Circular RNA HMGCS1 sponges MIR4521 to aggravate type 2 diabetes-induced vascular endothelial dysfunction
- Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang University, China
- Ningbo Innovation Center, Zhejiang University, China
Figures
Figure 1 with 1 supplement
circHMGCS1 upregulation and its association with PAHG-induced endothelial dysfunction.
(A) Heat map illustrating the differential expression of circRNAs in HUVECs treated with PAHG for 24 hours (n=3). (B) qRT-PCR validation of five circRNAs in HUVECs treated with PAHG compared to …
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Figure 1—source data 1
Uncropped and labeled gels for (Figure 1).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig1-data1-v1.zip
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Figure 1—source data 2
Raw unedited gels for (Figure 1).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig1-data2-v1.zip
Figure 1—figure supplement 1
Distinct expression profiles of circRNAs in endothelial dysfunction of HUVECs.
HUVECs exposed to palmitic acid and high glucose (PAHG) treatment for 24 hr were compared with untreated Control. (A) Alterations in NO content between the control and PAHG-treated groups (n=4). (B) …
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Figure 1—figure supplement 1—source data 1
Uncropped and labeled gels for (Figure 1—figure supplement 1).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Raw unedited gels for (Figure 1—figure supplement 1).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
circHMGCS1 overexpression aggravates PAHG-induced endothelial dysfunction.
(A) HUVECs were transfected with either circHMGCS1 overexpression lentivirus (pLV-circHMGCS1) or lentiviral circular RNA negative control vector (pLV-circNC). After 48 hours of infection, circHMGCS1 …
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Figure 2—source data 1
Uncropped and labeled gels for (Figure 2).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig2-data1-v1.zip
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Figure 2—source data 2
Raw unedited gels for (Figure 2).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig2-data2-v1.zip
Figure 2—figure supplement 1
A sketch map of circHMGCS1 plasmid construction.
Figure 3 with 1 supplement
MIR4521 prevents PAHG-induced endothelial dysfunction.
(A) Heat map depicting differentially expressed miRNAs in HUVECs with or without PAHG treatment for 24 hr (n=3). (B) Volcano plot illustrating significant changes in miRNAs between control and …
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Figure 3—source data 1
Uncropped and labeled gels for (Figure 3).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig3-data1-v1.zip
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Figure 3—source data 2
Raw unedited gels for (Figure 3).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig3-data2-v1.zip
Figure 3—figure supplement 1
MIR4521 inhibition aggravates diabetes-induced endothelial dysfunction.
(A) Relative expression of MIR98-5P, MIR3143, MIR181A-2–3 P and MIR4521 were detected in HUVECs after corresponding miRNA mimics treatment by qRT-PCR (n=4). (B, C) Relative expression level of MIR452…
Figure 4 with 1 supplement
circHMGCS1 regulates PAHG-induced endothelial dysfunction by targeting and sponging MIR4521.
(A) Luciferase reporter constructs containing wild-type (WT) or mutant (MUT) circHMGCS1 were cotransfected with MIR4521 mimics, MIR-NC, MIR4521 inhibitor, or MIR-NC inhibitor in HEK293T cells (n=4). …
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Figure 4—source data 1
Uncropped and labeled gels for (Figure 4).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig4-data1-v1.zip
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Figure 4—source data 2
Raw unedited gels for (Figure 4).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig4-data2-v1.zip
Figure 4—figure supplement 1
Verification of the binding site of MIR4521 and circHMGCS1.
HEK293T cells were used to package lentivirus overexpressing circHMGCS1, and then infected HUVECs cells, (A) Expression level of MIR4521 was assessed by qRT-PCR in pLV-circHMGCS1-transfected HEK293T …
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Figure 4—figure supplement 1—source data 1
Uncropped and labeled gels for (Figure 4—figure supplement 1).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Raw unedited gels for (Figure 4—figure supplement 1).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig4-figsupp1-data2-v1.zip
Figure 5 with 1 supplement
AAV9-mediated circHMGCS1 overexpression attenuates the protective effect of MIR4521 against diabetes-induced VED.
(A) Changes in body weight of mice from 0 to 14 weeks (n=8, ***p<0.001 DM versus control, #p<0.05 DM+agomir MIR4521 versus DM). (B) Alterations in fasting blood glucose in mice from 0 to 14 weeks …
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Figure 5—source data 1
Uncropped and labeled gels for (Figure 5).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig5-data1-v1.zip
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Figure 5—source data 2
Raw unedited gels for (Figure 5).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig5-data2-v1.zip
Figure 5—figure supplement 1
circHMGCS1 counteracts the protective role of MIR4521 in diabetes-induced VED.
(A) Timeline illustrating the HFHG feeding, AAV9 circHMGCS1, and MIR4521 agomir treatment schedule. (B, C) Quantification of the area under the curve (AUC) for glucose and insulin tests (n=8). (D) …
Figure 6 with 1 supplement
circHMGCS1 functions as a MIR4521 sponge in HUVECs to modulate ARG1 expression.
(A) Schematic depiction illustrating the intersection of predicted target genes of MIR4521 from TargetScan, miRWalk, mirDIP, and GeneCard. (B) Arg1 expression in thoracic aorta of DM by western blot …
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Figure 6—source data 1
Uncropped and labeled gels for (Figure 6).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig6-data1-v1.zip
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Figure 6—source data 2
Raw unedited gels for (Figure 6).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig6-data2-v1.zip
Figure 6—figure supplement 1
MIR4521 modulates the expression of ARG1.
(A) Schematic representation of the predicted binding sites and mutated sites for MIR4521 on ARG1 transcripts in both human cells and mice. (B) Western blot analysis assessing ARG1 and ARG2 …
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Figure 6—figure supplement 1—source data 1
Uncropped and labeled gels for (Figure 6—figure supplement 1).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Raw unedited gels for (Figure 6—figure supplement 1).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig6-figsupp1-data2-v1.zip
Figure 7 with 1 supplement
ARG1 is inseparable from circHMGCS1 and MIR4521 regulating diabetes-induced VED.
(A, B) circHMGCS1 and MIR4521 had no effect on NO content and ENOS activity expression in the absence of ARG1 (n=4). (C) ROS expression remained unaffected by circHMGCS1 and MIR4521 in the absence …
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Figure 7—source data 1
Uncropped and labeled gels for (Figure 7).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig7-data1-v1.zip
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Figure 7—source data 2
Raw unedited gels for (Figure 7).
- https://cdn.elifesciences.org/articles/97267/elife-97267-fig7-data2-v1.zip
Figure 7—figure supplement 1
Loss of functional regulation of circHMGCS1 and MIR4521 on HUVECs in the absence of ARG1 in vivo.
(A) Schematic outlining the timeline of HFHG feeding, AAV9 circHMGCS1 and MIR4521 agomir treatment, AAV9 ARG1 shRNA treatment, and experiment conclusion. (B, C) Blood glucose levels measured at week …
Author response image 1
The impact of circHMGCS1 knockdown on ARG1 and miR-4521 expression levels in HUVEC.
The cells were transfected with either circHMGCS1 shRNA1 or circHMGCS1 shRNA2, and the expressions levels of circHMGCS1 and HMGCS1 (A), miR-4521 (B) and ARG1 (C and D) in HUVECs were detected by …
Author response image 2
The impact of circHMGCS1 knockdown on ARG1 and miR-4521 expression levels in HUVEC.
The cells were transfected with either circHMGCS1 shRNA1 or circHMGCS1 shRNA2, and the expressions levels of circHMGCS1 and HMGCS1 (A), miR-4521 (B) and ARG1 (C and D) in HUVECs were detected by …
Author response image 3
The distribution of copy numbers for circHMGCS1, miR-4521 and ARG1 in HUVECs.
Author response image 4
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HUVEC | The Shanghai Cell Bank of the Chinese Academy of Sciences | RRID:CVCL_2959 | |
| Cell line (H. sapiens) | HEK-293T | The Shanghai Cell Bank of the Chinese Academy of Sciences | RRID:CVCL_0063 | |
| Gene (H. sapiens) | circHMGCS1 | circbase | circRNA ID: hsa_circ_0008621 | |
| Gene (H. sapiens) | HMGCS1 | GeneBank | Gene ID: 3157 | |
| Gene (H. sapiens) | MIR4521 | miRbase | miRNA ID: MIMAT0019058 | |
| Gene (Mus musculus) | ARG1 | GeneBank | Gene ID: 383 | |
| Gene (M. musculus) | Arg1 | GeneBank | Gene ID: 11846 | |
| Antibody | Anti- Liver Arginase (ARG1) (Rabbit monoclonal) | Abcam | ab133543 | WB (1: 1000) |
| Antibody | Anti- Arg2 (Rabbit monoclonal) | Abcam | ab264066 | WB (1: 1000) |
| Antibody | Anti- Endothelin 1(ET-1)(Mouse monoclonal) | Abcam | ab2786 | WB (1: 1000) |
| Antibody | Anti- VCAM1 (Rabbit monoclonal) | Abcam | ab134047 | WB (1: 1000) |
| Antibody | Anti-ICAM1 (Rabbit monoclonal) | Abcam | ab222736 | WB (1: 1000) |
| Antibody | Anti-AGO2 (Mouse monoclonal) | Proteintech | 67934–1-Ig | WB (1: 2000) |
| Antibody | Anti-beta ACTIN (Mouse monoclonal) | Abcam | ab8226 | WB (1: 2000) |
| Antibody | Anti-GAPDH (Mouse monoclonal) | Abcam | ab8245 | WB (1: 2000) |
| Recombinant DNA reagent | pCDH-CMV-MCS-EF1-Puro (plasmid) | Addgene | RRID:Addgene_73030 | |
| Recombinant DNA reagent | pLKO.1-GFP-shRNA (plasmid) | Addgene | RRID:Addgene_30323 | |
| Recombinant DNA reagent | psPAX2(plasmid) | Addgene | RRID:Addgene_12260 | |
| Recombinant DNA reagent | pMD2G(plasmid) | Addgene | RRID:Addgene_12259 | |
| Recombinant DNA reagent | pAAV-MCS (plasmid) | Ruipute | Cat#: 2212E4 | |
| Recombinant DNA reagent | pAAV-RC9 (plasmid) | Ruipute | Cat#: 23011 | |
| Recombinant DNA reagent | pHelper (plasmid) | Ruipute | Cat#: 230112 | |
| Recombinant DNA reagent | pAAV-MIR4521 sponge-zsGREEn1-shRNA(plasmid) | Ruipute | Cat#: 230113 | |
| Recombinant DNA reagent | pAAV-Arg1-zsGREEn1-shRNA(plasmid) | Ruipute | Cat#: 2212E1 | |
| Recombinant DNA reagent | pAAV-ARG1-zsGREEn1-shRNA(plasmid) | Ruipute | Cat#: 2212E2 | |
| Recombinant DNA reagent | pAAV-ZsGreen1 (plasmid) | Takara | Cat#: 6231 | |
| Recombinant DNA reagent | pAAV-ZsGreen1-shRNA (plasmid) | youbio | Cat#: VT8093 | |
| Commercial assay or kit | Fluorescent In Situ Hybridization Kit | RIBOBIO | Cat#: C10910 | |
| Commercial assay or kit | RNA pull down kit | GENESEED | Cat#: P0202 | |
| Commercial assay or kit | RNA Immunoprecipitation Kit | GENESEED | Cat#: P0102 | |
| Software, algorithm | ImageJ | National Institutes of Health | https://imagej.nih.gov/ij/ | |
| Software, algorithm | Prism | GraphPad v8.0 | RRID:SCR_002798 |
