Circular RNA circRSF1 binds to HuR to promote radiation-induced inflammatory phenotype in hepatic stellate cells / 南方医科大学学报

OBJECTIVE@#To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.@*METHODS@#Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.@*RESULTS@#Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).@*CONCLUSION@#circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.

Read-through circular RNA rt-circ-HS promotes hypoxia inducible factor 1α expression and renal carcinoma cell proliferation, migration and invasiveness / 北京大学学报(医学版)

OBJECTIVE@#To identify and characterize read-through RNAs and read-through circular RNAs (rt-circ-HS) derived from transcriptional read-through hypoxia inducible factor 1α (HIF1α) and small nuclear RNA activating complex polypeptide 1 (SNAPC1) the two adjacent genes located on chromosome 14q23, in renal carcinoma cells and renal carcinoma tissues, and to study the effects of rt-circ-HS on biological behavior of renal carcinoma cells and on regulation of HIF1α.@*METHODS@#Reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing were used to examine expression of read-through RNAs HIF1α-SNAPC1 and rt-circ-HS in different tumor cells. Tissue microarrays of 437 different types of renal cell carcinoma (RCC) were constructed, and chromogenic in situ hybridization (ISH) was used to investigate expression of rt-circ-HS in different RCC types. Small interference RNA (siRNA) and artificial overexpression plasmids were designed to examine the effects of rt-circ-HS on 786-O and A498 renal carcinoma cell proliferation, migration and invasiveness by cell counting kit 8 (CCK8), EdU incorporation and Transwell cell migration and invasion assays. RT-PCR and Western blot were used to exa-mine expression of HIF1α and SNAPC1 RNA and proteins after interference of rt-circ-HS with siRNA, respectively. The binding of rt-circ-HS with microRNA 539 (miR-539), and miR-539 with HIF1α 3' untranslated region (3' UTR), and the effects of these interactions were investigated by dual luciferase reporter gene assays.@*RESULTS@#We discovered a novel 1 144 nt rt-circ-HS, which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1α exon 2-6 and SNAPC1 exon 2-4. Expression of rt-circ-HS was significantly upregulated in 786-O renal carcinoma cells. ISH showed that the overall positive expression rate of rt-circ-HS in RCC tissue samples was 67.5% (295/437), and the expression was different in different types of RCCs. Mechanistically, rt-circ-HS promoted renal carcinoma cell proliferation, migration and invasiveness by functioning as a competitive endogenous inhibitor of miR-539, which we found to be a potent post-transcriptional suppressor of HIF1α, thus promoting expression of HIF1α.@*CONCLUSION@#The novel rt-circ-HS is highly expressed in different types of RCCs and acts as a competitive endogenous inhibitor of miR-539 to promote expression of its parental gene HIF1α and thus the proliferation, migration and invasion of renal cancer cells.

Circular RNA expression profiles and circRNA-miRNA-mRNA crosstalk in pre-eclamptic placenta / 中华妇产科杂志

Objective: To identify the expression profile of circular RNA (circRNA) in placenta of pre-eclampsia (PE) pregnant women by high-throughput sequencing, and to construct the circRNA-microRNA (miRNA)-messenger RNA (mRNA) interaction network, so as to reveal the related pathways and regulatory mechanisms of PE. Methods: The clinical data and placentas of 42 women with PE (PE group) and 30 normal pregnant women (control group) who delivered in West China Second University Hospital from November 2019 to June 2021 were collected. (1) High-throughput sequencing was used to establish the differentially expressed circRNA profiles in placental tissues of 5 pairs of PE group and the control group. (2) Real-time quantitative PCR (qRT-PCR) was used to verify the expression levels of 6 differentially expressed circRNAs in placental tissues of PE group and control group. (3) Bioinformatics analysis was used to predict the target miRNA and analyze the co-expressed mRNA to construct a competitive endogenous RNA (ceRNA) network. The differentially expressed circRNAs were analyzed by Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. (4) Logistic regression analysis, Pearson correlation and Kendall's tau-b correlation analysis were used to test the correlation between the three differentially expressed circRNAs and the risk of PE and clinical characteristics. (5) circRNA_05393 was selected for subsequent functional study. Small interfering RNA (siRNA) and overexpression plasmid were used to knock down or increase the expression level of circRNA_05393 in trophoblast cell line HTR-8/SVneo cells, respectively. Transwell assay was used to detect the migration and invasion ability of the trophoblasts in vitro. Cell counting kit-8 assay was used to detect the proliferation ability of the trophoblasts. Results: (1) Seventy-two differentially expressed circRNAs were identified by high-throughput sequencing, of which 35 were up-regulated and 37 were down-regulated. (2) qRT-PCR showed that compared with the control group, circRNA_00673 (1.306±0.168 vs 2.059±0.242; t=2.356, P=0.021) and circRNA_07796 (1.275±0.232 vs 1.954±0.230; t=2.018, P=0.047) were significantly increased, while circRNA_05393 (1.846±0.377 vs 0.790±0.094; t=3.138, P=0.002) was significantly decreased. (3) The circRNA-miRNA-mRNA interaction network contained 3 circRNAs, 8 miRNAs and 53 mRNAs. GO functional annotation analysis showed that the biological process was mainly enriched in iron ion homeostasis, membrane depolarization during action potential and neuronal action potential. In terms of cellular components, they were mainly enriched in cytoskeleton and membrane components. In terms of molecular function, they were mainly enriched in the activity of voltage-gated sodium channel and basic amino acid transmembrane transporter. KEGG pathway enrichment analysis showed that mRNAs in the interaction network were mainly enriched in complement and coagulation cascade, glycine, serine and threonine metabolism, p53 signaling pathway and peroxisome proliferators-activated receptors (PPAR) signaling pathway. (4) Logistic regression analysis showed that down-regulation of circRNA_05393 expression was a risk factor for PE (OR=0.044, 95%CI: 0.003-0.596; P=0.019). Correlation analysis showed that circRNA_05393 was significantly correlated with systolic blood pressure and diastolic blood pressure in PE pregnant women (both P<0.05). (5) Knock down or overexpression of circRNA_05393 significantly reduced or increased the migration and invasion abilities of HTR-8/SVneo cells (all P<0.05), but had no significant effect on the ability of tube formation and proliferation (all P>0.05). Conclusions: The construction of circRNA expression profile in placenta and the exploration of circRNA-miRNA-mRNA interaction network provide the possibility to reveal the regulatory mechanism of specific circRNA involved in PE. Inhibition of circRNA_05393 may induce the progression of PE by reducing the migration and invasion of trophoblasts.

circ_0003204 regulates the osteogenic differentiation of human adipose-derived stem cells via miR-370-3p/HDAC4 axis / 国际口腔科学杂志·英文版

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue regeneration. Circular RNAs (circRNAs) have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis. However, how circRNAs regulate hASCs in osteogenesis is still unclear. Herein, we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs. Knockdown of circ_0003204 by siRNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs. We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p. We predicted and confirmed that miR-370-3p had targets in the 3'-UTR of HDAC4 mRNA. The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis. Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model, while overexpression of circ_0003204 inhibited bone defect repair. Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.

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.

Progress in circular RNAs of plants / 生物工程学报

With the development of high-throughput sequencing technology, circular RNAs (circRNAs) have gradually become a hotspot in the research on non-coding RNA. CircRNAs are produced by the covalent circularization of a downstream 3' splice donor and an upstream 5' splice acceptor through backsplicing, and they are pervasive in eukaryotic cells. CircRNAs used to be considered byproducts of false splicing, whereas an explosion of related studies in recent years has disproved this misconception. Compared with the rich studies of circRNAs in animals, the study of circRNAs in plants is still in its infancy. In this review, we introduced the discovery of plant circRNAs, the discovery of plant circRNAs, the circularization feature, expression specificity, conservation, and stability of plant circRNAs and expounded the identification tools, main types, and biogenesis mechanisms of circRNAs. Furthermore, we summarized the potential roles of plant circRNAs as microRNA (miRNA) sponges and translation templates and in response to biotic/abiotic stress, and briefed the degradation and localization of plant circRNAs. Finally, we discussed the challenges and proposed the future directions in the research on plant circRNAs.

CircPlant: An Integrated Tool for circRNA Detection and Functional Prediction in Plants / 基因组蛋白质组与生物信息学报·英文版

The recent discovery of circular RNAs (circRNAs) and characterization of their functional roles have opened a new avenue for understanding the biology of genomes. circRNAs have been implicated to play important roles in a variety of biological processes, but their precise functions remain largely elusive. Currently, a few approaches are available for novel circRNA prediction, but almost all these methods are intended for animal genomes. Considering that the major differences between the organization of plant and mammal genomes cannot be neglected, a plant-specific method is needed to enhance the validity of plant circRNA identification. In this study, we present CircPlant, an integrated tool for the exploration of plant circRNAs, potentially acting as competing endogenous RNAs (ceRNAs), and their potential functions. With the incorporation of several unique plant-specific criteria, CircPlant can accurately detect plant circRNAs from high-throughput RNA-seq data. Based on comparison tests on simulated and real RNA-seq datasets from Arabidopsis thaliana and Oryza sativa, we show that CircPlant outperforms all evaluated competing tools in both accuracy and efficiency. CircPlant is freely available at http://bis.zju.edu.cn/circplant.

Discovery and molecular analysis of conserved circRNAs from cashmere goat reveal their integrated regulatory network and potential roles in secondary hair follicle

Background: Circular RNAs, a novel class in the eukaryotic transcriptome, are characterized by the 3' and 5' ends that are covalently joined in a covalently closed loop without free ends. Circular RNAs are considerably stable molecules and act as microRNA sponges with regulatory potential to the protein-coding genes. Results: Eight circular RNAs were found to be significantly upregulated at anagen skin tissue of cashmere goat compared with their counterparts at telogen. Rich and complex regulatory patterns were revealed among the eight upregulated circular RNAs at anagen and related miRNAs with their potential regulatory genes. The potential regulatory genes of eight upregulated circular RNAs at anagen were involved in several pathways related to the main physiological process of hair follicle, such as histone acetylation and axon. For chi_circ_1926, chi_circ_3541, chi_circ_0483, chi_circ_3196, and chi_circ_2092, overall, the relative expression in secondary hair follicle exhibited highly similar trends with their corresponding host genes during the different stages of the hair follicle cycle. However, the expression trends of chi_circ_0100, chi_circ_2829, and chi_circ_1967 were found to diverge from their corresponding host genes during the different stages of the hair follicle cycle. Conclusions: A total of eighteen circular RNAs were identified and characterized from skin tissue of cashmere goat. The eight upregulated circular RNAs at anagen might have significant roles in the secondary hair follicle of cashmere goat. Our results would provide a novel regulatory layer to elucidate the molecular mechanisms underlying the development of secondary hair follicle and the growth of cashmere fiber in cashmere goat.