Circular RNA: The evolving potential in the disease world

Circular RNAs(circRNAs),a new star of noncoding RNAs,are a group of endogenous RNAs that form a covalently closed circle and occur widely in the mammalian genome.Most circRNAs are conserved throughout species and fre-quently show stage-specific expression during various stages of tissue develop-ment.CircRNAs were a mystery discovery,as they were initially believed to be a product of splicing errors;however,subsequent research has shown that ci-rcRNAs can perform various functions and help in the regulation of splicing and transcription,including playing a role as microRNA(miRNA)sponges.With the application of high throughput next-generation technologies,circRNA hotspots were discovered.There are emerging indications that explain the association of circRNAs with human diseases,like cancers,developmental disorders,and in-flammation,and circRNAs may be a new potential biomarker for the diagnosis and treatment outcome of various diseases,including cancer.After the discoveries of miRNAs and long noncoding RNAs,circRNAs are now acting as a novel re-search entity of interest in the field of RNA disease biology.In this review,we aim to focus on major updates on the biogeny and metabolism of circRNAs,along with their possible/established roles in major human diseases.

Analysis of the Effects of Different Synthesis and Processing Methods on Circular RNA Integrity,Protein Expression,and Removal of Immunogenic Impurities

Circular RNAs(circRNAs)are emerging as a promising alternative to messenger RNAs(mRNAs)in gene delivery applications due to their enhanced stability and translation.Developing circRNA-based therapeutic platforms requires efficient manufacturing of circRNA with broad scalability.However,the permuted intron-exon(PIE)-based circRNA production commonly used to date involves complex RNA synthesis,circularization,precursor RNA digestion,and impurity removal steps that have limited practical applications.While co-transcriptional circularization could effectively streamline circRNA production,and both cellulose/phosphatase treatment and high-performance liquid chromatography(HPLC)have demonstrated their reliability in mRNA manufacturing,their potential effects on the quality,translation,and reactogenicity of circRNA remained to be fully investigated.Here,using circRNAs systematically manufactured through three independent workflows,we comprehensively examined the utilities of these RNA synthesis and processing methods in circRNA production by comparing the integrity,translation,and immunogenicity of their circRNA products.We began by manufacturing a mNeonGreen(mNG)-encoding circRNA through these workflows and subsequently assessed circRNA integrity via E-gel EX electrophoresis.Protein expression was then monitored in HEK 293T,A549,and DC2.4 cells at 72 hours post-transfection.Finally,we evaluated the immunogenicity of these circRNAs by measuring their interferon beta(IFN-β)induction in A549 cells at 4 hours post-transfection.Using HPLC purification over cellulose and phosphatase treatment resulted in 10-14%higher circRNA enrichment by reducing nicking associated with processing conditions.Protein expression remained consistent across circRNAs from different workflows(P>0.05),demonstrating that co-transcriptional circularization produces circRNA with translation levels comparable to those obtained from the conventional PIE method.Moreover,both cellulose/phosphatase treatment and HPLC purification effectively minimized IFN-βinduction of the purified circRNAs,confirming their reliability in removing immunogenic impurities introduced during in vitro transcription and their compatibility with the co-transcriptional circularization strategy.Collectively,our results provide valuable insights for improving the production efficiency and scalability of circRNA manufacturing that are crucial for addressing key bottlenecks in the development of circRNA-based therapeutic applications.

Knockdown of circular RNA (CircRNA)_001896 inhibits cervical cancer proliferation and stemness in vivo and in vitro

Objective:Previous studies indicated that aberrant circular RNA(circRNA)expression affects gene expression regulatory networks,leading to the aberrant activation of tumor pathways and promoting tumor cell growth.However,the expression,clinical significance,and effects on cell propagation,invasion,and dissemination of circRNA_001896 in cervical cancer(CC)tissues remain unclear.Methods:The Gene Expression Omnibus(GEO)datasets(GSE113696 and GSE102686)were used to examine differential circRNA expression in CC and adjacent tissues.The expression of circRNA_001896 was detected in 72 CC patients usingfluorescence quantitative PCR.Correlation analysis with clinical pathological features was performed through COX multivariate and univariate analysis.The effect of circRNA_001896 downregulation on CC cell propagation was examined using the cell counting kit-8(CCK-8)test,clonogenic,3D sphere formation,and in vivo tumorigenesis assays.Results:Intersection of the GSE113696 and GSE102686 datasets revealed an increased expression of four circRNAs,including circRNA_001896,in CC tissues.Fluorescence quantitative PCR confirmed circRNA_001896 as a circular RNA.High expression of circRNA_001896 was considerably associated with lymph node metastasis,International Federation of Gynecologists and Obstetricians(FIGO)stage,tumor diameter,and survival period in CC patients.Proportional hazards model(COX)univariate and multivariate analyses revealed that circRNA_001896 expressions are a distinct risk factor affecting CC patients’prognosis.Cellular functional experiments showed that downregulating circRNA_001896 substantially suppressed CC cell growth,colony formation,and 3D sphere-forming ability.In vivo,tumorigenesis analysis in nude mice demonstrated that downregulating circRNA_001896 remarkably reduced the in vivo proliferation capacity of CC cells.Conclusion:CircRNA_001896 is highly expressed in CC tissues and is substantially related to lymph node metastasis,FIGO stage,tumor size,and survival period in patients.Moreover,downregulating circRNA_001896 significantly inhibits both in vivo and in vitro propagation of CC cells.Therefore,circRNA_001896 might be used as a biomarker for targeted therapy in cervical cancer.

Circular RNA circ_0003609 ameliorates hypertrophied ligamentum flavum by regulating the miR-155/SIRT1 axis

Background:Hypertrophy of the ligamentumflavum(HLF)is a common contributor to spinal stenosis which results in significant neurological impairments.Circular RNA(circRNA)circ_0003609 has been linked to HLF;however,the exact mechanism by which it causes this disease is unclear.Methods:Circ_0003609 expressions were regulated in HLF cells by overexpression vectors and RNA interference.Cell proliferation andfibrosis-related gene expression were checked by the Cell Counting Kit-8(CCK-8)assay and western blotting.CircBank’s prediction of the association between miR-155 and circ_0003609 was supported by a dual-luciferase reporter experiment.The function of the miR-155/sirtuin 1(SIRT1)axis in controlling HLFfibrosis was further examined.Results:Overexpression of circ_0003609 suppressed HLF cell propagation andfibrosis compared to its silencing.It was found that circ_0003609 served as the sponge for miR-155 and that the circ_0003609/miR-155 axis controlled thefibrosis of HLF cells.It was found that circ_0003609 acted as a sponge for miR-155,regulating thefibrosis of HLF cells.Further,miR-155 targets SIRT1,and the miR-155/SIRT1 axis promotes HLF cellfibrosis.Conclusion:Circ_0003609 ameliorates hypertrophied ligamentumflavum(LF)by modulating the miR-155/SIRT1 axis,indicating a potential treatment approach for HLF.

Advances of N6-methyladenosine modification on circular RNA in hepatocellular carcinoma

N6-methyladenosine(m6A)is a reversible epigenetic modification, which is one of the most abundant modifiers in eukaryotic cells and has been commonly reported in messenger RNAs and non-coding RNAs. The processing modification of m6A regulates RNA transcription, processing, splicing, degradation, and translation, and plays an important role in the biological process of tumors. Circular RNA, which lacks the 5' cap structure, has been mistakenly regarded as a "junk sequence" generated by accidental shearing during the transcription process. However, it has been found that circRNAs can be involved in tumor invasion and metastasis through microRNAs, binding proteins, translated peptides, and m6A modifications. In this paper, we reviewed the role of m6A modifications in circRNA regulation and their functions in hepatocellular carcinoma and discussed their potential clinical applications and future development in this field.

Role of exosomal circular RNAs as microRNA sponges and potential targeting for suppressing hepatocellular carcinoma growth and progression

In this editorial,we comment on the article by Lyu et al published in the recent issue of the World Journal of Gastroenterology(2023;2219-2840).Hepatocellular carcinoma(HCC)is a frequently encountered and highly aggressive primary liver cancer,which remains the third-commonest cause of cancer-related death despite the current therapeutic modalities.There is urgency in developing novel thera-peutic approaches,such as by manipulating extracellular vesicles,which con-stitute a highly heterogeneous nanoparticle population that contains various cargoes.These cargoes have a pivotal role in cell-to-cell communication and can modify the functional level of the recipient cells via their uptake by other recipient cells.Exosomal non-coding RNAs have particular evolving significance in HCC,such as circular RNAs,which have been found differentially expressed in normal hepatic and HCC tissues.The aberrations in their expression levels have a key role in the HCC development and progression and the overall prognosis.In this editorial,we will shed light on the emerging role of exosomal circular RNAs in HCC development and progression,focusing on the oncogenic or potentially tumor suppressive effect of mesenchymal stem cells-derived exosomal non-coding RNAs.

Circular RNA_0015278与甲状腺乳头状癌及临床特征的相关性

目的探讨Circular RNA_0015278(circ_0015278)与甲状腺乳头状癌(PTC)及其临床病理特征的相关性。方法回顾分析手术切除的甲状腺乳头状癌患者200例,采用逆转录定量聚合酶链反应(RT-qPCR)检测Circular RNA_0015278在甲状腺乳头状癌(PTC)及癌旁组织的表达。结果与癌旁组织比较,甲状腺乳头状癌(PTC)中Circular RNA_0015278表达显著降低(Z=-12.122,P<0.001),ROC分析显示,以相对表达量0.743为截断值,可较好的鉴别PTC肿瘤及癌旁组织(AUC:0.903,95%CI:0.873~0.933)。而PTC肿瘤中Circular RNA_0015278的高表达与患者年龄<45岁(Z=-2.319,P=0.020)、无甲状腺外侵犯(Z=-2.042,P=0.041)、无淋巴结转移(Z=-2.494,P=0.013)及低pTNM分期(Z=-3.719,P=0.001)相关(P<0.05)。而未发现与性别(Z=-1.323,P=0.186)、肿瘤大小(Z=-1.273,P=0.203)具有相关性(P>0.05)。结论PTC肿瘤中Circular RNA_0015278的表达显著降低,而其在PTC肿瘤中高表达与患者年龄<45岁、无甲状腺外侵犯、无淋巴结转移及低pTNM分期具有相关性。

Circular RNA expression and the competitive endogenous RNA network in pathological,age-related macular degeneration events:A cross-platform normalization study

Age-related macular degeneration(AMD)causes irreversible blindness in people aged over 50 worldwide.The dysfunction of the retinal pigment epithelium is the primary cause of atrophic AMD.In the current study,we used the ComBat and Training Distribution Matching method to integrate data obtained from the Gene Expression Omnibus database.We analyzed the integrated sequencing data by the Gene Set Enrichment Analysis.Peroxisome and tumor necrosis factor-α(TNF-α)signaling and nuclear factor kappa B(NF-κB)were among the top 10 pathways,and thus we selected them to construct AMD cell models to identify differentially expressed circular RNAs(circRNAs).We then constructed a competing endogenous RNA network,which is related to differentially expressed circRNAs.This network included seven circRNAs,15 microRNAs,and 82 mRNAs.The Kyoto Encyclopedia of Genes and Genomes analysis of mRNAs in this network showed that the hypoxia-inducible factor-1(HIF-1)signaling pathway was a common downstream event.The results of the current study may provide insights into the pathological processes of atrophic AMD.

Circular RNAs in colorectal cancer:Possible roles in regulation of cancer cells

Colorectal cancer(CRC) is the third most commonly diagnosed cancer in the world and the fourth principal cause of cancer deaths worldwide. Currently, there is a lack of low cost and noninvasive screening tests for CRC, becoming a serious health problem. In this context, a potential biomarker for the early detection of CRC has recently gained attention. Circular RNAs(circ RNA), a re-discovered, abundant RNA specie, is a type of noncoding covalent closed RNAs formed from both exonic and intronic sequences. These circular molecules are widely expressed in cells, exceeding the abundance of the traditional linear m RNA transcript. They can regulate gene expression, acting as real sponges for mi RNAs and also regulate alternative splicing or act as transcriptional factors and inclusive encoding for proteins. However, little is known about circ RNA and its relationship with CRC. In this review, we focus on the biogenesis, function and role of these circ RNAs in relation to CRC, including their potential as a new biomarker.

Circular RNAs在肿瘤细胞程序性死亡中的研究进展

Circular RNAs(circRNAs)是由真核生物中数千个基因的前体mRNA反向剪接形成的一类共价闭合的连续环状结构的单链闭合RNA分子。高通量测序和生物信息学方法揭示了circRNAs在物种间的广泛表达。它们的高度稳定性、丰度和物种间的进化保守性表明它们具有独特的特性和多样的细胞功能,如高效与microRNA(miRNA)形成海绵机制。同时,circRNAs在调节转录和剪接方面也起着重要作用。细胞程序性死亡是维持人体正常发育和内环境稳态所必需的过程,它的主要功能是消除受损、感染或老化的细胞,一般有多种形式,包括细胞凋亡、细胞焦亡、铁死亡以及自噬等。研究显示,大多数circRNAs在病理条件下以组织特异性方式异常表达,如调节肿瘤细胞的程序性细胞死亡事件,参与肿瘤进展。本文概述circRNAs在肿瘤细胞程序性死亡中的研究进展以及circRNA作为新治疗靶点的未来应用。

Biological functions and potential implications of circular RNAs

Circular RNAs(circRNAs) are characterized by a covalent closed-loop structure with an absence of both 5′ cap structure and 3′ polyadenylated tail. Numerous studies have found that circRNAs play an important role in various diseases and have a variety of biological regulatory mechanisms, including acting as microRNA sponges,interacting with proteins, modulating the expression of related genes and translating into peptides or proteins.CircRNAs have also been used as biomarkers for a number of diseases, which could improve clinical practice.This review summarizes the most recent advances in biogenesis and knowledge of the biological functions of circRNAs as well as the related bioinformatics databases. We specifically describe developments in understanding of circRNA functions in the field of environmental exposure-induced diseases. Finally, we focus on potential clinical implications of circRNAs to facilitate their clinical transformation into disease treatment.

Regulatory RNAs,microRNA,long-non coding RNA and circular RNA roles in colorectal cancer stem cells

The properties of cancer stem cells(CSCs),such as self-renewal,drug resistance,and metastasis,have been indicated to be responsible for the poor prognosis of patients with colon cancers.The epigenetic regulatory network plays a crucial role in CSC properties.Regulatory non-coding RNA(ncRNA),including microRNAs,long noncoding RNAs,and circular RNAs,have an important influence on cell physiopathology.They modulate cells by regulating gene expression in different ways.This review discusses the basic characteristics and the physiological functions of colorectal cancer(CRC)stem cells.Elucidation of these ncRNAs will help us understand the pathological mechanism of CRC progression,and they could become a new target for cancer treatment.

Circular RNA LPAR3通过miR-143-5p/USP14通路调控食管癌细胞糖酵解的研究

目的探究调控食管癌细胞糖酵解的机制。方法逆转录实时定量PCR(quantitative real-time,qRT-PCR)法分别检测环状RNA溶血磷脂酸受体(circular RNA lysophosphatidic acid receptor 3,CircLPAR3),微小RNA-143-5(microRNA-143-5p,miR-143-5p)和泛素特异性蛋白酶14(ubiquitin-specific protease 14,USP14)的表达水平;使用Seahorse XF 24细胞外通量分析仪测量细胞外酸化率(extracellular acidification rate,ECAR)和耗氧率(oxygen comsumpition rate,OCR);Western印迹法检测糖酵解途径关键酶HK2的表达水平;使用Starbase数据库预测CircLPAR3与miR-143-5p以及miR-143-5p与USP14的靶向结合位点,双荧光素酶试验验证CircLPAR3和miR-143-5p以及miR-143-5p与USP14的靶向关系。结果与正常人食管上皮细胞(normal human esophageal epithelial cells,Het-1A)组比较,食管癌细胞系中CircLPAR3高表达,且KYSE450细胞系的表达最高,同时miR-143-5p低表达而USP14高表达。与Het-1A组比较,KYSE450组HK2的蛋白表达水平增加,同时细胞ECAR增加,整体糖酵解OCR减少;敲低CircLPAR3的表达导致细胞HK2的蛋白表达水平减少,ECAR减少,整体糖酵解OCR增加;在抑制CircLPAR3的表达情况下,抑制miR-143-5p的表达能够部分逆转细胞的糖酵解途径;双荧光素酶实验验证了CircLPAR3和miR-143-5p的靶向关系,以及miR-143-5p和USP14的靶向关系。结论CircLPAR3能够通过调控miR-143-5p/USP14通路调节食管癌细胞糖酵解途径。

Circular RNAs:Diagnostic and Therapeutic Perspectives in CNS Diseases

Circular RNAs(circRNAs)are a class of regulatory non-coding RNAs characterized by the presence of covalently closed ends.A growing body of evidence suggests that circRNAs play important roles in physiology and pathology.In particular,accumulating data on circRNA functions in various central nervous system(CNS)diseases and their correlations indicate that circRNAs are critical contributors to the onset and development of brain disorders.In this review,we focus on the regulatory and functional roles of circRNAs in CNS diseases,highlighting their diagnostic and therapeutic potential,with the aim of providing new insights into CNS diseases.

急性病毒性心肌炎血浆外泌体Circular RNA的转录及功能分析

目的:评估急性病毒性心肌炎(AVMC)患者外泌体circular RNA (circRNA)的表达差异并揭示其潜在功能。方法:于2021年7月至2023年3月在青岛大学附属医院采集9例临床确诊为AVMC的患者和9例健康对照(HC)的外周血。采用RNA高通量测序检测外泌体circRNA表达。我们进行了GO及KEGG富集分析预测circRNA在外泌体中的潜在分子、生物学功能和相关信号通路。我们选取了3个外泌体中差异性表达的circRNA进行了实时荧光定量聚合酶链反应(qRT-PCR)对测序结果进行了验证。结果:以P 2为截断值筛选具有差异性表达的circRNA。本研究共发现了外泌体中有84个差异性表达的circRNA,其中83个下调及1个上调。GO和KEGG富集分析结果表明,差异表达的circRNA可能与ATP酶活动、GTP酶结合、溶酶体膜、微管、内质网蛋白质加工等有关。PCR结果表明hsa_circ_0001546在AVMC患者中下调,与测序结果一致。结论:差异性表达的hsa_circ_0001546可能是AVMC的潜在诊断标志物;差异性表达的circRNA在AVMC中具有一定生物功能,可能参与AVMC的发病机制。

Rockburst process and strength-weakening effect of the high-stress circular tunnel under internal unloading

To investigate the influence of unloading effect of a circular tunnel face on rockburst process,by innovatively combining rock drilling unloading devices and triaxial systems,the strain rockburst simulation under the entire stress path of“high initial stressþinternal unloadingþstress adjustment”(HUS test)was realized for the intact cubic red sandstone samples(100 mm×100 mm×100 mm).Comparative tests were conducted on cubic red sandstone samples with prefabricated circular holes(425 mm)under the stress path of“prefabricated circular hole+þhigh initial stress+stress adjustment”(PHS test),thereby highlighting the influence of internal unloading on rockburst failure.The test results revealed that with an increase in vertical stress,the sidewalls in both the HUS and PHS tests suffered strain rockburst failure.Compared with the PHS test,the initial failure stress in the HUS test is lower,and it is easier to induce sidewall rockbursts.This indicates that the internal unloading influences the sidewall failure,causing an obvious strength-weakening effect,which becomes more significant with an increase in buried depth.The strain rockburst failure was more severe in the HUS test owing to the influence of internal unloading.V-shaped rockburst pits were formed in the HUS tests,whereas in the PHS test,arcshaped rockburst pits were produced.It was also found that strain rockburst failure may occur only when the rock has a certain degree of rockburst proneness.

Circular RNA and its potential as prostate cancer biomarkers

Advancing knowledge of the transcriptome has revealed that circular RNAs(circRNAs)are widely expressed and evolutionarily conserved molecules that may serve relevant biological roles.More interesting is the accumulating evidence which demonstrates the implication of circRNAs in diseases,especially cancers.This revelation has helped to form the rationale for many studies exploring their utility as clinical biomarkers.CircRNAs are highly stable due to their unique structures,exhibit some tissue specificity,and are enriched in exosomes,which facilitate their detection in a range of body fluids.These properties make circRNAs ideal candidates for biomarker development in many diseases.This review will outline the discovery,biogenesis,and proposed functions of circRNAs.

Down-regulating Circular RNA Prkcsh suppresses the inflammatory response after spinal cord injury

Circular RNAs(circRNAs)are a class of conserved,endogenous non-coding RNAs that are involved in transcriptional and post-transcriptional gene regulation and are highly enriched in the nervous system.They participate in the survival and differentiation of multiple nerve cells,and may even promote the recovery of neurological function after stroke.However,their role in the inflammatory response after spinal cord injury remains unclear.In the present study,we established a mouse model of T9 spinal cord injury using the modified Allen’s impact method,and identified 16,013 circRNAs and 960 miRNAs that were differentially expressed after spinal cord injury.Of these,the expression levels of circPrkcsh were significantly different between injured and sham-treated mice.We then treated astrocytes with tumor necrosis factor-αin vitro to simulate the inflammatory response after spinal cord injury.Our results revealed an elevated expression of circPrkcsh with a concurrent decrease in miR-488 expression in injured cells.We also found that circPrkcsh regulated the expression of the inflammationrelated gene Ccl2.Furthermore,in tumor necrosis factor-α-treated astrocytes,circPrkcsh knockdown decreased the expression of Ccl2 by upregulating miR-488 expression,and reduced the secretion of inflammatory cytokines in vitro.These findings suggest that differentially expressed circRNAs participate in the inflammatory response after spinal cord injury and act as the regulators of certain microRNAs.Furthermore,circPrkcsh may be used as an miR-488 sponge to regulate Ccl2 expression,which might provide a new potential therapy for SCI.The study was approved by the Animal Ethics Committee of Shandong University of China(approval No.KYLL-20170303)on March 3,2017.

Flow Dynamics around Two Side by Side Circular Cylinders with Alternating Movements

The flow dynamics is analyzed through two-dimensional numerical simulations around two circular cylinders arranged side by side, with 4 combinations of alternating motions. All simulations are performed for Re = 1000, amplitude of oscillation (A) equal to 3, frequency ratio (fr) of 0.5, specific rotation (α) equal to 0.5 and different values of spacing ratio (L/D). It is verified that the combination of the type of movement, together with the position of one cylinder in relation to the other, exerts significant influence on the flow dynamics, as well as on the pressure distribution around the cylinder surface and on the average values of the fluid dynamics coefficients. The smallest value of the average pressure coefficient (Cp = -3.3), is obtained for the oscillating cylinder when placed side by side with the clockwise rotation cylinder, case 3 and L/D = 1.5. On the other hand, the lowest mean drag coefficient (Cd = 1.0788), is obtained for the cylinder with counterclockwise rotation, located in the lower position in relation to oscillating cylinder in the upper position, with spacing between them of 1.5. Furthermore, it is observed that the rotation movement is more effective in reducing drag than the rotation-oscillation movement, for the studied frequency ratio.

Circular RNAs and human glioma

As a newly discovered type of RNA, circular RNAs(circRNAs) are widespread throughout the eukaryotic genome. The expression of circRNAs is regulated by both cis-elements and trans-factors, and the expression pattern of circRNAs is cell type-and diseasespecific. Similar to other types of non-coding RNAs, functions of circRNAs are also versatile. CircRNAs have been reported previously to function as microRNA(miRNA) sponges, protein sponges, coding RNAs or scaffolds for protein complexes.Recently, several circRNAs have been reported to play important roles in human malignancies, including glioma. Here, we reviewed several reports related to circRNAs and glioma, as well as the potential diagnostic and therapeutic applications of circRNAs in brain cancer. In general, some circRNAs, such as circSMARCA5 and circCFH, are found to be expressed in a gliomaspecific pattern, these circRNAs may be used as tumor biomarkers. In addition, some circRNAs have been found to play oncogenic roles in glioma(e.g., circNFIX and circNT5E), whereas others have been reported to function as tumor suppressors(e.g.,circFBXW7 and circSHPRH). Furthermore, circRNA is a good tool for protein expression because of its higher stability compared to linear RNAs. Thus, circRNAs may also be an ideal choice for gene/protein delivery in future brain cancer therapies. There are some challenges in circRNA research in glioma and other diseases. Research related to circRNAs in glioma is comparatively new and many mysteries remain to be solved.