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Objective To investigate the expression of circular-RNA DDX5(circ-DDX5)in breast cancer tissues and its relationship with the clinical stage of breast cancer patients,and to analyze the regulatory mechanism of circ-DDX5 on the proliferation and invasion of human breast cancer cell line.Methods The expression level of circ-DDX5 in breast cancer tissues and its correlation with the clinical stage of breast cancer patients were analyzed by TCGA database.Bioinformatics analysis and dual-luciferase reporter gene experiments verified the targeting rela-tionship between circ-DDX5 and miR-3940.The correlation between circ-DDX5 and miR-3940 expression in breast cancer tissues was analyzed by TCGA database.The expression level of circ-DDX5 in breast cancer SK-BR-3,MDA-MB-231,BT-549,MCF-7,and HCC-1937 cells was detected by RT-qPCR.The circ-DDX5 over-expression plasmid and negative control plasmid were transfected into MDA-MB-231 cells,which were named circ-DDX5 group and NC group,respectively.The proliferation and invasion of MDA-MB-231 cells in the circ-DDX5 group and the NC group were detected by colony formation assay and Transwell assay.The expressions of proliferation pheno-type protein and invasion phenotype protein of MDA-MB-231 cells were detected by Western blot.The expression level of miR-3940 in MDA-MB-231 cells of circ-DDX5 group and NC group was detected by RT-qPCR.Results The expression of circ-DDX5 in breast cancer tissues was lower than that in adjacent tissues(P<0.01)and the ex-pression level of circ-DDX5 was negatively correlated with the clinical stage of breast cancer patients(P<0.01).There was a targeting relationship between circ-DDX5 and miR-3940(P<0.01).The expression of circ-DDX5 and miR-3940 in breast cancer tissue was negatively correlated(P<0.01).The expression of circ-DDX5 in human breast cancer cell lines was lower than that in immortalized breast epithelial cells MCF-10A(P<0.05 or P<0.01).Compared with the NC group,the over-expression of circ-DDX5 could significantly inhibit the proliferation and in-vasion of MDA-MB-231 cells(P<0.01),as well as the proliferation phenotype proteins(cyclin C,CDK3)and in-vasion phenotype proteins(Snail,vimentin)expression(P<0.01)and miR-3940 expression(P<0.01).Conclu-sions The expression of circ-DDX5 in breast cancer tissues and cells is low.circ-DDX5 inhibits the proliferation and invasion of breast cancer MDA-MB-231 cells by targeting the expression of miR-3940.
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BACKGROUND:In recent years,there have been many studies on the mechanism of exosomal non-coding RNA in gestational diabetes mellitus,but there is a lack of the latest systematic review of exosomes from different sources,especially placental sources. OBJECTIVE:To summarize the changes and potential roles of microRNA(miRNA),long non-coding RNA(lncRNA),circular RNA(circRNA),and exosomes in gestational diabetes mellitus to provide potential targets for early screening and treatment of clinical gestational diabetes mellitus. METHODS:A literature search was conducted on PubMed,Web of Science,China National Knowledge Infrastructure,WanFang Data,and VIP databases to retrieve relevant articles on non-coding RNA or exosomal non-coding RNA in relation to gestational diabetes mellitus.A total of 74 articles were included for review. RESULTS AND CONCLUSION:(1)Non-coding RNAs play important pathological and physiological roles in the lifecycle activities,and increasing evidences suggest that non-coding RNAs are involved in the occurrence and development of gestational diabetes mellitus by regulating various physiological functions.This provides a new direction for the research of gestational diabetes mellitus.(2)Exosomes are widely present in the human body.Various cells can secrete exosomes,such as red blood cells,epithelial cells,and placental cells.Non-coding RNAs found in exosomes from different sources have been demonstrated to play a role in the pathogenesis,diagnosis,and treatment of gestational diabetes mellitus.(3)MiRNA and gestational diabetes mellitus:The role of peripheral blood miRNA in gestational diabetes mellitus is mainly to affect the functions of trophoblast cells,pancreatic beta cells and blood glucose levels in gestational diabetes mellitus;placental miRNA can reflect the severity of gestational diabetes and impair the function of trophoblast cells.(4)LncRNA and gestational diabetes mellitus:Peripheral blood lncRNA can induce insulin resistance through the phosphatidylinositol 3-kinase/protein kinase B pathway and may provide new insights for the diagnosis and treatment of gestational diabetes mellitus;placental lncRNA can regulate proliferation and migration of placental trophoblast cells,promoting the occurrence and development of gestational diabetes mellitus.(5)CircRNA and gestational diabetes mellitus:Peripheral blood and placental circRNA can induce the occurrence and development of gestational diabetes mellitus by impairing the proliferation,migration and metabolism of placental trophoblast cells.(6)Non-coding RNA in exosomes and gestational diabetes mellitus:Peripheral blood non-coding RNA in exosomes can affect gestational diabetes mellitus blood glucose levels and glucose homeostasis,and participate in the occurrence and development of gestational diabetes mellitus by influencing placental function.(7)Non-coding RNA has the potential to serve as biomarkers for early diagnosis of gestational diabetes mellitus.Additionally,engineered exosomes can better achieve targeted therapy for gestational diabetes mellitus.These latest findings provide a reference for both basic research and clinical translation of gestational diabetes mellitus.(8)In the future,improvements in the extraction and purification methods of peripheral blood exosomes should be improved,and factors such as race,diet and physical activity should be excluded to improve the reproducibility of results.Further prospective clinical studies are required to explore the clinical application of circulating non-coding RNA and exosomes in the prediction and diagnosis of gestational diabetes mellitus.
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AIM:To investigate the effect of circular RNA MYO9A-006(circMYO9A_006)on hypertrophic phenotype of cardiomyocytes and the underlying mechanism.METHODS:The effect of adenovirus-mediated overexpres-sion of circMYO9A_006 on the expression of hypertrophy-related proteins,including β-myosin heavy chain(β-MHC),skeletal muscle actin alpha 1(ACTA1)and atrial natriuretic peptide(ANP),was evaluated in neonatal mouse ventricular cardiomyocytes(NMVCs).Moreover,a neonatal rat ventricular cardiomyocyte(NRVC)model of phenylephrine(PE)-in-duced hypertrophy was established.The effect of circMYO9A_006 overexpression on NRVC size was ascertained using Phalloidin-iFluor 647 staining method.Dual-luciferase reporter assay was employed to measure the activity of potential in-ternal ribosome entry sites(IRES)in circMYO9A_006.The translation and intracellular location of the circMYO9A_006-translated protein,MYO9A-208aa,were verified using Western blot.To investigate the role of MYO9A-208aa in the ef-fect of circMYO9A_006 on the cardiomyocyte hypertrophic phenotype,we prepared and used the following adenoviruses:the recombinant circMYO9A_006-ORF adenovirus to express MYO9A-208aa,the recombinant circMYO9A_006-ATG-mut adenovirus that does not express MYO9A-208aa,the recombinant circMYO9A_006 adenovirus,and the adenovirus vector control.These were then employed to infect NRVCs.RESULTS:Successful adenovirus-mediated overexpression of circMYO9A_006 was observed in NMVCs.The increased expression of circMYO9A_006 notably reduced the expres-sion of hypertrophy-related proteins in NMVCs(P<0.01).Concurrently,overexpression of circMYO9A_006 substantially reduced the expression of hypertrophy-associated proteins and diminished the size of PE-induced NRVCs(P<0.05).Dual-luciferase reporter assay identified the activity of 2 IRES in circMYO9A_006.Western blot results indicated that circ-MYO9A_006 could produce the MYO9A-208aa protein with an anticipated molecular weight of 28 kD in NRVCs,primari-ly found in the cytoplasm.Elevated expression of both circMYO9A_006 and MYO9A-208aa consistently reduced the ex-pression of hypertrophy-associated proteins(P<0.01),and counteracted the enlarged size of PE-induced NRVCs(P<0.05).However,increased expression of circMYO9A_006-ATG-mut did not counteract the PE-induced hypertrophic phe-notype of NRVCs.CONCLUSION:circMYO9A_006 attenuates the hypertrophic phenotype of cardiomyocytes by synthe-sizing the MYO9A-208aa protein.
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Ovarian cancer has become the most lethal gynecological tumor due to the difficulty in early diagnosis,the late stage when diagnosed and the high recurrence rate.Resistance to platinum-based anti-tumor chemotherapy drugs is an important reason for the poor prognosis of ovarian cancer.circular RNA(circRNA)is more stable than mRNA in cells due to its special structure,and it is involved in the regulation of the occurrence,development and chemotherapy resistance of a variety of tumors.circRNA can be used as a competing endogenous RNA(ceRNA)of miRNA to bind to proteins,and regulates the phenotypic polarization of macrophages,it can also be used as an exosomal circRNA to regulate the sensitivity of platinum resistance in ovarian cancer.circRNA is expected to be a new marker of platinum resistance and a new target for the treatment of platinum resistance.In order to further explore the relationship between circRNA and platinum resistance in ovarian cancer,this article summarizes the recent literature related to circRNA and platinum resistance in ovarian cancer.
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Objective To explore the potential circular RNA(circRNA)biomarker of Uygur type 2 diabetes mellitus(T2DM).Methods A total of 120 T2DM patients and 120 subjects with normal glucose tolerance were recruited from the Department of Endocrinology and Metabolism of the First Affiliated Hospital of Shihezi University and Shihezi community from October 2020 to August 2021,and divided into four groups:60 Uygur T2DM patients(Uygur T2DM group),60 Uygur subjects with normal glucose tolerance(Uygur NC group),60 Han T2DM patients(Han T2DM group)and 60 Han subjects with normal glucose tolerance(Han NC group).Hsa_circRNA_0042817,hsa_circRNA_0006532 and hsa_circRNA_0004131 were selected as candidate circRNA,and the expression in peripheral blood were detected by RT-qPCR.Logistic regression was used to analyze the influencing factors for Uygur T2DM,and the receiver operating characteristic(ROC)curve was used to evaluate the biomarker value of circRNA in Uygur T2DM.Results The expressions of hsa_circRNA_0042817,hsa_circRNA_0006532 and hsa_circRNA_0004131 were higher in Uygur T2DM group than in Uygur NC group(P<0.05).The expression of hsa_circRNA_0042817 was higher in Uygur T2DM group than in Han T2DM group(P<0.05).Logistic regression analysis showed that hsa_circRNA_0042817 was an influencing factor for T2DM in Uygur population[OR(95%CI)3.420(1.567~7.465)].ROC curve analysis showed that the area under the curve was the largest(0.798)in hsa_circRNA_0042817.Conclusion There were up-regulated circRNA in peripheral blood in Uygur T2DM patients,and hsa_circRNA_0042817 may be a biomarker for T2DM in Uygur patients.
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Objective To explore the effect of circular RNA(circRNA)transcription factor 25(TCF25)-targeting microRNA-128b(miR-128b)on the proliferation and apoptosis of MCF-7 human breast cancer cells.Methods MCF-7 cells were cultured until the loga-rithmic growth stage.Cells were randomly divided into the blank(untransfected),si-NC(transfected si-NC),si-circRNA TCF25(transfected si-circRNA TCF25),pcDNA-circRNA TCF25(transfected pcDNA-circRNA TCF25),miR-NC(transfected miR-NC),miR-128b mimic(transfected miR-128b mimic),miR-128b inhibitor(transfected miR-128b inhibitor),and pcDNA-circRNA TCF25+miR-128b mimic(transfected with pcDNA-circRNA TCF25 and miR-128b mimic)groups.Each group included six multiple pores.Forty-eight hours after transfection,the expression of circRNA TCF25 and miR-128b in each group was determined using real-time reverse transcription-quanti-tative polymerase chain reaction(RT-qPCR).An RNase R enzyme digestion assay was used to identify circular RNA.Subcellular locali-zation of circRNA TCF25 was determined through cytoplasmic-nuclear separation.Cell proliferative activity was measured using the 2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Apoptosis was detected using flow cytometry.RT-qPCR was performed to determine the mRNA expression levels of phosphatase and tensin homolog(PTEN),proliferating nuclear antigen 67(Ki-67),andcaspase-3.Western blotting was performed to measure PTEN,Ki-67,caspase-3,and cleaved caspase-3 protein expression.The dual-luciferase reporter(DLR)assay was performed to analyze the relationship between circRNA TCF25 and miR-128b.Results Compared to the control group,the relative expression of circRNA TCF25 did not exhibit significant changes after RNase R enzyme treatment(P>0.05),whereas that of linear TCF25 decreased after RNase R enzyme treatment(P<0.05).The relative expression of circRNA TCF25 in the cytoplasm was higher than that in the nucleus(P<0.05).Compared with the blank and si-NC groups,the cell proliferation activity of the si-circRNA TCF25 group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleaved caspase-3mRNA and protein expression increased.In addition,cell proliferation activity increased and apoptosis rate decreased in the pcDNA-circRNA TCF25 group.Ki-67 mRNA and protein expression were increased,and PTEN,caspase-3,and cleaved caspase-3 mRNA and protein expression decreased,with statistical significance(all P<0.05).Compared with the blank and miR-NC groups,the cell proliferation activity of the miR-128b mimic group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleaved caspase-3 mRNA and protein expression were increased,whereas the cell proliferation activity increased and apoptosis rate decreased in the miR-128b inhibitor group.Ki-67mRNA and protein expression were increased,and PTEN,caspase-3,and cleavedcaspase-3mRNA and protein expression decreased,with statistical significance(all P<0.05).Compared with the pcDNA circRNA TCF25 group,the cell proliferation activity of the pcDNA circRNA TCF25+miR-128b mimic group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleavedcaspase-3mRNA and protein expression increased,with statistical significance(all P<0.05).The DLR assay results confirmed that circRNA TCF25 targets miR-128b.Conclusion CircRNAs may play a key role in promoting the proliferation of MCF-7 human breast cancer cells and inhibiting their apoptosis by targeting miR-128b expression;promoting Ki-67 expression;and inhibiting PTEN,caspase-3,and cleaved caspase-3 expression.
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Circular RNA(circRNA)is an emerging class of endogenous non-coding RNA,which is widely expressed in the brain and plays an important role in a variety of biological processes.Research has shown that circRNA plays a key role in physiological and pathological processes of the brain,such as neurodevelopment,synaptic plasticity and neurodegenerative diseases through a variety of mecha-nisms such as adsorption of microRNA,binding to proteins and translation of peptides.In the field of drug addiction,the expression of circRNA is significantly changed in animal models and brains of addicts,and the regulation involves neural adaptation in brain regions that form the reward circuit such as the nucleus accumbens and prefrontal cortex.Additionally,addiction-related circRNAs are closely associated with neurotransmitter systems,signaling pathways,and neuroinflammatory responses,and they influ-ence the formation and maintenance of drug addiction by modulating gene expression networks related to drug addiction.Here,the biogenesis and regulatory mechanism of circRNA as well as its important role in brain function and drug addiction are reviewed in order to provide a new perspective for explora-tions of the pathological mechanism of drug addiction.
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Objective @#To investigate the role and mechanism of circular RNA containing pleck strin homology do main of Pleck strin homology domain family M member 3 ( circRNA_PLEKHM3) in regulating epithelial mesenchy mal transition (EMT) behavior in cervical cancer cells through the miR-320 and KLF4 .@*Methods @#The expression levels of circRNA_PLEKHM3 in cervical cancer cells Hela and CaSki were detected by real time quantitative PCR (qRT PCR) . RNA fluorescence in situ hybridization was used to determine the localization of circRNA_PLEKHM3 in human cervical cancer epithelial cells CaSki . Dual luciferase reporter gene experiments were conducted to inves tigate the targeting relationship between circRNA_PLEKHM3 and miR-320 , as well as the targeting relationship be tween miR-320 and KLF4 . CaSki cells were overexpressed with circRNA_PLEKHM3. Additionally, three groups were set up: overexpression of miR-320 on the basis of circRNA_PLEKHM3 overexpression , silencing of KLF4 on the basis of circRNA_PLEKHM3 overexpression , and silencing of KLF4 on the basis of miR-320 overexpression . qRT PCR was performed to detect the expression levels of miR-320 in CaSki . Western blot experiments were con ducted to determine the expression of KLF4 and EMT markers including E-cadherin , N-cadherin , Vimentin , MMP-2 , and MMP-9 in CaSki cells . Transwell assays were performed to measure cell migration and invasion .@*Results@#The expression of circRNA_PLEKHM3 decreased in Hela and CaSki cells (P < 0 05) , mainly localized in the cytoplasm . The dual luciferase reporter gene experiment demonstrated a targeting relationship between miR-320 and circRNA_PLEKHM3 , as well as between KLF4 and miR-320 . Overexpression of circRNA_PLEKHM3 inhibited the protein expression of miR-320 , Ncadherin , Vimentin , MMP-2 , and MMP-9 , up regulated the protein expression of E-cadherin, and reduced cell migration and invasion ( P < 0.05) . Overexpression of miR 320 or silencing of KLF4 on the basis of circRNA_PLEKHM3 overexpression both promoted the protein expression of miR-320, N-cad herin , Vimentin , MMP-2 , and MMP-9 , down regulated the protein expression of E-cadherin , and increased cell migration and invasion (P < 0.05) . However , silencing of KLF4 on the basis of miR-320 overexpression inhibited the protein expression of KLF4 , N-cadherin , Vimentin , MMP-2 , and MMP-9 , up regulated the protein expression of E-cadherin , and reduced cell migration and invasion ( P < 0.05) . @*Conclusion @#Overexpression of circRNA _PLEKHM3 regulates EMT in cervical cancer cells through the miR -320/KLF4 axis .
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Objective @#To investigate the relationship between circular RNA homeodomain interacting protein kinase 3 (circHIPK3) and the activation of rat microglia (RM) cells.@*Methods @#In vitro , RM cells were cultured and randomized into normal and oxygen⁃glucose deprivation/reperfusion (OGD/R) groups , and the expression level of circHIPK3 in each group was detected by RT⁃qPCR. The circHIPK3 lentiviral vector with puromycin resistance was constructed , and the overexpression (OE) group and negative control (NC) group were set up. The optimal multiplicity of infection (MOI) for RM cells was determined based on fluorescence expression , and puromycin was used to screen RM cells stably expressing circHIPK3. The cells of OE and NC groups were treated with OGD/R , and the expression levels of ionized calcium binding adaptor molecule 1 (Iba ⁃1) and eukaryotic tumor necrosis factor receptor superfamily (CD40) were detected by Western blot. The circHIPK3 translational protein potential was analyzed by the circRNAdb database , while the potential binding microRNAs on circHIPK3 were predicted by circBank and Starbase databases.@*Results @#OGD/R down⁃regulated circHIPK3 in RM cells ( P < 0. 000 1) . The sequencing results were accurate and the lentiviral vector of circHIPK3 was constructed successfully. The optimal MOI of RM cells was 80 , puromycin at a concentration of 2 μg/ml was used to screen RM cell lines stably expressing circHIPK3. RT⁃qPCR results showed that the expression level of circHIPK3 was significantly higher in the OE group compared with the NC group (P < 0. 01) . Western blot results revealed that the expression levels of Iba and CD40 in the OE group were markedly lower than those in the NC group (P < 0. 05) . Protein translation analysis showed that circHIPK3 encoded a polypeptide of 404 amino acids with two internal ribosome entry sites (IRES) and an open reading frame (ORF) . Database analysis uncovered that circHIPK3 could target eight specific miRNAs , namely hsa⁃miR⁃3529⁃5p , hsa⁃miR⁃379⁃5p , hsa⁃miR⁃506⁃3p , hsa⁃miR⁃33 , hsa⁃miR⁃450b⁃5p , hsa⁃miR551b⁃3p , hsa⁃miR⁃193 , and hsa⁃miR⁃508 ⁃3p.@*Conclusion @#The overexpression of circHIPK3 effectively suppresses OGD/R⁃induced activation of RM cells. It has the potential to encode peptides and may act as a miRNA sponge. These findings provide a foundation for further study of circHIPK3 functions.
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ObjectiveTo investigate the regulatory effect of circular RNA circ_0120051 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved. MethodsThe expression of circ_0120051 and its host gene of solute carrier family 8 member A1(SLC8A1) mRNA in the myocardium of healthy organ donors (n=24) and heart failure (HF) patients (n=21) were assessed by real-time quantitative polymerase chain reaction (RT-qPCR) assay. RNA stability of circ_0120051 was identified by RNase R exonuclease digestion assay. The cytoplasmic and nuclear distribution of circ_0120051 in human cardiomyocyte AC16 was detected by RT-qPCR assay. The expression of fibrosis-related genes in mouse cardiac fibroblasts (mCFs) with adenovirus-mediated overexpression of circ_0120051 was detected by RT-qPCR and Western blot assay, respectively. The effect of overexpression of circ_0120051 on the migration activity of mCFs was evaluated by wound-healing assay. RNA co-immunoprecipitation (RIP) was conducted to detect the interaction between circ_0120051 and miR-144-3p. The binding site of miR-144-3p in the 3'-UTR of isocitrate dehydrogenase 2 (Idh2) mRNA was identified by the dual luciferase reporter gene assay. ResultsCirc_0120051 was significantly up-regulated in the myocardium of HF patients, while the mRNA expression of its host gene SLC8A1 was not changed. Circ_0120051 was mainly located in the cytoplasm of human AC16 cells. Results of RNase R exonuclease digestion revealed that circ_0120051 possesses the characteristic stability of circular RNA compared to the linear SLC8A1 mRNA. Overexpression of circ_0120051 could inhibit the expression of fibrosis-related gene in mCFs and mCFs migration. RIP assay confirmed the specific interaction between circ_0120051 and miR-144-3p. Transfection of miR-144-3p mimic could efficiently promote the expression of fibrosis-related genes in mCFs and reverse the inhibitory effect of circ_0120051 on the fibrotic phenotype of mCFs. Results of the dual luciferase reporter gene assay confirmed the interaction between miR-144-3p and the 3'-UTR of Idh2. Transfection of miR-144-3p transcriptionally inhibited Idh2 expression, and overexpression of circ_0120051 enhanced IDH2 expression in mCFs. MiR-144-3p mimic and Idh2 small interfering RNA (siRNA) could consistently reverse the inhibitory effects of circ_0120051 on fibrosis-related genes expression in mCFs and mCFs migration. ConclusionsCirc_0120051 inhibits the fibrotic phenotype of cardiac fibroblasts via sponging miR-144-3p to enhance the target gene of IDH2 expression.
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Background Pulmonary fibrosis currently lacks screening and diagnostic methods in the early stages and effective treatments in the later stages, so there is an urgent need to explore the mechanisms and develop targeted treatments. Objective To screen the expression of differentially expressed circular RNA (circRNA) hsa_circUCK2 under pathological conditions and to explore its effect on pulmonary fibrosis. Methods In the cell-based experiments, hsa_circUCK2 was knocked down in HPF-a cells using small interfering RNA (siRNA), and HPF-a cells were stimulated by TGF-β1. Four groups were set up: si-NC group, si-circUCK2 group, si-NC+TGF-β1-treated group, and si-circUCK2+TGF-β1-treated group. Western blot assay was used to detect the expression of fibronectin (FN1) in HPF-a cells of the four groups, scratch assay was used to detect the migration ability of HPF-a cells, and CCK-8 assay was used to detect the proliferation ability of HPF-a cells in the two groups with TGF-β1 stimulation, the si-NC+TGF-β1-treated group and the si-circUCK2+TGF-β1-treated group. In the animal experiments, forty-eighty healthy SPF-grade male C57BL/6 mice were randomly divided into four groups: saline+si-con group, saline+si-circ_0000115 group, SiO2+si-con group, and SiO2+si-circ_0000115 group. Mouse lung circRNA mmu_circ_0000115 (mouse homolog of hsa_circUCK2) was knocked down by tracheal drip injection of siRNA, and a mouse lung fibrosis model was constructed by tracheal drip injection of SiO2 suspension (0.2 g·kg−1, 50 mg·mL−1) after 48 h. Real-time fluorescence quantitative PCR was used to detect the knockout efficiency in each organ of the mouse, Western blot was applied to detect the expression of type I collagen α2 (COL1A2) in the lung tissues, and Sirius red was used to detect collagen synthesis in the lung tissues. Results In the cell-based experiments, after the knockdown of hsa_circUCK2, the Western blot results showed that the expression level of the FN1 protein in TGF-β1-stimulated HPF-a cells was significantly down-regulated (P <0.05); the CCK-8 assay and cell scratch assay showed that the down-regulation of hsa_circUCK2 gene significantly inhibited the proliferation and migration of HPF-a cells (P<0.01). In the animal experiments, the real-time fluorescence quantitative PCR results showed that among the detected organs, mmu_circ_0000115 was significantly knocked down only in the lung tissues (P<0.0001); the Western blot results showed that knocking down mmu_circ_0000115 significantly reduced the COL1A2 protein expression level when compared with the SiO2+si-con group (P<0.0001); the Sirius red results showed that knocking down mmu_circ_0000115 significantly reduced collagen production and deposition in lung tissues of mice in the model group. Conclusion Knockdown of hsa_circUCK2 inhibits fibroblast activation and reduces collagen deposition in lung fibrosis model mice. It is suggested that the hsa_circUCK2 is involved in the process of pulmonary fibrosis and may be a potential therapeutic target for pulmonary fibrosis.
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@#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.
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ObjectiveTo investigate the effect of circSLC8A1_005 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved. MethodsThe effect of adenovirus-mediated overexpression of circSLC8A1_005 on the expression of fibrosis-related genes, collagen type I alpha 1 chain (Col1a1), collagen type Ⅲ alpha 1 chain (Col3a1) and smooth muscle actin alpha 2 (Acta2), in mouse cardiac fibroblasts (mCFs) were detected. The proliferation and migration activities of mCFs were detected by EdU and wound-healing assay, respectively. Dual luciferase reporter gene assay was performed to detect the activity of potential internal ribozyme entry site (IRES) in circSLC8A1_005. CircSLC8A1_005-translated protein, SLC8A1-605aa, and its intracellular distribution was identified by Western blot assay. The effect of SLC8A1-605aa protein on transcription activity of Sod2 gene was detected by the dual luciferase reporter gene assay. RNA binding protein immunoprecipitation (RIP) was utilized to verify the interaction between SLC8A1-605aa and superoxide dismutase 2 (Sod2) mRNA. Actinomycin D treatment was used to detect the effect of SLC8A1-605aa on Sod2 mRNA stability in mCFs. ResultsAn efficient adenovirus-mediated overexpression of circSLC8A1_005 was achieved in mCFs. The enforced expression of circSLC8A1_005 suppressed proliferation and migration of mCFs, and inhibited the expression of fibrosis-related genes in mCFs. The dual luciferase reporter gene assay revealed the activities of 2 IRES in circSLC8A1_005. Results of Western blot assay showed that circSLC8A1_005 could translate protein SLC8A1-605aa with the prospected molecular weight of 70 ku, which is predominantly distributed in the nucleus. Overexpression of the circSLC8A1_005 and SLC8A1-605aa could consistently inhibit the fibrotic phenotype of mCFs. SLC8A1-605aa could up-regulate superoxide dismutase 2 (Sod2) expression, but not at the transcriptional level. RIP assay indicated that SLC8A1-605aa could specifically interact with Sod2 mRNA, and the results of actinomycin D assay showed that SLC8A1-605aa could enhance the stability of Sod2 mRNA in mCFs. ConclusionCircSLC8A1_005 inhibits the fibrotic phenotype of cardiac fibroblasts via translating SLC8A1-605aa protein, and SLC8A1-605aa may be a potential target for the treatment of myocardial fibrosis.
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Objective To analyze the core genes of lung ischemia-reperfusion injury and construct a competitive endogenous RNA (ceRNA) network. Methods Original data of GSE145989 were downloaded from the Gene Expression Omnibus (GEO) database as the training set, and the GSE172222 and GSE9634 datasets were used as the validation sets, and the differentially-expressed genes (DEG) were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. Protein-protein interaction (PPI) network was constructed, and the core genes were screened, and the diagnostic values of these core genes and the immune infiltration levels of immune cells were evaluated. The ceRNA network was constructed and validated. The targeted drugs based on ceRNA network were assessed. Results A total of 179 DEG were identified, including 61 down-regulated and 118 up-regulated genes. GO analysis showed that DEGs were associated with multiple biological processes, such as cell migration, differentiation and regulation, etc. They were correlated with cell components, such as vesicle membrane, serosa and membrane raft, etc. They were also associated with multiple molecular functions, such as chemokine receptor, G protein-coupled receptor, immune receptor activity and antigen binding, etc. KEGG pathway enrichment analysis revealed that DEG were involved in tumor necrosis factor (TNF), Wnt, interleukin (IL)-17 and nuclear factor (NF)-κB signaling pathways, etc. PPI network suggested that CD8A, IL2RG, STAT1, CD3G and SYK were the core genes of lung ischemia-reperfusion injury. The ceRNA network prompted that miR-146a-3p, miR-28-5p and miR-593-3p were related to the expression level of CD3G. The miR-149-3p, miR-342-5p, miR-873-5p and miR-491-5p were correlated with the expression level of IL-2RG. The miR-194-3p, miR-512-3p, miR-377-3p and miR-590-3p were associated with the expression level of SYK. The miR-590-3p and miR-875-3p were related to the expression level of CD8A. The miR-143-5p, miR-1231, miR-590-3p and miR-875-3p were associated with the expression level of STAT1. There were 13 targeted drugs for CD3G, 4 targeted drugs for IL-2RG, 28 targeted drugs for SYK and 3 targeted drugs for lncRNA MUC2. No targeted drugs were identified for CD8A, STAT1 and other ceRNA network genes. Conclusions CD8A, IL2RG, STAT1, CD3G and SYK are the core genes of lung ischemia-reperfusion injury. The research and analysis of these core genes probably contribute to the diagnosis of lung ischemia-reperfusion injury and providing novel research ideas and therapeutic targets.
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@#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.
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OBJECTIVES@#To investigate the role and mechanism of circRNA-SR-related CTD associated factor 8 (SCAF8) in regulating endothelial cell pyroptosis in high glucose environment.@*METHODS@#Human umbilical vein endothelial cells (HUVECs) were cultured and divided into six groups. The normal control group and high glucose control group were cultured in cell culture medium with 5 and 33 mmol/L glucose, respectively. The RNA control group, circRNA-SCAF8 inhibition group, miR-93-5p overexpression group and miR-93-5p inhibition group were added with non-functional siRNA, circRNA-SCAF8 inhibitor, miR-93-5p overexpression molecule and miR-93-5p inhibitor in high glucose environment, respectively. Cell viability and pyroptosis were detected by cell counting kit-8 (CCK-8) assay, flow cytometry and Hoechst 33342/propidium iodide fluorescence double staining. Western blotting and enzyme-linked immunosorbent assay were used to detect the expression of pyroptosis-related factors including apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartic acid specific protease-1 (caspase-1) and Gasdermin D (GSDMD), NOD like receptor protein 3 (NLRP-3), thioredoxin interacting proteins (TXNIP), IL-18 and IL-1β. The expression of circRNA-SCAF8, miR-93-5p and TXNIP was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Fluorescence in situ hybridization (FISH) was used to locate circRNA-SCAF8 and miR-93-5p. Dual luciferase assay was used to verify the targeted regulatory relationship between miR-93-5p and upstream and downstream molecules.@*RESULTS@#Compared with the RNA control group, the cell survival rate of circRNA-SCAF8 inhibition group and miR-93-5p overexpression group increased (both P<0.01), the pyroptosis decreased (both P<0.01), and the expressions of pyroptosis-related factors such as TXNIP, NLRP-3, caspase-1, GSDMD, ASC, IL-18 and IL-1β were significantly decreased (all P<0.05). The expression of miR-93-5p was significantly increased after inhibition of circRNA-SCAF8 (P<0.01), and the expression of circRNA-SCAF8 tended to decrease after overexpression of miR-93-5p, but with no statistical significance (P>0.05). Dual luciferase assay showed that miR-93-5p downre-gulated circRNA-SCAF8 expression by binding to the 3 ´ UTR region of circRNA-SCAF8, and miR-93-5p downregulated TXNIP expression by binding to the 3 ´ UTR region of TXNIP. FISH showed that circRNA-SCAF8 and miR-93-5p were both located in the cytoplasm and were highly associated in the cells. qRT-PCR showed that the relative expression of TXNIP increased or decreased after overexpression or inhibition of miR-93-5p compared with the RNA control group, respectively (both P<0.05), suggesting that miR-93-5p could regulate TXNIP gene expression.@*CONCLUSIONS@#CircRNA-SCAF8/miR-93-5p/TXNIP axis is involved in the regulation of pyroptosis in HUVECs under high glucose.
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Humans , Factor VIII , RNA, Circular , Endothelial Cells , Interleukin-18 , Pyroptosis , In Situ Hybridization, Fluorescence , Caspase 1 , MicroRNAs/genetics , Carrier Proteins/genetics , RNA-Binding ProteinsABSTRACT
Objective:To investigate the expression of serum circular RNA PUM1 (circPUM1) and microRNA-760 (miR-760) in patients with polycystic ovary syndrome (PCOS) , and their correlation with BMI and biochemical indexes.Methods:A total of 118 patients with PCOS who were diagnosed and treated in Zhengzhou Central Hospital Affiliated to Zhengzhou University from Feb. 2019 to Feb. 2022 were regarded as as the research subjects (PCOS group) . Another 120 women who underwent physical examination in our hospital were regarded as the control group. Real-time quantitative PCR (RT-qPCR) was performed to detect the expression levels of circPUM1 and miR-760 in serum, and to measure serum glucose and lipid metabolism indicators and sex hormone indicators; Pearson method was performed to analyze the correlation between the expression levels of circPUM1 and miR-760, and the correlation between circPUM1 and miR-760 and each index.Results:The expression level of BMI, FBG, FINS, HOMA-IR, TG, estradiol, LH, prolactin and circPUM1 was (23.81±2.85) kg/m 2, (4.87±0.73) ] mmol/L, (8.51±2.18) mIU/L, 1.85±0.69, (1.58±0.57) mmol/L, (182.34±30.47) pmol/L, (7.43±2.05) mIU/ml, (323.45±60.25) mIU/L and 1.05±0.21, while they were (25.24±2.36) kg/m 2, (5.35±0.65) mmol/L, (14.43±4.36) mIU/L, 3.21±1.05, (1.93±0.60) mmol/L, (193.75±38.42) pmol/L, (10.42±2.60) mIU/mL, (372.31±75.44) mIU/L and 1.83±0.45. The expression level of miR-760 was 1.01±0.23 in the control group, while it was 0.77±0.16 in PCOS group. Correlation analysis showed that serum circPUM1 in PCOS patients was negatively correlated with miR-760 ( r=-677, P<0.001) , and serum circPUM1 in PCOS patients was positively correlated with FBG, FINS, HOMA-IR, and TG ( P<0.05) , while was no greatly correlated with other indicators ( P>0.05) , and miR-760 was negatively correlated with FBG, FINS, HOMA-IR, and TG ( P<0.05) , while was no greatly correlated with other indicators ( P>0.05) . Conclusion:The expression level of circPUM1 in serum of patients with PCOS is increased, and the expression level of miR-760 is decreased, which is correlated with glucose and lipid metabolism indicators and can be used to predict the occurrence of metabolic diseases in PCOS patients.
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Schizophrenia is a complex and severe mental disorder,the precise cause of which remains unknown.Circular RNA(circRNA),a type of non-coding RNA with a loop-like structure,is abundant in eukaryotic cell transcriptomes.Recent advancements in molecular genetics have allowed researchers to gain a better understanding of circRNA's expression profile and biological functions.These molecules play critical roles in various biological processes,including regulation of gene expression,synaptic plasticity,and cognitive flexibility.Due to its high concentration in the mammalian brain,circRNA is involved in neurodevelopment and the maintenance of neural function.Changes in circRNA levels,both within and outside of cells,have been linked to numerous neuro-psychiatric disorders,including schizophrenia.As such,circRNA has a crucial and distinctive function in neuro-psychiatric disorders.Given its characteristics,biological functions,and regulatory roles in diseases,researchers are exploring the abnormal expression of circRNA in schizophrenia and its connection to the onset and progression of the disease.Among them,exosomal circRNA,due to its high stability,disease specificity,and abundant content,is poised to become a potential biological marker for diagnosing schizophrenia.This article provides an extensive overview of the recent circRNA and schizophrenia research,discussing the possibility of using exosomal circRNA as a diagnostic biomarker and therapeutic target for schizophrenia.
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Ovarian carcinoma(OC)is the most lethal malignant tumor among all gynecological tumors.Due to its low early diagnosis rate and high chemotherapy resistance,the mortality rate of OC has been high.Circular RNA(circRNA)is a kind of non-coding RNA that exists in various tissues and cells.Owing to the specific molecular structure,circRNAs are generally highly stable.In recent years,the roles of circRNAs in tumor diagnosis and treatment has become a research hotspot,including OC related circRNAs.circRNAs regulate gene expression at the transcriptional and post transcriptional level,indicating that circRNAs play an important role in the genesis and progression of tumors.Therefore,circRNAs are expected to be a potential diagnosis or prognosis marker for OC.This article will summarize the roles of circRNAs in the occurrence and development of OC and review the research progress of circRNAs in the diagnosis and treatment of OC.
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Objective:To study the influence of circular RNA hsa_circZDHHC21_004 on the proliferation of human small intestinal epithelial cells HIEC-6 after 60Co γ-rays exposure. Methods:HIEC-6 cells were exposed to 60Co γ-rays at 0, 5, 10, and 15 Gy with a dose rate of 1 Gy/min. The expression level of hsa_circZDHHC21_004 in the irradiated HIEC-6 cell was detected. Hsa_circZDHHC21_004 was knocked-down to investigate the influences of hsa_circZDHHC21_004 on the proliferation of irradiated HIEC-6 cells by CCK-8 assay and colony formation assay. Results:The expression level of hsa_circZDHHC21_004 in HIEC-6 cells was upregulated by (1.00±0.24), (1.34±0.28), (1.85±0.31), and (2.80±0.64) times of control after 0, 5, 10, and 15 Gy irradiation, respectively and there were significant difference between 10 or 15 Gy group and 0 Gy group ( F=10.86, P=0.008). Knockdown of hsa_circZDHHC21_004 significantly increased the proliferation rate of HIEC-6 cells at 24, 48, and 72 h after 10 Gy irradiation compared with non-irradiated control ( t=-6.25, -5.83, -7.75, P < 0.001). Under 2 and 5 Gy irradiation, the clone formation rates of the hsa_circZDHHC21_004 knockdown cells were significantly higher than those of the control ( t=-7.45, -8.83, P<0.01). Conclusions:Hsa_circZDHHC21_004 is increased after irradiation and influenced the proliferation of irradiated HIEC-6 cells.