CN111057760A - Coronary heart disease nucleic acid molecular marker and primer and application thereof - Google Patents

Coronary heart disease nucleic acid molecular marker and primer and application thereof Download PDF

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CN111057760A
CN111057760A CN202010032220.8A CN202010032220A CN111057760A CN 111057760 A CN111057760 A CN 111057760A CN 202010032220 A CN202010032220 A CN 202010032220A CN 111057760 A CN111057760 A CN 111057760A
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heart disease
coronary heart
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郑芳
梁宾
干学东
李梦兰
向阳
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Wuhan University WHU
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Abstract

The invention provides a coronary heart disease nucleic acid molecular marker, a primer and application thereof, wherein the marker is circular RNA hsa _ circ _0000615, the nucleotide sequence of the marker is shown in SEQ ID NO.1, and the marker can be used for auxiliary diagnosis of coronary heart disease. The coronary heart disease nucleic acid molecular marker provided by the invention has a higher diagnosis value for coronary heart disease, and the coronary heart disease auxiliary diagnosis kit can be prepared by using the primer of the coronary heart disease nucleic acid molecular marker, is used for detecting the expression level of the coronary heart disease nucleic acid molecular marker and assisting in diagnosing coronary heart disease, so that the diagnosis of coronary heart disease is more convenient and easier.

Description

Coronary heart disease nucleic acid molecular marker and primer and application thereof
Technical Field
The invention belongs to the field of biomedical specialty, and particularly relates to a coronary heart disease nucleic acid molecular marker, and primers and application thereof.
Background
Coronary heart disease (CAD) is also called coronary atherosclerotic heart disease, and is a group of diseases which cause plaque formation, even rupture and complete blockage of blood vessels due to atherosclerotic lesions of coronary arteries, cause myocardial ischemia, hypoxia and even necrosis, and cause angina pectoris, myocardial infarction and other serious symptoms clinically. The incidence of coronary heart disease is rising year by year, and the coronary heart disease becomes the first killer seriously threatening the health and the life quality of human beings due to high disability rate, high death rate, high recurrence rate and more complications.
The occurrence and development of coronary heart disease involve various pathological processes such as chronic inflammation, oxidative stress, lipid metabolism and the like, and are the result of the combined action of environmental factors and genetic factors. The diagnosis method of coronary heart disease mainly includes electrocardiogram, coronary artery CT, coronary artery angiography, etc. However, the sensitivity and specificity of electrocardiograms are low; coronary CT requires specialized instrumentation and diagnostics; coronary angiography has a high rate of certainty, but many low-income families and patients with mild symptoms and thought conservation are reluctant to receive such invasive examinations. Therefore, a low-cost and convenient method for assisting the early diagnosis of coronary heart disease is urgently needed. The nucleic acid biomarkers are rapidly developed, particularly non-coding RNA, and relative stability and detection convenience of the nucleic acid biomarkers are generally favored, so micro RNA kits for liver cancer auxiliary diagnosis approved by CFDA are already on the market.
Circular RNAs (circrnas) are a class of non-coding RNAs that are abundantly expressed in eukaryotic cells and are formed by reverse splicing of one or more exons, introns, or both. Because the RNA does not have a 5 'end cap structure and a 3' end poly (A) tail structure, the RNA can resist digestion of nuclease and has higher stability than linear RNA. Increasing research has demonstrated that circular RNA plays a very important role in gene expression and regulation. At present, the circRNA plays a great potential as a nucleic acid molecular marker in disease diagnosis due to the characteristics of abundance, conservation, stable structure and the like.
Disclosure of Invention
The invention aims to provide a coronary heart disease nucleic acid molecular marker, which is ring-shaped RNAhsa _ circ _0000615, has the expression quantity related to the occurrence of coronary heart disease and has important clinical significance.
The invention also aims to provide an application of the coronary heart disease nucleic acid molecular marker in auxiliary diagnosis of coronary heart disease, which has certain diagnostic value on coronary heart disease by detecting the expression level of peripheral blood leukocyte hsa _ circ _0000615 to assist in diagnosing coronary heart disease.
The invention also aims to provide a primer of the coronary heart disease nucleic acid molecular marker, which can be used for determining the hsa _ circ _0000615 content in peripheral blood leukocytes, so that the auxiliary diagnosis of the coronary heart disease is more convenient and easier.
The fourth purpose of the invention is to provide the application of the primer of the coronary heart disease nucleic acid molecular marker, and the primer is used for preparing an auxiliary diagnosis kit for coronary heart disease, and is used for quickly, simply and conveniently detecting the content of hsa _ circ _0000615 in peripheral blood leukocytes.
One of the purposes of the invention adopts the following technical scheme:
a coronary heart disease nucleic acid molecular marker is circular RNA hsa _ circ _0000615, and the nucleotide sequence of the marker is shown in SEQ ID NO. 1.
Further, the markers are from peripheral blood leukocytes.
The second purpose of the invention is realized by adopting the following technical scheme:
an application of the nucleic acid molecular marker for coronary heart disease in auxiliary diagnosis of coronary heart disease is disclosed.
Further, the hsa _ circ _0000615 expression level is increased, indicating that the patient has coronary heart disease.
The third technical scheme for realizing the purpose of the invention is as follows:
a primer of a coronary heart disease nucleic acid molecular marker is disclosed, and the sequence of the primer is shown as SEQ NO. 2 and SEQ NO. 3.
The fourth purpose of the invention is realized by adopting the following technical scheme:
an application of a primer of a coronary heart disease nucleic acid molecular marker, and reagents such as the primer are applied to the preparation of an auxiliary diagnostic kit for coronary heart disease.
Further, the coronary heart disease auxiliary diagnosis kit is used for detecting a coronary heart disease nucleic acid molecular marker, and comprises the following components:
(1) the primer of the coronary heart disease nucleic acid molecular marker;
(2) extracting peripheral blood leukocyte RNA reagent;
(3) reagents required for reverse transcription;
(4) reagents required for fluorescent quantitative PCR.
Wherein the reagent for extracting the RNA of the peripheral blood leukocytes comprises Trizol reagent, chloroform, isopropanol, 75% ethanol and RNase-free water; the reagent required for reverse transcription is from a reverse transcription kit; reagents required for the fluorescent quantitative PCR comprise GAPDH internal reference upstream and downstream primers, SYBR dye and RNase-free water.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the expression level of the circular RNA hsa _ circ _0000615 in the peripheral blood leukocyte is found to be related to the coronary heart disease for the first time through research, experiments prove that the mRNA expression level of hsa _ circ _0000615 in the peripheral blood leukocyte of a patient with the coronary heart disease is remarkably increased compared with that of a healthy population, and whether the subject suffers from the coronary heart disease can be judged in an auxiliary way by detecting the mRNA expression level of hsa _ circ _0000615 in the peripheral blood leukocyte, so that hsa _ circ _0000615 can be used as a nucleic acid molecular marker of the coronary heart disease for auxiliary diagnosis of the coronary heart disease, and has higher diagnostic value on the coronary heart disease; meanwhile, the coronary heart disease nucleic acid molecular marker primer is used for preparing a coronary heart disease auxiliary diagnosis kit, is used for detecting the expression level of a coronary heart disease nucleic acid molecular marker hsa _ circ _0000615, and assists in diagnosing the coronary heart disease, so that the diagnosis of the coronary heart disease is more convenient and easier.
Drawings
FIG. 1 is a graph showing the comparison between the expression levels of hsa _ circ _0000615 in peripheral blood leukocytes in a coronary heart disease group (CAD) and a normal control group (control) in the present example;
FIG. 2 is a ROC curve showing the expression level of hsa _ circ _0000615 in peripheral blood leukocytes of a coronary heart disease group (CAD) and a normal control group (control) in the present example.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
The experimental procedures in the following examples, in which specific conditions are not noted, are conventional procedures or conventional conditions well known in the art, or conditions as recommended by the manufacturer.
[ example ] hsa _ circ _0000615 differentially expressed in coronary heart disease and controls
First, experimental object
Peripheral blood was collected from 2016 to 2017, 5 months, for a total of 330 CAD patients (CAD) and 209 apparently healthy controls (controls) at the south hospital, university of wuhan. All patients and healthy controls were han nationality. According to the American Heart Association/American Heart Association (ACC/AHA) guidelines, the diagnostic criteria for coronary heart disease is a coronary angiographic evidence of stenosis in at least one coronary artery of greater than or equal to 50%. Subjects were excluded according to the following criteria: 1) heart diseases include congenital heart disease, myocardial bridge, cardiomyopathy, or severe non-coronary cardiovascular disease; 2) systemic acute and chronic infection or inflammatory disease; 3) a malignant tumor; 4) (ii) an autoimmune disease; 5) liver and kidney dysfunction; 6) endocrine diseases such as thyroid diseases.
The control group (control) was a physical examiner who was age and gender matched with coronary heart disease patients (CAD) from a physical examination center, and had no heart disease or any other disease mentioned above. The study was approved by the ethics committee of the south hospital, wuhan university, and informed consent was obtained from all participants.
Second, Experimental methods
1. Peripheral blood leukocyte RNA extraction (TRIzol method)
(1) 1mL of fresh EDTA-K2 anticoagulated whole blood was collected in a 1.5mL EP tube and centrifuged at 3000rpm in a 4 ℃ centrifuge for 8min to separate blood cells and plasma.
(2) Transferring the blood cells obtained by centrifugation into a 10mL centrifuge tube, adding 3mL of 1 × erythrocyte lysate according to the proportion of 1:3, turning upside down for 10-15 times, fully mixing, carrying out ice bath for 30-40 min, and turning upside down again for 3-5 times during the period.
(3) Centrifuging at 4 deg.C and 12000rpm for 10min, discarding supernatant, adding 2mL of 1 × erythrocyte lysate, shaking thoroughly, mixing, and ice-cooling for several minutes.
(4) Centrifuging at 4 ℃ and 12000rpm for 5min, removing the supernatant, collecting the white blood cells, adding 1mL of Trizol into an EP tube, repeatedly blowing until the white blood cells are completely dissolved, and standing at room temperature for 5-8 min.
(5) Adding 200 μ L chloroform, shaking vigorously with vortex oscillator for 30s, and standing at room temperature for 5 min.
(6) Centrifuge at 12000rpm for 15min at 4 ℃ and transfer the upper aqueous phase to a new 1.5mL EP tube.
(7) Adding isopropanol with the same volume, reversing the mixture up and down for about ten times, and standing the mixture for 1h in a refrigerator at the temperature of-20 ℃.
(8) The RNA was precipitated at the bottom of the tube by centrifugation at 12000rpm for 10min at 4 ℃.
(9) The supernatant was discarded, 1mL of pre-cooled 75% ethanol (prepared with RNase-free water) was added, the bottom of the tube was flicked until the RNA pellet floated, the pellet was washed thoroughly by flipping several times upside down, and centrifuged at 8500rpm for 5min at 4 ℃.
(10) And (5) repeating the step (9).
(11) Discarding supernatant, placing in an ultra-clean bench for naturally drying, adding 30 μ L of RNase-free water preheated at 65 deg.C, and storing in a refrigerator at-80 deg.C after RNA is completely dissolved.
2. Reverse transcription
cDNA was synthesized according to the protocol of TOYOBO reverse transcription kit ReverTraace qPCR RT Master Mix with gDNARemover. The method comprises the following steps: (1) RNA denaturation: for each sample, 1.0. mu.g of RNA was taken and the volume was made up to 12. mu.L with Nuclean-free water. Place on PCR machine, set program for 5min at 65 ℃.
(2) Removal of genomic DNA: 4.0. mu.L of 4 XDN Master Mix was added to (1), mixed by gentle shaking, spotted off and placed on a PCR instrument at 37 ℃ for 5 min.
(3) Reverse transcription: adding 4.0 μ L of 5 XTT Master Mix II into (2), shaking gently, mixing, spotting, placing on a PCR instrument, reacting at 37 deg.C for 15 min; 50 ℃ for 5 min; at 98 deg.C for 5 min.
3. Real-time fluorescent quantitative PCR (RT-PCR):
(1) RT-PCR reaction system (20. mu.L) is as follows in Table 1:
TABLE 1
Reagent Volume (μ L)
2×UltraSYBR Mixture 10
Forward Primer(10μM) 1
Reverse Primer(10μM) 1
cDNA 2
ddH2O 6
(2) The primer sequences are shown in Table 2 below:
TABLE 2
hsa _ circ _0000615 forward primer 5'-TTGGGAACTAAACCGGAGCC-3'(SEQ NO:2)
hsa _ circ _0000615 reverse primer 5'-TCAGACCTGCCACATTGGTC-3'(SEQ NO:3)
GAPDH reference specificity PCR forward primer 5'-TGTTGCCATCAATGACCCCTT-3'(SEQ NO:4)
GAPDH internal reference specificity PCR reverse primer 5'-CTCCACGACGTACTCAGCG-3'(SEQ NO:5)
Real-time fluorescent quantitative PCR reaction program: 5min at 95 ℃; 40 cycles (95 ℃ 30s, 60 ℃ 30s, 72 ℃ 30 s). SYBR Green was used as a fluorescent marker, PCR was performed on a Bio-Rad fluorescent quantitative PCR instrument, and GAPDH was used as an internal reference to calculate the relative expression level using the 2- Δ Ct method, as shown in the following formula: Δ Ct ═ Ct (hsa _ circ _0000615) -Ct (gapdh).
4. Statistical analysis:
the diagnostic value of hsa _ circ _0000615 for CAD was assessed by the Receiver-operator characterization (ROC). Data were analyzed using SPSS22.0 and Graphpad Prism 6.0 software. P <0.05 was considered statistically significant.
Third, experimental results
As shown in fig. 1, the expression level of peripheral blood leukocyte hsa _ circ _0000615 was significantly up-regulated in the CAD case group compared to the normal group (P < 0.0001).
As shown in FIG. 2, ROC curve analysis shows that hsa _ circ _0000615 has better diagnostic value (AUC 0.761P <0.0001) for coronary heart disease as a nucleic acid molecular marker, and the sensitivity and specificity are 61.5% and 80.4%, respectively.
In conclusion, the invention discovers that the expression level of the circular RNA hsa _ circ _0000615 in the peripheral blood leukocyte is related to the coronary heart disease for the first time through research, experiments prove that the mRNA expression level of hsa _ circ _0000615 in the peripheral blood leukocyte of a patient with the coronary heart disease is obviously increased compared with that of a healthy population, and whether the patient suffers from the coronary heart disease can be assisted and judged by detecting the mRNA expression level of hsa _ circ _0000615 in the peripheral blood leukocyte. The annular RNAhsa _ circ _0000615 can be used as a nucleic acid molecular marker of coronary heart disease, is used for auxiliary diagnosis of coronary heart disease, and has high diagnostic value on coronary heart disease. Meanwhile, the invention provides an auxiliary diagnosis kit for coronary heart disease, which is used for detecting the expression level of hsa _ circ _0000615 and assisting in diagnosing coronary heart disease, so that the diagnosis of coronary heart disease is more convenient and easier.
Sequence listing
<110> Wuhan university
<120> coronary heart disease nucleic acid molecular marker, and primer and application thereof
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>874
<212>DNA
<213>hsa_circ_0000615(hsa_circ_0000615)
<400>1
caatgatgtt gtccactggg catgtactga ccaatgtggc aggtctgaga acatagctga 60
agctgaaaat aggaaagctg ggggcaagga agagccttga atcttgaggt gggacgttga 120
ctctaagatg tccttgagca gtggagcctc cggagggaaa ggagtggatg caaacccggt 180
tgagacatac gacagtgggg atgaatggga cattggagta gggaatctca tcattgacct 240
ggacgccgat ctggaaaagg accagcagaa actggaaatg tcaggctcaa aggaggtggg 300
gataccggct cccaatgctg tggccacact accagacaac atcaagtttg tgaccccagt 360
gccaggtcct caagggaagg aaggcaaatc aaaatccaaa aggagtaaga gtggcaaaga 420
cactagcaaa cccactccag ggacttccct gttcactcca agtgaggggg cagctagcaa 480
gaaagaggtg caggggcgct caggagatgg tgccaatgct ggaggcctgg ttgctgctat 540
tgctcccaag ggctcagaga aggcggctaa ggcatcccgc agtgtagccg gttccaaaaa 600
ggagaaggag aacagctcat ctaagagcaa gaaggagaga agcgaaggag tggggacttg 660
ttcagaaaag gatcctgggg tcctccagcc agttcccttg ggaggacggg gtggtcagta 720
tgatggaagt gcaggggtgg atacaggagc tgtggagcca cttgggagta tagctattga 780
gcctggggca gcgctcaatc ctttgggaac taaaccggag ccagaggaag gggagaatga 840
gtgtcgcctg ctaaagaaag tcaagtctga aaag 874
<210>2
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ttgggaacta aaccggagcc 20
<210>3
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tcagacctgc cacattggtc 20
<210>4
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
tgttgccatc aatgacccct t 21
<210>5
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
ctccacgacg tactcagcg 19

Claims (7)

1. A coronary heart disease nucleic acid molecular marker, characterized in that: the marker is circular RNA hsa _ circ _0000615, and the nucleotide sequence of the marker is shown in SEQ ID NO. 1.
2. The coronary heart disease nucleic acid molecular marker of claim 1, wherein: the markers are derived from peripheral blood leukocytes.
3. The use of the nucleic acid molecular marker for coronary heart disease according to claim 1 or 2 in the auxiliary diagnosis of coronary heart disease.
4. The application of the nucleic acid molecular marker for coronary heart disease according to claim 3 in the auxiliary diagnosis of coronary heart disease, wherein: the hsa _ circ _0000615 expression level is increased, which indicates that the patient has coronary heart disease.
5. The primer of the coronary heart disease nucleic acid molecular marker of claim 1, wherein: the sequences of the primers are shown as SEQ NO. 2 and SEQ NO. 3.
6. The application of the primer of the coronary heart disease nucleic acid molecular marker in claim 5, wherein the primer comprises: the primer is applied to the preparation of an auxiliary diagnosis kit for coronary heart disease.
7. The use of the primer of the coronary heart disease nucleic acid molecular marker of claim 6, wherein: the coronary heart disease auxiliary diagnosis kit is used for detecting the coronary heart disease nucleic acid molecular marker and comprises the following components:
(1) the primer of the coronary heart disease nucleic acid molecular marker;
(2) extracting peripheral blood leukocyte RNA reagent;
(3) reagents required for reverse transcription;
(4) reagents required for fluorescent quantitative PCR;
wherein the reagent for extracting the RNA of the peripheral blood leukocytes comprises Trizol reagent, chloroform, isopropanol, 75% ethanol and RNase-free water; the reagent required for reverse transcription is from a reverse transcription kit; reagents required for the fluorescent quantitative PCR comprise GAPDH internal reference upstream and downstream primers, SYBR dye and RNase-free water.
CN202010032220.8A 2020-01-13 2020-01-13 Coronary heart disease nucleic acid molecular marker and primer and application thereof Withdrawn CN111057760A (en)

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CN111471762A (en) * 2020-05-29 2020-07-31 武汉大学 Coronary heart disease nucleic acid molecular marker and primer and application thereof
CN111850112A (en) * 2020-08-14 2020-10-30 蒋沁 Biomarker for detecting eye-base vascular diseases, detection kit and application
CN112899362A (en) * 2021-03-19 2021-06-04 福建医科大学 CircRNA marker for coronary heart disease risk assessment and diagnosis and detection reagent and kit thereof
CN113549688A (en) * 2021-08-27 2021-10-26 河北医科大学第二医院 Group of molecular markers for diagnosing coronary artery disease
CN113652479A (en) * 2021-08-27 2021-11-16 河北医科大学第二医院 Coronary artery disease diagnosis product based on molecular marker and application thereof
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111471762A (en) * 2020-05-29 2020-07-31 武汉大学 Coronary heart disease nucleic acid molecular marker and primer and application thereof
CN111850112A (en) * 2020-08-14 2020-10-30 蒋沁 Biomarker for detecting eye-base vascular diseases, detection kit and application
CN111850112B (en) * 2020-08-14 2021-04-23 蒋沁 Biomarker for detecting eye-base vascular diseases, detection kit and application
CN112899362A (en) * 2021-03-19 2021-06-04 福建医科大学 CircRNA marker for coronary heart disease risk assessment and diagnosis and detection reagent and kit thereof
CN113549688A (en) * 2021-08-27 2021-10-26 河北医科大学第二医院 Group of molecular markers for diagnosing coronary artery disease
CN113652479A (en) * 2021-08-27 2021-11-16 河北医科大学第二医院 Coronary artery disease diagnosis product based on molecular marker and application thereof
CN113549688B (en) * 2021-08-27 2023-08-04 河北医科大学第二医院 A set of molecular markers for diagnosing coronary artery disease
CN113652479B (en) * 2021-08-27 2023-10-20 河北医科大学第二医院 Diagnostic product for coronary artery disease based on molecular marker and application thereof
CN114058693A (en) * 2021-11-22 2022-02-18 中国人民解放军总医院第二医学中心 Coronary heart disease marker PRKAB2 and application thereof
CN115976208A (en) * 2022-10-24 2023-04-18 新乡医学院 Application of circular RNA circ _0000615 as marker in preparation of colorectal cancer detection kit

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Application publication date: 20200424