CN115851907A - Annular non-coding RNA-circZBTB46 and application thereof - Google Patents
Annular non-coding RNA-circZBTB46 and application thereof Download PDFInfo
- Publication number
- CN115851907A CN115851907A CN202211326866.2A CN202211326866A CN115851907A CN 115851907 A CN115851907 A CN 115851907A CN 202211326866 A CN202211326866 A CN 202211326866A CN 115851907 A CN115851907 A CN 115851907A
- Authority
- CN
- China
- Prior art keywords
- circzbtb46
- rna
- coronary
- cells
- atherosclerosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000029078 coronary artery disease Diseases 0.000 claims abstract description 25
- 230000014509 gene expression Effects 0.000 claims abstract description 22
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 17
- 206010003211 Arteriosclerosis coronary artery Diseases 0.000 claims abstract description 15
- 208000026758 coronary atherosclerosis Diseases 0.000 claims abstract description 15
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 239000003550 marker Substances 0.000 claims description 7
- 230000002441 reversible effect Effects 0.000 claims description 7
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 6
- 238000009007 Diagnostic Kit Methods 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims description 2
- 239000000032 diagnostic agent Substances 0.000 claims 1
- 229940039227 diagnostic agent Drugs 0.000 claims 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 abstract description 24
- 201000001320 Atherosclerosis Diseases 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 17
- 230000003143 atherosclerotic effect Effects 0.000 abstract description 9
- 230000005012 migration Effects 0.000 abstract description 8
- 238000013508 migration Methods 0.000 abstract description 8
- 238000011161 development Methods 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 7
- 230000035755 proliferation Effects 0.000 abstract description 7
- 210000004351 coronary vessel Anatomy 0.000 abstract description 5
- 208000019622 heart disease Diseases 0.000 abstract description 5
- 108091027963 non-coding RNA Proteins 0.000 abstract description 5
- 102000042567 non-coding RNA Human genes 0.000 abstract description 5
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- 239000000090 biomarker Substances 0.000 abstract 2
- 239000003147 molecular marker Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 41
- 238000002474 experimental method Methods 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- 108020004459 Small interfering RNA Proteins 0.000 description 9
- 239000004055 small Interfering RNA Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 230000030279 gene silencing Effects 0.000 description 7
- 238000003752 polymerase chain reaction Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000002131 composite material Substances 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000010839 reverse transcription Methods 0.000 description 5
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 239000012096 transfection reagent Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 230000000692 anti-sense effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 3
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 3
- 108091028075 Circular RNA Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 201000000057 Coronary Stenosis Diseases 0.000 description 2
- 108091092584 GDNA Proteins 0.000 description 2
- 108090000638 Ribonuclease R Proteins 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108091081021 Sense strand Proteins 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000534000 Berula erecta Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 208000034826 Genetic Predisposition to Disease Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000002586 coronary angiography Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000004049 epigenetic modification Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 210000000497 foam cell Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000007505 plaque formation Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000013826 regulation of smooth muscle cell migration Effects 0.000 description 1
- 230000004921 regulation of smooth muscle cell proliferation Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000006807 siRNA silencing Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 230000026799 smooth muscle cell apoptotic process Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a circular non-coding RNA circZBTB46, and an application of a preparation for detecting the influence and the expression quantity of the circular non-coding RNA circZBTB46 on the progression of coronary atherosclerotic heart disease in the preparation of a coronary atherosclerotic diagnostic reagent. According to the invention, by researching the regulation and control function of the circZBTB46 on the proliferation and migration of human coronary artery smooth muscle cells, the circZBTB46 is found to play an important role in the whole occurrence and development process of atherosclerosis 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 timely and effective intervention can be performed on atherosclerotic patients through screening the target spot in clinic, and clinical events are reduced. Therefore, the invention provides a new molecular marker and an intervention target for diagnosing and treating atherosclerosis.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a circular non-coding RNA circZBTB46, and an application of a preparation for detecting the influence and expression quantity of the circular non-coding RNA circZBTB46 on the progression of coronary atherosclerotic heart disease in the preparation of a coronary atherosclerotic diagnostic reagent.
Background
Coronary heart disease (CAD) is one of the most major cardiovascular diseases affecting human health worldwide and has been shown to be a genetically predisposed disease with a number of genes involved in the development of the disease. There is increasing evidence that gene expression, transcriptional control, and structural stability are closely related to epigenetic modifications. Atherosclerosis (AS) is a chronic complex disease involving multiple cells by multiple factors. It is characterized by the initiation of vascular endothelial injury, accompanied by inflammation, immune response, lipid deposition in the vessel wall, and involved in inflammatory and proliferative cascades of major functional cells including smooth muscle cells, endothelial cells, and immune cells. The existing research methods and means have already provided certain cognition and understanding on the occurrence and development of AS, and have substantially improved the diagnosis and treatment of cardiovascular diseases, but the factors and mechanisms for controlling the formation of advanced atherosclerotic lesions are still poorly understood.
Smooth Muscle Cells (SMC) play an important role in the formation, progression, and rupture of AS plaques, and studies have shown that about 70% of the various cells contained within AS plaques are derived from SMC. The migration of SMC through abnormal proliferation and the synthesis of extracellular matrix at the early stage of AS is the key of early damage of AS; in addition, SMC can secrete a plurality of proinflammatory and proliferation promoting factors to activate SMC and recruit macrophages; SMC also phagocytose lipid uptake, forming foam cells, and as lesions progress, increased SMC apoptosis is the leading cause of plaque rupture. Therefore, the abnormal proliferation and migration of SMC are indistinguishable from the occurrence and development of AS.
Circular RNA (circular RNA), an emerging class of non-coding RNAs, is ubiquitous in eukaryotes, expression-stable, and highly conserved across species. Research shows that the circRNA has different levels of changes in the conditions of atherosclerosis, coronary plaque formation, plaque rupture and the like, the AS is used AS a genetic susceptibility disease, the research on the development mechanism of the AS at the transcription level can deeply explain the AS, the research is helpful for better understanding of the pathogenesis of the AS, and a new diagnosis and treatment target point is provided for the AS from the RNA level.
Disclosure of Invention
The invention aims to provide a non-coding circular RNA circZBTB46 which is remarkably high expressed in Peripheral Blood Mononuclear Cells (PBMC) of patients with coronary heart disease and influences the functions of Human Coronary Artery Smooth Muscle Cells (HCASMCs) in vitro, and provides application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme: the circular non-coding RNA-circZBTB46 has a nucleotide sequence shown in SEQ ID NO. 1.
Use of a preparation for detecting the expression level of circular non-coding RNA-circZBTB46 in the preparation of a diagnostic reagent for diagnosing or predicting the risk of developing coronary atherosclerosis, wherein the nucleotide sequence of the RNA-circZBTB46 is shown as SEQ ID No. 1.
Further, the test agent is used for judging whether the subject has or is at risk of developing coronary atherosclerosis by detecting the expression of RNA-circZBTB46 in PBMC cells of the subject, and the expression is obviously up-regulated compared with a healthy control group.
A marker for diagnosing and predicting the risk of coronary atherosclerosis, wherein the marker is circular non-coding RNA-circZBTB46, and the nucleotide sequence is shown as SEQ ID NO. 1.
A diagnostic reagent for coronary atherosclerosis comprising: a preparation for detecting the expression quantity of 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 and development process of atherosclerosis by researching the regulation and control function of the circZBTB46 on the proliferation and migration of vascular smooth muscle cells, and provides a standard gene and a molecular identification method suitable for the molecular identification of coronary atherosclerosis by detecting the expression level of the circZBTB46 in PBMCs of patients with coronary heart disease and a control group. The invention can find patients with coronary heart disease at early stage, reduce death rate of coronary heart disease and improve prognosis of patients by screening the target spot in clinic. At present, the main diagnostic method of the coronary atherosclerotic heart disease has the risks of high surgical risk coefficient, large radiation and the like, and the peripheral blood of a patient is collected for detection, so that the method has the advantages of small wound, good repeatability and the like, and is easier to accept.
Drawings
FIG. 1 shows the cyclic structure of circZBTB46 by forward and reverse primer PCR, RNase R treatment and sanger sequencing.
FIG. 2 shows siRNA silencing effect.
FIG. 3 is a CCK8 experiment demonstrating that silencing the expression of circZBTB46 in human coronary artery smooth muscle cells in vitro inhibits the proliferative activity of smooth muscle cells.
FIG. 4 is a cell scratch experiment demonstrating that in vitro silencing of circZBTB46 expression in human coronary smooth muscle cells inhibits smooth muscle migration. (ii) a
FIG. 5 is a characterization of circZBTB46 expression in clinical specimens;
FIG. 6 shows the results of the analysis of the working characteristic curve of circZBTB46 in clinical specimens.
Detailed Description
In order to make the technical solutions of the present application better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection 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 this application and the above-described drawings, 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, but may include other steps or elements not 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 and cultured using the Vascular Smooth Muscle Cell Growth Kit. The culture conditions were 37 ℃ and 5% CO2.
1.2 reagents
TRIzol Reagent, reverse transcription kit
III RT SuperMix for qPCR (+ gDNA wiper), fluorescent Real-time (Real-time) quantitative PCR (polymerase chain reaction from Biotech, inc., of Kinzyme Biotech Co., ltd.) agarose was purchased from Sigma, gelred was purchased from Bio-ium, inc., DNA Marker and Loading Buffer were purchased from Beijing Tiangen, inc. PCR specific primers were designed and synthesized by Biotechnology, inc. primers are as follows:
2. method of producing a composite material
2.1 RNA extraction
Cell RNA was extracted using Trizol reagent according to the instructions of the reagents and cells were placed in 1mL Trizol reagent. After standing at room temperature for 5min, cell nuclei were lysed sufficiently. Adding 0.2mL of chloroform, vortexing vigorously while shaking for 15 seconds, standing at room temperature for 5-10min, 4 ℃,12000 rpm, centrifuging for 15min, then observing the liquid to separate into three layers, transferring the colorless aqueous phase to another clean 1.5mL centrifuge tube (note that the middle layer cannot be touched), adding equal amount of isopropanol (about 0.4 mL), mixing by gentle inversion, and incubating at room temperature for 5-10min to precipitate RNA. Centrifuging at 12000 rpm at 4 deg.C for 10min, discarding supernatant, adding 1mL 75% ethanol (diluted with DEPC water, prepared as before), washing precipitate, and repeatedly beating. Centrifuging at 4 deg.C and 12000 r/min for 5-6min, discarding supernatant, adding 1mL of anhydrous ethanol, washing precipitate, centrifuging at 4 deg.C for 5-6min, and discarding supernatant. And (5) automatically drying the white RNA precipitate for 5-10min. 20-50uL DEPC water is added to dissolve the precipitate, and the total RNA concentration and purity are measured by ultraviolet spectrophotometry.
2.2 reverse transcription
Reverse transcription reaction was performed using reverse transcription kit (HiScript III RT Supermix for qPCR (+ gDNA wiper)) as per the instructions.
2.3 PCR
The PCR reaction was carried out according to the following system (10 ul system) according to the instruction of the ChamQ SYBR qPCR Master Mix kit of Novozam
SYBR Green Master 5μl
Forward primer(F) 0.2μl
Reverse primer(R) 0.2μl
RNase Free water 3.6μl
After the system is prepared, pre-denaturation is carried out at 95 ℃ for 30s, and PCR results are obtained by cycling 40 times according to the denaturation at 95 ℃ for 10s and the annealing process at 60 ℃ for 30 s.
2.4 agarose gel electrophoresis
Preparing glue: 1.5% agarose gel, 1.05g agarose was weighed, dissolved in 70ml 0.5 x TBE solution, 2. Mu.l Gelred dye was added, shaken and mixed well, heated for 2min to dissolve agar powder sufficiently. And (6) pouring the glue. Standing for about 30min, and slightly pulling off the comb after the gel is completely solidified; electrophoresis: DNA marker I6. Mu.l, DNA product 5. Mu.l + loading buffer 1. Mu.l, mixed well, 120v constant voltage electrophoresis. Imaging: and imaging by using a Berle gel imager and taking a picture.
3. Results
The cyclic 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 researching the functions of the circZBTB 46.
Example 2: construction of silencing model of smooth muscle cell circZBTB46
1. Material
1.1 cells and siRNA
The cells and culture conditions used in this example were as in example 1. The design and synthesis of small interfering RNA (siRNA) was accomplished by Suzhou Jima Gen GmbH. Silencing circZBTB46 (siRNA) sequence, RNAi-1 Sense strand (Sense): CCACUCGCUGUCCAGUUCUTT, antisense strand (Antisense): AGACUGGGACAGCGAGGUGGTT, RNAi-2 Sense strand (Sense): UCGCUGCCAGUCUGUAGTT, antisense strand (Antisense): CUACAGACAGUCGGAGCAGCGATT
2. Method of producing a composite material
2.1 Expression of circZBTB46 in smooth muscle cells
After the cells in vigorous growth are counted, cell inoculation is carried out according to the required cell amount, and the cell density can reach 50-80% after the cells grow for 3-4 days. Using Lipofectamine TM 2000 transfection reagent for cell transfection experiment, the preparation and process of transfection reagent mixed solution are as follows, lipofectamine TM 2000, preparation: a sterile Eppendorf tube (EP tube) of 1.5ml was added first 250. Mu.l of serum-free and antibiotic-free DMEM medium and then 5. Mu.l of Lipofectamine TM 2000 transfection reagent is dissolved in DMEM medium and kept standing for 5min at room temperature to fully mix the transfection reagent in DMEM medium. Preparation of circRNA: a1.5 ml sterile Eppendorf tube was taken, 250. Mu.l of sterile DMEM medium (without serum and antibiotics) was added first, and then 5. Mu.l of a circRNA solution was added to the medium in an appropriate amount, and allowed to stand at room temperature for 5min to sufficiently mix the circRNA in the DMEM medium. After 5min, DMEM medium containing Lipofectamine TM 2000 transfection reagent and culture medium containing circRNA were addedThe culture medium was mixed, and left at room temperature for 20min to allow them to be fully combined, and 500. Mu.l of DMEM medium was added to prepare a circRNA transfection solution. The original medium in the six-well plate was removed, the cells were gently washed 1-2 times along the walls of the wells with pre-warmed PBS solution, PBS was then dropped directly onto the cells, and the PBS was removed. The circRNA transfection solution was slowly added to the prepared cells along the pore wall, the solution was aspirated after 8h at 37 ℃ and 2ml of complete DMEM medium was added to continue the culture. After culturing the cells for 48-72h, cell density growth to 90% was observed and the expression of circZBTB46 in the cells was detected by Real-time PCR.
3. Results
The extracted total RNA of smooth muscle cells was analyzed for the expression of circZBTB46 by Real-time PCR. Compared with the si-NC transfected group, the expression level of the circZBTB46 in the si-RNA transfected group is obviously reduced, which indicates that the cell silencing model of the circZBTB46 is successfully modeled (figure 2), thereby laying a foundation for further research on the function of the circZBTB46 in the smooth muscle cell function.
Example 3: regulation of smooth muscle cell proliferation by circZBTB46
1 Material
1.1 cells
The cells and culture method used in the experiment 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 was purchased from APEXBIO.
2. Method of producing a composite material
2.1 CCK8 value-added experiment
Transfecting siRNA according to example 2, inoculating transfected cells to a 96-well plate, inoculating the cells at a cell density of 5000 cells per well, and placing the culture plate into a constant temperature incubator at 37 ℃ for pre-culture (wall adhesion is 6-8 h); after the cells were treated for a while, the original medium was replaced with a pre-prepared medium containing 10% of CCK-8, cultured for 2 hours, and the absorbance (OD value) at 450nm of a 96-well plate was measured with a microplate reader. And continuously measuring for several days, and drawing a growth curve.
3 results
According to the invention, the proliferation condition of smooth muscle cells after in vitro transfection siRNA silenced circZBTB46 is detected through a CCK8 experiment, and the cell proliferation of the circZBTB46 silencing group is found to be obviously inhibited (figure 3), which indicates that the circZBTB46 has the capacity of promoting the proliferation of human coronary artery smooth muscle cells.
Example 4: regulation of smooth muscle cell migration by circZBTB46
1. Cells
The cells and culture method used in the experiment were the same as in example 1.
1.2 reagents
The siRNA used in the experiment was the same as in example 2.
2. Method of producing a composite material
2.1 cell scratch test
Cells were routinely digested to a density of 5X 10 cells per ml 5 And (3) suspending the cells, inoculating the cells into a 6-well culture plate, and culturing for 16-24 h in a conventional mode until a monolayer of cells is formed. Culturing in serum-free culture medium for 4h. Scratch is carried out along the bottom of the culture plate in a straight line shape, and the relative distance of a scratch area is recorded under a mirror. The cells were washed 3 times with PBS and serum-free medium was added. 37 deg.C, 5% CO2, and culturing in incubator. Samples were taken at 0h, 24h and photographed.
3. Results
In order to verify whether the circZBTB46 affects the migration ability of smooth muscle cells, the invention detects the change of the cell migration ability after siRNA transfection of cells for 24h through a cell scratch experiment, and finds that the migration level of the circZBTB46 silencing group cells is obviously inhibited (figure 4), which indicates that the circZBTB46 has the ability of promoting the migration of smooth muscle cells.
Example 5: judging the efficacy of circZBTB46 as a coronary heart disease diagnostic marker
1. Patients were enrolled: 125 patients with continuous coronary heart disease confirmed by coronary angiography and 33 patients with continuous coronary heart disease confirmed by normal control group are used. Based on the results of the imaging, at least one subject with > 50% degree of coronary stenosis was selected as the patient group, and all subjects with <50% degree of coronary stenosis were selected as the control group.
2. Method of producing a composite material
2.1 isolating the sample
PBMCs were separated by density gradient centrifugation with sample to lymphocyte separation volume of 1:1,2000rpm 20min, after layering, inserting into a cloud layer by using a liquid shifter, sucking a mononuclear cell, then storing the mononuclear cell in a TRIzol reagent, centrifuging total RNA for 15min by using 12000g of chloroform, centrifuging for 12000g of 10min by using equal volume of isopropanol, precipitating, centrifuging for 7500g of 5min by using 75% ethanol, purifying, and finally dissolving in water without RNase.
2.2 design of primers
The same Divergent (circular) primer as in example 1.
2.3 reverse transcription
The same as in example 1.
PCR amplification
The same as in example 1.
4. Results
Statistical analysis results show that circZBTB46 has 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 subject shows that the area under the curve is more than 0.5 (figure 5), and the gene has certain diagnostic capacity for the coronary atherosclerosis.
The results show that the circZBTB46 is involved in the process of coronary atherosclerosis, can obviously influence the functions of human coronary artery smooth muscle cells, and has feasibility for diagnosing and identifying patients with coronary atherosclerosis by detecting the expression level of the molecule.
It is to be understood that this invention has been described by way of example only and that modifications may be made within the scope and spirit of the invention. The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (7)
1. A circular non-coding RNA-circZBTB46, characterized by: the nucleotide sequence of the RNA-circZBTB46 is shown as SEQ ID NO. 1.
2. Use of an agent for detecting the expression level of circular non-coding RNA-circZBTB46 for the preparation of a diagnostic agent 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.
3. Use according to claim 2, characterized in that: the detection preparation is used for judging whether the subject has or is at risk of developing coronary atherosclerosis by detecting the expression of RNA-circZBTB46 in PBMC cells of the subject and obviously increasing the expression compared with a healthy control group.
4. Marker for diagnosing and predicting the risk of developing coronary atherosclerosis characterized in that: the marker is circular non-coding RNA-circZBTB46, and the nucleotide sequence is shown in SEQ ID NO. 1.
5. A diagnostic reagent for coronary atherosclerosis, comprising: a preparation for detecting the expression quantity of RNA-circZBTB46, wherein the nucleotide sequence of the RNA-circZBTB46 is shown as SEQ ID NO. 1.
6. A diagnostic kit for coronary atherosclerosis, characterized in that: the diagnostic kit comprising the diagnostic reagent according to claim 4.
7. The diagnostic kit of claim 6, wherein: 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211326866.2A CN115851907B (en) | 2022-10-26 | 2022-10-26 | Annular non-coding RNA-circZBTB46 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211326866.2A CN115851907B (en) | 2022-10-26 | 2022-10-26 | Annular non-coding RNA-circZBTB46 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115851907A true CN115851907A (en) | 2023-03-28 |
| CN115851907B CN115851907B (en) | 2023-07-25 |
Family
ID=85661959
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211326866.2A Active CN115851907B (en) | 2022-10-26 | 2022-10-26 | Annular non-coding RNA-circZBTB46 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115851907B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017211999A1 (en) * | 2016-06-08 | 2017-12-14 | Aalborg Universitet | Antisense oligonucleotides for modulation of long noncoding rnas |
| CN114032237A (en) * | 2021-10-11 | 2022-02-11 | 哈尔滨医科大学 | Circular non-coding RNA circSTK39 and application thereof in preventing and treating atherosclerosis |
| CN115058420A (en) * | 2022-06-09 | 2022-09-16 | 哈尔滨医科大学 | Circular non-coding RNA-circSP3, interference RNA thereof and application thereof |
-
2022
- 2022-10-26 CN CN202211326866.2A patent/CN115851907B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017211999A1 (en) * | 2016-06-08 | 2017-12-14 | Aalborg Universitet | Antisense oligonucleotides for modulation of long noncoding rnas |
| CN114032237A (en) * | 2021-10-11 | 2022-02-11 | 哈尔滨医科大学 | Circular non-coding RNA circSTK39 and application thereof in preventing and treating atherosclerosis |
| CN115058420A (en) * | 2022-06-09 | 2022-09-16 | 哈尔滨医科大学 | Circular non-coding RNA-circSP3, interference RNA thereof and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115851907B (en) | 2023-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103316342A (en) | Applications of miR-31 inhibitor in inhibition of angiostenosis after damage | |
| CN107012256A (en) | Abdominal aneurvsm diagnosis and treatment mark | |
| CN106244688B (en) | A kind of marker for assessing adenocarcinoma of colon risk | |
| CN115851907B (en) | Annular non-coding RNA-circZBTB46 and application thereof | |
| CN107312851A (en) | Myocardial infarction biomarker miR 1283 | |
| CN113981064B (en) | Biomarker for detecting diabetic retinopathy, detection kit and application | |
| CN110317878A (en) | A kind of long-chain non-coding RNA and its application for bladder cancer diagnosis and treatment monitoring | |
| CN111979315A (en) | Application of annular TP63 as lung squamous carcinoma diagnosis or treatment target | |
| CN116064513A (en) | Atherosclerosis plasma exosome miRNA marker group, acquisition method and application thereof | |
| CN105603117B (en) | MiR-3613 is used to distinguish lung squamous cancer transfer and non-diverting miRNA marker | |
| CN118562800A (en) | Annular non-coding RNA circTEX10 and application thereof | |
| CN109161596B (en) | Application of miR-129 and target gene thereof in detection of lung adenocarcinoma | |
| CN105664163A (en) | Application of mir-5010 and mature miRNA (micro ribonucleic acid) of mir-5010 in preparation of OSA (osteosarcoma) diagnosis and treatment preparation | |
| CN112813158B (en) | MiRNA marker related to myocardial fibrosis disease auxiliary diagnosis and application thereof | |
| CN107254537A (en) | The application of miR 1912 and its target gene in diagnosis and treatment myocardial infarction | |
| CN117007816B (en) | Application of target Nesfatin-1 in preventing or treating vascular calcification | |
| CN111455081B (en) | Application of lncRNA147410.3 in toxoplasma encephalopathy | |
| CN112941183B (en) | Application of non-coding gene miR-187-5p in primary liver cancer diagnosis and treatment | |
| CN115948546B (en) | Exosome miRNA biomarker for breast cancer and application thereof | |
| CN115074444B (en) | Application of miR-5189-3p in head and neck squamous cell carcinoma diagnosis and treatment | |
| CN109337926B (en) | Method for constructing haplotype chicken myoblasts with high let-7b mature body expression | |
| CN114767703B (en) | Application of miR-4311 mimic in preparation of lung cancer treatment drug | |
| CN113403390B (en) | Application of lncRNA in diagnosis and treatment of children myocarditis | |
| CN108676892B (en) | Colorectal cancer diagnosis marker METTL11A and application thereof | |
| CN107012257A (en) | Biomarker for diagnosis and treatment abdominal aneurvsm |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |