Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: The shRNA sequences.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: shRNA

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: Primer sequence for RT-qPCR.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: Sequencing

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: MIAT upregulates ENC1 expression through MYC. (A) The subcellular localization of MIAT. CN RCI < 0 indicates that lncRNA is expressed in the nucleus, and CN RCI > 0 indicates that lncRNA is expressed in the cytoplasm. (B) After the nucleus and cytoplasm of HBEC-5i cells were separated, MIAT expression was detected by RT-qPCR. GAPDH was a marker of cytoplasm and U6 was a marker of nucleus. (C) The downstream TFs of MIAT analyzed by PPI analysis. The redder the color, the higher the core degree was Vice versa, the bluer the color, the lower the core degree was. (D) RNA pull down to detect the binding of MYC with MIAT. (E) RIP to detect the binding of MYC with MIAT. (F) The Venn map of the differentially expressed genes in the microarray GSE75436 and the downstream genes of TF MYC, and the intersection was ENC1. (G) The enrichment of MYC binding to the promoter region of ENC1 by ChIP. (H) MIAT expression in endothelial cells after overexpressing or silencing MIAT detected by RT-qPCR. (I) MYC mRNA expression in endothelial cells after overexpressing or silencing MIAT detected by RT-qPCR. (J) ENC1 expression in endothelial cells after overexpressing or silencing MIAT detected by RT-qPCR. (K) Protein expression of MYC and ENC1 in endothelial cells after overexpressing or silencing MIAT determined by Western blot analysis. (L) The regulatory effect on ENC1 by MIAT and MYC detected by dual luciferase reporter gene assay. * p < 0.05, ns p > 0.05. The measurement data were expressed as mean ± standard derivation, comparisons among multiple groups were analyzed by one-way ANOVA and followed by Tukey’s post hoc test, and the cell experiment was conducted three times independently.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Marker, Binding Assay, Microarray, Western Blot, Luciferase, Reporter Gene Assay

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: ENC1 expression is augmented in IA patients, and ENC1 silencing negates MIAT-induced endothelial cell apoptosis. (A) ENC1 expression in ruptured IA, unruptured IA and control samples determined by RT-qPCR. (B) Analysis of the correlation between ENC1 expression and MIAT expression. (C) The correlation between the expression of ENC1 and disease-free survival of patients with IA. (D) The correlation between the expression of ENC1 and overall survival of patients with IA. (E) The protein expression of ENC1 in vascular endothelial cells in vitro in response to oe-MIAT and sh-ENC1 alone or in combination measured by Western blot analysis. (F) Flow cytometry analysis for apoptosis of endothelial cells in response to oe-MIAT and sh-ENC1 alone or in combination. (G) The expression of apoptosis-related factors (cleaved Caspase-3, cleaved PARP1, Bax, and Bcl-2) in response to oe-MIAT and sh-ENC1 alone or in combination measured by Western blot analysis. * p < 0.05. The measurement data were expressed as mean ± standard derivation. Comparisons between two groups were analyzed by unpaired t test, while the correlation between the expression of ENC1 in patients with IA and their disease-free survival and overall survival was analyzed by Kaplan–Meier method (long-rank test). Comparisons among multiple groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test, and the cell experiment was conducted three times independently.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing, Control, Quantitative RT-PCR, In Vitro, Western Blot, Flow Cytometry

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: Silencing ENC1 reduces endothelial cell apoptosis in vivo to protect rats from IA. Rats were induced with IA model and treated with sh-ENC1 or sh-NC, with the sham-operated rats as control. (A) The expression of ENC1 in rats determined by RT-qPCR. (B) HE staining (×200) for the ACA/OA branch sections of rats. (C) The representative images of TUNEL staining (×200) for apoptosis of rats (arrows indicate the arterial wall). (D) Quantitative analysis of cell apoptosis in rats evaluated by TUNEL staining. (E) The expression of apoptosis-related factors (cleaved Caspase-3, cleaved PARP1, Bax, and Bcl-2) as determined by Western blot analysis and quantified by Image J software. * p < 0.05. The measurement data were expressed as mean ± standard derivation. Comparisons between two groups were analyzed by unpaired t test, and comparisons among multiple groups were analyzed by one-way ANOVA and followed by Tukey’s post hoc test. The cell experiment was conducted three times independently. The sham group: n = 12, the IA + sh-NC group: n = 12, and the IA + sh-ENC1 group: n = 6.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: In Vivo, Control, Expressing, Quantitative RT-PCR, Staining, TUNEL Assay, Western Blot, Software

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: The mechanism diagram illustrating the effects of the MIAT/MYC/ENC1 axis on endothelial cell apoptosis in IA. MIAT enhanced the expression of ENC1 through MYC, thereby promoting vascular endothelial cell apoptosis and further inducing the pathogenesis of IA.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing