CN114344468B - Use of lncRNA non-coding repressor of NFAT in vascular related diseases - Google Patents

Abstract

The invention belongs to the technical field of medical research, and particularly relates to application of an lncRNA non-coding repressor of NFAT in vascular related diseases. The invention mainly relates to: application of the Nron knock-down preparation in preparing a medicament for preventing and treating arterial vascular diseases. Use of a knock-down formulation of Nron in the preparation of an atherosclerosis-inhibiting formulation and/or a plaque-stabilizing formulation. Use of Nron in the preparation of inhibitors of NFATc3 activity and/or formulations for inhibiting NFATc3 translocation. The application of the Nron and/or the Nron expression promoter in preparing a preparation for promoting VSMCs to secrete VEGFA. Use of Nron and/or an Nron expression promoter in the preparation of a formulation for promoting atherosclerotic plaque rupture. The invention provides a new treatment scheme for some blood vessel related diseases, provides a direction for the development of new treatment products, and is expected to research related effects.

Description

Use of lncRNA non-coding repressor of NFAT in vascular related diseases

Technical Field

The invention belongs to the technical field of medical research, and particularly relates to application of an lncRNA non-coding repressor of NFAT in vascular related diseases.

Background

Atherosclerosis is a type of cell in which systemic diseases, characterized by the interplay of multiple diseases, lead to local inflammation of the arterial wall. Even with the greatly improved understanding of mechanisms that contribute to sustainable development, the overall reduction in atherosclerosis and cardiovascular mortality remains a large absolute disease burden. The major clinical consequences of atherosclerosis, such as myocardial infarction or stroke, are not a disease lumen that is progressively reduced in function, but rather, are unstable plaques due to thrombotic events associated with acute rupture or erosion of blood vessels. The occurrence and progression of atherosclerosis, the critical role of Vascular Smooth Muscle Cells (VSMCs) in cardiovascular disease, has been considered to contribute to plaque stability, as they are the major cellular component damage of the intracellular protective fibrous cap and the synthesis of extracellular matrix is an important integral part of the extracellular matrix. Thus, VSMC or its loss of function in advanced plaques has been shown to have deleterious effects, leading to thinning of the fibrous cap, necrotic core formation, and plaque erosion.

Long non-coding RNAs (lncRNAs) are a group of non-protein coding RNAs that are greater than 200 nucleotides in length. Although most studies have focused on the increasing evidence of the role of lncRNAs in cell development and differentiation, lncRNAs play an additional role in tumors in cardiovascular diseases such as atherosclerosis. Recently, an increasing number of lncRNAs have been implicated in the development of atherosclerosis in the activation of atherosclerotic cells, such as VSMC, endothelial Cells (ECs) and monocytes/macrophages. Modulation of lncRNA may provide new diagnostic and therapeutic strategies to reduce the burden of atherosclerosis. The lncRNA non-coding repressor (Nron) of NFAT is approximately 2.7KB in length, and consists of three exons, and can be alternatively spliced to give transcripts varying in size from 0.8 to 3.7 KB. It is an RNA/protein complex that acts as a cytoplasmic trap for phosphorylated proteins, the nuclear factor of activated T cells (NFAT) protein in T lymphocytes. Although initially thought to be limited to the immune system and to play a central role in the immune system in the differentiation of T cells, nron has been shown to regulate different cellular differentiation pathophysiological processes and to be involved in many other diseases. This work explored the role of Nron in the progression of atherosclerosis and revealed that previously unexpected effects of Nron in smooth muscle function and function contribute to modulation of plaque stability in plaque angiogenesis.

Disclosure of Invention

In view of the above problems, the present invention provides the use of the lncRNA non-coding repressor of NFAT in vascular-related diseases, mainly for some vascular-related disorders, and for some of the adaptation of the regulatory mechanisms.

In order to solve the problems, the invention adopts the following technical scheme:

application of the Nron knock-down preparation in preparing a medicament for preventing and treating arterial vascular diseases.

In some forms, the arterial vascular disease comprises acute coronary syndrome; the acute coronary syndrome comprises unstable angina, non-ST elevation myocardial infarction and ST elevation myocardial infarction.

Use of a knock-down formulation of Nron in the preparation of an atherosclerosis-inhibiting formulation and/or a plaque-stabilizing formulation.

Use of Nron in the preparation of inhibitors of NFATc3 activity and/or formulations for inhibiting NFATc3 translocation.

In some forms, the NFATc3 is derived from VSMCs cytoplasm.

The Nron and/or the Nron expression promoter is applied to the preparation of a preparation for promoting VSMCs to secrete VEGFA.

Use of Nron and/or an Nron expression promoter in the preparation of a formulation for promoting atherosclerotic plaque rupture.

Use of Nron and/or an Nron expression promoter in the preparation of a preparation for promoting angiogenesis in atherosclerotic plaques.

In some forms, the Nron is an overexpressed Nron.

Formulations for inducing NFATc3 translocation, including Nron knock-down formulations, ox-LDL pretreatment formulations.

The beneficial effects of the invention are:

provides a new treatment scheme for some blood vessel related diseases, provides a direction for the development of new treatment products, and is expected to research related effects. And regulating reagents required by biological model making are also provided, so that some pathological conditions and the like can be better simulated in vitro.

Drawings

FIGS. 1A-B show down-regulation of Nron expression in human and mouse atherosclerotic lesions;

FIGS. 2A-D show that Nron overexpression induces highly characteristic structurally more vulnerable plaques;

FIGS. 3A-D show that knocking down Nron inhibits the development of atherosclerosis and increases the stability of atherosclerotic plaques;

A-G in FIG. 4 show that Nron binds to NFATc3 in VSMC;

FIGS. 5A-D show NFATc3 activation and translocation into the nucleus of the cell in atherosclerotic lesion VSMC;

FIGS. 6A-D show that Nron knockdown in combination with ox-LDL treatment induces translocation of nuclear NAFTc3 in VSMC;

FIGS. 7A-F show that the effects of Nron knockdown on cell proliferation, migration, and apoptosis VSMC is dependent on NFATc3 activation;

FIGS. 8A-I show that Nron contributes to neovascularization of atherosclerotic plaques;

FIG. 9 is a primer sequence;

FIG. 10 is a graph of body weight and blood lipid profiles unaffected by Nron gene transfer;

FIG. 11 is a graph of a decreased rate of weight gain, decreased plasma triglyceride levels, and decreased cholesterol levels compared to the control group in Nron KD mice after 4 months on an atherogenic diet;

FIG. S1: in ApoE ^-/- Detection of adenovirus and adeno-associated virus infection efficiency in mice. A. 6 week old Male ApoE ^-/- Or wild type mice were fed Western Diet (WD) or Standard Control Diet (SCD) for 16 weeks. Relative expression of Nron at different time points in the aorta. B. Nron expression in aorta after 1,2,3 weeks of adenovirus administration (Ad-SM 22-EV or Ad-SM 22-Nron), respectively. C. And (5) animal experiments. 6 week Male ApoE ^-/- Mice were fed with WD for 16 weeks. Mice were injected intravenously with adenovirus (Ad-SM 22-EV or Ad-SM 22-Nron) every two weeks for the next 8 weeks. D. Expression of Nron in aorta 3 weeks after adeno-associated virus infection (rAAV 2/8-shNC or rAAV 2/8-shNron). E. Expression of Nron in aorta and heart 3 weeks after adeno-associated virus (rAAV 2/9-shNC or rAAV 2/9-shNron) infection. F. And (5) animal experiments. 6 week Male ApoE ^-/- Mice were fed with WD for 16 weeks. Mice received one intravenous injection of adeno-associated virus 2/8 (rAAV shNC or rAAV shNron) 4 weeks after feeding. Data are expressed as mean ± standard error (n =6 per group). Graph A was subjected to Student-t test using Dunnett-t test, and data in panels B, D, and E. * Number denotes P<0.05；

FIG. S2: nron does not bind to NFATc1, NFATc2 and NFATc4 in VSMC. Analyzing binding of NFATc1, NFATc2 and NFATc4 and biotinylated Nron in VSMC by using western immunoblotting;

FIG. S3: localization of the NFAT family in normal blood vessels and atherosclerotic lesion areas. Immunofluorescence assay C57BL/6 mice (Normal) and ApoE ^-/- NFATc1, NFATc2 and NFATc4 in the aortic sinus of mice (plaques). Co-localization analysis: sections were co-stained for NFAT family (green) and alpha-SMA (red; smooth muscle cell marker). DAPI for nuclear staining (blue);

FIG. S4: NFATc3 of human atherosclerotic lesion areas translocates into the nucleus of VSMC. A. Immunofluorescence measures NFATc3 expression in normal vascular and carotid atherosclerotic lesion regions in humans. For co-localization analysis, sections were analyzed for NFATc3 (green) and α -SMA (red; smooth muscle cell marker) co-staining. DAPI was used for nuclear staining (blue). Arrows indicate nuclear NFATc3 and α -SMA staining double positive cells. B. Percentage of VSMCs that are nuclear NFATc3 positive;

FIG. S5: nron regulates expression of VEGFA in VSMC. A and B, T/G HA-VSMC cultured in vitro was transfected with NRON expression plasmid or NRON siRNA. mRNA (A) and protein (B) levels of VEGFA were determined. Data represent mean ± sem of three independent experiments. * Indicates P compared with EV group or disturbed siRNA group<0.05. Statistical analysis used Student-t test. C and D,6 week Male ApoE ^-/- The mice were fed a western diet for 16 weeks. Mice were injected with adenovirus (Ad-SM 22-EV or Ad-SM 22-Nron) every two weeks for the next eight weeks. Aortic sinus transection was immunostained with VEGFA antibody (C) and the positive stained area was quantified as a percentage of total plaque stained area (D). Data are expressed as mean ± standard error (n =8 per group). Statistical analysis was performed using Student-t test. * Indicates P in comparison with Ad-SM22-EV group<0.05。

Detailed Description

Definitions of partial nouns

The term "knock-down": the gene expression is reduced (including knockout), and the purpose is to reduce the expression of the corresponding gene as required, but the degree of reduction is not limited.

The term "overexpresses Nron": the expression "over-expression" is relative, but not limited to a specific degree, and any expression degree of Nron which can enable the Nron to have related functions shall belong to the over-expression of the invention.

The term "Nron expression promoter": agents that promote the expression of Nron, both now known and later developed, are intended to be within the scope of the present invention, provided they are used for purposes related to the present invention. Wherein promotion is a relative expression, without limiting its extent, primarily acting in a positive regulatory role on the expression of Nron.

The term "control": refers to the medical management of a patient with the intent to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed to the amelioration of a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed to the removal of the cause of the associated disease, pathological condition, or disorder. Moreover, the term also includes palliative treatments, i.e., treatments designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder; prophylactic treatment, i.e. treatment aimed at minimising or partially or completely inhibiting the development of the relevant disease, pathological condition or disorder; and supportive treatment, i.e. treatment for supplementing another specific therapy directed to an improvement in the relevant disease, pathological condition or disorder. Meanwhile, in commercial activities, if a product containing the same components as the present invention is produced, the product specification does not describe the same similar uses as the present invention, but it should be regarded as an application when it gives an indication that the corresponding product is used for achieving the same similar purpose as the present invention.

The definitions of the terms used herein are to be construed as being given their ordinary meaning in the art unless otherwise indicated. In the absence of counterexamples, no diminutive explanation is made, i.e., except for the case where evidence of counterexamples exists, the rest should fall within the scope.

In a first aspect of this section is described the use of agents for knock-down of Nron expression as a medicament in the treatment of disease：

First, the application of the knock-down preparation of Nron in preparing the medicine for preventing and treating the artery disease.

In the aforementioned diseases, the arterial vascular disease includes acute coronary syndrome; the acute coronary syndrome comprises unstable angina, non-ST elevation myocardial infarction and ST elevation myocardial infarction. Of course, the foregoing is only a few of them, and other similar mechanisms should be equally within the scope of the present invention.

Secondly, the application of the knock-down preparation of the Nron in preparing an atherosclerosis inhibiting preparation and/or a plaque stabilizing preparation. By knocking down the Nron expression, the inhibition of the Nron expression realizes the inhibition of the further development of atherosclerosis and the improvement of the plaque stability.

In a second aspect of this section, certain uses of Nron and its expression promoter are described：

First, the use of Nron in the preparation of inhibitors of NFATc3 activity and/or formulations for inhibiting NFATc3 translocation and/or formulations for inducing NFATc3 translocation. Nron has a function of binding to NFATc3 to inhibit its activity.

In some embodiments, the Nron is an overexpressed Nron that can produce a better effect.

In other embodiments, the NFATc3 is derived from VSMCs cytoplasm, and modulating the VSMCs is achieved by modulating NFATc 3.

One form of the aforementioned modulation is the use of Nron and/or Nron expression promoters in the preparation of a formulation that promotes secretion of VEGFA from VSMCs.

Secondly, the application of the Nron and/or the Nron expression promoter in preparing a preparation for promoting the rupture of atherosclerotic plaques. In some commercial experiments, the corresponding mechanisms can be adjusted by the Nron and/or the Nron expression promoter, so that some biological environments are simulated, and some biological models are prepared. For example, the extent of atherosclerotic plaque rupture in an organism or model is modulated by Nron (a Nron expression promoter) to simulate different conditions.

Thirdly, the Nron and/or the Nron expression promoter is applied to the preparation of the preparation for promoting the angiogenesis of the atherosclerotic plaque. The simulation of some research project models is realized by simulating the development of a strain block by promoting the formation of new blood vessels of atherosclerotic plaques.

A third aspect of this section introduces a regulatory formulation related to NFATc3：

Formulations that induce NFATc3 translocation, including Nron knock-down formulations, ox-LDL pretreatment formulations. The formulation comprises at least an ox-LDL pretreatment formulation and a Nron knock-down formulation, wherein the ox-LDL pretreatment formulation acts as a pretreatment agent and is then modified by the Nron knock-down formulation. The specific pretreatment principle adopts the prior art in some modes, so that the corresponding treatment effect can be realized, and meanwhile, the specific proportioning scheme and the preparation form can be adjusted according to the needs.

It should also be noted that whether the Nron knock-down formulation, ox-LDL pretreatment formulation are combined into one agent for use, or combined in two separate packages in one product, the product should be in the conditioning formulation of the present invention.

The fourth aspect of this section is presented in connection with specific research projects：

Materials and methods

Materials: all human studies and animal experiments were in compliance with the National Institute of Health (NIH) guidelines and were approved by the university of science and technology in china in conjunction with the ethical committee of hospitals. According to the declaration of helsinki, written informed consent was obtained from all participants.

The method comprises the following steps: human atherosclerotic tissue: 8 patients with atherosclerosis lesions and 10 patients with coronary artery bypass surgery were collected as non-atherosclerotic control arteries.

Preparing recombinant adenovirus and adeno-associated virus: adenovirus SM22 alpha promoter (Ad-SM 22-Nron) carrying control GFP (Ad-SM 22-EV) or Nron cDNA for inducing Overexpression (OE) Nron in ApoE ^-/- Expression in mouse VSMC. The recombinant adeno-associated virus 2/8[ rAAV2/8 ], AAV2 Inverted Terminal Repeat (ITR) DNA binds to AAV serotype 8 to the capsid]Carrying scramble shRNA or Nron shRNA was used to knock down Nron in ApoE ^-/- Expression in mice. All recombinant adenoviruses and AAV are supplied by GeneChem Technologies.

Animal study: 6-week-old Male C57BL/6 mice and ApoE ^-/- Mice were purchased from Beijing university (Beijing, china). All mice were housed in a light-controlled facility at appropriate temperature and humidity, with water ad libitum. Mice were fed with Standard Control Diet (SCD) or Western Diet (WD) for 16 weeks, respectively. ApoE ^-/- Mice were given an intravenous injection of adenovirus (Ad-SM 22-EV or Ad-SM22-Nron; n =12 per group) every two weeks 8 weeks prior to sacrifice, or a single intravenous injection of adeno-associated virus 2/8 (rAAV shNC or rAAV shNron; n =12 per group) after four weeks on western diet.

Histological analysis and quantification of atherosclerotic lesions: mice were fasted for 4 hours and then anesthetized. Thoracic-abdominal aorta for fat accumulationen faceOil red O staining method. Briefly, the mouse aorta was dissected longitudinally using a very fine Vanna microdissection muscle and pinned flat to black with a stainless steel pin 0.2mm in diameterOn the surface of the wax. The fixed aorta was stained with oil red O and the images were taken with a standard image digital camera. Microscopic evaluation of aortic sinus lesions: hearts were fixed in 4% paraformaldehyde, cryopreserved in 15% sucrose, followed by 30% sucrose. After implantation of OCT compound (Sakura Finetek, tollens, california), the heart was cryosectioned and 6 μm sections were collected at 80 μm intervals from 100 μm from the appearance of the aortic valve. Aortic sinus section oil red O staining showed lipid accumulation, hematoxylin-eosin (HE) staining showed morphology, collagen content was measured by Masson trichrome staining (Masson), and elastic fiber was measured by elastic van Gieson staining (EVG). Immunohistochemistry measures the relative amounts of macrophages and smooth muscle cells.

Freezing and continuously slicing: 0.3% in PBS ₂ O ₂ Treatment to block endogenous peroxidase activity, followed by blocking in 4-vol bsa (sigma). A first antibody: f4/80 (ab 100790, abcam), α -SMA (ab 7817, abcam) or VEGFA (ab 1316, abcam). All sections were stained with biotin secondary antibody and detected using ABC reagent (Vector Laboratories). Images were collected by light microscopy and quantitative morphometric measurements were performed with Image Pro Plus. The lesion area was determined by calculating the average lesion area of 4 slices at 80 μm intervals. To assess the relative amounts of the staining components, the percentage of total plaque area was determined for blue (Masson stained collagen) and DAB stained (immunohistochemistry) positive areas. The fiber cap area is quantified as a percentage of the total plaque area. The fibrous cap is defined as the VSMC and proteoglycan-rich region, overlying the cholesterol-rich, matrix-deficient and acellular regions of the necrotic core. The elastic panel failure of the middle membrane was evaluated by quantifying the breakage (i.e., discontinuity or breakage) of the elastic fibers. To detect intimal capillaries, sections of sinus aortic with substantial lesions were incubated with anti-cd 31 antibody (ab 182981, abcam). Luminal and adventitial capillary endothelium positive staining served as a control. Intimal vessels were identified under a high power microscope (x 400) and counted when endothelial nuclei and lumens were found and vessels were also observed in adjacent sections.

RNA fluorescence in situ hybridization: tissue sections were fixed in 4% paraformaldehyde for 30 minutes and infiltrated with 0.1% triton. Cells were then washed three times with Phosphate Buffered Saline (PBS) and treated with prehybridization buffer (2 x saline sodium citrate, 10% formamide). The Nron (Exiqon) FISH (fluorescence in situ hybridization) probe was resuspended in hybridization buffer (2 × saline sodium citrate, 10% formamide, 10% dextran sulfate) to a final concentration of 250 nM/probe set. Hybridization was carried out for 16 h in a 37 ℃ humidification chamber. After incubation of the Nron probe, the cells were washed three times with PBS, treated with α -SMA antibody (ab 7817, abcam) for 2 hours at 37 ℃, washed three times with PBS and treated with fluorescently labeled secondary antibody for 1 hour at 37 ℃. The process continues through several rounds of washing (including the optional 4', 6-diamino-2-phenylindole staining step) and finally the coverslip is mounted to a microscope slide with a fade-resistant mounting medium.

Immunofluorescence: immunofluorescence studies of human carotid and mouse aortic sinus sections. Briefly, for double staining, sections were incubated with anti- α -SMA antibodies (ab 7817, abcam), and anti-NFATc 1 antibodies (ab 25916, abcam), anti-NFATc 2 antibodies (sc-7296, santa Cruz), anti-NFATc 3 antibodies (sc-8405, santa Cruz) OR anti-NFATc 4 antibodies (ab 99431, abcam) at 4 ℃ overnight, followed by incubation with AlexaFluor 568 (red) and AlexaFluor 488 (green) (molecular probes, eugene, OR) bound secondary antibodies for 30 minutes. Single and pooled images of the signal antigen were detected using fluorescence microscopy (olympus, japan) and Axiovision 4.8 software.

RNA extraction and qRT-PCR: total RNA was extracted from cells or tissues using TRIzol reagent (D9108A, takara Bio). RNA was reverse transcribed using RNA PCR kit (RR 036A, takara Bio). Quantitative polymerase chain reaction (qPCR) amplification using ABI PRISM 7900 sequence detector system (applied biosystem, foster, ca) the use followed the manufacturer's instructions. Relative gene expression (normalization of endogenous control gene β -actin) was calculated using the Δ Ct method. The primer sequences are shown in FIG. 9.

Protein immunoblotting: cells were harvested at the indicated time points and homogenized in cold suspension buffer (Sigma-Aldrich) to which protease inhibitor cocktail was added. Protein concentration was determined using BCA protein assay kit (Thermo). Equivalent amounts of protein were separated by SDS polyacrylamide gel and immunoblotted with anti-NFATc 3 antibody (sc-8405, santa Cruz) or anti-VEGFA antibody (ab 1316, abcam). The membrane was incubated with peroxidase-conjugated secondary antibody, and specific bands were generated and detected using the Bio-Rad imaging System (Hercules, calif.).

RNA pull down and mass spectrometry: biotin-labeled RNA was transcribed in vitro using Biotin RNA-labeled Mix and T7 RNA polymerase (Ambion) and purified by DNA digestion on an RNeasy Mini Kit (QIAGEN) column. Biotinylated Nron or antisense Nron were incubated with cell lysates (containing Rnasin) overnight at 4 ℃. The interacting complexes were purified with streptavidin beads at room temperature for 3 hours and visualized by silver staining (Pierce silver stain kit, thermo Scientific). In mass spectrometry, experimental group-specific bands were extracted for further analysis.

RNA immunoprecipitation: cells were treated with 0.3% formaldehyde for 10 min at 37 ℃. Glycine was added to a final concentration of 0.125M for 5 minutes at room temperature. The cells were then collected after washing twice with PBS. The cells were resuspended in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.5mM DTT and 1mM PMSF mixture) and vortexed for 30 minutes on ice. NFATc3 or IgG antibody was added and incubated overnight at 4 ℃. The RNA/protein complex was recovered with protein G magnetic beads and washed several times with PBS. RNA was recovered using Trizol and analyzed by RT-PCR.

VSMC proliferation assay: VSMC proliferation was detected using EdU (5-ethynyl-20-deoxyuridine) (Cell Light EdU Apollo 567 in vitro kit (RiboBio, guangzhou, china)) method. VSMC were seeded in six-well plates. After the designated treatment, the medium was replaced with DMEM containing 50. Mu.M EdU and cultured for 2 hours. Cells were fixed in PBS containing 4% paraformaldehyde for 30 minutes and then stained according to the manufacturer's instructions. Five randomly selected areas (magnification, × 200) were counted for EdU positive cells.

VSMC migration analysis: using Transwell thinThe cell culture chamber measures SMC migration. VSMC (5X 10 per well) ⁴ Individual cells) were suspended in 100 μ l DMEM and added to the upper chamber. The lower compartment was supplemented with DMEM with 10% FBS to induce migration. After 5 hours of incubation, cells on both sides of the membrane were fixed with 1% toluidine blue (Sigma-Aldrich) and stained. Cells on top of the membrane were removed with a cotton swab. Randomly selected 5 high resolution (200X) fields, and counted the average number of cells under the membrane.

VSMC Tunel analysis: tunel assay was performed using Promega (Madison, wis.) kit. VSMC was performed at 2X 10 on glass coverslips of 12 well plates ⁵ Density culture of cells/well. After ox-LDL and siRNA treatment, VSMCs were washed with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. The fixed cells were then incubated on ice for 2 minutes in a permeation solution (0.1% Triton X-100 in 0.1% sodium citrate). Slides were rinsed with PBS before adding rTdT incubation. After incubation for 60 min at 37 ℃, nuclei were counterstained with DAPI, rinsing 3 times with 2 × SSC. 5 fields (. Times.200) were randomly selected to count the number of tunel-positive cells.

Test tube formation test: the test tube formation assay was performed as described previously. Mouse Aortic Endothelial Cells (MAECs) at 4X 10 ⁴ Speed/well plating in 96-well plates, plates were coated with ECMatrix (BD bioscience). Cells VSMC were then treated with Nron OE medium alone or in combination with VEGFR2 siRNA. Manifold length and branch points were calculated and analyzed using the angiogenisis Analyzer tool of imagesoftware.

And (3) cell culture: primary VSMCs were obtained from aortas of 6-8 week old C57 male mice by collagenase-elastase digestion as follows: aorta was excised, washed with PBS, incubated in DMEM containing 1 mg/mL collagenase type II (Worthington Biochemical Co.) for 10-15 minutes, then the adventitia was dissected under a microscope, minced with scissors and digested with collagenase 0.5 mg/mL (Sigma type I, C-0130) and elastase 0.125 mg/mL (Sigma type III, E-0127) in serum-free DMEM at 37 ℃ until the majority of the cell suspension. The cell suspension was recovered, centrifuged at 400g for 5min, resuspended in DMEM (Invitrogen) containing 20% fetal bovine serum (FBS, hyClone) and 2% penicillin streptomycin, and plated. Cells from passage 3 to passage 5 were used for the experiments.

Statistical analysis: statistical analysis was performed using GraphPad Prism software (GraphPad software inc., la Jolla, CA). The grayscale of the Western blot Image was quantified using Image J Software. Values are expressed as mean ± sem of at least three independent experiments. The statistical significance of the differences between the two groups was assessed by student tests. For the multiple groups, significance was assessed using one-way ANOVA with Bonferroni test (homogeneity of variance) or Tamhanes s T2 test (heterogeneity of variance). P < 0.05 is statistically significant. Random and blind methods are used where possible. The determination of the size of the cohort of animals was based on previous similar studies.

As a result, the

Nron expression down-regulated in human and mouse atherosclerotic lesions: to elucidate the role of Nron in atherogenesis, its expression in human carotid atherosclerotic plaques and mammary arteries (normal blood vessels) was first examined. Fluorescence In Situ Hybridization (FISH) coupled with immunostaining indicated that Nron is abundantly expressed in healthy vascular SMCs and co-localizes with α -SMA; however, in the intimal-proliferating SMCs of atherosclerotic lesions, nron expression was barely detectable (fig. 1A). Notably, the fluorescence signal of Nron, albeit relatively weak, was observed in the medium of the diseased vessel (fig. 1A). Further detecting Nron in ApoE ^-/- Expression of mouse aortic sinus (fig. 1B). Accordingly, nron is highly expressed in normal blood vessels and hardly expressed in atherosclerotic plaques. In addition, it was observed that the Nron signal in the myocardium was weak. The dynamic changes of Nron in the progression of atherosclerosis were next analyzed. ApoE ^-/- Aortic Nron levels in mice declined with age and were more pronounced when fed Western Diet (WD), while Nron levels remained unchanged in wild-type C57/BL6 mice on either Standard Control Diet (SCD) or WD (fig. S1A). These results indicate that the expression of Nron is closely related to the severity of the disease, regardless of age or diet.

Overexpression of Nron in VSMC induces highly characteristic structures of more vulnerable plaques: the reduction of Nron in atherosclerosis prompted the study of NronAnd atherosclerosis. ApoE was treated with an adenovirus vector with SM22 alpha promoter-driven Nron cDNA (Ad-SM 22-Nron) ^-/- Mice, induced overexpression of Nron (OE) in VSMCs. Experiments showed that transgene expression was evident in the aorta 1 week after intravenous injection of recombinant adenovirus vectors, followed by a gradual decline to basal levels (B in fig. S1). Given the short term expression of the transgene, adenovirus was injected every 2 weeks for 8 weeks prior to mouse sacrifice, as shown in figure S1C.

Body weight and blood lipid profiles were not affected by Nron gene transfer (see figure 10).

The lesion areas were not significantly different, as shown below: thoraco-abdominal aorta and aortic sinus oil red O staining was determined between Ad-SM22-Nron treated mice and control group (Ad-SM 22-EV) (fig. 2A and B). The atherosclerotic plaques were further studied for complex (F4/80 positive) infiltration in macrophages, smooth muscle cell (α -SMA positive) proliferation, collagen (masson trichrome stain) content, fibrous cap and necrotic core region, and for assessment of elastic layer (Verhoeff's Van gieseon stain) destruction (C and D in fig. 2). The Nron OE group mice developed VSMC-deficient fibrous caps with thinner plaques, loose collagen, and matrix-covered lipid-rich necrotic cores compared to the control group. In addition, macrophage content was increased and degradation of elastic fibers in the injured region of the Nron OE mice was more pronounced. These results indicate that the Nron OE induces a highly characteristic more vulnerable plaque structure.

Knock-down of the Nron gene inhibits the development of atherosclerosis and increases the stability of atherosclerotic plaques: to achieve higher Knockdown (KD) efficiency in the aorta, recombinant adeno-associated virus (rAAV) vectors carrying a Nron-shRNA (rAAV-shNron) were synthesized and used. The infection efficiency of different vector types is detected, and the intravenous injection of rAAV2/8 effectively inhibits ApoE ^-/- Expression of mouse aortic Nron (D and E in fig. S1). Since rAAV mediates long-term expression of transgenes in vivo, apoE ^-/- Mice were given rAAV2/8 at 10 weeks and were fed continuously with WD for 3 months prior to sacrifice as shown in figure S1F.

Oil red O staining showed a significant decrease (29.4%) in the thoracic and abdominal aortic lipid accumulation in the Nron KD mice compared to the control group (rAAV-shNC treatment) (fig. 3A). Lipid accumulation in the aortic sinus was also consistently reduced by 31.6% (fig. 3B). Next, plaques were examined for plaque vulnerability morphological features (fig. 3C and D), and as a result, nron KD mice were found to have significantly increased collagen content and fibrous cap area and a higher proportion of smooth muscle cells compared to the control group. In addition, macrophage content and necrotic core regions within foci were significantly reduced in Nron KD mice. These results indicate that rAAV2/8 mediated Nron KD not only reduced plaque area, but also increased plaque stability. Notably, nron KD mice fed the atherogenic diet for 4 months had a slower rate of weight gain, decreased plasma triglyceride levels, and decreased cholesterol levels, but had no statistical significance, compared to the control group, see figure 11.

Nron in VSMC binds to NFATc3: VSMC, an important component of atherosclerotic lesions, play an important role in the development of disease in atherosclerosis. Moderate proliferation of VSMCs favors stable late lesions of the plaque and increased apoptosis rates leading to plaque rupture. Vascular smooth muscle cells play an important role in the development of disease as an important component of atherosclerotic lesions. Moderate proliferation favors the stabilization of late-stage lesion plaques, and increased apoptosis rate leads to plaque rupture 3,4. Fluorescent staining of aortic sinus lesions showed Nron OE ApoE compared to control group ^-/- Mice had decreased proliferative (ki 67 positive) VSMCs (fig. 4A and B) and increased apoptotic (tune positive) VSMCs (fig. 4C and D). In order to understand the molecular mechanism of Nron in regulating plaque stability and VSMC function, RNA pull down analysis and mass spectrometry were performed, and it was found that interleukin enhanced binding factor 2 (ILF 2), a subunit of NFAT, was associated with Nron in VSMCs (fig. 4E). The independent immunoblot assay further identified the NFAT 3 protein, but not other NFAT proteins, as a specific nron binding protein (fig. 4F and S2). This finding was confirmed by RNA Immunoprecipitation (RIP) assay (fig. 4G).

In atherosclerotic lesions, NFATc3 is activated and translocated into the nucleus of VSMC: although initially considered limited toModulating immune function, since then, different NFAT family members have been shown to modulate different behavioral pathophysiological processes and participate in various diseases. The expression of NFAT protein in mouse normal and atherosclerotic blood vessels was next examined. Immunofluorescent staining showed (fig. 5A and fig. S3) that NFATc2, NFATc3 and NFATc4 are abundantly expressed in normal blood vessels, all localized in the cytoplasm of VSMCs, whereas NFATc1 is mainly localized in myocardium. Notably, in atherosclerotic lesions, NFATc3 is abundantly expressed in the fibrous cap and migrates from the cytoplasm to the nucleus, while NFATc2 and NFATc4 are both retained in the cytoplasm. Consistent results were observed in atherosclerotic plaques in humans (fig. S4A and B). The protein level of NFATc3 in aorta of animal models was further measured using western blot. NFATc3 is expressed in the cytoplasm and hardly in the nucleus of control C57 mice, whereas WD-fed ApoE ^-/- The aorta of the mice developed progressive atherosclerotic lesions showing an increase in the active form of NFATc3 (shown by faster migration within the gel). More significantly, the Nron OE in VSMCs effectively inhibited ApoE ^-/- Nuclear translocation of mouse aortic NFATc3 (B and D in fig. 5).

Nron knockdown in combination with ox-LDL treatment induced nuclear translocation of NFATc3 in vsmc: since Nron has been shown to bind to NFATc3 in VSMCs and its expression is reduced in plaques, it is speculated that direct regulation of NFATc3 activity by Nron is reasonable. Transfection of primary VSMCs with siRNA induced Nron KD in vitro and treated with ox-LDL. Neither ox-LDL nor Nron KD induced NFATc3 nuclear translocation, however, when used together, a large accumulation of NFATc3 in the nucleus was clearly observed (FIG. 6A). Western blot analysis further confirmed this result. NFATc3 is phosphorylated and retained in the cytoplasm, and Nron KD in combination with ox-LDL leads to dephosphorylation and nuclear translocation of NFATc3 (fig. 6B and C). At the same time, the activity of the nfat-driven luciferase reporter in the Nron KD VSMCs was also increased (fig. 6D). These results indicate that the reduction of Nron expression and ox-LDL excitation may be a cause of NFATc3 activation, both of which are indispensable.

The effect of Nron KD on VSMC cell proliferation, migration and apoptosis was dependent on NFATc3: due to the fact thatNron regulation of ApoE ^-/- Proliferation and apoptosis of VSMCs in mouse atherosclerotic plaques, whether this effect is mediated by the interaction of Nron with NFATc3 was examined. Treatment with Nron KD in combination with ox-LDL promoted proliferation of VSMCs in vitro as measured by EdU, and this effect disappeared after NFATc3 knockdown (fig. 7A and B). In addition, the acceleration of VSMCs migration by Nron KD was also completely blocked by the combined knock-down of NFATc3 (fig. 7C and D). Next, VSMCs were analyzed for apoptosis using the TUNEL method. Nron KD caused a significant decrease in Tunel-positive VSMCs, and this anti-apoptotic effect was abolished by NFATc3 knockdown (fig. 7E and F). Taken together, these data indicate that the pro-proliferative and anti-apoptotic effects of Nron KD are dependent on nuclear translocation and activation of NFATc 3.

Nron contributes to neovascularization within the atherosclerotic intima: the neovasculature within the plaque has been considered to be a possible cause of vessel rupture-the process by which asymptomatic fibroatheromatous plaque becomes diseased. Nron OE ApoE was observed compared to the control group ^-/- The number of CD 31-positive microangioses in aortic sinus plaques increased in mice (fig. 8A). To determine the molecular and cellular mechanisms responsible for the action of Nron in angiogenesis, the expression of angiogenic factors secreted by VSMCs in culture was examined. The Nron OE resulted in elevated primary mouse VSMCs Vegfa and Vegfc mRNA levels, whereas the Nron KD reversed (fig. 8B and C). Furthermore, western blot analysis found that protein levels of VEGFA were constantly changing (fig. 8D and E). Similar results were also observed in the human aortic smooth muscle cell line T/G HA-VSMC (A and B in FIG. S5). Since Nron had no effect on the activity of nfat-driven luciferase reporter (fig. 6D, fig. 8F, G). The regulation of VEGFA expression by Nron is independent of NFATc3 activation. The immunohistochemical detection results showed that Nron OE ApoE ^-/- VEGFA expression was increased in mouse atherosclerotic plaques (B and C in fig. 5). Next, a test tube formation experiment was performed with Mouse Aortic Endothelial Cells (MAECs). Cultured MAECs were treated with conditioned media from VSMCs and total branch length and branch point were measured to assess EC angiogenesis. The results showed that both branch length and branch number were significantly higher for the Nron OE VSMCs treated group than the control group. However, in MAECs VEGFR2 (the major part of VEGFA) is inhibitedReceptors) blocked the pro-angiogenic effect of Nron OE VSMCs (fig. 8H and I). These findings support the concept that VSMCs-derived VEGFA regulated by Nron can be involved in the angiogenic process as a paracrine factor.

Discussion of the related Art

There is a large body of evidence that lncRNA is associated with atherosclerosis. In this study, lncRNA Nron was found to be specifically expressed in VSMCs of atherosclerotic plaque samples decreased. This results in activation of NFATc3 to promote VSMCs proliferation and inhibit apoptosis, and results in inhibition of plaque angiogenesis independent of NFATc 3. All of these effects induce a highly characteristic, more stable atherosclerotic plaque structure. Studies revealing a role for Nron in VSMC function and the development of atherosclerosis.

Nron was identified as a non-coding RNA repressor of the transcription factor NFAT. There are five members of the NFAT family, NFAT1 through NFAT5, with the exception of NFAT5, which are calcium-regulated transcription factors. In resting T cells, NFAT is phosphorylated by the Nron/protein complex and remains in the cytoplasm. Following T cell antigen receptor (TCR) signaling, NFAT proteins are dephosphorylated and transferred to the nucleus to activate gene expression. Previous studies found that knock-down of Nron leads to nuclear translocation of NFATc1 in immune cells. In this study, a significant reduction in Nron expression with activation of NFATc3 was observed in atherosclerotic lesions, providing evidence that Nron is specifically associated with NFATc3 and regulates its nuclear transport in vsmc. However, knock-down of Nron induced NFATc3 translocation only in the following cases: pretreatment with ox-LDL. These results indicate that the reduction of Nron expression, together with other pathogenic factors such as ox-LDL, leads to activation of NFATc3 in atherosclerotic lesions. Notably, no activation of other NFAT members in the plaques was observed, suggesting functional heterogeneity of the Nron and NFAT families in different cell types and diseases.

Vascular smooth muscle cells play a key role in the development of atherosclerosis. Generally speaking, intimal VSMCs are thought to play a beneficial role by promoting a fibrous cap, thereby stabilizing atherosclerotic plaques. The loss of vascular smooth muscle cells by apoptosis results in thinning of the fibrous cap and promotes the formation of necrotic cores. The role of NFAT in vsmc migration and proliferation has been proposed in previous articles. Here, it was found that the combined knock-down of NFATc3 completely abolished the proliferative and anti-apoptotic effects of Nron KD in ox-LDL induced VSMCs. This provides direct evidence for the involvement of the Nron-NFATc3 interaction in VSMC behavioral regulation.

Microvessels rarely occur in healthy intima, but are often observed in pathological conditions such as atherosclerosis. Intra-plaque angiogenesis is an important trigger for plaque progression and is also associated with plaque erosion, bleeding and coronary thrombosis. Since newly formed blood vessels are immature to grow into the plaque, they are inherently leaky, allowing inflammatory cell infiltration and blood components to flow in (including red blood cells and platelets), making them important hallmarks of plaque instability. VEGFA is released by a variety of cells, including SMCs, ECs and macrophages, and is a potent promoter of neovascularisation. VEGFA contributes to the maintenance and regeneration of arterial endothelium under physiological conditions, however, the elevation of VEGFA in advanced atherosclerotic plaques clearly leads to high permeability and leakage of microvessels that fail to mature normally. In this study, more microvessels were observed in the Nron OE mouse plaques and evidence that Nron promoted VEGFA expression in vsmc was provided. VEGFA secreted by vascular endothelial cells is used as a paracrine factor and is involved in angiogenesis of intimal EC of atherosclerotic plaques. This may be one of the important factors causing plaque instability. Notably, the regulation of VEGFA expression by Nron is independent of NFATc 3. More research is needed to identify novel signaling proteins that may mediate the effects of Nron on angiogenesis.

To investigate the function of Nron in VSMCs during the development of atherosclerosis, an adenoviral vector driven by the SM22 α promoter was used to control the expression of Nron in the aorta. In addition, stable Nron knockdown in the aorta was achieved successfully using tail vein delivered rAAV2/8 vectors carrying the Nron shRNA, although this effect was not vsmc specific. Surprisingly, rAAV2/8 mediated Nron KD not only increased plaque stability, but also significantly reduced plaque area in the aorta and aortic sinus (the Nron OE of VSMC had no effect on plaque area). Notably, the blood lipid levels of the Nron KD mice improved and the body weight decreased again. This finding suggests a potential role for Nron in other metabolic diseases, and is worthy of further investigation. Furthermore, in future studies, it may be more meaningful and considerable to elucidate the specific role of Nron in different types of cells in the pathogenesis of atherosclerosis using conditional knockdown or transgenic mice. Taken together, the reduction in lncRNA Nron expression increases atherosclerotic plaque stability by modulating VSMC function in advanced lesions and inhibiting neovascularization. The research provides important clues for clarifying pathogenesis of atherosclerosis and suggests that Nron is a potential therapeutic target of atherosclerosis.

It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims (3)

1. The application of the knockdown preparation of the Nron in preparing the medicine for treating atherosclerosis, wherein the knockdown preparation of the Nron is rAAV carrying Nron shRNA.
2. 2. The use of claim 1, wherein the knocked-down preparation of a Nron shRNA is a rAAV2/8 carrying the Nron shRNA.
3. 3. The use of claim 1, wherein the knockdown formulation of the Nron shRNA appears to increase atherosclerotic plaque stability.

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