CircRNA recurrence IF=18.88 high score article--Circle mechanism research summary
2024-02-12 13:07:45
CircRNA re-issue IF=18.88 high score article - you want to know the circRNA mechanism research, are here...
Article introduction
Super-proton-regulated circRNA-Nfix deletion induces post-myocardial infarction in adult mice
CircRNAs are becoming potent regulators of heart development and disease, but their role in cardiac regeneration remains unknown. In view of this, the author and his team explored the function of circRNA-Nfix associated with super-enhancer (SEs) in the post-myocardial infarction regeneration in mice, and explored its molecular mechanism of regulating cardiac remodeling, and in 2019 On April 5, the results were published in the circulation magazine (IF=18.88). In this study, the authors first used bioinformatics to analyze RNA sequencing data and combined SE catalogues to identify SE-related circRNAs, and used qPCR and in situ hybridization techniques to detect the discovery of circNfix in human, rat, and mouse adult hearts. Overexpression. Then, by interfering with or overexpressing circNfix in myocardial cells (CM) after myocardial infarction (MI), its role in cell proliferation and myocardial repair was explored, and it was found that down-regulation of circNfix can promote CM proliferation and angiogenesis after myocardial infarction, and Inhibit CM apoptosis, reduce cardiac dysfunction, and improve prognosis. Finally, the authors used chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSAs) to determine the transcription factor Meis1 binding to SE with circ-nfix; RNA pull-down and luciferase reporter assays for circRNA Interaction with proteins or with miRNAs; found that down-regulation of circNfix enhances the interaction of Ybx1 with Nedd4l (E3 ubiquitin ligase) and induces ubiquitination of Ybx1, inhibiting the expression of downstream cyclin-A2 and cyclin-B1. In addition, circNfix also acts as a ceRNA, adsorbs mir-214 and promotes Gsk3β expression and inhibits β-catenin activity. That is, SE-regulated circNfix deletion may inhibit the Ybx1 ubiquitin-dependent degradation, increase miR-214 activity, promote cardiac regeneration and functional recovery after myocardial infarction, and may be a promising strategy to improve the prognosis of myocardial infarction.
Technical route
CircRNAs are becoming potent regulators of heart development and disease, but their role in cardiac regeneration remains unknown. In view of this, the author and his team explored the function of circRNA-Nfix associated with super-enhancer (SEs) in the post-myocardial infarction regeneration in mice, and explored its molecular mechanism of regulating cardiac remodeling, and in 2019 On April 5, the results were published in the circulation magazine (IF=18.88). In this study, the authors first used bioinformatics to analyze RNA sequencing data and combined SE catalogues to identify SE-related circRNAs, and used qPCR and in situ hybridization techniques to detect the discovery of circNfix in human, rat, and mouse adult hearts. Overexpression. Then, by interfering with or overexpressing circNfix in myocardial cells (CM) after myocardial infarction (MI), its role in cell proliferation and myocardial repair was explored, and it was found that down-regulation of circNfix can promote CM proliferation and angiogenesis after myocardial infarction, and Inhibit CM apoptosis, reduce cardiac dysfunction, and improve prognosis. Finally, the authors used chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSAs) to determine the transcription factor Meis1 binding to SE with circ-nfix; RNA pull-down and luciferase reporter assays for circRNA Interaction with proteins or with miRNAs; found that down-regulation of circNfix enhances the interaction of Ybx1 with Nedd4l (E3 ubiquitin ligase) and induces ubiquitination of Ybx1, inhibiting the expression of downstream cyclin-A2 and cyclin-B1. In addition, circNfix also acts as a ceRNA, adsorbs mir-214 and promotes Gsk3β expression and inhibits β-catenin activity. That is, SE-regulated circNfix deletion may inhibit the Ybx1 ubiquitin-dependent degradation, increase miR-214 activity, promote cardiac regeneration and functional recovery after myocardial infarction, and may be a promising strategy to improve the prognosis of myocardial infarction.
Technical route
1 Filter and identify CircNfix
CircNfix is ​​a conservative heart circrna. RPM expression level of CircRNA (A). Expression of CircNfi and circSlc8a1 in P7 mouse and rat cardiomyocytes (CMs) and fibroblasts (CFs), respectively (B). CF. was identified as a circular RNA. qPCR was used to detect various cells (H) in mouse, primary cells (H) analyzed by mouse heart, mouse heart tissue (I) at different time periods, and cardiomyocytes isolated from mouse heart tissue at different time periods (J ). ISH (K) and corresponding analysis (LM) were used to detect the expression of CircNfix in human, rat, and mouse hearts. Nuclear separation PCR (N) and FISH (O) detected the subcellular localization of CircNfix. 
2 Explore the function of CircNfix
(1) Down-regulation of CircNfix promoted the proliferation of CM: Ki67 immunofluorescence (A), EdU staining (B), PH3 immunofluorescence (C) and Aurora B immunofluorescence (D) were used to detect the effect of CircNf on the proliferation of P7 CM. The effect of the fluorescent mitochondrial dye tetramethylrhodamine ethyl ester (TMRE) on the ability of CircNf to interfere with CM splitting (E). Ki67 immunofluorescence (F), EdU staining (G) and PH3 immunofluorescence (H) were used to detect the effect of overexpressing CircNf on the proliferative capacity of P0 CM. Immunofluorescence staining of Nkx2.5, α-SMA, RUNX1, DAB2 was used to detect the dedifferentiation ability of P7 CM (I). Flow cytometry analysis interfered with the effect of CircNf on the cell cycle of CM (J).
(2) Down-regulation of circNfix can induce cardiac regeneration after myocardial infarction: echocardiographic analysis of cardiac function before myocardial infarction, 1, 14 and 28 days after infarction (A). Quantitative analysis (EF) and shortening score (FS) (BC) of ejection fraction. Mouse hearts scored after 28 days of transfection of sh-circnfixo in mice (D). Mouse ventricular transverse section TTC staining (E) at 14 and 28 days after myocardial infarction. Representative images of Masson-stained heart sections at 14 and 28 days after myocardial infarction (F). Quantitative analysis of the fibrotic area in the heart section (G). Kaplan-Meier survival curve analysis of the shcircNfix group after myocardial infarction (H).
3 Explore the molecular mechanism of CircNfix
Transcription factor:
(1) The transcription factor Meis1 drives circNfix expression by SE binding to the Nfix site: ChIP-seq spectra of H3k27ac, H3k4me1 and H3k27me3 at the Nfix site of human cardiac tissue (the cloud sequence organism provides this service) (A). Conservative scores of H3k27ac, H3k4me1, H3k27me3, P300, DNA hypersensitivity (DHS), Meis1 and PhastCons in the Nfix site of mouse cardiac tissue, and H3k27ac ChIP-seq in mouse CMs (the cloud order organism provides this service) (B ). 3C-qPCR analysis of the looping event between SE and circNfix promoter region (C). The pgl3 promoter reporter vector luciferase assay (D) constructed with four component enhancers (E1, E2, E3, E4). Luciferase activity (E) of wild-type E2 and mutant E2 in P7 CMs after Meis1 silencing. EMSA results (F) of nuclear proteins extracted from P7 CMs after incubation of the Meis1 binding site in circNfix-SE by DIG-ddUTP-labeled oligonucleotide probes. ChIP-qPCR (provided by the cloud-sequence organism) assay showed that the Meis1 binding site was amplified in circNfix-SE (G). QRT-PCR was used to detect the expression levels of circNfix and Nfix mRNA in P7 CMs after Meis1 gene knockout (H). RNA FISH was used to detect the subcellular localization of circNfix in P7 CMs after Meis1 knockout (I). EdU staining was used to detect the effect of Meis1 and circNfix interference on the proliferation of P7 CMs (J).
(1) The transcription factor Meis1 drives circNfix expression by SE binding to the Nfix site: ChIP-seq spectra of H3k27ac, H3k4me1 and H3k27me3 at the Nfix site of human cardiac tissue (the cloud sequence organism provides this service) (A). Conservative scores of H3k27ac, H3k4me1, H3k27me3, P300, DNA hypersensitivity (DHS), Meis1 and PhastCons in the Nfix site of mouse cardiac tissue, and H3k27ac ChIP-seq in mouse CMs (the cloud order organism provides this service) (B ). 3C-qPCR analysis of the looping event between SE and circNfix promoter region (C). The pgl3 promoter reporter vector luciferase assay (D) constructed with four component enhancers (E1, E2, E3, E4). Luciferase activity (E) of wild-type E2 and mutant E2 in P7 CMs after Meis1 silencing. EMSA results (F) of nuclear proteins extracted from P7 CMs after incubation of the Meis1 binding site in circNfix-SE by DIG-ddUTP-labeled oligonucleotide probes. ChIP-qPCR (provided by the cloud-sequence organism) assay showed that the Meis1 binding site was amplified in circNfix-SE (G). QRT-PCR was used to detect the expression levels of circNfix and Nfix mRNA in P7 CMs after Meis1 gene knockout (H). RNA FISH was used to detect the subcellular localization of circNfix in P7 CMs after Meis1 knockout (I). EdU staining was used to detect the effect of Meis1 and circNfix interference on the proliferation of P7 CMs (J).
Binding protein:
(2) CircNfix binds to Ybx1 and promotes its degradation: protein profiling of circNfix and control (A). RIP experiments were performed using Ybx1, Nedd4l or negative IgG antibodies (cloud order organisms provide this service) (B). Ybx1 protein expression level (CD) after overexpression of circNfix in P0 CMs. qRT-PCR was used to detect Ybx1 mRNA expression level (E) in P0 CMs. Subcellular colocalization of circNfix and Ybx1 (F) when RNA FISH overexpresses and does not express circNfix in P0 CMs. Fractionation experiments showed that circNfix overexpression promoted the translocation of Ybx1 in the cytoplasm and increased the degradation of Ybx1 in the cytoplasm (G). Expression levels of cyclin A2 and cyclin B1 after circNfix and Ybx1 interference (H). ChIP-qPCR (provided by the cloud-sequence organism) assay showed amplification of the Ybx1 binding site in the cyclin A2 and cyclin B1 promoters (I). The cell lysate was immunoprecipitated with an anti-Ybx1 antibody, and subjected to WB analysis by immunoblot analysis using a ubiquitin (Ub)-specific antibody, an anti-Ybx1 antibody or an anti-nedd4l antibody. Ybx1 protein expression level (KL) in CMs overexpressing circNfix.
(2) CircNfix binds to Ybx1 and promotes its degradation: protein profiling of circNfix and control (A). RIP experiments were performed using Ybx1, Nedd4l or negative IgG antibodies (cloud order organisms provide this service) (B). Ybx1 protein expression level (CD) after overexpression of circNfix in P0 CMs. qRT-PCR was used to detect Ybx1 mRNA expression level (E) in P0 CMs. Subcellular colocalization of circNfix and Ybx1 (F) when RNA FISH overexpresses and does not express circNfix in P0 CMs. Fractionation experiments showed that circNfix overexpression promoted the translocation of Ybx1 in the cytoplasm and increased the degradation of Ybx1 in the cytoplasm (G). Expression levels of cyclin A2 and cyclin B1 after circNfix and Ybx1 interference (H). ChIP-qPCR (provided by the cloud-sequence organism) assay showed amplification of the Ybx1 binding site in the cyclin A2 and cyclin B1 promoters (I). The cell lysate was immunoprecipitated with an anti-Ybx1 antibody, and subjected to WB analysis by immunoblot analysis using a ubiquitin (Ub)-specific antibody, an anti-Ybx1 antibody or an anti-nedd4l antibody. Ybx1 protein expression level (KL) in CMs overexpressing circNfix.
ceRNA mechanism:
(3) Interaction of circNfix with miR-214: miR-214 and miR-761 were predicted to bind to circNfix (A). Expression of miR-214 and miR-761 after down-regulation of circNfix in adult mouse hearts (B). The dual fluorescence reporter assay detects that miR-214 and circNfix can bind directly (C). RNA pull-down (the cloud sequencing organism provides this service) experiments to detect circNfix can pull down miR-214 (D). In RNA FISH assays, miR-124 and circNfix were colocalized in the cytoplasm (E) in P0 CMs. In RNA FISH assays, miR-124 and Gsk3β were colocalized in the cytoplasm (F) in P0 CMs. The dual fluorescence reporter assay detects direct binding (G) of miR-214 and Gsk3β 3'UTR. WB detects the expression level of Gsk3β protein after interference with circNfix (H). WB detects the expression level of Gsk3β protein after interference or overexpression of miR-124 (I). WB detects the expression level of Gsk3β protein after interference with circNfix and miR-214 (J). In CMs, the expression level of β-catenin after circ Nfix was disturbed (K). In CMs, the expression level of Meis1 and Gsk3β after Meis1 was interfered (L). In CMs, the expression level of GSK3β (M) after interference with Meis1 and circNfix. After interference with circNfix, Ybx1, miR-214 and Gsk3β, P7 CMs were subjected to pH3 and Aurora B immunostaining (N).
(3) Interaction of circNfix with miR-214: miR-214 and miR-761 were predicted to bind to circNfix (A). Expression of miR-214 and miR-761 after down-regulation of circNfix in adult mouse hearts (B). The dual fluorescence reporter assay detects that miR-214 and circNfix can bind directly (C). RNA pull-down (the cloud sequencing organism provides this service) experiments to detect circNfix can pull down miR-214 (D). In RNA FISH assays, miR-124 and circNfix were colocalized in the cytoplasm (E) in P0 CMs. In RNA FISH assays, miR-124 and Gsk3β were colocalized in the cytoplasm (F) in P0 CMs. The dual fluorescence reporter assay detects direct binding (G) of miR-214 and Gsk3β 3'UTR. WB detects the expression level of Gsk3β protein after interference with circNfix (H). WB detects the expression level of Gsk3β protein after interference or overexpression of miR-124 (I). WB detects the expression level of Gsk3β protein after interference with circNfix and miR-214 (J). In CMs, the expression level of β-catenin after circ Nfix was disturbed (K). In CMs, the expression level of Meis1 and Gsk3β after Meis1 was interfered (L). In CMs, the expression level of GSK3β (M) after interference with Meis1 and circNfix. After interference with circNfix, Ybx1, miR-214 and Gsk3β, P7 CMs were subjected to pH3 and Aurora B immunostaining (N).
to sum up
How circNfix regulates cardiac regeneration by binding to Ybx1 and miR-214: the binding of the transcription factor Meis1 to the SE of circNfix, the expression of the inhibitor circNfix, and the deletion of circNfix enhances the interaction of Ybx1 with Nedd4l (E3 ubiquitin ligase) And induced ubiquitination degradation of Ybx1, and further inhibited the expression of downstream cyclin-A2 and cyclin-B1. At the same time, the missing circNfix can reduce the adsorption of miR-214, further inhibit the expression of Gsk3β and VEGF, and finally promote cardiac regeneration and functional recovery after myocardial infarction.
Full text link:
https://
https://
Cloud order related product recommendation
Whole transcriptome sequencing circular RNA sequencing
RNA pull down
RIP sequencing
ChIP sequencing
M6A RNA whole transcriptome methylation sequencing
m6A RNA circular RNA sequencing
M5C RNA whole transcriptome methylation sequencing
m5C RNA circular RNA sequencing
m1A RNA whole transcriptome methylation sequencing
m1A RNA circular RNA sequencing ultra-micro circular RNA methylation sequencing
RNA pull down
RIP sequencing
ChIP sequencing
M6A RNA whole transcriptome methylation sequencing
m6A RNA circular RNA sequencing
M5C RNA whole transcriptome methylation sequencing
m5C RNA circular RNA sequencing
m1A RNA whole transcriptome methylation sequencing
m1A RNA circular RNA sequencing ultra-micro circular RNA methylation sequencing
Cloud order customer published sequencing article
1.Whole-genome and Transcriptome Sequencing of Prostate Cancer Identify New Genetic Alterations Driving Disease Progression
2.SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function
3.CircHLA-C Plays an Important Role in Lupus Nephritis by Sponging miR-150
4.Identification of Circular RNAs and Their Targets in Leaves of Triticum aestivum L. under Dehydration Stress
5.Circular RNA alterations are involved in resistance to avian leukosis virus subgroup-J-induced tumor formation in chickens
6.Identification of circular RNAs and their alterations involved in developing male Xenopus laevis chronically exposed to atrazine
7.Identification and characterization of circular RNAs in zebrafish
8.Differential Expression of Circular RNAs in Glioblastoma Multiforme and Its Correlationwith Prognosis
9.Circular RNA Signature Predicts Gemcitabine Resistance of Pancreatic Ductal Adenocarcino
10.Circular RNA Vav3 sponges gga-miR-375 to promote epithelial-mesenchymal transition
11.RNA-Seq profiling of circular RNAs in human colorectal Cancer liver metastasis and the potential biomarkers
12.Analysis of changes to lncRNAs and their target mRNAs in murine jejunum after radiation treatment
13.circular RNA expression alteration in exosomes from the brain extracellular space after traumatic brain injury in mice
Shanghai Yunxu Biological Technology Co., Ltd.
Shanghai Cloud-seq Biotech Co., Ltd.
Address: 3rd Floor, Building 20, No. 518, Zhangzhu Road, Songjiang District, Shanghai
Telephone Fax Website:
mailbox:
Shanghai Cloud-seq Biotech Co., Ltd.
Address: 3rd Floor, Building 20, No. 518, Zhangzhu Road, Songjiang District, Shanghai
Telephone Fax Website:
mailbox:
X Ray Single Side Protection Lead Apron,Xray Radiation Lead Aprons,X-Ray Front Protection Lead Apron,X Ray Radiation Proof Lead Apron
Longkou Kangxie Medical Instrument Co., Ltd , https://www.lkkangxiemedical.com