Long Noncoding RNA Implicated in Cardiovascular Disease and Use Thereof

Abstract

The present invention relates to a composition for diagnosing cardio-cerebrovascular disease and a composition for preventing or treating cardio-cerebrovascular disease. The present invention may provide important clinical information that enables early establishment of treatment strategies by highly reliable prediction of not only the development of cardio-cerebrovascular diseases, including arteriosclerosis, but also the likelihood of future development of cardio-cerebrovascular diseases, based on the expression of lncRNA HSPA7. In addition, the composition for treating cardio-cerebrovascular disease according to the present invention inhibits HSPA7 expression, thereby inhibiting the migration of smooth muscle cells and decreasing the expression of inflammatory factors to block the development and progression of atherosclerotic plaques themselves, and thus it may be effectively used as a fundamental therapeutic composition that goes beyond symptomatic therapy such as administration of antithrombotic agents.

Claims

1. A method for predicting or diagnosing cardio-cerebrovascular disease comprising measuring an expression level of lncRNA HSPA7.

2. The method of claim 1, wherein measuring the expression level of lncRNA HSPA7 is carried out using a primer or a probe that binds specifically to the nucleotide sequence of SEQ ID NO: 1.

3. The method of claim 1, wherein the cardio-cerebrovascular disease is selected from the group consisting of myocardial infarction, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina, stroke, vascular stenosis, vascular restenosis, aortic aneurysms, and acute ischemic arteriovascular events.

4. A method for preventing or treating cardio-cerebrovascular disease comprising administering an inhibitor for lncRNA HSPA7 expression a subject in need thereof.

5. The method of claim 4, wherein the inhibitor for lncRNA HSPA7 expression is at least one selected from the group consisting of shRNA, siRNA, miRNA, ribozyme, PNA, and antisense oligonucleotides, which bind specifically to the nucleotide sequence of SEQ ID NO: 1.

6. The method of claim 4, wherein the inhibitor for lncRNA HSPA7 expression inhibits migration of smooth muscle cells.

7. The method of claim 4, wherein the inhibitor for lncRNA HSPA 7 expression reduces secretion of interleukin (IL)-1β and IL-6.

8. The method of claim 4, wherein the cardio-cerebrovascular disease is selected from the group consisting of myocardial infarction, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina, stroke, vascular stenosis, vascular restenosis, aortic aneurysms, and acute ischemic arteriovascular events.

9. A method for screening a composition for preventing or treating cardio-cerebrovascular disease, the method comprising steps of: (a) bringing a candidate substance into contact with a biological sample containing lncRNA HSPA 7-expressing cells; and (b) measuring an expression level of lncRNA HSPA 7 in the sample, wherein the candidate substance is determined as the composition for preventing or treating cardio-cerebrovascular disease, when the expression level of lncRNA HSPA 7 has decreased.

10. The method of claim 9, wherein the lncRNA HSPA7-expressing cells are smooth muscle cells.

11. The method of claim 9, wherein the cardio-cerebrovascular disease is selected from the group consisting of myocardial infarction, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina, stroke, vascular stenosis, vascular restenosis, aortic aneurysms, and acute ischemic arteriovascular events.

12. A composition for predicting or diagnosing cardio-cerebrovascular disease containing, as an active ingredient, an agent for measuring an expression level of lncRNA HSPA7.

13. The composition of claim 12, wherein the agent for measuring the expression level of lncRNA HSPA7 is a primer or a probe that binds specifically to the nucleotide of SEQ ID NO: 1.

14. The composition of claim 12, wherein the cardio-cerebrovascular disease is selected from the group consisting of myocardial infarction, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina, stroke, vascular stenosis, vascular restenosis, aortic aneurysms, and acute ischemic arteriovascular events.

15. A composition for preventing or treating cardio-cerebrovascular disease containing an inhibitor for lncRNA HSPA7 expression as an active ingredient.

16. The composition of claim 15, wherein the inhibitor for lncRNA HSPA7 expression is at least one selected from the group consisting of shRNA, siRNA, miRNA, ribozyme, PNA, and antisense oligonucleotides, which bind specifically to the nucleotide sequence of SEQ ID NO: 1.

17. The composition of claim 15, which inhibits migration of smooth muscle cells.

18. The composition of claim 15, which reduces secretion of interleukin (IL)-1β and IL-6.

19. The composition of claim 15, wherein the cardio-cerebrovascular disease is selected from the group consisting of myocardial infarction, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina, stroke, vascular stenosis, vascular restenosis, aortic aneurysms, and acute ischemic arteriovascular events.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIGS. 1A and 1B, together with Table 1, show that HSPA7 expression is upregulated in human atherosclerotic plaques and induced by atherogenic stimuli. FIG. 1A is a Heatmap of genes differentially expressed in human atherosclerotic plaques compared to control tissues. Table lshows a list of high-ranking lncRNAs with up- or downregulated expression in plaques. FIG. 1B shows the results of performing hierarchical clustering and multidimensional scaling of data from plaques and controls. FIG. 1C shows the results of verifying differently expressed lncRNA through RNA-sequencing. Among the three top-ranked genes, only HSPA7 showed a significant elevation of expression upon proatherogenic stimuli, particularly oxLDL. Experiments were conducted in duplicates, and data were obtained from three independent experiments.

[0057] FIGS. 2A-2E show that knockdown of HSPA7 attenuates migration and inflammatory changes in HASMC. FIGS. 2A and 2B show the results of Transwell assay performed to assess cell migration of HASMCs transfected with siHSPA7 or control siRNA for 24 hours. FIG. 2C shows the results of ELISA and qPCR analyses performed to examine the secretion patterns of IL-1β and IL-6 upon treatment with oxLDL and siHSPA7. FIG. 2D shows the results of immunofluorescence staining performed to examine the expression patterns of CD68, SM22α and calponin1 upon treatment with oxLDL and siHSPA7. FIG. 2E shows the result of qPCR performed to evaluate the effect of HSPA7 on phenotypic markers, particularly CNN1 and CD68. Data were obtained from three independent experiments.

[0058] FIGS. 3A-3F show that HSPA7 promotes inflammatory changes in HASMCs by sponging miR-223. FIG. 3A shows the results of bioinformatics analysis using miRcode, which predicted that the HSPA7 sequence contains a potential miR-223 binding site. FIG. 3B shows qPCR results indicating that FOXO1 expression was upregulated in HASMCs treated with a miR-223 inhibitor, but diminished after siHSPA7 treatment. NF-κB activity was elevated upon miR-223 inhibition, but attenuated after siHSPA7 treatment. FIG. 3C shows that, after the same treatment of HASMCs, the secretion of IL-1β and IL-6 was increased by a miR-223 inhibitor, but decreased after siHSPA7 treatment, as verified by qPCR. FIG. 3D shows immunofluorescence staining results indicating that the expression of markers of the contractile SMC phenotype was not changed by a miR-223 inhibitor, but was increased after siHSPA7 addition. FIG. 3E shows the results of verifying the above results using qPCR for TAGLN and CNN1. FIG. 3F shows that miR-223 is a target of HSPA7 in an AGO2-dependent manner. AGO2 expression was not different in HASMCs regardless of the presence of oxLDL. RIP assays showed that HSPA7 and miR-223 were more enriched in AGO2-containing miRNPs than IgG immunoprecipitates. HSPA7 had increased binding to AGO2 in the presence of oxLDL. Data were obtained from three independent experiments.

[0059] *: p<0.05, AngII: Angiotensin II, HASMC: human aortic smooth muscle cell; oxLDL: oxidized low-density lipoprotein; C: control; AGO: argonaute-2; RIP: RNA immunoprecipitation; miRNP: miRNA ribonucleoprotein; ELISA: enzyme-linked immunosorbent assay; qPCR: quantitative real-time polymerase chain reaction.

DETAILED DESCRIPTION

[0060] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for illustrating the present invention in more detail, and it will be obvious to those skilled in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

EXAMPLES

Experimental Methods

Study Subjects and Aortic Tissue Extraction

[0061] The study protocol (no. 4-2013-0688) was approved by the Institutional Review Board of Severance Hospital, Seoul, Korea. All participants provided written informed consent. Aortic samples were obtained from patients who underwent aortic graft replacement surgery for a thoracic aortic aneurysm. Samples were classified by an experienced pathologist according to the presence of atherosclerotic plaques. The lesions were classified according to the modified classification of the American Heart Association (Virmani, ATVB 2000) without any knowledge of the specimens.

RNA Sequencing and Classification of lncRNAs

[0062] RNA was extracted from cells using a Ribospin RNA Extraction Kit (GeneAll, Seoul, Korea). RNA concentration and purity were assessed using a NanoDrop ND1000 spectrophotometer. Total RNA sequencing libraries were prepared using a TruSeq RNA sample preparation kit (Illumina, San Diego, Calif., USA) according to the manufacture's instruction. The differentially expressed genes between samples with and without atherosclerotic plaques were compared using Cuffdiff. Genes with q values <0.05 and >two-fold changes were identified.

Human Aortic Smooth Muscle Cells (HASMCs) and Other Reagents

[0063] HASMCs were purchased from Lonza (Basel, Switzerland) and cultured in SMC basal growth medium (containing growth factor and supplemented with 2% fetal bovine serum and penicillin/streptomycin) at 37° C. Lipopolysaccharide (LPS) and angiotensin II (AngII) were purchased from Sigma-Aldrich (St. Louis, Mo., USA) and used for HASMC stimulation. Low-density lipoprotein (LDL) was isolated from the plasma of healthy donors using sequential ultracentrifugation. LDL was dialyzed for 24 hours at 4° C. with phosphate-buffered saline and oxidized for 24 hours using 5 μM CuSO.sub.4 at 37° C. Ethylenediaminetetraacetic acid was added to stop the reaction, and thiobarbituric acid reactive substance (TBARS) assays were used to analyze the oxidation state of LDL before each experiment.

Migration Assay

[0064] For analysis and comparison of HASMC migration, the cells were added to the upper Transwell chamber (Neuro Probe, Inc., Gaithersburg, Md., USA) in serum-free medium after transfection with siHSP A7 or control siRNA for 24 hours. The lower chamber was filled with SMC growth basal medium with fetal bovine serum. Next, LPS (10 ng/mL), oxLDL (50 μg/mL), or AngII (300 nM) was added to the upper chamber and incubated for another 24 hours. Thereafter, the cells were stained with a Diff Quik staining kit (Kobe, Japan), and those on the lower surface of the filter were photographed and counted under a fluorescence microscope. All treatments were performed in duplicate wells.

RNA Sequencing and Analysis of lncRNAs

[0065] The fragmentation step resulted in an RNA-Seq library that included inserts of approximately 100 to 400 bp. The average insert size in an Illumina TruSeq library was approximately 200 bp. cDNA fragments underwent an end repair process: the addition of a single ‘A’ base to the 3′ end and then ligation of adapters. Finally, the products were purified and enriched with polymerase chain reaction (PCR) to obtain double-stranded cDNA libraries. Libraries were quantified using KAPA Library Quantification kits for the Illumina HiSeq 2500 platform according to the protocol guide KK4855 (KAPA Biosystems, Wilmington, Mass., USA).

Enzyme-Linked Immunosorbent Assay (ELISA)

[0066] Cell culture supernatants were collected, and the amount of IL-1β or IL-6 was quantified using an ELISA kit (R&D Systems, Minneapolis, Minn., USA) according to the manufacturer's protocol. 96-well plates were coated with 1 mg/well capture antibody. The coated plates were washed twice with phosphate-buffered saline containing 0.05% Tween-20 and were exposed to biotin-conjugated secondary antibodies. The plates were read at an absorbance of 450 nm. Target proteins were analyzed according to the manufacturer's protocol.

Immunoblot Assay

[0067] Cells were harvested and lysed in cell lysis buffer containing 1M HEPES (pH 7.5), 5M NaCl, 0.5M EDTA, 1% Triton X-100 and protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentration was measured by bicinchoninic acid protein assay (Pierce Biotechnology Inc., Waltham, Mass., USA). Cell lysates (20 mg/lane) were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore, Burlington, Mass., USA). The membranes were incubated with 5% bovine serum albumin in Tween 20-containing TBS (Tris-buffered saline) solution for 1 hour at room temperature and incubated with primary antibody overnight at 4° C. The next day, the membranes were washed with TBS and incubated with horseradish peroxidase-conjugated secondary antibody at room temperature. Proteins were detected with an enhanced chemiluminescence system.

Quantitative Real-Time PCR (RT-qPCR)

[0068] RNA was extracted from cells using a Ribospin RNA Extraction Kit (GeneAll, Seoul, Korea). The integrity of the extracted RNA was analyzed with a NanoDrop and quantified using spectrophotometric absorbance at 260 nm. Next, 1 μg of RNA was synthesized into cDNA using an iScript™ cDNA Synthesis kit (Bio-Rad, Hercules, Calif., USA). RT-qPCR was performed with a SYBR Green dye system on a LightCycler 480 real-time PCR machine (Roche, Basel, Switzerland). LightCycler software was used to analyze gene expression based on cycle threshold values normalized to β-actin expression. Amplified gene expression was assessed by melting curve analysis, and no reverse transcriptase or template controls were included. Analyses were performed in duplicates.

RNA-Binding Protein Immunoprecipitation (RIP) Assay

[0069] The RIP assay was conducted using an EZ-Magna RIP kit (Merck Millipore, Burlington, Mass., USA) according to the manufacturer's instructions. HASMCs were harvested and lysed in RIP lysis buffer. Cell extracts were then incubated with RIP buffer containing magnetic beads conjugated with anti-AGO2 antibody and IgG (Merck Millipore). Immunoprecipitated RNA was isolated, and qPCR analysis was performed to detect HSPA7 and miR-223.

Statistical Analysis

[0070] All data are presented as the mean ±standard error. Analysis of variance, followed by Tukey's test, was used to compare values of continuous variables between groups with post hoc analysis. Differences were considered statistically significant when the p value was p<0.05. The software package Prism 5.0 was used for all data analyses (GraphPad Software Inc., San Diego, Calif., USA).

Experimental Results

Identification of lncRNAs Associated With Human Atherosclerosis

[0071] RNA sequencing was performed to identify lncRNAs associated with atherosclerotic plaques. A total of 380 RNAs were found to be differentially expressed between plaques and controls (FIG. 1A). Hierarchical clustering and multidimensional scaling were conducted based on fragments per kilobase of transcripts per million mapped reads values (FPKM) (fold change >2; p<0.05), and the gene expression pattern in plaques was distinct from that of the controls. Table 1 shows high-ranking lncRNAs with up- or downregulated expression in plaques.

TABLE-US-00001 TABLE 1 IncRNA Fold accession Chromosome Start Stop change ρ Up- HSPA7 1 161606291 161608217 6.13 4.50E−02 regulated TYROBP 19 35904401 35908309 2.85 2.70E−02 LIPE-AS1 19 42397148 42652355 2.79 1.50E−03 PRDM16 1 3059617 3067725 2.36 2.30E−02 LOC102724659 3 46364660 46407059 2.18 4.64E−03 LAIR1 19 54353624 54370556 2.09 2.11E−02 SLC7A7 14 22773222 22619811 2.04 3.91E−04 Down- MIR4697HG 11 133696435 133901740 2.07 1.88E−03 regulated LINC00982 1 3059617 3067725 2.37 1.00E−03 LINC00312 3 8571782 8574688 2.40 1.44E−02 NAV2-A56 11 19710934 19714672 2.86 1.00E−03

[0072] Multidimensional scaling visualized differences in gene expression between the groups (FIG. 1B). Three lncRNAs (HSPA7, LOC102724659, and LINC00982) showing significantly different expression were selected and subjected to additional experiments. When HASMCs were incubated with LPS, oxLDL, or AngII, only HSPA7 showed upregulated expression, whereas the expression of the other two lncRNAs was not substantially changed (FIG. 1C).

Knockdown of HSPA7 Attenuates Migration and Inflammatory Changes in HASMCs

[0073] To examine the effect of HSPA7 knockdown on HASMC migration, the cells were transfected with siHSPA7 or control siRNA for 24 hours and plated in the upper Transwell chamber with or without LPS, oxLDL, or AngII. After another 24 hours, analysis of the cells in the lower chamber revealed that migration of HASMCs promoted by oxLDL was significantly inhibited after siHSPA7 treatment (FIGS. 2A and 2B). After transfection with siHSPA7 or control siRNA, HASMCs were treated with or without oxLDL for 24 hours. ELISAs and qPCR showed that the oxLDL-promoted secretion and expression of IL-1β and IL6 were suppressed by siHSPA7 (FIG. 2C). Immunofluorescence staining showed that the expression of markers of the contractile SMC phenotype, SM22α, and calponin1, was decreased upon oxLDL treatment, and this change was reversed by siHSPA7. The expression of CD68, a marker of macrophage-like cells, was upregulated upon oxLDL treatment, whereas this change was partly inhibited by siHSPA7 (FIG. 2D). The effects of HSPA7 on phenotype markers, particularly CNNI and CD68, were validated using qPCR (FIG. 2E).

HSPA7 Promotes Inflammatory Changes in HASMCs by Sponging miR-223

[0074] miRcode (http://www.mircode.org/) was used to search for candidate miRNAs interacting with HSPA7, and as a result, it was shown that miR-223 had an optimal site capable of binding to the HSPA7 sequence (FIG. 3A). miRDB (http://mirdb.org/) revealed FOXO1 as a binding target of miR-223 (FIG. 3A). Furthermore, miR-223 has been reported to be associated with cardiac insufficiency and vascular inflammation. HASMCs were transfected with a miR-223 inhibitor and/or siHSPA7 or control siRNA and then treated with 50 mg/mL oxLDL for 24 hours. After 48 hours, luciferase activity was measured. qPCR showed that upregulated expression of FOXO1 by the miR-223 inhibitor was diminished after siHSPA7 treatment. (FIG. 3B) FOXO1 is a transcriptional activator of NF-κB. Although NF-κB activity was elevated due to miR-223 inhibition, this parameter was attenuated upon HSPA7 knockdown (FIG. 3D). 24 hours after the same treatment of HASMCs, the secretion of IL-1β and IL-6 was increased by the miR-223 inhibitor. Increased secretion of chemokines, particularly IL-6, was attenuated after siHSPA7 treatment, which was confirmed again by qPCR (FIG. 3C). In immunofluorescence staining, SM22α and calponin1 expression were not changed upon miR-223 inhibitor treatment, whereas the expression of these markers increased after the addition of siHSPA7 (FIG. 3D). These results were verified using qPCR, particularly for TAGLN gene (FIG. 3E).

HSPA7 Targets miR-223 in an AGO2-Dependent Manner

[0075] miRNA exists in the form of a miRNA ribonucleoprotein complex (miRNP) including AGO2, which is a key component of the RNA-induced silencing complex. To evaluate whether HSPA7 is associated with miRNA, the present inventors conducted an RIP assay in HASMCs using an AGO antibody. AGO2 expression was not different in the cells irrespective of the presence of oxLDL. RIP assays showed that HSPA7 and miR-223 were enriched in the AGO2-containing miRNPs compared to the IgG immunoprecipitates. Furthermore, HSPA7 binding to AGO2 was enhanced in the presence of oxLDL (FIG. 3F).

[0076] The features and advantages of the present invention are summarized as follows:

[0077] (a) The present invention provides a composition for diagnosing cardio-cerebrovascular disease and a composition for preventing or treating cardio-cerebrovascular disease.

[0078] (b) The present invention may provide important clinical information that enables early establishment of treatment strategies by highly reliable prediction of not only the development of cardio-cerebrovascular diseases, including arteriosclerosis, but also the likelihood of future development of cardio-cerebrovascular diseases, based on the expression of lncRNA HSPA7.

[0079] (c) In addition, the composition for treating cardio-cerebrovascular disease according to the present invention may inhibit HSPA7 expression, thereby inhibiting the migration of smooth muscle cells and decreasing the expression of inflammatory factors to block the development and progression of atherosclerotic plaques themselves, and thus it may be effectively used as a fundamental therapeutic composition that goes beyond symptomatic therapy such as administration of antithrombotic agents.

[0080] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.