CN110699442A

CN110699442A - Application of LncRNA PEBP1P2, kit for diagnosing heart diseases and medicine for treating heart diseases

Info

Publication number: CN110699442A
Application number: CN201910930479.1A
Authority: CN
Inventors: 于涛; 辛辉; 杨艳艳; 何兴强
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2020-01-17
Anticipated expiration: 2039-09-29
Also published as: CN110699442B

Abstract

The invention discloses an application of LncRNA PEBP1P2, a kit for diagnosing heart diseases and a medicine for treating the heart diseases, and relates to the technical field of biology. qRT-PCR analysis shows that LncRNA PEBP1P2 is down-regulated in carotid artery tissues of injured rats and the purpose of diagnosing heart diseases can be achieved by specifically detecting LncRNA PEBP1P 2. Meanwhile, the excessive proliferation and phenotype transformation of vascular smooth muscle cells can be effectively inhibited by over-expressing LncRNA PEBP1P 2. The LncRNA PEBP1P2 is expected to provide meaningful biological indexes for the diagnosis and treatment of clinical heart diseases, and provides new action targets and new ideas for the design of anti-heart diseases.

Description

Application of LncRNA PEBP1P2, kit for diagnosing heart diseases and medicine for treating heart diseases

Technical Field

The invention relates to the technical field of biology, in particular to application of LncRNA PEBP1P2, a kit for diagnosing heart diseases and a medicine for treating the heart diseases.

Background

With the improvement of living standard, the change of living habits, the aging of population and the change of environment, the prevalence rate of Cardiovascular diseases (CVD) in China continuously rises. According to the calculation, the number of CVD patients in China is 2.9 million, wherein the number of CVD patients is 2.7 million, the number of cerebral apoplexy is 1300 million, the number of coronary heart disease is 1100 million, the number of pulmonary heart disease is 500 million, the number of heart failure is 450 million, the number of rheumatic heart disease is 250 million, and the number of congenital heart disease is 200 million. In China, the mortality rate of CVD is higher than that of tumors and other diseases, and the mortality rate is the top. CVD death accounted for approximately 43.8% of all causes of death, with 2 out of 5 deaths being CVD. Accordingly, the hospitalization costs for cardiovascular diseases have also increased rapidly, with annual growth rates far higher than GDP growth rates since 2004. The cardiovascular diseases have wide prevalence, long course of disease, large burden and high lethality disability rate. Therefore, prevention and treatment of CVD has become an important public health problem in the present society, and enhancement of prevention and treatment of cardiovascular diseases has been difficult and slow.

Atherosclerosis (AS) is the leading cause of CVD. AS a chronic progressive inflammatory disease, atherosclerosis is characterized by multifocal structural changes in the walls of the middle and large arteries, ultimately leading to the formation of AS plaques. The pathogenesis of the disease comprises: endothelial dysfunction and activation; monocyte and macrophage adhesion, activation and migration; local oxidative stress; lipid deposition; synthesizing extracellular matrix; smooth muscle cell migration; the proliferation of plaque and the formation of new blood vessels. Vascular Smooth Muscle Cells (VSMCs) play an important role in the initiation and progression of atherosclerosis. Smooth muscle cells of normal arteries express a range of smooth muscle cell markers including smooth muscle cell myosin heavy chain (SMHC), 22kDa smooth muscle cell lineage limiting protein (SM22 α), α -SMA, CNN1, and the like. Expression of these markers in vascular smooth muscle cells is reduced in vitro culture and atherosclerosis, and the ability to proliferate, migrate and secrete a variety of extracellular matrix proteins and cytokines is obtained. Macrophage markers and properties have also been obtained by phenotypic switching of vascular smooth muscle cells, which has long been recognized as critical for atherosclerosis. Therefore, the research on the phenotypic transformation of vascular smooth muscle cells plays an important role in preventing and treating atherosclerosis.

Long non-coding RNAs (lncrnas) are transcripts with a length of more than 200 nucleotides, have no protein coding ability, and are widely involved in various biological processes such as development, differentiation, and carcinogenesis. There is increasing evidence that long non-coding RNAs have an important role in maintaining normal physiological functions of cardiovascular system, and abnormal expression of LncRNA in cardiovascular system is closely related to occurrence and development of many cardiovascular diseases. Therefore, the LncRNA is used as a target point for treating cardiovascular diseases, and the development of related medicaments has potential clinical application value. One LncRNA can act on a plurality of targets, influence a plurality of signal paths, regulate the expression of a plurality of genes participating in the regulation and control of cardiovascular diseases, and achieve the aim of treating diseases on the whole. In addition, since LncRNA is differentially expressed in pathological states, a disease can be predicted and diagnosed by detecting its expression level.

The pathogenesis and the current treatment situation of the cardiovascular-related diseases are still in a great unknown field, and continuous exploration and research are still needed, so that the invention is particularly provided.

Disclosure of Invention

In view of the drawbacks of the prior art, a first object of the present invention is to provide the use of LncRNA PEBP1P2 in the preparation of a product for diagnosing and/or treating heart diseases, which at least alleviates one of the technical problems of the prior art.

It is a second object of the present invention to provide a kit for diagnosing heart diseases, so as to achieve effective diagnosis of heart diseases.

The third purpose of the invention is to provide a medicine for treating heart diseases, so as to realize targeted treatment of the heart diseases.

The invention provides application of LncRNA PEBP1P2 in preparing products for diagnosing and/or treating heart diseases, wherein the LncRNA PEBP1P2 contains a cDNA sequence shown as SEQ ID NO. 1.

Further, the heart disease includes heart diseases caused by vascular smooth muscle proliferation and phenotypic transformation.

Further, the heart disease includes coronary heart disease or atherosclerosis.

Further, the product includes a kit or a medicament.

The present invention also provides a kit for diagnosing heart diseases, which includes a marker recognizing LncRNAPEBP1P 2;

the LncRNA PEBP1P2 contains a nucleotide sequence shown as SEQ ID NO. 1.

Further, the marker recognizing LncRNA PEBP1P2 includes at least one of the following a) or b):

a) a primer that binds LncRNA PEBP1P 2;

b) a biomacromolecule that binds LncRNA PEBP1P2, said biomacromolecule comprising: an antibody or functional fragment of an antibody, or, an RNA binding protein or functional fragment thereof.

Further, the primer binding to LncRNA PEBP1P2 has the nucleotide sequence shown in SEQ ID No.2 and SEQ ID No. 3.

In addition, the invention also provides a medicine for treating heart diseases, which comprises one or more of the following I) to IV):

Ⅰ)LncRNA PEBP1P2；

II) a recombinant vector containing a gene encoding LncRNA PEBP1P 2;

III) a recombinant virus containing a gene encoding LncRNA PEBP1P 2;

IV) a recombinant viral vector containing a gene encoding LncRNA PEBP1P 2;

the LncRNA PEBP1P2 contains a nucleotide sequence shown as SEQ ID NO. 1.

Further, the medicament also comprises a pharmaceutically acceptable carrier;

preferably, the carrier comprises one or more of chitosan, cholesterol, liposomes, and nanoparticles.

Further, the dosage form of the medicament comprises an oral preparation or an injection preparation.

The cDNA sequence of the long-chain non-coding RNA PEBP1P2 related to heart diseases is shown in SEQ ID NO. 1. The inventor screens out long-chain non-coding RNA-PEBP1P2 which is differentially expressed in a human coronary artery vascular smooth muscle cell line (HCASMC) by a high-throughput sequencing technology, qRT-PCR analysis shows that LncRNA PEBP1P2 is down-regulated in coronary heart disease patients and injured rat carotid artery tissues, and the purpose of diagnosing heart diseases can be achieved by carrying out specific detection on LncRNA PEBP1P 2. Meanwhile, the proliferation of vascular smooth muscle cells can be effectively inhibited by over-expressing LncRNA PEBP1P 2. The LncRNAPEBP1P2 is expected to provide meaningful biological indexes for the diagnosis and treatment of clinical heart diseases, and provide new action targets and new ideas for the design of anti-heart diseases. Different products can be developed aiming at the long non-coding RNA, for example, a novel kit for detecting LncRNA PEBP1P2 is developed to diagnose heart diseases, or a medicament taking LncRNA PEBP1P2 as a target to treat heart diseases and the like, and the product has important influence on the diagnosis and treatment of heart diseases.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

Fig. 1A is a graph illustrating the result of the coding probability of LncRNAPEBP1P2 obtained by the coding potential evaluation tool CPAT according to an embodiment of the present invention;

fig. 1B is a histogram of coding probabilities of LncRNAPEBP1P2 obtained by a coding potential evaluation tool CPAT according to an embodiment of the present invention;

FIG. 2A is a graph showing the results of down-regulation of LncRNA PEBP1P2 in tissues of patients with coronary heart disease according to an embodiment of the present invention;

FIG. 2B is a graph showing the results of down-regulation of LncRNA PEBP1P2 in injured rat carotid artery tissue, provided by an example of the present invention;

FIG. 2C is a graph showing the results of enrichment of LncRNA PEBP1P2 in vascular smooth muscle provided by an example of the present invention;

FIG. 2D is a graph showing the results of enrichment of LncRNA PEBP1P2 in rat aorta, which is provided in the present invention;

FIG. 3 is a graph showing the results of CCK8 analysis of vascular smooth muscle cell proliferation after PEBP1P2 knock-down according to an embodiment of the present invention;

FIG. 4A is a graph showing the results of a vascular smooth muscle cell scratch test performed after the knockdown of PEBP1P2 according to an embodiment of the present invention;

FIG. 4B is a graph showing the results of a Transwell experiment on vascular smooth muscle after the knockdown of PEBP1P2 according to an embodiment of the present invention;

FIG. 5A is a graph showing the results of the mRNA expression levels of the vascular smooth muscle contraction markers a-SMA, CNN1 and SMMHC after the knockdown of PEBP1P2 according to the embodiment of the present invention;

FIG. 5B is a graph showing the results of the protein expression levels of the vascular smooth muscle contraction markers a-SMA, CNN1 and SMMHC after the knockdown of PEBP1P2 according to the embodiment of the present invention;

FIG. 6 is a graph showing the results of CCK8 analysis of vascular smooth muscle cell proliferation after overexpression of PEBP1P2 according to an embodiment of the present invention;

FIG. 7A is a graph showing the results of a vascular smooth muscle cell scratch test performed after overexpression of PEBP1P2 according to an embodiment of the present invention;

FIG. 7B is a graph showing the results of a Transwell experiment on vascular smooth muscle performed after the overexpression of PEBP1P2 according to the present invention;

FIG. 8A is a graph showing the results of the expression levels of mRNA for vascular smooth muscle contraction markers a-SMA, CNN1, and SMMHC after overexpression of PEBP1P2 according to the present invention;

FIG. 8B is a graph showing the results of the protein expression levels of vascular smooth muscle contraction markers a-SMA, CNN1, and SMMHC after overexpression of PEBP1P2 according to the present invention;

FIG. 9 is a graph showing the results of the expression level of PEBP1P2 in vascular smooth muscle cells treated with PDGF-BB (20 ng/mL);

FIG. 10A is a graph of the results of a CCK8 assay for PDGF-BB induced vascular smooth muscle cell proliferation after overexpression of PEBP1P2, provided by an example of the present invention;

FIG. 10B is a graph showing the results of PDGF-BB-induced vascular smooth muscle cell scratch assay after overexpression of PEBP1P2 according to an embodiment of the present invention;

FIG. 10C is a graph showing the results of PDGF-BB induced vascular smooth muscle Transwell experiments following overexpression of PEBP1P2 according to the present invention;

FIG. 10D is a graph showing the results of the protein expression levels of vascular smooth muscle contraction markers a-SMA, CNN1, and SMMHC after overexpression of PEBP1P2, according to an embodiment of the present invention;

FIG. 11A is a graph showing the results of RIP provided in the examples of the present invention demonstrating the direct binding of PEBP1P2 to the downstream target CDK 9;

FIG. 11B is a graph showing the results of CDK9 expression levels after knocking down and overexpressing PEBP1P2, according to the present invention;

FIG. 12 is a graph showing the results of CDK9 expression in injured rat carotid artery tissue, according to an embodiment of the present invention;

FIG. 13A is a graph of the partial recovery of knocked-down CDK9 due to knocking-down PEBP1P2 according to embodiments of the invention;

FIG. 13B is a graph showing the partial recovery of decreased cell proliferation due to knockdown of CDK9 due to knockdown of PEBP1P2, provided by an example of the present invention;

FIG. 14A is a graph showing the results of a cell scratch test in which the reduction in migration of cells following knock-down of CDK9 is partially restored by knock-down of PEBP1P2, according to an embodiment of the present invention;

FIG. 14B is a graph showing the results of a Transwell experiment in which the reduction in migration of cells following knock-down of CDK9 was partially restored by knock-down of PEBP1P2, as provided by an example of the present invention;

FIG. 15A is a graph showing the results of the protein expression levels of the vascular smooth muscle contraction markers a-SMA, CNN1 and SMMHC after knock-down of CDK9, according to the example of the present invention;

FIG. 15B is a graph showing the results of protein expression of CDK9 after knockdown of CDK9 and treatment with PDGF-BB, according to an embodiment of the present invention;

FIG. 16A is a graph showing the results that knockdown CDK9 provided by the examples of the present invention significantly reduced PDGF-BB-induced proliferation of vascular smooth muscle cells;

FIG. 16B is a graph showing the results of a PDGF-BB-induced vascular smooth muscle cell migration significantly reduced by knockdown CDK9 provided by example of the present invention;

FIG. 17A is a graph showing the partial recovery of overexpressed CDK9 from knockdown of PEBP1P2, according to an embodiment of the invention;

FIG. 17B is a graph showing the results of partial recovery of increased proliferation of cells overexpressing CDK9 by overexpression of PEBP1P2, provided by an example of the present invention;

FIG. 18A is a graph showing the results of a cell scratch test in which the reduction in migration of cells after overexpression of CDK9 is partially restored by overexpression of PEBP1P2 according to an embodiment of the present invention;

FIG. 18B is a graph showing the results of a Transwell experiment in which cells with reduced migration after overexpression of CDK9 were partially restored by overexpression of PEBP1P2, according to an embodiment of the present invention;

FIG. 19 is a graph showing the results of protein expression levels of vascular smooth muscle contraction markers a-SMA, CNN1, and SMMHC after overexpression of CDK9, according to an embodiment of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

According to an aspect of the present invention, there is provided a use of LncRNA PEBP1P2 containing a cDNA sequence as shown in SEQ ID No.1 for preparing a product for diagnosing and/or treating heart diseases.

The inventor screens out long-chain non-coding RNA-PEBP1P2 differentially expressed in a human coronary artery vascular smooth muscle cell line (HCASMC) by a high-throughput sequencing technology, and qRT-PCR analysis shows that LncRNAPEBP1P2 is down-regulated in carotid artery tissues of a coronary heart disease patient and an injured rat, and the purpose of diagnosing heart diseases can be achieved by specifically detecting LncRNA PEBP1P 2. Meanwhile, the proliferation of vascular smooth muscle cells can be effectively inhibited by over-expressing LncRNAPEBP1P 2. The LncRNA PEBP1P2 is expected to provide meaningful biological indexes for the diagnosis and treatment of clinical heart diseases, and provides new action targets and new ideas for the design of anti-heart diseases.

In the present invention, the LncRNA PEBP1P2 has the nucleotide sequence shown in SEQ ID No.1, which means that the LncRNA PEBP1P2 may have other functional sequences, such as a tag sequence or a linker sequence, in addition to the nucleotide sequence shown in SEQ ID No. 1.

The diagnosis and/or treatment of heart disease means that the LncRNA PEBP1P2 provided by the present invention can be used for the diagnosis of heart disease, or for the treatment of heart disease, or for both the diagnosis of heart disease and the treatment of heart disease.

In some preferred embodiments, the cardiac disorder comprises a cardiac disorder caused by vascular smooth muscle proliferation.

Vascular smooth muscle cells, which are important components of the vascular wall, regulate the systolic and diastolic functions of blood vessels, and also secrete various cytokines and intercellular substances, which are major factors causing vulnerable plaque rupture, thus playing an important role in atherosclerosis or vascular stenosis. The heart diseases caused by the vascular smooth muscle hyperplasia, such as atherosclerosis, coronary heart disease and the like, also play an important role in the vascular smooth muscle hyperplasia in the diseases such as hypertension, pulmonary hypertension and the like.

In some preferred embodiments, the product comprises a kit or a medicament.

the LncRNA PEBP1P2 contains a nucleotide sequence shown as SEQ ID NO. 1.

The expression of LncRNA PEBP1P2 in normal human tissues or cells is high, but the expression level of LncRNA PEBP1P2 in carotid artery tissues of patients with coronary heart disease and injured rats is remarkably reduced, and is usually reduced by 40-60% compared with a control group.

In some preferred embodiments, the marker recognizing LncRNA PEBP1P2 comprises at least one of the following a) or b):

a) a primer that binds LncRNA PEBP1P 2;

Wherein, the antibody or the antibody functional fragment is preferably a fluorescence-labeled antibody or antibody functional fragment; the RNA binding protein or functional fragment thereof is preferably a fluorescently labeled RNA binding protein or functional fragment thereof.

Using the above-mentioned markers, the expression level of LncRNA PEBP1P2 in the cells can be quantitatively detected.

In some preferred embodiments, the primer binding to LncRNA PEBP1P2 has a nucleotide sequence as shown in SEQ ID No.2 and SEQ ID No. 3.

Preferably, the kit further comprises a positive control, wherein the positive control is Nlrp3, and the forward primer sequence of the positive control is as follows: 5'-GGAGAGACCTTTATGAGAAAGCAA-3' (SEQ ID NO.4), and the reverse primer sequence is: 5'-GCTGTCTTCCTGGCATATCACA-3' (SEQ ID NO. 5). By setting the positive control, the validity of the detection result can be judged more accurately.

Ⅰ)LncRNA PEBP1P2；

II) a recombinant vector containing a gene encoding LncRNA PEBP1P 2;

III) a recombinant virus containing a gene encoding LncRNA PEBP1P 2;

IV) a recombinant viral vector containing a gene encoding LncRNA PEBP1P 2;

the LncRNA PEBP1P2 contains a nucleotide sequence shown as SEQ ID NO. 1.

It is to be noted that the drug for treating heart diseases provided by the present invention may include one or two or more of LncRNAPEBP1P2, a recombinant virus containing a gene encoding LncRNA PEBP1P2, a recombinant virus containing a gene encoding LncRNA PEBP1P2 or a recombinant viral vector containing a gene encoding LncRNA PEBP1P2, such as a recombinant viral vector containing only LncRNA PEBP1P2 or only a recombinant vector containing a gene encoding LncRNAPEBP1P2, or a recombinant viral vector containing LncRNAPEBP1P2 and a recombinant vector containing a gene encoding LncRNAPEBP1P2, or a recombinant viral vector containing a gene encoding LncRNAPEBP1P2, a recombinant virus containing a gene encoding LncRNAPEBP1P2 and a recombinant viral vector containing a gene encoding LncRNAPEBP1P 6853 or both LncRNAPEBP1P2 and a recombinant viral vector containing LncRNAPEBP1P 73742.

In some preferred embodiments, the medicament further comprises a pharmaceutically acceptable carrier;

The carrier can effectively wrap one or more of LncRNA PEBP1P2, recombinant virus containing coding genes of LncRNA PEBP1P2, recombinant virus containing coding genes of LncRNA PEBP1P2 or recombinant virus vector containing coding genes of LncRNA PEEBP 1P2, and carry the active ingredients into the body and release the active ingredients so as to achieve the purpose of administration and treatment. Preferably, the carrier can further play a role in targeted drug delivery by further modifying the carrier, such as connecting binding sites and the like.

In some preferred embodiments, the dosage form of the drug may be an oral preparation or an injectable preparation.

When administered orally, the above-mentioned drugs may be formulated into any orally acceptable formulation form, for example, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions.

Among these, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate may be added. Diluents for use in capsules typically include lactose and dried corn starch. Oral suspensions are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents.

Optionally, some sweetener, aromatic or colorant may be added into the above oral preparation.

When the medicine is administered in the form of injection, the medicine can be prepared into any preparation form acceptable for injection, such as, but not limited to, injection solution or powder injection.

Among the carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The main biological samples and experimental operation information used in the examples of the present invention are as follows, and unless otherwise specified, the biological samples and experimental operations used in the examples of the present invention are performed as follows:

1. clinical samples

All blood samples were taken from coronary patients (n-27) confirmed by coronary angiography and were collected without stent implantation or before stent implantation, and healthy persons without any disease (n-29) served as a control group. Exclusion criteria were: diabetes, tumors, immune system diseases, blood system diseases or infections. Serum was extracted from whole blood samples and stored at-80 ℃ prior to testing. All patients need to sign informed consent. The study was approved by the ethical committee of the research in Qingdao, and all experiments were performed according to the principles announced by Helsinki.

2. Laboratory animal

Male SD rats (300-350g) were purchased from Wittisley, Inc. and a rat carotid balloon injury model was constructed according to previous literature reports. Rats were sacrificed on

days

3, 7 and 21, respectively, and aortic tissue was collected for subsequent testing, with the right carotid artery as a negative control. All experimental animals and protocols were approved by the animal protection committee of the university of Qingdao.

3. Cell culture, transfection

Human aortic smooth muscle cell line (HASMC) was cultured in DMEM medium containing 10% fetal bovine serum and penicillin (50U/ml) and streptomycin (50. mu.g/ml). Cells were transfected using Lipofectamine 2000 and according to the manufacturer's instructions.

4. Construction of PEBP1P2 and CDK9 knockdown or over-expression cell lines

Small interfering RNA (siRNA) specifically targeting PEBP1P2 or CDK9 was designed and synthesized by Gima gene corporation. PEBP1P2 or CDK9 were knocked down in HASMC cell lines by stable transfection of si-PEBP1P2 or si-CDK9, with non-silent siRNA transfection as a control. The PEBP1P2 sequence is biosynthesized by Huada gene company and cloned into pcDNA3.0 vector, CDS region of CDK9 is amplified by PCR and cloned into pAsRed2 vector, and PEBP1P2 and CDK9 overexpression vectors are respectively constructed. Overexpression of PEBP1P2 or CDK9 was achieved by stable transfection of pcDNA3.0-PEBP1P2 or pAsRed2-CDK9, and HASIMC was transfected with empty pcDNA3.0, pAsRed2 vectors, respectively, as controls.

5. RNA extraction, reverse transcription and real-time quantitative PCR (qRT-PCR)

Total RNA was extracted from blood, tissue and cell samples using TRIzol reagent. Then, cDNA was synthesized by PrimeScriptTMRT kit. qRT-PCR was performed using Hieff UNICON Power qPCR SYBR GreenMaster Mix. GAPDH was used as an endogenous control. The Delta-Delta Ct method was used to analyze relative gene expression data. All experimental procedures were performed according to the manufacturer's instructions.

6. Western blot analysis

Cells were washed 2 times in PBS solution and lysed with RIPA buffer as indicated. Protein concentration was determined using the BCA method. Then, the protein was heated at 95 ℃ for 5 minutes in SDS-PAGE loading buffer, an equal amount of protein was separated by 10% SDS-PAGE, and the gel was blotted onto a 0.45. mu.mVDF membrane. The membranes were blocked in 5% skim milk for 1 hour, incubated overnight for the primary antibody, rinsed 3 times (10 min/time) with TBS-T, incubated for one hour for the secondary antibody, rinsed again with TBS-T and developed.

7. Cell proliferation and migration assay

Cells were seeded in 96-well plates and then tested for cell proliferation by EdU and CCK-8 kits. Cell migration was detected using wound healing and Transwell assays. Wound healing experiments: HASMC were seeded in 6-well plates for wound healing assays. After 12 hours of cell treatment, a monolayer of cells was scratched using a 1000 μ l tip and photographs were taken using an inverted phase contrast microscope at different time points. Transwell experiment: HASMC was cultured in DMEM medium containing 10% FBS for 24 hours. Then, the treated cells (1X 105 cells) were trypsinized and inoculated into the upper chamber of DMEM medium (200. mu.l) without FBS, and then the upper chamber was immersed for 24 hours in the lower chamber containing 10% FBS medium (500. mu.l) or/and PDGF-BB (20 ng/ml). Then, the medium was removed, washed with PBS, and the cells in the lower membrane were fixed with 4% paraformaldehyde for half an hour, and then stained with 0.1% gentian violet for half 5 hours before photographing.

8. RNA Immunoprecipitation (RIP)

Approximately 1 × 107 HASMCs were used per sample. The cells were washed twice with PBS and lysed with RIPA buffer containing RNase inhibitor for 5 minutes. The lysate was centrifuged at 12000rpm at 4 ℃ for 4 minutes, the supernatant was collected, and the protein concentration was determined using the BCA method. Proteins (500. mu.g) and magnetic beads (30. mu.l) were then incubated with CDK9 antibody (5. mu.g) or control IgG (5. mu.g), respectively, overnight at 4 ℃. RNA was extracted from immunoprecipitated magnetic beads, proteins and RNA complexes using TRIzol, followed by reverse transcription, RT-PCR and agarose gel electrophoresis.

9. Cellular immunofluorescence

HASMC were seeded on slides for 24 hours. Then, the cells were washed twice in PBS solution and fixed by 4% paraformaldehyde for 15 minutes. Cells were then incubated overnight with CDK9 antibody followed by 1 hour incubation with Fluorescent (FITC) secondary antibody. DAPI was used to stain nuclei. Photographs were taken using a Leica TCS SP8 laser confocal microscope.

10. Statistical analysis

Continuous and categorical variables are expressed as mean ± standard deviation and quantity (percentage), respectively. Statistical analysis was performed using GraphPadPrism 8.0 and SPSS 25.0. Analysis was performed using unpaired t-test, one-way analysis of variance, or Mann-Whitney test, according to variable distribution. P <0.05 was considered statistically significant. Each experiment was repeated a minimum of three times.

Example 1 basic Properties study of LncRNA PEBP1P2

(1) The coding potential evaluation tool CPAT shows that the coding probability of PEBP1P2 is 0.0029434043899346, and the result is shown in FIG. 1 and FIG. 2, which indicates that the LncRNA PEBP1P2 is a non-coding transcript and has no protein coding capacity.

(2) Results as shown in fig. 2A, 2B, 2C and 2D, qRT-PCR analysis showed that PEBP1P2 was down-regulated in coronary heart disease patients, injured rat carotid artery tissue, enriched in vascular smooth muscle, rat aorta.

Example 2 study of function and mechanism of LncRNA PEBP1P2

After knockdown of PEBP1P2 by siRNA:

as shown in fig. 3, the increase in proliferation of vascular smooth muscle was suggested by CCK8 analysis.

As shown in fig. 4A and 4B, the scratch test and the Transwell results indicate enhanced migration of vascular smooth muscle.

As shown in fig. 5A and 5B, following PEBP1P2 knockdown, vascular smooth muscle contraction markers a-SMA, CNN1, SMMHC were decreased at the mRNA and protein levels, while the cycle-related protein CCDN1 was not significantly changed.

After overexpression of PEBP1P 2:

as shown in fig. 6, analysis by CCK8 suggested a decrease in vascular smooth muscle proliferation.

As shown in fig. 7A and 7B, the scratch test and the Transwell results showed a decrease in migration of vascular smooth muscle.

As shown in fig. 8A and 8B, after PEBP1P2 overexpression, vascular smooth muscle contraction markers a-SMA, CNN1, SMMHC were elevated at mRNA and protein levels, while cycle-related protein CCDN1 was not significantly changed.

As shown in FIG. 9, the expression of PEBP1P2 was reduced after PDGF-BB (20ng/mL) treatment of vascular smooth muscle cells.

As shown in fig. 10A, 10B, 10C, and 10D, overexpression of PEBP1P2 significantly reduced PDGF-BB-induced proliferation, migration, and phenotypic shift of vascular smooth muscle cells.

As shown in fig. 11A and 11B, this example found PEBP1P2 to function by binding directly to the downstream target CDK9 by bioinformatics approach and confirmed by RIP; CDK9 expression was up-regulated after knocking down PEBP1P2, CDK9 expression was down-regulated after overexpression PEBP1P 2.

As shown in fig. 12, CDK9 was upregulated in injured rat carotid artery tissue.

As shown in fig. 13A and 13B, in vascular smooth muscle, knockdown CDK9 was partially restored by knockdown of PEBP1P 2; the reduction in cell proliferation following CDK9 knockdown was partially restored by the knockdown of PEBP1P 2.

As shown in fig. 14A and 14B, cell migration was reduced following knock-down of CDK9, and migration was partially restored by knock-down of PEBP1P 2.

As shown in fig. 15A and 15B, vascular smooth muscle contraction markers a-SMA, CNN1, SMMHC were elevated at mRNA and protein levels, while cycle-related protein CCDN1 was not significantly changed following CDK9 knock-down. The profile of CDK9 increased with time after PDGF-BB treatment.

As shown in fig. 16A and 16B, knockdown CDK9 significantly reduced PDGF-BB-induced proliferation and migration of vascular smooth muscle cells.

As shown in fig. 17A and 17B, in vascular smooth muscle, overexpressed CDK9 was partially restored by knock-down of PEBP1P 2; cell proliferation is increased after overexpression of CDK9 and can be partially restored by overexpression of PEBP1P 2.

As shown in fig. 18A and 18B, migration of cells was reduced after overexpression of CDK9, and migration was partially restored by overexpression of PEBP1P 2.

As shown in figure 19, after overexpression of CDK9, the vascular smooth muscle contraction markers a-SMA, CNN1, SMMHC were reduced at mRNA and protein levels and recovered after overexpression of PEBP1P2, while the cycle-related protein CCDN1 was not significantly changed.

In conclusion, this example identifies long-chain non-coding RNA-PEBP1P2 differentially expressed in vascular smooth muscle, and demonstrates that it inhibits proliferation, migration and phenotypic shift of vascular smooth muscle cells by negatively regulating downstream target CDK9, suggesting that PEBP1P2 is an important target for regulating vascular smooth muscle development. In view of its regulatory properties on vascular smooth muscle, PEBP1P2 is useful in the treatment of atherosclerotic diseases.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims

Use of LncRNA PEBP1P2 in the preparation of a product for diagnosing and/or treating heart diseases, wherein lncrnaapebp 1P2 comprises a cDNA sequence as shown in SEQ ID No. 1.
2. The use of claim 1, wherein the cardiac disorder comprises a cardiac disorder caused by vascular smooth muscle proliferation.
3. Use according to claim 1 or 2, wherein the heart disease comprises coronary heart disease or atherosclerosis.
4. The use of claim 1, wherein the product comprises a kit or a medicament.
5. A kit for diagnosing heart diseases, comprising a marker recognizing LncRNA PEBP1P 2;

the LncRNA PEBP1P2 contains a nucleotide sequence shown as SEQ ID NO. 1.
6. The kit of claim 5, wherein the marker recognizing the LncRNA PEBP1P2 comprises at least one of the following a) or b):

a) a primer that binds LncRNA PEBP1P 2;

b) a biomacromolecule that binds LncRNA PEBP1P2, said biomacromolecule comprising: an antibody or functional fragment of an antibody, or, an RNA binding protein or functional fragment thereof.
7. The kit of claim 6, wherein the primer binding to the LncRNA PEBP1P2 has a nucleotide sequence as set forth in SEQ ID No.2 and SEQ ID No. 3.
8. A medicament for the treatment of heart disease, said medicament comprising one or more of the following i) -iv):

Ⅰ)LncRNA PEBP1P2；

II) a recombinant vector containing a gene encoding LncRNA PEBP1P 2;

III) a recombinant virus containing a gene encoding LncRNA PEBP1P 2;

IV) a recombinant viral vector containing a gene encoding LncRNA PEBP1P 2;

the LncRNA PEBP1P2 contains a nucleotide sequence shown as SEQ ID NO. 1.
9. The medicament of claim 8, further comprising a pharmaceutically acceptable carrier;

preferably, the carrier comprises one or more of chitosan, cholesterol, liposomes, and nanoparticles.
10. The medicament of claim 8 or 9, wherein the dosage form of the medicament comprises an oral preparation or an injection preparation.