Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

Research progress on the role of circular RNA in neonatal hypoxic-ischemic encephalopathy / 中国临床药理学与治疗学

Hypoxic-ischemic encephalopathy (HIE) is a major cause of newborn mortality and childhood disability. Despite hypothermia treatment being the current standard method, it has its limitations and often produces unsatisfactory outcomes. Additionally, due to time and equipment constraints, hypothermia treatment cannot be promptly administered, leading to high mortality rates or varying levels of neurological impairments even after treatment. Hence, the exploration of alternative and effective treatment methods for HIE has become a challenging and highly researched topic in the field of neonatology. Research has shown that HIE induces intricate changes in the neurological system at the physiological, cellular, and molecular levels. Circular RNA (circRNA) exhibits high expression in the central nervous system and plays a role in regulating physiological and pathophysiological processes. Therefore, circRNA holds promise as a potential therapeutic target for HIE. This article provides a comprehensive overview of the regulatory effects of circRNA on different types of neural cells in HIE, aiming to offer new theoretical foundations for the treatment of HIE.

Peranan RNA Bulat dalam Kanser Kolorektal / Jurnal Sains Kesihatan Malaysia

@#Circular RNAs (circRNAs) are a class of non-coding RNAs formed by covalently closed loops through backsplicing. From previous studies, RNA was found to be involved in the development and formation of colorectal cancer. By emphasizing on the true function of circRNAs through focusing on its biogenesis, roles in disease treatment and roles as biomarkers, we are able to utilize circRNAs as therapy for cancer. Cancer is a chronic disease that contributes to high mortality worldwide. Colorectal cancer is one of the most common cancer in the world and in Malaysia in general. The incidence and mortality rates of colorectal cancer worldwide show a drastic and alarming increase. Colorectal cancer occurs due to mutations from certain genes involved in proliferation regulation, cell survival and cell death. This article aims to discuss the role and importance of Circular RNA in colorectal cancer. By identifying the true function of circRNAs, it can help us to develop our understanding on the real biological roles of circRNAs in colorectal cancer.

The aberrant overexpression of hsa_circ_0078607 in colorectal cancer tissues and serum and its clinical significance / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究hsa_circ_0078607在结直肠癌组织和患者血清中的表达水平及其与结直肠癌患者临床病理特征的关系，评价其能否作为结直肠癌潜在的分子诊断标志物及治疗靶标。方法：收集2018年6月至2022年1月于柳州市人民医院胃肠外科接受结直肠癌切除手术患者的58对癌及癌旁组织标本，收集2020年1月至2022年12月于柳州市人民医院初次确诊的结直肠癌患者、结直肠息肉患者及健康人体检血清共152例；从结直肠癌差异表达circRNA谱中挑选特异性高表达的hsa_circ_0078607作为候选标志物，采用qPCR法检测其在结直肠癌细胞、组织、患者血清及结直肠息肉患者血清中的相对表达量，分析其与临床病理特征的关系。采用ROC曲线评估hsa_circ_0078607对结直肠癌及结直肠息肉的诊断价值。通过Circular RNA Interactome数据库预测与hsa_circ_0078607结合的miRNA，并用Cytoscape 3.9.1软件构建circRNA-miRNA-mRNA调控网络，同时通过GO/KEGG富集分析进一步了解其功能。结果：与癌旁组织或健康人血清相比，hsa_circ_0078607在结直肠癌细胞、组织和血清及息肉患者血清中呈高表达（P<0.001），其中有52例（89.7%）患者癌组织中表达上调，6例（10.3%）表达下调。结直肠癌组织中hsa_circ_0078607的相对表达量与肿瘤位置（P=0.029）、分化程度（P=0.046）和远处转移（P=0.043）有关联。ROC结果显示，在结直肠癌组织和血清中其诊断结直肠癌的AUC分别为0.845 7[95%CI（0.772 8，0.918 6），P<0.000 1]和0.868 3[95%CI（0.790 7，0.945 9），P<0.000 1]；在息肉患者血清中，hsa_circ_0078607诊断结直肠息肉的AUC为0.710 1 [95%CI（0.610 0，0.810 1）]。GO/KEGG富集分析结果表明，hsa_circ_0078607下游的miRNA可能参与RNA聚合酶Ⅱ启动子转录调控、蛋白K48-连锁泛素化、Wnt、Hippo及MAPK信号通路调控等多个生物过程。结论：Hsa_circ_0078607在结直肠癌细胞、组织和血清中呈高表达，其在结直肠癌组织中的表达水平与肿瘤位置、分化程度和远处转移有关联，提示其可作为结直肠癌潜在的分子诊断标志物；其还可能介导结直肠癌的发生发展过程，对发现结直肠癌潜在的治疗靶点有重要意义。

Research Progress on Regulatory Role of CircPVT1 in Tumors / 肿瘤防治研究

Circular RNA (circRNA) is involved in tumor progression. CircPVT1 is an oncogene that is abnormally expressed and correlated with a variety of tumors. It can regulate tumors' malignant behavior and affect the survival and prognosis of patients. This article reviews research on the regulatory roles of circPVT1 in tumors to provide references for accurate treatment.

Co-expression of circRNA in oral leukoplakia and oral lichen planus / 口腔疾病防治

Objective@# To find any differentially expressed circRNAs in oral leukoplakia (OLK) and oral lichen planus (OLP), to investigate the possible role of circRNAs in the pathogenesis of these two diseases.@*Methods@# This study obtained hospital ethical approval. High-throughput sequencing was used to detect differentially expressed circRNAs in OLK, OLP, oral squamous cell carcinoma and normal oral mucosal tissues. CircRNAs were verified by qRT-PCR, enzyme tolerance assays and Sanger sequencing. GO functional analysis and KEGG pathway analysis were performed to predict the functions of circRNAs in OLP. TargetScan and miRanda were applied to predict targeted miRNAs and mRNAs of circRNAs, and ceRNA networks were mapped. @*Results@#A total of 49 circRNAs were differentially expressed in OLK and OLP together, including 30 upregulated and 19 downregulated circRNAs. The five circRNAs confirmed with RT-qPCR, including circHLA-C, circRNF13, circTTN, circSEPN2 and circALDH3A2, were all abnormally expressed in OLK and OLP, among which circHLA-C was a key circRNA with trans splice sites, which was validated by expanding the sample size. ROC curve analysis showed that the area under the circHLA-C curve for predicting OLK was 0.955, and the area under the circHLA-C curve for predicting OLP was 0.988. GO functional analysis showed enrichment of many biological processes related to the immune process. The KEGG pathway with the highest enrichment score was "Natural killer cell mediated cytotoxicity". HLA-C was significantly enriched in these processes/pathways. CeRNA network analysis showed that circHLA-C interacted with a variety of miRNAs, such as hsa-miR-26a-5p, hsa-miR-129-5p, and hsa-miR-29a-3p.@*Conclusion@#Many circRNAs were differentially expressed in both OLK and OLP, circHLA-C being the most elevated. CircHLA-C is valuable for the early diagnosis of OLK and OLP and may serve as a potential biomarker for the diagnosis and prognosis of OLK and OLP.

Analysis of circRNA expression profile and functional enrichment of myocardial mitochondria in mice with diabetic cardiomyopathy / 西安交通大学学报(医学版)

【Objective】 To explore the differential expression and functional analysis of circRNA from myocardial mitochondria in diabetes cardiomyopathy (DCM) mice. 【Methods】 The DCM mice model was established in 16-week-old db/db mice, and C57BL/KsJ mice were used as controls. RNA was extracted from the myocardium of two groups of mice, high-throughput sequencing was used to screen mitochondrial circRNA differentially expressed in the two groups, RT-qPCR was used to verify the sequencing results of the first 10 circRNAs with significant differential expression, and functional enrichment analysis was performed on the differentially expressed circRNA target genes, and miRNA target prediction software was used to analyze the circRNA-miRNA co-expression network. 【Results】 There were 147 mitochondrial circRNAs differentially expressed in the myocardium of DCM mice, including 89 highly expressed and 58 low expressed. The expression pattern of differentially expressed circRNAs in tissues was consistent with those of sequencing results. The enrichment analysis of GO and KEGG showed that the differentially expressed circRNA target genes were mainly enriched in cGMP/PKG, glucagon pathways, which were related to mitochondrial energy metabolism and cardiac hypertrophy. circRNA-miRNA co-expression analysis found that the most significantly up-regulated circRNA, chrM:1207-1536+, was associated with miR-491-3p, miR-99a-3p, and miR-99b-3p, and the most significantly down-regulated circRNA, chrM:1453-3205+, was associated with miR-181b-1-3p, miR-181b-2-3p, and miR-672-5p. 【Conclusion】 Compared to the control mice, there is differential expression of circRNAs in myocardial mitochondria of DCM mice. The differentially expressed circRNAs may interact with the corresponding miRNA to affect myocardial fibrosis and hypertrophy through regulation of energy metabolism, apoptosis and other pathways, thus participating in the pathogenesis of DCM.

Role of circRNA-miRNA-mRNA networks in exosomes in gastric cancer development and its treatment / 中国肿瘤生物治疗杂志

@#[摘 要] 复杂的肿瘤微环境（TME）是导致胃癌高度异质性的主要原因之一。外泌体是多囊泡体和质膜融合后释放到体液中形成的纳米级生物囊泡，是TME中的重要组成部分。外泌体携带的生物分子通过促进细胞间的信号交流，在一定程度上参与调控癌症的发生和转移。环状RNA（circRNA）是稳定存在于外泌体中的非编码RNA，其作为miRNA分子海绵，抑制miRNA与mRNA的结合，进而调节下游靶基因mRNA的表达，并由此形成circRNA-miRNA-mRNA网络。外泌体中circRNA-miRNA-mRNA网络通过参与调节胃癌的增殖、迁移和侵袭，诱导胃癌细胞上皮间质转化（EMT），介导胃癌血管生成，调节胃癌转移，调控胃癌的化疗耐药以及放射敏感性，在胃癌的发生发展中发挥重要作用。此外，以外泌体中circRNA为靶点或者靶向circRNA-miRNA-mRNA网络可能为胃癌的治疗提供新的治疗选择。

CircFhit Modulates GABAergic Synaptic Transmission via Regulating the Parental Gene Fhit Expression in the Spinal Dorsal Horn in a Rat Model of Neuropathic Pain / 神经科学通报·英文版

Effective treatments for neuropathic pain are lacking due to our limited understanding of the mechanisms. The circRNAs are mainly enriched in the central nervous system. However, their function in various physiological and pathological conditions have yet to be determined. Here, we identified circFhit, an exon-intron circRNA expressed in GABAergic neurons, which reduced the inhibitory synaptic transmission in the spinal dorsal horn to mediate spared nerve injury-induced neuropathic pain. Moreover, we found that circFhit decreased the expression of GAD65 and induced hyperexcitation in NK1R+ neurons by promoting the expression of its parental gene Fhit in cis. Mechanistically, circFhit was directly bound to the intronic region of Fhit, and formed a circFhit/HNRNPK complex to promote Pol II phosphorylation and H2B monoubiquitination by recruiting CDK9 and RNF40 to the Fhit intron. In summary, we revealed that the exon-intron circFhit contributes to GABAergic neuron-mediated NK1R+ neuronal hyperexcitation and neuropathic pain via regulating Fhit in cis.

Promising roles of non-exosomal and exosomal non-coding RNAs in the regulatory mechanism and as diagnostic biomarkers in myocardial infarction / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Non-exosomal non-coding RNAs (non-exo-ncRNAs) and exosomal ncRNAs (exo-ncRNAs) have been associated with the pathological development of myocardial infarction (MI). Accordingly, this analytical review provides an overview of current MI studies on the role of plasma non-exo/exo-ncRNAs. We summarize the features and crucial roles of ncRNAs and reveal their novel biological correlations via bioinformatics analysis. The following contributions are made: (1) we comprehensively describe the expression profile, competing endogenous RNA (ceRNA) network, and "pre-necrotic" biomarkers of non-exo/exo-ncRNAs for MI; (2) functional enrichment analysis indicates that the target genes of ncRNAs are enriched in the regulation of apoptotic signaling pathway and cellular response to chemical stress, etc.; (3) we propose an updated and comprehensive view on the mechanisms, pathophysiology, and biomarker roles of non-exo/exo-ncRNAs in MI, thereby providing a theoretical basis for the clinical management of MI.

Cangxi Tongbi Capsules promote chondrocyte autophagy by regulating circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit development of knee osteoarthritis / 中国中药杂志

To investigate the mechanism by which Cangxi Tongbi Capsules promote chondrocyte autophagy to inhibit knee osteoarthritis(KOA) progression by regulating the circRNA＿0008365/miR-1271/p38 mitogen-activated protein kinase(MAPK) pathway. The cell and animal models of KOA were established and intervened with Cangxi Tongbi Capsules, si-circRNA＿0008365, si-NC, and Cangxi Tongbi Capsules combined with si-circRNA＿0008365. Flow cytometry and transmission electron microscopy were employed to determine the level of apoptosis and observe autophagosomes, respectively. Western blot was employed to reveal the changes in the protein levels of microtubule-associated protein light chain 3(LC3)Ⅱ/Ⅰ, Beclin-1, selective autophagy junction protein p62/sequestosome 1, collagen Ⅱ, a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5), and p38 MAPK. The mRNA levels of circRNA＿0008365, miR-1271, collagen Ⅱ, and ADAMTS-5 were determined by qRT-PCR. Hematoxylin-eosin staining was employed to reveal the pathological changes of the cartilage tissue of the knee, and enzyme-linked immunosorbent assay to measure the levels of interleukin-1β(IL-1β) and tumor necrosis factor-alpha(TNF-α). The chondrocytes treated with IL-1β showed down-regulated expression of circRNA＿0008365, up-regulated expression of miR-1271 and p38 MAPK, lowered autophagy level, increased apoptosis rate, and accelerated catabolism of extracellular matrix. The intervention with Cangxi Tongbi Capsules up-regulated the expression of circRNA＿0008365, down-regulated the expression of miR-1271 and p38 MAPK, increased the autophagy level, decreased the apoptosis rate, and weakened the catabolism of extracellular matrix. However, the effect of Cangxi Tongbi Capsules was suppressed after interfering with circRNA＿0008365. The in vivo experiments showed that Cangxi Tongbi Capsules dose-dependently inhibited the p38 MAPK pathway, enhanced chondrocyte autophagy, and mitigated articular cartilage damage and inflammatory response, thereby inhibiting the progression of KOA in rats. This study indicated that Cangxi Tongbi Capsules promoted chondrocyte autophagy by regulating the circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit the development of KOA.

Effects of titanium dioxide nanoparticles on circRNA expression profiles in human hepatocellular carcinoma cells HepG2 / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effect of titanium dioxide nanoparticles (TiO2 NPs) on the expression profile of circular ribonucleic acid (circRNA) in human hepatocytes through in vitro cell experiments, and to attempt to understand the potential mechanism of hepatotoxicity through bioinformatics analysis.@*METHODS@#TiO2 NPs were characterized from the aspects of particle size, shape and agglomeration state. The cell counting kit-8 (CCK8) was used to detect the cytotoxicity of TiO2 NPs against human hepatocellular carcinoma cells (HepG2) after exposure to 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 mg/L TiO2 NPs for 24 h or 48 h. The cells were treated at doses of 0 mg/L TiO2 NPs (control group) and 100 mg/L TiO2 NPs (treatment group), and collected after exposure for 48 h, and then RNA from the extracted cell samples was collected and sequenced. The differential circRNAs between the control and the TiO2 NPs treatment groups were screened, and then the enrichment pathway of the differential circRNA target gene was analyzed by multivariate statistics. According to the sequencing results, significantly altered genes and important genes in the significant enrichment pathways were screened, and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) was performed to verify the results.@*RESULTS@#TiO2 NPs were spherical anatase with a hydrated particle size of (323.50±85.44) nm and a Zeta potential of (-21.00±0.72) mV in a serum-free medium. The results of the CCK8 cytotoxicity assay showed that with the increase of TiO2 NPs concentration, cell viability gradually decreased. A total of 11 478 circRNAs were found by RNA sequencing. Compared with the control groups, TiO2 NPs treatment groups (100 mg/L) had a total of 89 differential circRNAs, of which 59 were up-regulated and 30 were down-regulated. Analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway showed that the targeted genes of differential circRNAs were mainly enriched in fatty acid degradation, Fanconi anemia pathway, and fatty acid metabolism. The expression levels of circRNA.6730, circRNA.3650 and circRNA.4321 were significantly different between the TiO2 NPs treatment group and the control group, which were consistent with the sequencing results.@*CONCLUSION@#TiO2 NPs can induce changes in circRNA expression profile, and epigenetics may play an important role in the mechanism of hepatotoxicity.

Expression and diagnostic value of plasma exosomal hsa_circ_0064910 in gastric cancer / 中华检验医学杂志

Objective:This study aimed to investigate the expression of plasma exosomal hsa_circ_0064910 in gastric cancer (GC) patients, explore its correlation with the clinical pathological characteristics and evaluate its diagnostic efficacy in GC.Methods:Sixty patients with GC and 30 patients with Chronic Gastritis (disease control group) admitted to The First Affiliated Hospital of Henan University from October 2019 to December 2020 were selected. Meanwhile, 30 healthy subjects (healthy control group) who underwent physical examination were also enrolled. General data of GC patients were collected, including tumor size, degree of differentiation, TNM stage, lymph node metastasis, etc. Blood samples were collected before treatment and the expression levels of plasma exosomal hsa_circ_0064910 were detected via quantitative reverse transcription PCR (qRT-PCR). The serum concentrations of traditional biomarker (CEA and CA19-9) were measured via a chemiluminescent detection system. The receiver operating characteristic (ROC) curve and area under curve (AUC) were used to estimate the diagnostic capacity of different index in GC. Then, the expression difference of plasma exosomal hsa_circ_0064910 in GC patients before and after operation was analyzed, and its relationship with clinicopathological features of GC patients was also investigated.Results:RT-PCR results revealed that compared with Chronic gastritis patients and healthy control, the expression of plasma exosomal hsa_circ_0064910 was upregulated in the gastric cancer group(0.47±0.06, 0.43±0.05, 0.97±0.12, all P<0.001). The area under the ROC curve was 0.778, and AUC of the combination of CEA and CA19-9 for the diagnosis of gastric cancer was 0.841. which was higher than the diagnostic accuracies of CEA (AUC=0.673)and CA 19-9(AUC=0.653). The expression level of exosomal hsa_circ_0064910 was also significantly correlated with tumor size( χ2=7.545, P<0.01), TNM stage( χ2=4.571, P<0.05)and lymph node metastasis( χ2=6.907, P<0.01). The postoperative expression levels of exosomal hsa_circ_0064910 were lower compared with those of preoperative levels(1.21±0.21 vs 0.62±0.11, P<0.01). Conclusion:Our data demonstrated that exosomal hsa_circ_0064910 is highly expressed in GC patients and might be a potential noninvasive biomarker for the auxiliary diagnosis of GC.

Research status and prospect of circular RNAs in diabetic retinopathy / 中华眼底病杂志

The mechanisms behind diabetic retinopathy (DR) can be ascribed primarily to retinal microvascular abnormalities, excessive inflammatory response and neurodegeneration. Circular RNA (circRNA) is a type of endogenous non-coding RNA with a special circular structure, which is mainly composed of precursor RNA after shearing and processing. It is widely present in the retina and participates in the occurrence and development of various fundus diseases. CircRNAs express in an abnormal way in retina, serving as "the sponge" for miRNA so as to play roles in dysfunction of retinal vascular, inflammatory response and neurodegeneration in the development of DR. Further studies for circRNAs in DR will illustrate pathophysiology of DR more deeply, shedding light on circRNAs becoming novel biomarkers and molecular targets for diagnosis and treatment, thus achieving the goal of early diagnosis and precise therapy of DR.

Expressions and clinical significance of plasma exosomal hsa_circ_0022417 in gastric cancer / 中华普通外科杂志

Objective:To investigate expressions and clinical significance of plasma exosomal hsa_circ_0022417 in gastric cancer (GC).Methods:Sixty gastric cancer patients, 30 chronic gastritis patients (disease control group) and 30 healthy volunteers (healthy control group) were enrolled in this study. The expression levels of plasma exosomal hsa_circ_0022417 and serum CEA and CA19-9 were detected. The ROC curve and AUC were used to estimate the diagnostic capacity.Results:Compared with chronic gastritis patients and healthy control, the expression of plasma exosomal hsa_circ_0022417 was significantly upregulated in the gastric cancer group ( F=9.96, P<0.05). The expression level of hsa_circ_0022417 in GC tissue was significantly higher than that in adjacent tissue ( t=6.08, P<0.05). The AUC of hsa_circ_0022417, serum CEA and CA 19-9 was 0.79, 0.68 and 0.66, respectively. The combined detection of three indicators had the highest AUC (0.86) ( P<0.05). The expression level of exosomal hsa_circ_0022417 was significantly correlated with tumor size ( χ2=6.42, P<0.01), differentiation degree ( χ2=5.83, P=0.05), TNM stage ( χ2=7.14, P<0.05) and lymph node metastasis ( χ2=5.17, P<0.05). Conclusion:Exosome hsa_circ_0022417 is highly expressed in the plasma of GC patients, which is of great significance for clinical auxiliary diagnosis and early screening of GC.

Role of circular RNAs in immune-related diseases / 南方医科大学学报

Objective Circular RNAs (circRNAs) are non-coding RNAs (ncRNA) circularized without a 3' polyadenylation [poly-(A)] tail or a 5' cap, resulting in a covalently closed loop structure. circRNAs were first discovered in RNA viruses in the 1970s, but only a small number of circRNAs were discovered at that time due to limitations in traditional polyadenylated transcriptome analyses. With the development of specific biochemical and computational methods, recent studies have shown the presence of abundant circRNAs in eukaryotic transcriptomes. circRNAs play vital roles in many physiological and pathological processes, such as acting as miRNA sponges, binding to RNA-binding proteins (RBPs), acting as transcriptional regulatory factors, and even serving as translation templates. Current evidence has shown that circRNAs can be potentially used as excellent biomarkers for diagnosis, therapeutic effect evaluation, and prognostic assessment of a variety of diseases, and they may also provide effective therapeutic targets due to their stability and tissue and development-stage specificity. This review focuses on the properties of circRNAs and their immune relationship to disease, and explores the role of circRNAs in immune-related diseases and the directions of future research.

Triptolide inhibits inflammatory response and migration of fibroblast like synovial cells in rheumatoid arthritis through the circRNA 0003353/JAK2/STAT3 signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of triptolide (TPL) on inflammatory response and migration of fibroblast like synovial cells (FLS) in rheumatoid arthritis (RA-FLS) and the mechanism of circular noncoding RNA (circRNA) 0003353 for mediating this effect.@*METHODS@#We collected peripheral blood mononuclear cells (PBMCs) and serum samples from 50 hospitalized RA patients and 30 healthy individuals for detecting the expression of circRNA 0003353, immune and inflammatory indexes (ESR, CRP, RF, anti-CCP, IgA, IgG, IgM, C3, and C4) and DAS28 score. Cultured RA-FLS was treated with 10 ng/mL TPL and transfected with a circRNA 0003353 overexpression plasmid, and cell counting kit-8 (CCK-8) assay and Transwell assay were used to detect the changes in the viability and migration of the cells. Enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokines IL-4, IL-6, and IL-17, and real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the expression of circRNA 003353; Western blotting was used to detect the expressions of p-JAK2, pSTAT3, JAK2 and STAT3 proteins in the treated cells.@*RESULTS@#The expression of circRNA 0003353 was significantly increased in PBMCs from RA patients and showed a good performance in assisting the diagnosis of RA (AUC=90.5%, P < 0.001, 95% CI: 0.83-0.98). CircRNA 0003353 expression was positively correlated with ESR, RF and DAS28 (P < 0.05). Treatment with TPL significantly decreased the expression of circRNA 0003353, suppressed the viability and migration ability, decreased the expressions of IL-6 and IL-17, and increased the expression IL-4 in cultured RA-FLS in a time-dependent manner (P < 0.01). TNF-α stimulation of RA-FLS significantly increased the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, which were obviously lowered by TPL treatment (P < 0.01). TPL-treated RA-FLS overexpressing circRNA 0003353 showed significantly increased cell viability and migration ability with decreased IL-4 expression and increased IL-6 and IL-17 expressions and ratios of p-JAK2/ JAK2 and p-STAT3/STAT3 (P < 0.01).@*CONCLUSION@#The expression of circRNA 0003353 is increased in PBMCs in RA patients and in RA-FLS. TPL treatment can regulate JAK2/STAT3 signal pathway and inhibit the inflammatory response and migration of RA-FLS through circRNA 0003353.

Analysis of m / 中国生物化学与分子生物学报

In this study, we performed high-throughput sequencing technology methylated RNA immunoprecipitation sequencing (MeRIP-seq), transcriptome sequencing (RNA-seq) and bioinformatics to analyze the differentially m6A-methylated and differentially expressed profile of circular RNA (circRNA) in middle cerebral artery occlusion/reperfusion (MCAO/R) model, which provided some scientific evidences for revealing the relationship between RNA epigenetic modification and cerebral ischemia reperfusion injury. The neurological deficit scores of mice were evaluated by the Longa score standard. TTC staining was used to detect cerebral infarction volumes, and dot blot was used for the quantification of m

Porcine Circular RNA Insulin-like Growth Factor 1 Receptor Promotes Adipocyte Differentiation / 中国生物化学与分子生物学报

Circular RNA (circRNA), as a competitive endogenous RNA (ceRNA), plays an importantrole in the regulation of cell differentiation. The purpose of this study was to identify and analyze porcinecircular RNA insulin-like growth factor 1 receptor (circIGF1R), explore its expression patterns, construct a ceRNA regulatory network related to circIGF1R, and explore the regulation of its ectopicexpression on adipogenic differentiation of mouse mesenchymal stem cells (C3H10T1 / 2) effect. Forwardand reverse PCR, Sanger sequencing, RNase R enzyme digestion tests, and qRT-PCR were used toverify that circIGF1R is a circRNA formed by the second exon of insulin-like growth factor 1 receptor(IGF1R). It was expressed in all tissues of pigs, and its expression level increased with age in adiposetissues. miRDB, TargetScan and miRWalk online software were used to predict circIGF1R target genes. RNAhybrid software was used for binding site prediction. DAVID bioinformatics functional analysissoftware was used to perform GO and KEGG enrichment analysis on candidate target genes. Cytoscapesoftware was used to construct the ceRNA network diagram. Based on the gene expression correlation andpredicted target relationship, the GO and KEGG enrichment analysis was drawn and the ceRNA networkwas constructed; the dual luciferase reporter gene test was used, and we found that circIGF1R andFABP4 can bind to ssc (Sus scrofa chromosome) -miR-133a-5p. The circIGF1R overexpression vectorwas successfully constructed and expressed in C3H10T1/ 2 cells. It was found that after overexpression ofcircIGF1R, the expression of key adipogenic regulatory factors CEBPa, CEBPß, FABP4 and PPAR? increased significantly(P<0. 01), and the number of lipid droplets increased significantly. The results ofthis study show that circIGF1R exists in pig adipose tissues, and may positively regulate the adipogenicdifferentiation of C3H10T1/ 2 cells through the ceRNA mechanism, which lays a theoretical foundation forfurther research on circIGF1R regulating the adipogenic differentiation of pig precursor intramuscularadipocytes.

The Role of m6A Modification of Circular RNA in Tumors / 中国生物化学与分子生物学报

Circular RNA (circRNA) is a novel RNA with circular structures. It is conserved in various species, and characterized by high stability, high expression levels and tissue specificity. Meanwhile, it could serve as microRNA (miRNA) sponges, bind to RNA-binding proteins, or regulate transcription and protein translation. With the development of high-throughput sequencing and bioinformatics, circRNA was reported to participate in the pathogenesis of cancer. N6-methyladenosine (m6A) modification is the most common type of RNA modification in eukaryote RNA, which is a dynamic and reversible process regulated by m6A methyltransferase, m6A demethylase and m6A-binding proteins. M6A modification isinvolved in the regulation of RNA nuclear export, splicing, stability, translation and degradation, thus playing a key role in the occurrence and development of multiple human diseases, such as cancers, cardiovascular diseases. Recently, some researches demonstrated that m6A modification of circRNA was essential in the occurrence and development of malignant tumors, such as cervical cancer, colorectal carcinoma, hepatocellular carcinoma, non-small cell lung cancer, poorly differentiated adenocarcinoma of the stomach. In the current manuscript, we summarized the mechanism of m6A RNA modification, the roles of m6A modification in regulation of circRNA, and the effects of circRNA m6A modification on tumor. The potential clinical application value of m6A-modified circRNA was further discussed, as to provide some new ideas and ways for early diagnosis, clinical treatment and prognosis of tumors.