CN115851907B

CN115851907B - Annular non-coding RNA-circZBTB46 and application thereof

Info

Publication number: CN115851907B
Application number: CN202211326866.2A
Authority: CN
Inventors: 贾恩志; 符亚红; 贾侨伟; 张胜; 别恒杰
Original assignee: Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
Current assignee: Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
Priority date: 2022-10-26
Filing date: 2022-10-26
Publication date: 2023-07-25
Anticipated expiration: 2042-10-26
Also published as: CN115851907A

Abstract

The invention discloses a circular non-coding RNA circZBTB46, and an influence thereof in the progress of coronary atherosclerosis heart disease and an application of an expression quantity detection preparation thereof in preparing a coronary atherosclerosis diagnostic reagent. The invention discovers that the circZBTB46 plays an important role in the whole process of atherosclerosis occurrence and development by exploring the regulation and control function of the circZBTB46 on proliferation and migration of human coronary artery smooth muscle cells, and can be used as a biomarker for diagnosing coronary atherosclerotic heart disease. The invention provides a biomarker for diagnosing coronary atherosclerosis, which is used for biological identification and clinical application of coronary atherosclerotic heart disease, so that an atherosclerosis patient can be effectively intervened in time by screening the target in clinic, and clinical events are reduced. Thus, the present invention provides new molecular markers and targets for intervention for diagnosis and treatment of atherosclerosis.

Description

Annular non-coding RNA-circZBTB46 and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a circular non-coding RNA circZBTB46, and an influence of the circular non-coding RNA circZBTB46 on the progress of coronary atherosclerotic heart disease and application of an expression quantity detection preparation thereof in preparation of a coronary atherosclerosis diagnostic reagent.

Background

Coronary heart disease (coronary artery disease, CAD) is one of the most prominent cardiovascular diseases affecting global human health, and has been shown to be a genetically susceptible disease, with a large number of genes involved in the development and progression of the disease. There is increasing evidence that gene expression, transcriptional control and structural stability are closely related to epigenetic modification. Atherosclerosis (AS) is a chronic complex disease involving multiple cells by multiple factors. It is characterized in that, starting from vascular endothelial injury, with accompanying inflammation, immune response, lipid deposition in the vessel wall, involves inflammatory and proliferative cascades of mainly functional cells including smooth muscle cells, endothelial cells and immune cells. Existing research methods and means have had a certain knowledge and understanding of the occurrence and development of AS, and have substantially improved diagnosis and treatment of cardiovascular diseases, but little is known about the factors and mechanisms controlling the formation of advanced atherosclerotic lesions.

Smooth muscle cells (smooth muscle cell, SMC) play an important role in AS plaque formation, progression and rupture, and studies have revealed that about 70% of various cells contained in AS plaque are derived from SMC. SMC migration through abnormal proliferation and synthesis of extracellular matrix during early AS is critical for early AS injury; in addition, SMCs can secrete a variety of pro-inflammatory, pro-proliferative factors to activate SMCs and recruit macrophages; SMC can also phagocytose lipids to form foam cells, and as lesions progress, increased SMC apoptosis is the primary cause of plaque rupture. Thus, abnormal proliferation and migration of SMCs are indispensible from the development of AS.

Circular RNAs (circrnas), an emerging class of non-coding RNAs, are ubiquitous in eukaryotes, stable in expression and highly conserved among species. Research shows that the circRNA has different levels of change in the conditions of atherosclerosis, coronary plaque formation, plaque rupture and the like, AS is taken AS a genetic susceptibility disease, and the development mechanism of AS can be further elaborated by researching the development mechanism of AS at the transcription level, so that the pathogenesis of the AS can be better understood, and a new diagnosis and treatment target is provided for AS from the RNA level.

Disclosure of Invention

The invention aims to provide non-coding circular RNA circZBTB46 which is remarkably high in peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) of patients with coronary heart disease and influences the functions of human coronary smooth muscle cells (human coronary artery smooth muscle cells, HCASMCs) in vitro, and provides application thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme: a circular non-coding RNA-circZBTB46, wherein the nucleotide sequence of the RNA-circZBTB46 is shown in SEQ ID NO. 1.

The application of a preparation for detecting the expression level of annular non-coding RNA-circZBTB46 in preparing a diagnostic reagent for diagnosing or predicting the risk of coronary atherosclerosis is characterized in that the nucleotide sequence of the RNA-circZBTB46 is shown as SEQ ID NO. 1.

Further, the test agent detects the risk of coronary atherosclerosis or coronary atherosclerosis in a subject by detecting the expression of RNA-circZBTB46 in the PBMC cells of the subject, wherein the expression is significantly up-regulated compared with a healthy control group.

A marker for diagnosing and predicting the risk of coronary atherosclerosis, wherein the marker is annular non-coding RNA-circZBTB46, and the nucleotide sequence of the marker is shown as SEQ ID NO. 1.

A diagnostic agent for coronary atherosclerosis comprising: and (3) detecting the preparation of the expression quantity of the RNA-circZBTB46, wherein the nucleotide sequence of the RNA-circZBTB46 is shown as SEQ ID NO. 1.

A diagnostic kit for coronary atherosclerosis comprising the diagnostic reagent of claim 4.

Further, the diagnostic reagent comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 3.

Compared with the prior art, the invention has the beneficial effects that: the invention discovers that the circZBTB46 plays an important role in the development process of atherosclerosis by exploring the regulation and control function of the circZBTB46 on vascular smooth muscle cell proliferation and migration, and provides a standard gene and a molecular identification method suitable for the identification of coronary atherosclerosis molecules by detecting the expression level of the circZBTB46 in coronary heart disease patients and PBMC of a control group. The invention can be used for screening the target spot in clinic, so that the patients with coronary heart disease can be found early, the death rate of the coronary heart disease can be reduced, and the prognosis of the patients can be improved. The main diagnosis method of the coronary atherosclerotic heart disease at present has the risks of high operation risk coefficient, large radiation and the like, and has the advantages of small wound, good repeatability and the like by collecting peripheral blood of a patient for detection, and is more easily accepted.

Drawings

FIG. 1 shows the circular structure of circZBTB46 by forward and reverse primer PCR, RNase R treatment, and sanger sequencing.

FIG. 2 shows siRNA silencing effect.

FIG. 3 shows that CCK8 experiments verify that silencing human coronary smooth muscle cell circZBTB46 expression in vitro inhibits smooth muscle cell proliferation activity.

FIG. 4 shows that in vitro silencing of human coronary smooth muscle cells by expression of circZBTB46 inhibits smooth muscle migration. The method comprises the steps of carrying out a first treatment on the surface of the

FIG. 5 shows the expression profile of circZBTB46 in clinical specimens;

FIG. 6 shows the results of analysis of the working characteristics of the subjects in clinical specimens by the circZBTB 46.

Detailed Description

In order to make the present application solution better understood by those skilled in the art, the following description will be made in detail and with reference to the accompanying drawings in the embodiments of the present application, it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.

It should be noted that the terms "comprises" and "comprising," and any variations thereof, in the description and claims of the present application and in the foregoing figures, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or apparatus.

Example 1: structural identification of circZBTB46

1. Material

1.1 cells

Human coronary smooth muscle cells (HCASMC) used in the present invention were purchased from Sigma, applied in cell culture Vascular Smooth Muscle Cell Growth Kit. The culture conditions were 37℃and 5% CO2.

1.2 reagents

TRIzol Reagent, reverse transcription kitIII RT SuperMix for qPCR (gDNA wind), fluorescent Real-time (Real-time) quantitative PCR (polymerase chain reaction from Nanjinovirginal Biotech Co., ltd.) agarose from Sigma Co., USA, gelred from biological Co., USA, DNA Marker and Loading Buffer from Beijing Tiangen Co., beijing Tiangen:

：Divergent(circular)primers；/>：Convergent(linear)primers.

2. method of

2.1 RNA extraction

Cell RNA was extracted using Trizol reagent according to the reagent instructions and cells were placed in 1mL Trizol reagent. Standing at room temperature for 5min, and fully lysing the cell nuclei. 0.2mL of chloroform was added while vigorously vortexing for 15 seconds, left at room temperature for 5-10min, 4 ℃, at 12000 rpm, centrifuged for 15min, after which the observable liquid was separated into three layers, the colorless water was phase shifted to another clean 1.5mL centrifuge tube (note that the middle layer was not touchable), and equal amount of isopropanol (about 0.4 mL) was added, gently inverted and mixed, and incubated at room temperature for 5-10min to precipitate RNA. Centrifugation was performed at 12000 rpm at 4℃for 10min, the supernatant was discarded, and 1mL of 75% ethanol (diluted with DEPC water, as-prepared) was added to wash the pellet, during which time the pipetting was repeated. Centrifuging at 12000 rpm at 4deg.C for 5-6min, discarding supernatant, adding anhydrous ethanol 1mL, washing precipitate, centrifuging at 4deg.C for 5-6min, and discarding supernatant. And (5) drying the white RNA precipitate for 5-10min. The precipitate was dissolved in 20-50uL DEPC water and the total RNA concentration and purity were measured by UV spectrophotometry.

2.2 reverse transcription

Reverse transcription reactions were performed using a reverse transcription kit (HiScript III RT SuperMix for qPCR (+gDNA wind)) according to instructions.

2.3 PCR

PCR was performed according to the following protocol (10 ul system) of the Novain ChamQ SYBR qPCR Master Mix kit

cDNA template 1 μl

SYBR Green Master 5μl

Forward primer(F) 0.2μl

Reverse primer(R) 0.2μl

RNase Free water 3.6μl

After the system preparation is finished, the PCR result is obtained by carrying out denaturation at 95 ℃ for 30s and carrying out 40 times of annealing process at 60 ℃ for 10 s.

2.4 agarose gel electrophoresis

And (3) glue preparation: 1.5% agarose gel, 1.05g agarose was weighed and dissolved in 70ml of 0.5 x TBE solution, 2. Mu.l Gelred dye was added, and the mixture was stirred and mixed well, and heated for 2min to dissolve the agar powder sufficiently. Pouring the glue. Standing for about 30min, and slightly pulling off the comb after the glue is completely solidified; electrophoresis: 6. Mu.l of DNA marker I and 5. Mu.l of DNA product and 1. Mu.l of loading buffer were mixed well and subjected to 120v constant pressure electrophoresis. Imaging: and (5) imaging by using a Berle gel imager, and taking a picture.

3. Results

The circular structure characteristics of the circZBTB46 are determined through forward and reverse primer PCR, RNase R treatment and sanger sequencing experiments, and a foundation is laid for further exploration of the functions of the circZBTB 46.

Example 2: construction of a silencing model for smooth muscle cell circZBTB46

1. Material

1.1 cells and siRNA

The cells and culture conditions used in this example are as described in example 1. The design and synthesis of small interfering RNAs (sirnas) was done by su Ji Ma gene inc. Silencing the circZBTB46 (siRNA) sequence, RNAi-1 Sense strand (Sense): CCACUCGCUGUCCCAGUCUTT, antisense strand (Antisense): AGACUGGGACAGCGAGUGGTT, RNAi-2 Sense strand (Sense): UCGCUGUCCCAGUCUGUAGTT, antisense strand (Antisense): CUACAGACUGGGACAGCGATT

2. Method of

2.1 Expression of circZBTB46 in smooth muscle cells

After the cells growing vigorously are counted, the cells are inoculated according to the required cell quantity, and after the cells grow for 3-4 days, the cell density can reach 50-80%. Using Lipofectamine ^TM 2000 transfection reagent for cell transfection experiment, and the preparation of transfection reagent mixed solution comprises Lipofectamine ^TM 2000 preparation: a1.5 ml sterile Eppendorf tube (EP tube) was taken, 250. Mu.l serum-free and antibiotic-free DMEM medium was added followed by 5. Mu.l Lipofectamine ^TM 2000 transfection reagent was dissolved in DMEM medium and allowed to stand at room temperature for 5min to allow complete mixing of the transfection reagent in DMEM medium. Preparing circRNA: a1.5 ml sterile Eppendorf tube was taken, 250. Mu.l of double free DMEM medium (without serum and antibiotics) was added first, then 5. Mu.l of a proper amount of the circRNA solution was added to the medium, and the mixture was allowed to stand at room temperature for 5min, so that the circRNA was thoroughly mixed in the DMEM medium. After 5min, DMEM medium containing Lipofectamine 2000 transfection reagent and the culture medium containing circRNA were mixed and left at room temperature for 20min to allow the two to combine sufficiently, and 500. Mu.l of DMEM medium was added to prepare a circRNA transfection solution. The original culture medium in the six-hole plate is removed, the cells are gently washed 1-2 times along the hole wall by a pre-temperature PBS solution, PBS is not directly dripped onto the cells, and the PBS is removed. The circRNA transfection solution was slowly added to the prepared cells along the walls of the wells, after 8h at 37℃the solution was aspirated and 2ml of complete DMEM medium was added for further culture. After culturing the cells for 48-72 hours, the cell density was observed to grow to 90%, and the expression of circZBTB46 in the cells was detected by Real-time PCR.

3. Results

The total RNA of the extracted smooth muscle cells was analyzed for expression of circZBTB46 by Real-time PCR. Compared with the si-NC transfection group, the expression quantity of the circZBTB46 is obviously reduced, which proves that the cell silencing model of the circZBTB46 is successfully modeled (figure 2), thereby laying a foundation for further researching the function of the circZBTB46 in smooth muscle cells.

Example 3: regulatory effects of circZBTB46 on smooth muscle cell proliferation

1 Material

1.1 cells

The cells used in the experiment and the culture method were the same as in example 1.

1.2 reagents

The siRNA used in the experiment was the same as in example 2.CCK-8 is available from APEXBIO.

2. Method of

2.1 CCK8 proliferation assay

siRNA was transfected as in example 2, the transfected cells were inoculated into 96 well plates, cells were inoculated at 5000 cell densities per well, and the plates were placed in a 37℃incubator for preculture (adherence for 6-8 h); after a period of time of treatment, the cells were subjected to pipetting and the original medium was replaced with a pre-prepared medium containing 10% CCK-8 and incubated for 2 hours, and absorbance (OD value) at 450nm was measured in a 96-well plate using a microplate reader. And continuously measuring for a plurality of days, and drawing a growth curve.

3 results

After the CCK8 experiment detects the proliferation condition of smooth muscle cells after siRNA is transfected in vitro to silence the circZBTB46, the invention discovers that the proliferation of the cells in the circZBTB46 silencing group is obviously inhibited (figure 3), which suggests that the circZBTB46 has the capacity of promoting the proliferation of human coronary smooth muscle cells.

Example 4: regulating action of circZBTB46 on smooth muscle cell migration

1. Cells

1.2 reagents

The siRNA used in the experiment was the same as in example 2.

2. Method of

2.1 cell scratch experiments

Cells were routinely digested to a density of 5X 10 per ml ⁵ The suspension of individual cells is inoculated into a 6-well culture plate and routinely cultured for 16-24 hours until a monolayer of cells is formed. The serum-free culture solution is cultured for 4 hours. The culture plate is scratched along the bottom of the culture plate in a straight line, and the relative distance of the scratch area is recorded under a mirror. Washing cells 3 times with PBS, and culturing in serum-free mediumA base. Culturing in an incubator at 37 ℃ and 5% CO2. Sampling for 0h and 24h, and photographing.

3. Results

To verify whether the smooth muscle cell migration ability was affected by the circZBTB46, the invention detects the change of the cell migration ability of the siRNA transfected cells for 24 hours through a cell scratch experiment, and discovers that the migration level of the cells of the circZBTB46 silent group is obviously inhibited (fig. 4), which suggests that the circZBTB46 has the ability to promote the smooth muscle cell migration.

Example 5: judging efficacy of circZBTB46 as coronary heart disease diagnosis marker

1. Group-in patients: the serial coronary heart disease patients confirmed by coronary angiography are 125 cases and 33 cases of normal control patients. According to the contrast results, at least one subject with a degree of coronary artery stenosis > 50% is selected as a patient group, and all subjects with a degree of coronary artery stenosis <50% are selected as a control group.

2. Method of

2.1 separation of samples

PBMCs were isolated by density gradient centrifugation with sample and lymphocyte separation volumes of 1:1,2000 rpm for 20min, inserting into cloud foggy layer by using a liquid transfer device after layering, sucking mononuclear cells, then preserving the mononuclear cells in TRIzol reagent, extracting total RNA by using chloroform 12000g for 15min, centrifuging 12000g for 10min by using isopropanol with equal volume, precipitating by using 7500g for 5min by using ethanol with 75%, purifying by centrifugation, and finally dissolving in RNase-free water.

2.2 design of primers

The primer was identical to Divergent (circular) of example 1.

2.3 reverse transcription

As in example 1.

PCR amplification

As in example 1.

4. Results

Statistical analysis results show that the circZBTB46 has a significant expression difference (p < 0.05) between the two groups, and the gene has high expression in the coronary heart disease group. The analysis result of the working characteristic curve of the test subject shows that the area under the curve is larger than 0.5 (figure 5), and the gene has certain diagnosis capability on coronary atherosclerosis.

The results show that the circZBTB46 participates in the coronary atherosclerosis process and can significantly influence the function of human coronary smooth muscle cells, and the detection of the molecular expression level is feasible for diagnosing and identifying coronary atherosclerosis patients.

It will be appreciated that the invention has been described by way of example only and that modifications may be made while remaining within the scope and spirit of the invention. The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by a person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims

1. The use of a formulation for detecting the expression level of circular non-coding RNA-circZBTB46 for the preparation of a diagnostic reagent for diagnosing or predicting the risk of developing coronary atherosclerosis, characterized in that: the nucleotide sequence of the RNA-circZBTB46 is shown as SEQ ID NO. 1.

2. The use according to claim 1, characterized in that: the detection agent detects the expression of RNA-circZBTB46 in PBMC cells of a subject, and the expression is up-regulated compared with a healthy control group, so as to judge that the subject has coronary atherosclerosis or is at risk of developing coronary atherosclerosis.

3. The application of a preparation for detecting the expression level of annular non-coding RNA-circZBTB46 in preparing a kit for diagnosing coronary atherosclerosis is characterized in that: the nucleotide sequence of the RNA-circZBTB46 is shown as SEQ ID NO. 1.