CN110734971A - group biomarker for diagnosing metabolic fatty liver mediated vascular diseases and application thereof - Google Patents
group biomarker for diagnosing metabolic fatty liver mediated vascular diseases and application thereof Download PDFInfo
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Abstract
The invention provides groups of markers for diagnosing metabolic fatty liver mediated vascular diseases, which comprise miR-240, miR-197, miR-2125, miR-923, miR-1796, miR-1367 and miR-1839, wherein the nucleotide sequences of the markers are shown in SEQ ID No. 1-7.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to groups of biomarkers for diagnosing cardiovascular diseases and application thereof.
Background
In recent years, the prevention and control situation of cardiovascular diseases in China is still severe, the mortality rate of the cardiovascular diseases is high and is in an ascending trend, data show that the number of the cardiovascular diseases in China is about 2.9 hundred million, the number of the cardiovascular diseases in China is increased from more than 250 ten thousand to nearly 400 ten thousand in 1990-2016, and the gross mortality rate of the cardiovascular diseases in China is increased from 220.8/10 ten thousand to 290.8/10 ten thousand in 1990-2016.
Cardiovascular diseases have no obvious clinical symptoms in early stage, patients are usually diagnosed only after physical examination or typical symptoms appear, and the optimal period of treatment is often missed. If the cardiovascular disease can be found in time in the early stage and the prognosis effect of the patient can be greatly improved by changing the life style or intervening with drugs. The current clinical diagnosis indexes of cardiovascular diseases mainly comprise: homocysteine (HCY), hypersensitive C-reactive protein (hs-CRP), Creatine Kinase (CK), myoglobin (Mb/Myo), Brain Natriuretic Peptide (BNP), Endothelin (ET) and the like, but the indexes are mostly used for diagnosing and typing the cardiovascular diseases, and the early and different stages of the cardiovascular diseases cannot be diagnosed.
With the intensive step of research on the action mechanism of miRNA and the research on the relation between miRNA and diseases by using the latest high-throughput technical means such as miRNA chips, the network understanding of higher eukaryotic gene expression regulation is improved to new levels, so that miRNA can become a new biological marker for disease diagnosis, molecule can become a drug target, or a new drug is developed by simulating molecule, and new means can be provided for the treatment of human diseases.
The blood contains abundant miRNA, which is convenient for detection, has sensitive index and has obvious relevance with specific diseases. Therefore, by a noninvasive means, a rapid, sensitive and specific quantitative detection method, miRNA with specific expression or abnormal expression in blood of a cardiovascular disease patient is screened as a molecular marker, and an auxiliary diagnosis kit for early and different stages of cardiovascular disease is developed, is used for detecting susceptible people of cardiovascular disease, evaluating the risk of suffering from cardiovascular disease and taking effective intervention measures, and has significant social value for inhibiting the current increasing cardiovascular disease incidence trend.
Disclosure of Invention
Based on the above purpose, the inventors found out group miRNAs with expression difference between metabolic fatty liver mediated vascular disease mice and normal mice through a large number of experiments, and suggested that the miRNAs can be used as markers for diagnosing cardiovascular diseases.
Specifically, the group of miRNAs are used as markers for cardiovascular disease diagnosis, the markers are miR-240, miR-197, miR-2125, miR-923, miR-1796, miR-1367 and miR-1839, and nucleotide sequences of the markers are shown in SEQ ID Nos. 1-7.
Table 1: the nucleotide sequence of miRNA used as cardiovascular disease diagnosis marker
| miRNAs | Gene sequences | SEQ ID No. |
| miR-240 | AGGCUGGAGAGAUGGCUCAGGUC | 1 |
| miR-97 | GGGACUGGUGAGAUGGCUCAGUGGG | 2 |
| miR-2125 | CUGCUUCUUGGGCUGUUUCAGGGGU | 3 |
| miR-923 | AGGGGCUGGUGAGAUGGCUCAGUG | 4 |
| miR-1796 | GUGGGUGCUGGGAACUGAACC | 5 |
| miR-1367 | CUGCUUCUUGGGCUGUUUCAGGGC | 6 |
| miR-1839 | GUGGGUGCUGGGAACUGAACC | 7 |
The invention also provides reagents for detecting miRNAs sequences, wherein the sequences comprise SEQ ID Nos. 1-7.
Preferably, the reagents are amplification primers for the sequences of the miRNAs.
Preferably, the amplification primer sequence is shown in SEQ ID No. 8-14.
The invention also provides application of the reagent in preparing a kit for diagnosing or evaluating the prognosis of the metabolic fatty liver-mediated vascular disease.
The miRNAs composition can be used for diagnosing the metabolic fatty liver mediated vascular heart disease, and experiments prove that compared with a normal control group, mRNA levels of miR-240, miR-197, miR-923, miR-1796 and miR-1839 in blood of a mouse with the metabolic fatty liver mediated vascular disease are remarkably increased, and mRNA levels of miR-2125 and miR-1367 are remarkably reduced, so that the miRNAs composition can be used as a molecular marker for diagnosing and evaluating the metabolic fatty liver mediated vascular disease, and has definite accuracy.
Drawings
FIG. 1 is a graph showing a plurality of vascular lesion indexes (A. represents vascular permeability after staining Ewensa blue; B. represents copolymerization coefficient of endo-splenic nitric oxide synthase having vasodilator function; C. copolymerization coefficient of vascular endothelial-link-associated transmembrane protein 1; D. copolymerization coefficient of vascular endothelial-link-associated transmembrane protein 2) of normal Control group (Control) and metabolic fatty liver-mediated vascular disease group (Model) mice.
FIG. 2: miR-240 in blood of mice in a normal Control group (Control) and a cardiovascular disease group (Model); miR-197; miR-2125; miR-923; miR-1796; miR-1367; the mRNA expression level of miR-1839 is shown in the figure (in the figure, novel-miR-240 is miR-240, novel-miR-197 is miR-197, novel-miR-2125 is miR-2125, novel-miR-923 is miR-923, novel-miR-1796 is miR-1796, novel-miR-1367 is miR-1367, and novel-miR-1839 is miR-1839).
Detailed Description
To more clearly and clearly demonstrate the technical solutions, objects and advantages of the present invention, the following detailed description is provided in conjunction with the specific embodiments and the accompanying drawings.
Example 1
The main reagents are absolute ethanol (LOT: 2013928, Daojing chemical Co., Ltd., Tianjin), DEPC (Sigma, LOT: WXBB3108V), chloroform (LOT: 20150601, Hongda chemical Co., Ltd., Tianjin), isopropanol (LOT: 20150410-2, chemical Co., Ltd., ), SuperReal Pre Mix × plus (SYBR Green) (TIANGEN BIOTECH (BEIJING) CO., LTD., LOT: 03120), MiScript SYBRGreen PCR Kit (Qiagen, Lot #218073), MiScript II RTKit (Qiagen, 218160) Trizol Reagent (Thermo Life technology, LOT: 28218).
Experimental methods
1. Blood collection
The C57 male mice were randomly divided into a normal group to which MCS feed (feed with normal content of choline methionine) was given and a model group to which MCD feed (feed lacking choline methionine) was given, and were fed for 8 weeks, and the model group caused cardiovascular disease in the mice. Blood was collected from the mice using a heart bleed method, approximately 500-. 2000g, centrifuged at 4 ℃ for 30min and the supernatant was aspirated. 10000g, centrifugation at 4 ℃ for 30min, and supernatant aspiration. Filtering with a 0.22 μm microporous filter membrane. 110000g, centrifugation at 4 ℃ for 70min to take the precipitate, washing with PBS and resuspending, 110000g, centrifugation at 4 ℃ for 70min to take the precipitate, and resuspending in 100ul PBS.
2. Total RNA extraction from blood
And (3) adding 1ml of Trizol into the sample, uniformly blowing and then standing for 5 min. Add 200. mu.l chloroform, shake repeatedly by hand about 15-20 times, then centrifuge at 12000g for 15min at 4 ℃. After completion of the centrifugation, 150. mu.l of the RNA-containing solution in the upper layer was taken out, 500. mu.l of isopropanol was added thereto, and the mixture was allowed to stand at room temperature for 10min to separate RNA, followed by 12000g, 10min, centrifugation at 4 ℃ and precipitation of RNA. After removing the supernatant, 1ml of pre-cooled 75% ethanol was added and the mixture was inverted repeatedly until the RNA pellet floated to remove impurities such as residual phenol. 7500g, 5min, centrifuging at 4 deg.C, removing supernatant, and blowing in a super clean bench for 15 min. Add 20. mu.l of DEPC water to dissolve the RNA. And (3) diluting the sample according to the ratio of 1:10 to detect the RNA content and purity. The A260/A280 is 1.8-2.0 as a qualified sample.
3. cDNA Synthesis
Qualified RNA samples were assayed and the concentration adjusted to 500 ng/. mu.l using DEPC water. Mu.l of the sample was subjected to reverse transcription to give cDNA. The system and the scheme are shown in the table 1.
Table 1: amplification system and method
4. Real-time fluorescent quantitative RT-qPCR gene expression detection
According to the results of the preliminary test, all samples were diluted 5-fold for gene expression amount measurement. The amplification sequences used in the real-time fluorescent quantitative RT-qPCR are shown in table 2, and the amplification systems and amplification conditions are shown in tables 3 and 4. The real-time fluorescent quantitative RT-qPCR amplification program is operated according to the fluorescent quantitative detection kit and amplified on a Bio-rad fluorescent quantitative PCR instrument. Analyzing the result to obtain Ct values of the reference gene and the target gene in each group, and applying 2-△△Ct is calculated by the formula.
Table 2: amplification primers for miRNAs
| miRNAs | Gene sequences | SEQ ID No. |
| miR_240 | aggctggagagatggctca | 8 |
| miR_197 | gggactggtgagatggctc | 9 |
| miR_2125 | actgcttcttgggctgtttcag | 10 |
| miR_923 | ttattatggggggggatggggtg | 11 |
| miR_1796 | gtgggtgctgggaactgaa | 12 |
| miR_1367 | cctgcttcttgggctgtttcag | 13 |
| miR_1839 | gtgggtgctgggaactgaa | 14 |
Table 3: amplification system
Table 4: amplification conditions
II, experimental results:
SPSS20.0 statistical software is adopted, and the group comparison is analyzed by a least significant difference method (LSD); as shown in FIG. 2, mRNA levels of novel-miR-240, novel-miR-197, novel-miR-923, novel-miR-1796 and novel-miR-1839 in the cardiovascular disease group (Model) are obviously higher than those of the normal control group (Ctrl) (P < 0.01); mRNA levels of novel-miR-2125 and novel-miR-1367 in the cardiovascular disease group are obviously lower than those in a normal control group (P < 0.01). Therefore, mRNA levels of novel-miR-240, novel-miR-197, novel-miR-923, novel-miR-1796 and novel-miR-1839 are obviously increased in the blood of patients with cardiovascular diseases, and mRNA levels of novel-miR-2125 and novel-miR-1367 are obviously reduced in the blood of patients with cardiovascular diseases, so that the mRNA level can be used as a molecular index for cardiovascular disease detection.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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<400>13
cctgcttctt gggctgtttc ag 22
<210>14
<211>19
<212>DNA
<213> Artificial Synthesis
<400>14
Claims (5)
- The group of markers for diagnosing metabolic fatty liver mediated cardiovascular diseases is characterized by comprising miR-240, miR-197, miR-2125, miR-923, miR-1796, miR-1367 and miR-1839, and the nucleotide sequences of the markers are shown in SEQ ID Nos. 1-7.
- 2, reagent for detecting miRNAs sequences, which is characterized in that the sequences comprise SEQ ID No. 1-7.
- 3. The reagent of claim 2 wherein said reagent is an amplification primer for said miRNAs sequence.
- 4. The reagent of claim 3, wherein the amplification primer sequence is shown as SEQ ID Nos. 8-14.
- 5. Use of the reagent of any one of claims 2 to 5 to in the preparation of a kit for diagnosing or assessing the prognosis of a metabolic fatty liver mediated cardiovascular disease.
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