Circular RNA Expression Profiles Research Articles

Comprehensive analysis of circRNA-miRNA-mRNA in oral squamous cell carcinoma

ObjectivesThe aim of the present study was to explore expression profiles of circular RNAs (circRNAs) and potential molecular mechanisms in oral squamous cell carcinoma (OSCC). DesignIn this study, high-throughput microarray was used to detect the expression profiles of circRNAs in OSCC. Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of dysregulated circRNAs in microarray. The validated microRNAs sponged by circRNAs and targeted mRNA were collected from literatures and miRTarBase database. and Gene Ontologypathways were analyzed on DAVID platform. Furthermore, network of circRNA-miRNA-mRNA and protein-protein interaction were constructed by Cytoscape 6.0 software. ResultsThe results revealed that 1540 circRNAs were differentially expressed in 4 paired OSCC tissues and adjacent tissues significantly. Among them, 6 abundant circRNAs which have been reported in literatures were identified through RT-qPCR. According to the hypothesis of competing endogenous RNAs, 14 miRNAs sponged by the validated circRNAs and 62 downstream target protein coding genes were identified. Upon Gene Ontologyanalysis, 62 target genes were found to play roles in positively regulation of cell migration, cell division, angiogenesis and response to hypoxia. For Kyoto Encyclopedia of Genes and Genomes pathways, target genes were found to be involved in oncogenic signaling pathways (hsa05200), PI3K-Akt signaling pathway (hsa04151), focal adhesion (hsa04510), microRNAs in cancer (hsa05206), ECM-receptor interaction (hsa04512). These results suggested that circRNAs may participate in the tumorigenesis and development of OSCC via sponging miRNAs. ConclusionsThis study indicated that circRNA may play vital role in tumorigenesis and metastasis in OSCC.

Circular RNA profiles of osteoarthritic synovium.

To identify the circular RNA (circRNA) expression profile in the synovium of patients with osteoarthritis (OA) and explore their potential regulatory mechanism. Transcriptome high-throughput sequencing was used to detect the expression profiles of circRNA and mRNA. We performed real-time PCR for the validation of circRNAs and used bioinformatics analysis to predict their possible biological functions. The conservation of circRNAs was evaluated, a circRNA-miRNA-mRNA interaction network was constructed and the receiver operating characteristic (ROC) curves of target genes were also drawn. We found 136 differentially expressed circRNAs, 64 upregulated and 72 downregulated. We also found 2035 differentially expressed mRNAs, 1216 upregulated and 819 downregulated. It was verified by qRT-PCR that hsa_circ_0072697 was significantly upregulated. The GO analysis results showed that the parental genes were mainly enriched in organelle organization, cytosol and anion binding. The most enriched pathways for these circRNAs participated in cellular senescence. And hsa_circ_0072697 might act as a sponge of hsa-miR-6736-5p, which could therefore lead to increased LEP and ULK1 mRNA expression. CircRNAs are significantly expressed in the knee synovium of OA patients and may play an important role in the occurrence and development of OA. The potential mechanism could be sponging miRNAs to increase mRNA expression.

Comprehensive analysis of circRNA expression profiles in rat cerebral cortex after moderate traumatic brain injury.

Traumatic brain injury is a medical event of global concern, and a growing body of research suggests that circular RNAs can play very important roles in traumatic brain injury. To explore the functions of more novel and valuable circular RNA in traumatic brain injury response, a moderate traumatic brain injury in rats was established and comprehensive analysis of circular RNA expression profiles in rat cerebral cortex was done. As a result, 301 up-regulated and 284 down-regulated circular RNAs were obtained in moderate traumatic brain injury rats, the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed based on the circular RNA's host genes, and a circRNA-miRNA interaction network based on differentially expressed circular RNAs was constructed. Also, four circular RNAs were validated by RT-qPCR and Sanger sequencing. This study showed that differentially expressed circular RNAs existed between rat cerebral cortex after moderate traumatic brain injury and control. And this will provide valuable information for circular RNA research in the field of traumatic brain injury.

Expression Profiles of Circular RNA in Aortic Vascular Tissues of Spontaneously Hypertensive Rats.

Background: Circular RNAs (circRNAs), as a kind of endogenous non-coding RNA, have been implicated in ischemic heart diseases and vascular diseases. Based on theirs high stability with a closed loop structure, circRNAs function as a sponge and bind specific miRNAs to exert inhibitory effects in heart and vasculature, thereby regulating their target gene and protein expression, via competitive endogenous RNA (ceRNA) mechanism. However, the exact roles and underlying mechanisms of circRNAs in hypertension and related cardiovascular diseases remain largely unknown.Methods and Results: High-throughput RNA sequencing (RNA-seq) was used to analyze the differentially expressed (DE) circRNAs in aortic vascular tissues of spontaneously hypertensive rats (SHR). Compared with the Wistar-Kyoto (WKY) rats, there were marked increases in the levels of systolic blood pressure, diastolic blood pressure and mean blood pressure in SHR under awake conditions via the tail-cuff methodology. Totally, compared with WKY rats, 485 DE circRNAs were found in aortic vascular tissues of SHR with 279 up-regulated circRNAs and 206 down-regulated circRNAs. Furthermore, circRNA-target microRNAs (miRNAs) and the target messenger RNAs (mRNAs) of miRNAs were predicted by the miRanda and Targetscan softwares, respectively. Additionally, real-time RT-PCR analysis verified that downregulation of rno_circRNA_0009197, and upregulation of rno_circRNA_0005818, rno_circRNA_0005304, rno_circRNA_0005506, and rno_circRNA_0009301 were observed in aorta of SHR when compared with that of WKY rats. Then, the potential ceRNA regulatory mechanism was constructed via integrating 5 validated circRNAs, 31 predicted miRNAs, and 266 target mRNAs. More importantly, three hub genes (NOTCH1, FOXO3, and STAT3) were recognized according to PPI network and three promising circRNA-miRNA-mRNA regulatory axes were found in hypertensive rat aorta, including rno_circRNA_0005818/miR-615/NOTCH1, rno_circRNA_0009197/ miR-509-5p/FOXO3, and rno_circRNA_0005818/miR-10b-5p/STAT3, respectively.Conclusions: Our results demonstrated for the first time that circRNAs are expressed aberrantly in aortic vascular tissues of hypertensive rats and may serve as a sponge linking with relevant miRNAs participating in pathogenesis of hypertension and related ischemic heart diseases via the circRNA-miRNA-mRNA ceRNAnetwork mechanism.

The circular RNA expression profile in ovarian serous cystadenocarcinoma reveals a complex circRNA\u2013miRNA regulatory network

BackgroundOvarian serous cystadenocarcinoma is one of the most serious gynecological malignancies. Circular RNA (circRNA) is a type of noncoding RNA with a covalently closed continuous loop structure. Abnormal circRNA expression might be associated with tumorigenesis because of its complex biological mechanisms by, for example, functioning as a microRNA (miRNA) sponge. However, the circRNA expression profile in ovarian serous cystadenocarcinoma and their associations with other RNAs have not yet been characterized. The main purpose of this study was to reveal the circRNA expression profile in ovarian serous cystadenocarcinoma.MethodsWe collected six specimens from three patients with ovarian serous cystadenocarcinoma and adjacent normal tissues. After RNA sequencing, we analyzed the expression of circRNAs with relevant mRNAs and miRNAs to characterize potential function.Results15,092 unique circRNAs were identified in six specimens. Approximately 46% of these circRNAs were not recorded in public databases. We then reported 353 differentially expressed circRNAs with oncogenes and tumor-suppressor genes. Furthermore, a conjoint analysis with relevant mRNAs revealed consistent changes between circRNAs and their homologous mRNAs. Overall, construction of a circRNA–miRNA network suggested that 4 special circRNAs could be used as potential biomarkers.ConclusionsOur study revealed the circRNA expression profile in the tissues of patients with ovarian serous cystadenocarcinoma. The differential expression of circRNAs was thought to be associated with ovarian serous cystadenocarcinoma in the enrichment analysis, and co-expression analysis with relevant mRNAs and miRNAs illustrated the latent regulatory network. We also constructed a complex circRNA–miRNA interaction network and then demonstrated the potential function of certain circRNAs to aid future diagnosis and treatment.

Circular RNA Expression and Regulation Profiling in Testicular Tissues of Immature and Mature Wandong Cattle (Bos taurus).

Wandong cattle are an autochthonous Chinese breed used extensively for beef production. The breed tolerates extreme weather conditions and raw feed and is resistant to tick-borne diseases. However, the genetic basis of testis development and sperm production as well as breeding management is not well established in local cattle. Therefore, improving the reproductive efficiency of bulls via genetic selection is crucial as a single bull can breed thousands of cows through artificial insemination (AI). Testis development and spermatogenesis are regulated by hundreds of genes and transcriptomes. However, circular RNAs (circRNAs) are the key players in many biological developmental processes that have not been methodically described and compared between immature and mature stages in Bovine testes. In this study, we performed total RNA-seq and comprehensively analyzed the circRNA expression profiling of the testis samples of six bulls at 3 years and 3 months of developmental age. In total, 17,013 circRNAs were identified, of which 681 circRNAs (p-adjust < 0.05) were differentially expressed (DE). Among these DE circRNAs, 579 were upregulated and 103 were downregulated in calf and bull testes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the identified target genes were classified into three broad functional categories, including biological process, cellular component, and molecular function, and were enriched in the lysine degradation, cell cycle, and cell adhesion molecule pathways. The binding interactions between DE circRNAs and microRNAs (miRNAs) were subsequently constructed using bioinformatics approaches. The source genes ATM, CCNA1, GSK3B, KMT2C, KMT2E, NSD2, SUCLG2, QKI, HOMER1, and SNAP91 were found to be actively associated with bull sexual maturity and spermatogenesis. In addition, a real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed a strong correlation with the sequencing data. Moreover, the developed model of Bovine testes in the current study provides a suitable framework for understanding the mechanism of circRNAs in the development of testes and spermatogenesis.

Circular RNA expression profiles following negative pressure wound therapy in burn wounds with experimental Pseudomonas aeruginosa infection

ABSTRACT Infections of burn wounds, especially those caused by Pseudomonas aeruginosa, could trigger sepsis or septic shock, which is the main cause of death after burn injury. Compared with traditional saline-wet-to-dry dressings, negative pressure wound therapy (NPWT) is more effective for the prevention and treatment of wound infections. However, the mechanism by which NPWT controls infection and accelerates wound healing remains unclear. Accordingly, in this study, the molecular mechanisms underlying the effects of NPWT were explored using a murine model of P. aeruginosa-infected burn wounds. NPWT significantly reduced P. aeruginosa levels in wounds, enhanced blood flow, and promoted wound healing. Additionally, NPWT markedly alleviated wound inflammation and increased the expression of wound healing–related molecules. Recent evidence points to a role of circular RNAs (circRNAs) in wound healing; hence, whole-transcriptome sequencing of wound tissues from NPWT and control groups was performed to evaluate circRNA expression profiles. In total, 12 up-regulated and 25 down-regulated circRNAs were identified between groups. Among these, five significant differentially expressed circRNAs acting as microRNA sponges were identified, and their predicted targets were verified by reverse transcription-quantitative polymerase chain reaction. These results further support the roles of circRNAs in wound healing by NPWT and the prevention of P. aeruginosa infection, providing key molecular targets for further functional analyses.

Circular RNA Expression Profiles and Bioinformatic Analysis in Mouse Models of Obstructive Sleep Apnea-Induced Cardiac Injury: Novel Insights Into Pathogenesis.

Circular RNAs (circRNAs) participate in the development of various kinds of diseases. However, the function and roles of circRNAs in obstructive sleep apnea (OSA)-induced cardiovascular disease remain poorly understood. Therefore, we sought to explore the circRNA expression profiles and predict their functions in OSA-induced cardiac injury with the use of bioinformatics analysis. The model of OSA was established in mouse treated by chronic intermittent hypoxia (CIH) exposure. Then, we screened the circRNA profile using circRNA microarray. By comparing circRNA expression in three matched pairs of CIH-treated cardiac tissues and controls, differentially expressed circRNAs were identified in the CIH groups. Comparison of the selected circRNAs expression levels was performed between qRT-PCR and microarray. Meanwhile, we employed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to predict the functions of these selected circRNAs. Finally, we constructed a circRNA-miRNA-mRNA network based on the target prediction. It was found that a total of 124 circRNAs were differentially expressed in CIH-treated cardiac tissues (p ≤ 0.05, fold-change ≥ 1.5). Among them, 23 circRNAs were significantly down-regulated, and the other 101 were up-regulated. Then, ten circRNAs were randomly selected to validate the reliability of the microarray results by using qRT-PCR. Next, we conducted the GO and KEGG pathway analysis to explore the parental genes functions of differentially expressed circRNA. Finally, two significantly differentially expressed circRNAs (mmu_circRNA_014309 and mmu_circRNA_21856) were further selected to create a circRNA-miRNA-mRNA regulation network. Our study did first reveal that the differentially expressed circRNAs played a vital role in the pathogenesis of OSA-induced cardiac damage. Thus, our findings bring us closer to unraveling the pathophysiologic mechanisms and eliciting novel therapeutic targets for the treatment of OSA-associated cardiovascular diseases.

Exploring the Role of CircRNA in Diabetic Kidney Disease from a Novel Perspective: Focusing on Both Glomeruli and Tubuli.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease, but the molecular mechanisms of disease remain not very clear and there is no curative therapeutic strategy so far. This study was carried out to identify the expression profile of circular RNA (circRNA) in human DKD and explore circRNA regulatory function in glomeruli and tubuli simultaneously. As a result, a total of 40 upregulated and 23 downregulated differentially expressed circRNAs (DEcircRNAs) were detected. Six candidate DEcircRNAs were verified by quantitative real-time polymerase chain reaction in high glucose-treated human mesangial cells and human proximal renal tubular epithelial cells, respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that both in glomeruli and in tubuli the DEcircRNAs-targeted genes participated in many pathophysiological processes of DKD. Correlation analysis with renal function showed that expression level of DEcircRNA-targeted hub gene was related to renal function. In conclusion, this is the first study to report expression profiles of circRNAs in kidney of DKD patients, and further analysis demonstrated that circRNA probably played a significant regulatory role, providing help for understanding the pathogenesis of DKD and investigating novel diagnostic and therapeutic strategy.

RNA sequencing reveals the circular RNA expression profiles of the infrapatellar fat pad/synovium unit.

BackgroundThe infrapatellar fat pad (IPFP) and synovium are reported to act as a functional unit, with emerging roles in the pathophysiology of knee osteoarthritis (KOA). Circular RNAs (circRNAs) are involved in the pathogenesis of KOA, especially in cartilage homeostasis regulation. Nevertheless, the regulatory mechanisms of circRNAs in the KOA IPFP/synovium unit remain to be elucidated. Therefore, the current study aimed to investigate alterations in the expression of circRNAs and predict their functions in the KOA IPFP/synovium unit using bioinformatics analysis.MethodsIn brief, 6 synovium and IPFP specimens were collected, in which 3 from patients with KOA and 3 from controls. Then, circRNA sequencing was conducted on 2 KOA synovium and IPFP specimens as well as 1 control to investigate the expression profiles of circRNAs. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome signaling pathway analyses were employed to predict the functions of the differentially expressed circRNAs. Based on the miRNA sponge theory, we constructed a circRNA-miRNA network to predict the molecular regulatory mechanism of these circRNAs. Venn analysis was performed to confirm the circRNAs and miRNAs expressed in both synovium and IPFP. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was also used to validate the expression levels of circRNAs that were co-expressed in both synovium and IPFP.ResultsA total of 65 and 72 circRNAs were differentially expressed in KOA synovium and IPFP, respectively (fold change ≥2, P<0.05). After obtaining the parental genes of differentially expressed circRNAs, the top 10 enrichment GO entries, KEGG pathways, and Reactome pathways were annotated. Furthermore, hsa_circ_0005265 was found to be down-regulated in both synovium and IPFP, which was validated by qRT-PCR. The circRNA-miRNA network was created to annotate the probable regulatory mechanisms of the differentially expressed circRNAs, which consequently confirmed 2 target miRNAs (hsa-miR-6769b-5p and hsa-miR-1249-5p) associated with hsa_circ_0005265 in both synovium and IPFP.ConclusionsOur outcomes bring us closer to understanding the potential mechanism of the IPFP/synovium unit in the progression of KOA and finding new molecular targets for KOA therapy.

Expression Profile and Potential Function of Circular RNAs in Peripheral Blood Mononuclear Cells in Male Patients With Primary Gout.

Circular RNAs (circRNAs) are non-coding RNAs (ncRNAs) with a single-stranded covalently closed-loop structure, and their abnormal expression may participate in the pathogenesis of various human diseases. Currently, knowledge of circRNAs in gout is limited. In this case-control study, human circRNA microarrays were used to identify differentially expressed circRNAs in peripheral blood mononuclear cells (PBMCs) from patients with primary gout (n = 5) and healthy controls (HC; n = 3). Bioinformatics methods were used to analyze significantly different circRNAs (fold change >1.5, p < 0.05). In addition, four significantly differentially expressed circRNAs were selected for quantitative real-time polymerase chain reaction to detect expression levels in 90 gout patients and 60 HC. Subsequently, circRNA-miRNA-mRNA network was established to predict the function of circRNAs of interest. Microarray analysis indicated that 238 circRNAs were upregulated and 41 circRNAs were down-regulated in the gout group (fold change >1.5, p < 0.05). Bioinformatics analysis showed that differentially expressed circRNAs were involved in the pathogenesis of gout via various pathways. Moreover, the expression levels of hsa_circRNA_103657 and hsa_circRNA_000241 were significantly higher in the gout group than those in the HC group, and both correlated significantly with lipid metabolism parameters. Furthermore, the area under the curve of hsa_circRNA_103657 was 0.801 (95% confidence interval (CI): 0.730–0.871; p < 0.001). Our results provide novel insights into the pathogenesis of primary gout. Differentially expressed circRNAs were identified in the PBMCs of gout patients, and these differential circRNAs may play important roles in the development and progression of gout.

Integrating analysis of circular RNA and mRNA expression profiles in doxorubicin induced cardiotoxicity mice.

Doxorubicin (DOX)-induced cardiotoxicity impedes its clinical application, but the mechanisms have not been thoroughly elucidated. Based on circRNA and mRNA expression profiles, we illustrated RNA expression signature changes during DOX-induced cardiotoxicity; mechanism exploration and biomarkers screening were also conducted. Twelve mice were randomly divided into two groups, induction group was treated with doxorubicin, and the control group was given an equal quantity of saline. After the confirmation of myocardial injury in induction group, the heart tissues from both groups were isolated for RNA high-throughput sequencing. The expression profiles were compared between the two groups; a total of 295 mRNAs and 11 circRNAs were shown as biased expression in DOX-induced cardiotoxicity mouse hearts. The dysregulation of three circRNAs were validated by quantitative real-time PCR: mmu_circ_0015773, mmu_circ_0002106, and mmu_circ_001606. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the differentially expressed RNAs were performed; the results implied that DOX might cause cardiotoxicity by interfering hemoglobin-based oxygen delivery and DNA-associated signal pathways. We integrated the differential expressed mRNA and validated circRNAs by constructing a competing endogenous RNA (ceRNA) network, which indicated that the alteration of the three circRNAs could activate apoptosis process of myocardial cells. This study provided novel insight into the mechanisms of DOX induced cardiotoxicity, and potential biomarkers or therapeutic targets were also proposed.

Circular RNAs in Hedgehog Signaling Activation and Hedgehog-Mediated Medulloblastoma Tumors.

Simple SummaryHere the expression profile of circular RNAs in Hedgehog signaling-dependent cell lines and medulloblastoma cells was interrogated. Using stringent criteria, a reduced expression of seven circular RNAs in Hedgehog-dependent medulloblastoma versus cerebellum was clearly established. Depletion and/or overexpression of these deregulated RNA circles in two medulloblastoma cell lines revealed minimal effects in cellular proliferation based on two independent assays. These findings highlight the complexity of gene expression outcomes and the possibility that gene products may not necessarily have an obvious phenotypic impact on the cellular context where they are present. It is not inconceivable that a substantial number of differentially expressed circular RNAs may represent “passenger molecules” with little impact on a cell, reflecting the stochasticity of the gene expression and splicing processes.Within the past decade, circular RNAs have largely emerged as novel regulators of human biology, including brain function and cancer development. On the other hand, the Hedgehog pathway has established roles in regulating biological processes, including tumorigenesis. Here, the circular RNA transcriptome, in the context of Hedgehog signaling activation of medulloblastoma Daoy and human embryonic palatal mesenchyme HEPM cells, was determined. In total, 29 out of the 30 selected circular RNAs were validated by Sanger sequencing, with some regulated to a limited extent by Hedgehog signaling. Interestingly, back-spliced junctions, the marker of exonic RNA circles, were also identified at a low frequency within poly (A) mRNAs, reflecting exon repetition events. Thirteen circular RNAs had reduced expression in human medulloblastoma tumors in comparison to normal cerebellum. For seven out of these thirteen RNA circles, the linear mRNAs originating from the same genes did not exhibit a reduced expression. Depletion and/or overexpression of these seven circular RNAs minimally affected medulloblastoma cell proliferation. These findings highlight that differential expression of a gene product may not necessarily elicit an obvious phenotypic impact. Consequently, further analysis is required to determine the possible subtle contributions to the development of this cerebellar tumor.

Construction of a competing endogenous RNA network related to the prognosis of cholangiocarcinoma and comprehensive analysis of the immunological correlation.

Cholangiocarcinoma (CCA) is a malignant tumor of the digestive system, with occult onset in the early stage, a high degree of malignancy in the late stage, and poor prognosis. At present, the pathogenesis of CCA is not clear, and there is a lack of effective immunotherapy. The purpose of this study was to identify the potential regulatory mechanism of CCA and analyze the possibility of its related immunotherapy. The circular RNAs (circRNAs) expression profile data of CCA was downloaded from the Gene Expression Omnibus (GEO) database; the miRNA and mRNA expression profile data of CCA were downloaded from The Cancer Genome Atlas (TCGA) database. Prognostic factors were screened by univariate Cox regression analysis, and the competing endogenous RNA (ceRNA) network was constructed via survival analysis. Multivariate Cox analysis was used to screen the independent prognostic factors and construct a prognostic correlation subnetwork. Analyzing the tumor microenvironment of CCA and survival analysis were performed according to the score of the microenvironment, and the distribution of tumor infiltrating immune cells (TICs) in CCA was calculated using the CIBERSORT algorithm. We explored the expression pattern of the target genes in pan-cancer, and the correlation between the key genes in the ceRNA subnetwork, TICs and immune checkpoints was analyzed using an online database. Finally, the expression levels of target genes were validated based on the Human Protein Atlas (HPA) databases. We screened four circRNAs, 10 miRNAs, and 17 mRNAs with significant differences, and constructed the ceRNA network. Independent prognostic factors were screened by multivariate Cox regression analysis, and a subnetwork containing five nodes (hsa_circ_0002073→hsa-mir-4524a-3p→SLC16A3/SLC35E4/DDX4) was constructed. Further analysis showed that SLC16A3 was not only an independent posterior factor of CCA, but was also closely correlated with immune cells, immune checkpoints, and immunotherapy, and had a certain regulatory effect on the tumor microenvironment. Our study identified a novel prognostic marker of CCA, SLC16A3, and revealed the regulatory role of SLC16A3 in the tumor microenvironment, which is expected to provide new insights for the early diagnosis, prognosis, and targeted therapy of CCA.

CircCOL1A1 Promotes the Progression of Gastric Cancer Cells through Sponging miR-145 to Enhance RABL3 Expression.

Circular RNA has been reported to be a new noncoding RNA which plays important roles in tumor progression. One of the most common functions of circular RNA is to regulate microRNA expression by acting as a microRNA sponge. However, the circular RNA expression profile and function remain mostly unclear in gastric cancer. In the study, we explored the expression and function of circCOL1A1 (hsa_circ_0044556) in gastric cancer. We performed RT-PCR with divergent primers, mRNA stability assay, and RNase R digestion assay to characterize circCOL1A1 in gastric cancer cell lines. qRT-PCR was applied to detect the level of circCOL1A1 in both gastric cancer cell lines and tissues. Gain- and loss-of-function studies were carried out to detect the influence of circCOL1A1 on gastric cancer cells by performing CCK8, migration, and invasion assays. The regulation of the downstream genes was identified by qRT-PCR, western blot assay, dual luciferase assay, and RNA pull-down assay. The results showed that circCOL1A1 was highly expressed in both gastric cancer cells and tissues. Silence of circCOL1A1 inhibited the proliferation, migration, and invasion of gastric cancer cells. circCOL1A1 regulated the expression of miR-145 by acting as a microRNA sponge, and the influence of circCOL1A1 could be abrogated by miR-145 mimics. Our research shows that miR-145 plays its functions through targeting and regulating RABL3. Inhibition of circCOL1A1/miR-145/RABL3 could effectively suppress gastric cancer cell proliferation, migration, and invasion. circCOL1A1 also promote the transformation of M1 into M2 macrophage. Our study identified circCOL1A1 as a novel oncogenic circRNA and will provide more information for gastric cancer therapy.

Circular RNAs Repertoire and Expression Profile during Brassica rapa Pollen Development.

Circular RNAs (circRNAs) are covalently closed RNA molecules generated by the back-splicing of exons from linear precursor mRNAs. Though various linear RNAs have been shown to play important regulatory roles in many biological and developmental processes, little is known about the role of their circular counterparts. In this study, we performed high-throughput RNA sequencing to delineate the expression profile and potential function of circRNAs during the five stages of pollen development in Brassica rapa. A total of 1180 circRNAs were detected in pollen development, of which 367 showed stage-specific expression patterns. Functional enrichment and metabolic pathway analysis showed that the parent genes of circRNAs were mainly involved in pollen-related molecular and biological processes such as mitotic and meiotic cell division, DNA processes, protein synthesis, protein modification, and polysaccharide biosynthesis. Moreover, by predicting the circRNA–miRNA network from our differentially expressed circRNAs, we found 88 circRNAs with potential miRNA binding sites, suggesting their role in post-transcriptional regulation of the genes. Finally, we confirmed the back-splicing sites of nine selected circRNAs using divergent primers and Sanger sequencing. Our study presents the systematic analysis of circular RNAs during pollen development and forms the basis of future studies for unlocking complex gene regulatory networks underpinning reproduction in flowering plants.

Whole Transcriptome Analysis Revealed a Stress Response to Deep Underground Environment Conditions in Chinese Hamster V79 Lung Fibroblast Cells.

Background: Prior studies have shown that the proliferation of V79 lung fibroblast cells could be inhibited by low background radiation (LBR) in deep underground laboratory (DUGL). In the current study, we revealed further molecular changes by performing whole transcriptome analysis on the expression profiles of long non-coding RNA (lncRNA), messenger RNA (mRNA), circular RNA (circRNA) and microRNA (miRNA) in V79 cells cultured for two days in a DUGL.Methods: Whole transcriptome analysis including lncRNA, mRNAs, circ RNA and miRNA was performed in V79 cells cultured for two days in DUGL and above ground laboratory (AGL), respectively. The differentially expressed (DE) lncRNA, mRNA, circRNA, and miRNA in V79 cells were identified by the comparison between DUGL and AGL groups. Quantitative real-time polymerase chain reaction(qRT-PCR)was conducted to verify the selected RNA sequencings. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was analyzed for the DE mRNAs which enabled to predict target genes of lncRNA and host genes of circRNA.Results: With |log2(Fold-change)| ≥ 1.0 and p < 0.05, a total of 1257 mRNAs (353 mRNAs up-regulated, 904 mRNAs down-regulated), 866 lncRNAs (145 lncRNAs up-regulated, 721 lncRNAs down-regulated), and 474 circRNAs (247 circRNAs up-regulated, 227 circRNAs down-regulated) were significantly altered between the two groups. There was no significant difference in miRNA between the two groups. The altered RNA profiles were mainly discovered in lncRNAs, mRNAs and circRNAs. DE RNAs were involved in many pathways including ECM-RI, PI3K-Akt signaling, RNA transport and the cell cycle under the LBR stress of the deep underground environment.Conclusion: Taken together, these results suggest that the LBR in the DUGL could induce transcriptional repression, thus reducing metabolic process and reprogramming the overall gene expression profile in V79 cells.

Pepsinogen C expression-related lncRNA/circRNA/mRNA profile and its co-mediated ceRNA network in gastric cancer.

The expression of pepsinogen C (PGC) is considered an ideal negative biomarker of gastric cancer, but its pathological mechanisms remain unclear. This study aims to analyze competing endogenous RNA (ceRNA) networks related to PGC expression at a post-transcriptional level and build an experimental basis for studying the role of PGC in the progression of gastric cancer. RNA sequencing technology was used to detect the differential expression (DE) profiles of PGC-related long non-coding (lnc)RNAs, circular (circ)RNAs, and mRNAs. Ggcorrplot R package and online database were used to construct DElncRNAs/DEcircRNAs co-mediated PGC expression-related ceRNA networks. In vivo and in vitro validations were performed using quantitative reverse transcription-PCR (qRT-PCR). RNA sequencing found 637 DEmRNAs, 698 DElncRNAs, and 38 DEcircRNAs. The PPI network of PGC expression-related mRNAs consisted of 503 nodes and 1179 edges. CFH, PPARG, and MUC6 directly interacted with PGC. Enrichment analysis suggested that DEmRNAs were mainly enriched in cancer-related pathways. Eleven DElncRNAs, 13 circRNAs, and 35 miRNA-mRNA pairs were used to construct ceRNA networks co-mediated by DElncRNAs and DEcircRNAs that were PGC expression-related. The network directly related to PGC was as follows: SNHG16/hsa_circ_0008197-hsa-mir-98-5p/hsa-let-7f-5p/hsa-let-7c-5p-PGC. qRT-PCR validation results showed that PGC, PPARG, SNHG16, and hsa_circ_0008197 were differentially expressed in gastric cancer cells and tissues: PGC positively correlated with PPARG (r = 0.276, P = 0.009), SNHG16 (r = 0.35, P = 0.002), and hsa_circ_0008197 (r = 0.346, P = 0.005). PGC-related DElncRNAs and DEcircRNAs co-mediated complicated ceRNA networks to regulate PGC expression, thus affecting the occurrence and development of gastric cancer at a post-transcriptional level. Of these, the network directly associated with PGC expression was a SNHG16/hsa_circ_0008197-mir-98-5p/hsa-let-7f-5p/hsa-let-7c-5p - PGC axis. This study may form a foundation for the subsequent exploration of the possible regulatory mechanisms of PGC in gastric cancer.

Circular RNA expression profiles in human bronchial epithelial cells treated with beryllium sulfate.

Circular RNAs (circRNAs), is a novel type of endogenous non-coding RNAs (ncRNAs) participated in the pathogenesis of many diseases. Beryllium is one of the carcinogenesis elements. However, the mechanism and function of circRNAs in human bronchial epithelial cells (16HBE) induced by beryllium sulfate (BeSO4) was rarely reported. Therefore, the high-throughput RNA sequencing analysis was performed to detect the circRNA profiles between control groups and BeSO4-induced groups. Furthermore, circRNA-miRNA-mRNA network, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and PPI network analysis were used for bioinformatics analysis. CircRNA sequencing analysis revealed that 36 circRNAs were up-regulated and 35 circRNAs were down-regulated in the BeSO4-exposed groups. The selected circRNAs were verified by real-time fluorescent quantitative PCR (qRT-PCR). Hsa_circ_0004214 and hsa_circ_0003586 were validated to be up-regulated, hsa_circ_0047958, hsa_circ_0001944, and hsa_circ_0008982 were down-regulated. The circRNA-miRNA-mRNA network annotated the key signaling pathway including cellular senescence, TNF signaling pathway, NF-kappa B signaling pathway, HIF-1 signaling pathway, and Hippo signaling pathway. The PPI network indicated the most circRNAs might participate in the BeSO4 toxicity by acting as a sponge for the miR-663b through JAK-STAT signaling pathway. In summary, our study suggests that circRNAs may play roles in the mechanism of beryllium toxicity.

Identification of Circular RNA Expression Profiles in White Adipocytes and Their Roles in Adipogenesis.

Obesity and its related metabolic diseases have become great public health threats worldwide. Although accumulated evidence suggests that circRNA is a new type of non-coding RNAs regulating various physiological and pathological processes, little attention has been paid to the expression profiles and functions of circRNAs in white adipose tissue. In this study, 3,771 circRNAs were detected in three stages of white adipogenesis (preadipocyte, differentiating preadipocyte, and mature adipocyte) by RNA-seq. Experimental validation suggested that the RNA-seq results are highly reliable. We found that nearly 10% of genes which expressed linear RNAs in adipocytes could also generate circRNAs. In addition, 40% of them produced multiple circRNA isoforms. We performed correlation analysis and found that a great deal of circRNAs (nearly 50%) and their parental genes were highly correlated in expression levels. A total of 41 differential expression circRNAs (DECs) were detected during adipogenesis and an extremely high ratio of them (80%) were correlated with their parental genes, indicating these circRNAs may potentially play roles in regulating the expression of their parental genes. KEGG enrichment and GO annotation of the parental genes suggesting that the DECs may participate in several adipogenesis-related pathways. Following rigorous selection, we found that many up-regulated circRNAs contain multiple miRNAs binding sites, such as miR17, miR-30c, and miR-130, indicating they may potentially facilitate their regulatory functions by acting as miRNA sponges. These results suggest that plenty of circRNAs are expressed in white adipogenesis and the DECs may serve as new candidates for future adipogenesis regulation.