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

Journal: The FASEB Journal

Article Title: Using an Unbiased Coexpression Network to Reveal Cross‐Talking Pathways of Phosphoinositide‐3‐Kinase Regulatory Subunit 1 in Skin Aging and Rejuvenation

doi: 10.1096/fj.202402347RRRR

Figure Lengend Snippet: Gene expression and differentially expressed genes. (A) Comparison of PIK3R1 expression in aged untreated, aged treated and young groups. Aged untreated versus aged treated group ( n = 5, p = 0.0201, t = 2.895, degree of freedom = 8, 95% confidence interval: −1.198 to −0.1356), aged treated versus young group ( n = 5, p = 0.0084, t = 3.474, degree of freedom = 8, 95% confidence interval: −1.840 to −0.3719), aged untreated versus young group ( n = 5, p = 0.0002, t = 6.526, degree of freedom = 8, 95% confidence interval: −2.400 to −1.147). Error bar indicated Mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001. Volcano plot of differentially expressed genes in aged/young (B) and PIK3R1‐low/high (C) groups: Red and blue represent up‐regulation and down‐regulation, respectively. (D) Heatmap of overlapping DEGs. DEGs, Differentially expressed genes. Green to red represents a change in gene expression from downregulated to upregulated.

Article Snippet: The lentiviruses packaging PIK3R1 shRNA were purchased from Shanghai Genechem Co. Ltd. Each of these sequences was cloned into the corresponding vector.

Techniques: Gene Expression, Comparison, Expressing

Journal: The FASEB Journal

Article Title: Using an Unbiased Coexpression Network to Reveal Cross‐Talking Pathways of Phosphoinositide‐3‐Kinase Regulatory Subunit 1 in Skin Aging and Rejuvenation

doi: 10.1096/fj.202402347RRRR

Figure Lengend Snippet: Molecular regulatory network and module‐pathway subnetwork in vitality. Scatterplots of module membership versus gene significance for vitality (A) and PIK3R1 (B). (C) Protein–protein interaction network of co‐expression module genes: Low expression of PIK3R1 is highlighted in yellow; red and blue represent up‐regulation and down‐regulation, respectively; larger node reflects higher gene connectivity. (D) Cross‐talking pathways of PIK3R1 in vitality.

Article Snippet: The lentiviruses packaging PIK3R1 shRNA were purchased from Shanghai Genechem Co. Ltd. Each of these sequences was cloned into the corresponding vector.

Techniques: Expressing

Journal: The FASEB Journal

Article Title: Using an Unbiased Coexpression Network to Reveal Cross‐Talking Pathways of Phosphoinositide‐3‐Kinase Regulatory Subunit 1 in Skin Aging and Rejuvenation

doi: 10.1096/fj.202402347RRRR

Figure Lengend Snippet: Functional identification of PIK3R1 in senescence. (A) Sample dendrogram and heatmap of senescence trait. (B) Relationships of co‐expression modules with senescence and PIK3R1. (C) Cross‐talking pathways of PIK3R1 in senescence. (D) ROC analysis of PIK3R1 in senescence. AUC, area under the curve; ROC, receiver operating curve.

Article Snippet: The lentiviruses packaging PIK3R1 shRNA were purchased from Shanghai Genechem Co. Ltd. Each of these sequences was cloned into the corresponding vector.

Techniques: Functional Assay, Expressing

Journal: The FASEB Journal

Article Title: Using an Unbiased Coexpression Network to Reveal Cross‐Talking Pathways of Phosphoinositide‐3‐Kinase Regulatory Subunit 1 in Skin Aging and Rejuvenation

doi: 10.1096/fj.202402347RRRR

Figure Lengend Snippet: Functional identification of PIK3R1 in rejuvenation. (A) Sample dendrogram and heatmap of rejuvenation trait. (B) Relationships of co‐expression modules with rejuvenation and PIK3R1. (C) Cross‐talking pathways of PIK3R1 in rejuvenation. (D) ROC analysis of PIK3R1 in rejuvenation. AUC, area under the curve; ROC, receiver operating curve.

Article Snippet: The lentiviruses packaging PIK3R1 shRNA were purchased from Shanghai Genechem Co. Ltd. Each of these sequences was cloned into the corresponding vector.

Techniques: Functional Assay, Expressing

Journal: The FASEB Journal

Article Title: Using an Unbiased Coexpression Network to Reveal Cross‐Talking Pathways of Phosphoinositide‐3‐Kinase Regulatory Subunit 1 in Skin Aging and Rejuvenation

doi: 10.1096/fj.202402347RRRR

Figure Lengend Snippet: Detection of PIK3R1 mRNA and protein levels in different groups. (A, B) TaqMan Real‐time RT‐PCR showed that PIK3R1 mRNA expression levels were dramatically decreased in both young and aged treated groups, compared with the aged untreated group respectively. Aged untreated versus aged treated group ( n = 11, fold change = 1.52, 95% confidence interval: −0.4709 to −0.2071). (C, D) Western blot assay revealed that PIK3R1 protein levels were significantly declined in both young and aged treated groups, compared with the aged untreated group respectively. Aged untreated versus young group ( n = 11, fold change = 2.7, 95% confidence interval: −0.7737 to −0.4863). Aged untreated versus aged treated group ( n = 11, fold change = 1.69, 95% confidence interval: −0.5881 to −0.2319). All results are either representative data or the Mean ± SE of the values obtained in three independent experiments. Error bar indicated Mean ± SE. *** p < 0.001, **** p < 0.0001.

Article Snippet: The lentiviruses packaging PIK3R1 shRNA were purchased from Shanghai Genechem Co. Ltd. Each of these sequences was cloned into the corresponding vector.

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: The FASEB Journal

Article Title: Using an Unbiased Coexpression Network to Reveal Cross‐Talking Pathways of Phosphoinositide‐3‐Kinase Regulatory Subunit 1 in Skin Aging and Rejuvenation

doi: 10.1096/fj.202402347RRRR

Figure Lengend Snippet: Knockdown of PIK3R1 in HDFs for cell senescence, proliferation, apoptosis, collagen (I and III) expression and potential targets detection. (A, B) β‐galactosidase staining showed that knockdown of PIK3R1 brought more resistance to UVA induced cell senescence in HDFs. Scale bars, 200 μm. NS, no significance compared with blank group. ns, no significance compared with NC group. *** p < 0.001 compared with NC group. (C, D) EdU staining showed that HDFs with PIK3R1 knockdown brought more proliferation in the no UVA treatment group and more resistance to UVA induced cell proliferation inhibition in the UVA treated group. Scale bars, 100 μm. ** p < 0.01 compared with NC group. (E, F) Western blot assay showed that knockdown of PIK3R1 partly reversed collagen I and III expression decline caused by UVA in HDFs. PIK3R1 knockdown decreased the cellular senescence marker p16 in both no UVA and UVA treated groups. ns, no significance compared with NC group. Error bar indicated Mean ± SE. * p < 0.05, ** p < 0.01. (G, H) 7‐AAD and Annexin V staining indicated that knockdown of PIK3R1 decreased HDFs apoptosis in the no UVA group and partly reversed HDFs apoptosis caused by UVA. Error bar indicated Mean ± SE. * p < 0.05, ** p < 0.01. (I, J) Western blot assay showed that PIK3R1 knockdown increased p‐AKT (Ser473) and decreased p‐FOXO1 (Ser256) in both no UVA and UVA treated groups. Meanwhile, PIK3R1 knockdown increased Bcl‐2 and decreased MMP‐1 in UVA treated HDFs. NS, no significance compared with no UVA‐NC group. ns, no significance compared with NC groups respectively. Error bar indicated Mean ± SE. ** p < 0.01. All results are either representative data or the Mean ± SE of the values obtained in three independent experiments.

Article Snippet: The lentiviruses packaging PIK3R1 shRNA were purchased from Shanghai Genechem Co. Ltd. Each of these sequences was cloned into the corresponding vector.

Techniques: Knockdown, Expressing, Staining, Inhibition, Western Blot, Marker