circRNA expression changes occurring early in pre-symptomatic AD, and in autosomal dominant AD. We also observed AD-associated circRNAs co-expressing with known AD genes. Finally, we identified potential microRNA binding sites in AD-associated circRNAs for microRNAs predicted to target AD genes. Together, these results highlight the importance of analyzing non-linear RNAs and support future studies exploring the potential roles of circRNAs in AD pathogenesis.

Identification and characterization of circRNAs as competing endogenous RNAs for miRNA-mRNA in colorectal cancer.

BackgroundRecent studies showed that circRNAs are involved in the biological process of some human cancers. However, little is known about their functions in colorectal cancer (CRC).MethodsHere we first revealed the expression profiles of circRNAs in the CRC tissues and the adjacent non-tumorous tissues using high-throughput sequencing. The sequence feature, chromosome location, alternative splicing and other characteristics of the circRNAs were also explored. The miRNA and mRNA expression profiles were then obtained by analyzing relevant CRC data retrived from the TCGA database. We obtained and analyzed the competing endogenous RNA (ceRNA) network of the top three pairs of the largest up-regulated and down-regulated circRNAs.ResultsIn this study, we obtained 50,410 circRNAs in the CRC tissue and the adjacent non-tumor tissues, of which 33.7% (16,975) were new, and revealed differential changes in circRNA expression during colorectal carcinogenesis. We have identified six potential key circRNAs (circPIEZO1-3, hsa_circ_0067163, hsa_circ_0140188, hsa_circ_0002632, hsa_circ_0001998 and hsa_circ_0023990) associated with CRC, which play important roles in carcinogenesis as ceRNA for regulation of miRNA-mRNA network. In the subsequent KEGG analysis, several CRC-related pathways were found.ConclusionsOur findings advance the understanding of the pathogenesis of CRC from the perspective of circRNAs and provide some circRNAs as candidate diagnostic biomarkers or potential therapeutic targets.

Comprehensive circular RNA profiling reveals theregulatory role of the hsa_circ_0137606/miR‑1231 pathway in bladder cancer progression.

Bladder cancer (BC) is one of the most common malignant tumors in males globally. Its progression imposes a heavy burden on patients; however, the expression profile of circular (circ)RNAs in BC progression remains unclear. This study explored changes in circRNA expression during BC progression by sequencing different grade BC samples and normal controls to reveal the circRNA expression profiles of different BC grades. Gene Ontology (GO) and Kyoto Encyclopedia of Gens and Genomes (KEGG) pathway analyses, and protein-protein interaction network construction were used to predict pathways that the differentially expressed circRNAs may participate in. circRNA expression levels were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and dual-luciferase reporter assays were used to investigate the interactions between circRNA and microRNA (miR). Cell Counting Kit-8 and Transwell assays were also performed to detect cell proliferation, migration, and invasion. In total, 244 circRNAs were found to be differentially expressed in high-grade BC compared to low-grade BC, whilst 316 dysregulated circRNAs were detected in high-grade BC compared with normal urothelium. Furthermore, 42 circRNAs overlapped between the two groups, seven of which were randomly selected and detected by RT-qPCR to validate the sequencing results. GO analysis and KEGG pathway analyses revealed that the differentially expressed circRNAs may participate in BC via 'GTPase activity regulation', 'cell junction', and 'focal adhesion' pathways. Of note, we proposed that a novel circRNA in BC progression, hsa_circ_0137606, could suppress BC proliferation and metastasis by sponging miR-1231. Through bioinformatics analysis, we predicted that PH domain and leucine rich repeat protein phosphatase 2 could be a target of the hsa_circ_0137606/miR-1231 axis in BC progression. Using high-throughput sequencing, this study revealed the circRNA expression profiles of different grades of BC and proposed that the novel circRNA, hsa_circ_0137606, suppresses BC proliferation and metastasis by sponging miR-1231. Our findings may provide novel insight into potential therapeutic targets for treating BC.

Circular RNA expression profile of liver tissues in an EtOH-induced mouse model of alcoholic hepatitis

Alcoholic hepatitis (AH) is a widely prevalent liver-related disease that results from long-term alcohol consumption. However, there is still a lack of effective treatment. Previous studies have reported that circular RNAs (circRNAs) are related to the development of various diseases. However, the function of circRNAs and their roles in AH are largely unknown. Therefore, we used bioinformatics analysis to investigate changes in circRNA expression and predict their functions in AH. An AH model was established in C57BL/6J mouse treated by Gao-binge modeling method, and then the circRNA profile of liver tissues was screened by Next Generation Sequencing. By comparing circRNA expression in liver tissues in AH model groups and normal controls, we identified that circRNAs were differently expressed during AH pathogenesis, and then differential expression levels of selected circRNAs were validated by qRT-PCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were employed to predict the functions of these circRNAs. A total of 20 circRNAs were found to be differentially expressed in AH model groups (p ≤ 0.05) compared with the expression of the normal controls respectively. Among them, 9 circRNAs were significantly up-regulated, and the other 11 were down-regulated. Ten circRNAs were randomly selected to verify the reliability of these profiles by qRT-PCR. After obtaining the parental genes of the differentially expressed circRNAs, the top 30 enrichment GO entries and KEGG pathways were annotated. Then, we selected significantly differentially expressed circRNA (mm9_ circ_018725) for further analysis in vitro. Although the exact mechanisms and biological functions of these circRNAs in the development of AH need further exploration, our findings do suggest that knockdown of mm9_ circ_018725 could inhibit hepatocyte apoptosis induced by EtOH in vitro. In addition, suppression of mm9_ circ_018725 reduced the release of pro-inflammatory cytokines from EtOH-stimulated Raw264.7 cells. Thus, our study brings us closer to understanding the pathogenic mechanisms and finding new molecular targets for the clinical treatment of alcoholic hepatitis.

Changes in the Circular RNA Transcriptome During Inorganic Arsenic‐Induced Epithelial‐to‐Mesenchymal Transition

Inorganic arsenic (iAs) found in the environment is associated with diseases such as cancer. Chronic, low dose exposure to iAs induces cellular transformation and epithelial‐to‐mesenchymal transition (EMT), a hallmark of metastasis. Since iAs is not genotoxic, we hypothesize that iAs drives carcinogenesis by altering gene expression though epigenetic mechanisms. One class of epigenetic gene regulators recently discovered are circular RNAs (circRNAs), which are covalently closed and highly stable isoforms derived from precursor messenger RNAs (mRNAs). They are generated from actively transcribed genes and their biogenesis competes with that of mRNA. circRNAs are highly conserved across eukaryotes however the function of this new class of regulators is still unclear. Our lab and others have shown alterations to the epigenome as driving iAs‐mediated carcinogenesis. However the relevance of the expression of circRNAs in this process is not understood. Interestingly, our preliminary data suggest that circRNAs are differentially expressed during this process. We therefore aim to identify and profile the changes in circRNA expression that occur during iAs‐induced EMT. We have treated BEAS‐2B cells with iAs over a time‐course, measuring changes in EMT‐markers over time. We already have shown that cells move from pre‐EMT to mid EMT to late‐EMT and finally back through MET during iAs‐mediated carcinogenesis. This model was used to profile differential expression of circRNAs. To accomplish this, total RNA was isolated and enriched for circRNAs with an RNase R digest. For gene‐specific analyses, divergent primers designed to amplify across the circRNA backsplice junctions were used in reverse transcription quantitative polymerase chain reaction to identify differentially expressed circRNA transcripts. In our preliminary gene‐specific approach, we have identified a circRNA containing the 3′ untranslated region of neural cell adhesion molecule 2 (NCAM2) in BEAS‐2B cells. NCAM2 is a cell‐to‐cell adhesion molecule involved in neuronal growth and migration, and is known to be upregulated during EMT. We observed that during iAs‐mediated EMT, while the expression of the linear NCAM2 transcript increased, there was a concurrent decrease in the expression of the 3′ untranslated region. These results indicate that circRNA expression is altered during iAs‐induced EMT and suggests that iAs promotes carcinogenesis by altering steady state competition between linear and circular RNA production. We are currently using next generation sequencing to fully profile the circRNA transcriptome as cells undergo iAs‐induced EMT to gain a broader understanding of the functional significance of circRNA in iAs‐induced EMT.Support or Funding InformationThis work was supported by NIEHS grant (R01‐ES024478).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RETRACTED ARTICLE: Circular RNA hsa_circ_0055538 regulates the malignant biological behavior of oral squamous cell carcinoma through the p53/Bcl-2/caspase signaling pathway

BackgroundOral squamous cell carcinoma (OSCC) is a common oral and maxillofacial malignant tumor with high rates of metastasis and mortality. Circular RNAs (circRNAs), a type of non-coding RNA, are involved in the development of a variety of tumors. The roles of circRNAs in OSCC are unclear; in this study, the correlation between the circRNA hsa_circ_0055538, previously identified by high-throughput sequencing, and the biological behavior of OSCC was evaluated.MethodscircRNA expression was evaluated using patient tissue samples and various OSCC cell lines. The effects of overexpression and knockdown were evaluated by lentiviral infection and siRNA transfection of the SCC9 and CAL27 cell lines. Migration, invasion, apoptosis, and the expression of proteins in the p53 signaling pathway were evaluated. Infected cells were injected into nude mice to evaluate tumorigenesis.ResultsLow hsa_circ_0055538 expression levels were verified in tumor tissues and OSCC cell lines. Clinical data analysis showed that the expression level is related to the degree of tumor differentiation. Lentiviral infection and siRNA transfection of SCC9 and CAL27 cell lines revealed that changes in circRNA expression significantly affected the malignant biological behavior of OSCC cells. Importantly, nude mouse experiments showed that high expression of hsa_circ_0055538 inhibited tumor growth. Finally, hsa_circ_0055538 may affect the development of OSCC via the p53/Bcl-2/caspase signaling pathway.ConclusionsOur results indicated that hsa_circ_0055538 is involved in OSCC via the p53 signaling pathway and may be a diagnostic and/or prognostic marker as well as a therapeutic target.