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

Abstract

Intracranial aneurysm (IA) is vascular enlargement occurred on the wall of cerebral vessels and can result in fatal subarachnoid hemorrhage when ruptured. Recent studies have supported the important role of long non-coding RNAs (lncRNAs) in IA treatment. This study identified functional significance of lncRNA myocardial infarction associated transcript (MIAT) in IA. Myocardial infarction associated transcript and ectodermal-neural cortex 1 (ENC1) expression was detected by reverse transcription quantitative polymerase chain reaction. Cell counting kit 8 assay flow cytometry were conducted to detect cell viability and apoptosis of endothelial cells in IA. The interaction among MIAT, ENC1, and myelocytomatosis oncogene (MYC) was analyzed by RNA pull down, RNA immunoprecipitation assay, chromatin immunoprecipitation assay, and dual luciferase reporter assay. Intracranial aneurysm was induced by ligating the left carotid artery and the bilateral posterior branch of the renal artery in rats for studying the role of MIAT and ENC1 in vivo. Myocardial infarction associated transcript and ENC1 were upregulated in IA. Endothelial cells in IA presented a decreased cell viability and an increased apoptotic rate. Myocardial infarction associated transcript could regulate the expression of ENC1, and MYC could bind to the promoter region of ENC1. High expression of MIAT increased endothelial cell apoptosis and vascular endothelial injury, while MIAT knockdown was identified to reduce the risk of IA both in vitro and in vivo through regulating ENC1. To sum up, MIAT silencing is preventive for IA occurrence by decreasing the MYC-mediated ENC1 expression, which represents a novel therapeutic target for IA.

Keywords: apoptosis; ectodermal-neural cortex 1; intracranial aneurysm; long non-coding RNA MIAT; myelocytomatosis oncogene.

FIGURE 1

The high expression of MIAT and damaged endothelial cell continuity are observed in patients with IA. (A) The volcano plot of GSE75436 analyzed by R language. Red dots represented the upregulated genes, and green dots represented the downregulated genes (LogFC > 2, p < 0.01). (B) The Venn diagram of the differentially expressed lncRNAs in GSE75436 and the total lncRNAs obtained from lncMAP, and the intersections included lncRNA SFTA1P, CTD-2165H16.3, NAPSB, RP3-525N10.2, CTB-12O2.1, FLVCR2, EMX2OS, EGFEM1P, and MIAT. (C) The expression of MIAT in ruptured IA, unruptured IA, and control samples determined by RT-qPCR. (D) The correlation between the expression of MIAT and disease-free survival of patients with IA. (E) The correlation between the expression of MIAT and overall survival of patients with IA. (F) The HE staining (×100, ×200) for carotid arteries near branch or IA lesions, and immunofluorescence staining (×200) for vascular endothelial cells labeled with CD34 antibody. *p < 0.05 vs. the control samples. 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 MIAT in patients with IA and their disease-free survival and overall survival was analyzed by Kaplan–Meier method (long-rank test).

FIGURE 2

The endothelial cell apoptosis can be induced by overexpression of MIAT in vitro. (A) Representative images of TUNEL staining (×200) and quantitative analysis of TUNEL-positive cells in patients with IA. (B) The expression of cleaved Caspase-3, cleaved PARP1, Bax, and Bcl-2 in patients with IA measured by Western blot analysis. (C) The expression of MIAT in endothelial cells after overexpressing MIAT determined by RT-qPCR. (D) Viability of endothelial cells in response to oe-MIAT detected by CCK-8 assay. (E) Flow cytometry analysis of apoptosis of endothelial cells in response to oe-MIAT. (F) The expression of apoptosis-related factors (cleaved Caspase-3, cleaved PARP1, Bax, and Bcl-2) in response to oe-MIAT 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, and the cell experiment was conducted three times independently.

FIGURE 3

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.

FIGURE 4

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.

FIGURE 5

MIAT silencing inhibits endothelial cell apoptosis and maintains endothelial cell continuity in IA rats. Rats were induced with IA model and treated with sh-MIAT or sh-NC, with the sham-operated rats as control. (A) The expression of MIAT in rats determined by RT-qPCR. (B) HE staining (× 200) for the ACA/OA branch sections of rats. (C) The representative images of the IA wall of ACA/OA branch of rats (×200) (arrows indicate the arterial wall). (D) Quantitative analysis of cell apoptosis in rats assessed 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 the Image J software. *p < 0.05. The measurement data were expressed as mean ± standard derivation. 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. The sham group: n = 12, the IA + sh-NC group: n = 12, and the IA + sh-MIAT group: n = 6.

FIGURE 6

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.

FIGURE 7

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.