CN108796070B - Application of miR-125a-3p in preparation of cardiovascular disease diagnosis kit - Google Patents
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention belongs to the field of biomedical specialty, and particularly relates to application of miR-125a-3p in preparation of a cardiovascular disease diagnosis kit. The diagnostic kit uses miR-125a-3p protein as a marker to detect the expression quantity of the protein in blood plasma, and performs early diagnosis on cardiovascular diseases. Meanwhile, the function regulation and control effect on the mononuclear macrophages is remarkable, the phagocytosis of lipid components in blood vessels is effectively and quickly promoted, the inflammation level of hyperlipidemia and arteriosclerosis is further controlled, and the application prospect is very good.
Description
Technical Field
The invention belongs to the field of biomedical specialty, and particularly relates to application of miR-125a-3p in preparation of a cardiovascular disease diagnosis kit.
Background
Cardiovascular diseases are serious diseases endangering human health and are one of the main causes of death. The medicine has high morbidity, high disability rate, high mortality, high recurrence rate and many complications, and seriously threatens the life and health of human beings. Currently, cardiovascular disease has become second killer to malignant tumors in our national resident's death spectrum, and millions of other people suffer from the disease every year, resulting in disability. Atherosclerosis (AS) is a common disease and frequently encountered disease in the world, is a main cause of various diseases such AS coronary heart disease and cerebral apoplexy, seriously harms human health and life quality, and has become a focus of attention in the cardiovascular field. The main cause of the atherosclerosis is that excessive fat is deposited on the inner wall of a blood vessel due to various reasons, so that the blood vessel wall is thickened, the blood flow is influenced, and meanwhile, excessive lipid in the blood can activate inflammatory reaction to gradually erode the blood vessel wall. With the progress of arteriosclerosis, the vessel wall is further hardened, and the excessive lipid in the blood can block the blood vessel, so that the blood cannot normally pass through to form thrombus, thereby causing stroke and myocardial infarction. When the cardiovascular disease is developed to the later stage, due to the long-term poor blood pumping of the heart, the organs of the whole body can be damaged to different degrees due to blood stasis and oxygen deficiency, such as various complications of lung infection, liver cirrhosis, hypertension, renal failure and the like occur, and the symptoms can aggravate the condition of the cardiovascular disease and cause more serious damage to the body.
MicroRNAs (miRNAs, miRs) are endogenous non-coding small RNAs with a length of 18-25 single-stranded nucleotides, and are generated by processing precursors with enzymes, and the sequence of the precursors is generally 70-120 nucleotides. The mechanism of action of mirnas is mainly manifested by degradation or inhibition of target genes. 2-8 bases at the' end of miRNA5 are called miRNA seed sequences. Through the interaction of the seed sequence and the 5' -UTR and 3' -UTR ' regions of the target mRNA, the gene or protein expression is regulated at the transcription or translation level, and the gene or protein is involved in various biological processes, and the evolutionary process is conserved. Each miRNA can localize to multiple mrnas, inhibiting their transcription or causing their degradation, and thus many transcripts may be finely regulated by mirnas, allowing efficient operation of the transcription-translation system, and thus allowing cells to rapidly and effectively control various physiological or pathological processes. It is currently believed that mirnas function in essential processes of life such as growth and development, organogenesis, hematopoiesis, cell proliferation and apoptosis, stress response, and tumorigenesis. Since miRNA exists in human body fluid and blood system stably, it has repeatability, can be detected stably from peripheral blood, and can be used as biological marker of disease.
The discovery of miRNA associated with cardiovascular disease may have a significant impact on gene therapy associated with the disease. Through the miRNA differential expression analysis of patients with cardiovascular diseases and normal people, related sensitive miRNA can be identified, and further, the expression quantity of related miRNA or precursor thereof is regulated and controlled by introducing specific miRNA complementary antisense nucleotide inhibition or a method of virus vector or plasmid vector overexpression, so that the purpose of treating cardiovascular diseases is achieved. Therefore, the discovery of specifically expressed mirnas is of great importance for the diagnosis and treatment of cardiovascular diseases. Research shows that various serum microRNAs are closely related to the development process of arteriosclerosis, and the expression level of the microRNAs presents organ specificity and tissue specificity. Changes in specific microRNA expression profiles can indicate specific pathological and physiological processes in vivo. Research proves that miR-21-3p can promote vascular smooth muscle proliferation so as to promote atherosclerosis; miR-155 is closely related to vascular inflammatory reactions such as oxidized low density lipoprotein and the like; miR-126-3p is closely related to vascular stability, integrity and expression of adhesion molecules; miR-210 can promote vascular proliferation in an anoxic state and is anoxic specific microRNA; the miR-221-3p and miR-222-3p have the effects of resisting vascular proliferation and inhibiting endothelial cell migration proliferation and angiogenesis. However, the effect of miR-125a-3p in cardiovascular diseases, particularly hyperlipidemia and atherosclerosis, is not reported in the literature.
For patients with hyperlipidemia and atherosclerosis, due to high blood lipid content, inflammatory cells in blood such as monocyte macrophage are focused on damaged vascular wall by action of chemotactic factor, phagocytose lipid components, and form foam cells. When the mononuclear macrophage is affected by environmental factors and the immune function is weakened and lipid components in blood vessels cannot be effectively removed in time, lipid accumulation and arteriosclerosis are accelerated to develop, and severe consequences such as stroke, myocardial infarction and the like are finally caused. The preliminary research of the subject group of the inventor shows that the expression level of miR-125a-3p in peripheral blood mononuclear cells of patients with hyperlipidemia and atherosclerosis is specifically reduced, and the characteristic expression can be used as a marker of hyperlipidemia and atherosclerosis; because miR-125a-3p has an obvious regulation effect on the function of mononuclear macrophages, the miR-125a-3p expression level in the mononuclear macrophages of a patient can be increased by using a virus vector or plasmid vector overexpression method, so that the mononuclear macrophages of the patient can phagocytize and treat lipid components in blood vessels normally, and the development of hyperlipidemia and arteriosclerosis can be effectively controlled. The discovery of the function of the miR-125a-3p has extremely important significance for diagnosing and treating cardiovascular diseases such as hyperlipidemia, atherosclerosis and the like.
The mature bodies of human miR-125a-3p (has-miR-125 a-3p, MIMAT 0004602) and murine miR-125a-3p (mmu-miR-125 a-3p, MIMAT 0004528) provided by the invention both comprise the following sequences:
5’- ACAGGUGAGGUUCUUGGGAGCC -3’(SEQ ID1)。
the mir-125a (MI 0000151) stem-loop structure sequence is as follows:
CUGGGUCCCUGAGACCCUUUAACCUGUGAGGACGUCCAGGGUCACAGGUGAGGUUCUUGGGAGCCUGG(SEQ ID2)。
disclosure of Invention
The invention aims to provide a new application of miR-125a-3 p. Through previous researches, the inventor discovers that the following SEQ ID1 and SEQ ID2 can be used as markers of diseases related to hyperlipidemia and atherosclerosis, and particularly miR-125a-3p imic has a good technical effect on improving the microenvironment of hyperlipidemia and atherosclerosis inflammation, so that the invention is completed.
The invention adopts the following technical scheme:
the application of miR-125a-3p in preparing a cardiovascular disease diagnosis kit is characterized in that miR-125a-3p protein is used as a marker to detect the expression quantity of the miR-125a-3p protein in blood plasma, and early diagnosis of cardiovascular diseases is carried out.
The diagnostic kit comprises the following components: 10 × miRNA reverse transcription primer, 20 × miRNA upstream primer, 20 × miRNA universal primer, 4 × One step miRNA RT Solution, 5 × SYBR Green qPCR Mix and 50 × ROX Reference Dye for miR-125a-3 p.
The miR-125a-3p can inhibit the expression of mononuclear macrophage inflammatory factors, and can be used for preparing a medicament for treating cardiovascular diseases, wherein the cardiovascular diseases are hyperlipidemia or atherosclerosis.
Specifically, the invention uses human peripheral blood mononuclear cells, C57BL/6 wild type mice (WT mice), ApoE gene knockout mice (ApoE) -/- Mouse) heart and aorta blood vessel of the hyperlipemia model are taken as materials to carry out real-time fluorescence quantitative PCR analysis of miRNA molecules. The general molecular biology technology is adopted to extract total RNA from cells and tissues, and the total RNA is subjected to reverse transcription and PCR amplification through specific primers. The detection result of the fluorescence real-time quantification (qRT-PCR) technology shows that the expression level of the miR-125a-3p is obviously reduced in a high-fat state; the expression of miR-125a-3p is inhibited by using specific complementary antisense nucleotide, and the level expression of monocyte inflammatory factor is up-regulated. And the expression of miR-125a-3p in cells is increased by using the synthesized miR-125a-3p precursor RNA, so that the expression of inflammatory factors can be obviously reduced.
The hsa-miR-125a-3p target genes are subjected to predictive analysis through websites such as miRBase, targetScan and mirpathDB, and the result shows that the miR-125a-3p regulated target genes mainly comprise: cytokines related to inflammation, such as CCL4, NCR2, IL10, TLR9, IL-33, IL-1R1, CCR3 and IL-4R, CCL 25.
Therefore, when the sequences SEQ ID1 and SEQ ID2 of miRNA are respectively applied to a subject, the function control effect on the mononuclear macrophage is remarkable, the phagocytosis of lipid components in blood vessels is effectively and quickly promoted, and the inflammation level of hyperlipidemia and arteriosclerosis is controlled.
Drawings
FIG. 1 shows the expression of miR-125a-3p in peripheral blood mononuclear cells of patients with hyperlipidemia and atherosclerosis.
FIG. 2 shows ApoE -/- Expression of mouse model blood vessel and heart tissue miR-125a-3 p.
FIG. 3 shows the expression of miR-125a-3p in mononuclear macrophages under high-fat environment.
FIG. 4 is a graph showing the effect of miR-125a-3p imic on the level of inflammatory factors in mononuclear macrophages in a high-fat environment.
Detailed Description
In preparing a cardiovascular disease diagnostic kit, the kit comprises the following components: 10 XmiRNA reverse transcription primer, 20 XmiRNA upstream primer, 20 XmiRNA universal primer, 4 Xone step miRNA RT Solution, 5 XSYBR Green qPCR Mix and 50 XROX Reference Dye aiming at miR-125a-3 p.
The invention is further illustrated by the following examples.
The RNA sequences disclosed in the invention can be obtained by artificial synthesis or other biological methods.
Example 1: comparing the expression level of miR-125a-3p in peripheral blood mononuclear cells of healthy people and hyperlipidaemia arteriosclerosis patients.
1. Human peripheral blood mononuclear cell separation
The method adopts a Ficoll density gradient centrifugation method to separate peripheral blood mononuclear cells, and comprises the following specific operation modes:
(1) transferring 10ml of heparin anticoagulated venous whole blood into a 50ml centrifuge tube, adding 10ml of Phosphate Buffer Solution (PBS) solution for dilution, and gently mixing uniformly;
(2) two 15ml centrifuge tubes were taken and 5ml of Ficoll solution was added first. Then, gently adding the diluted blood to the upper layer of the Ficoll liquid of the two centrifuge tubes, wherein the Ficoll liquid must be gentle so as to avoid mixing the two solutions together, and 10ml of the diluted blood is added into each centrifuge tube;
(3) and (3) centrifuging for 30 minutes at 800g, wherein no break is set in the speed reduction setting, and after the centrifugation is finished, the tube is divided into three layers, wherein the upper layer is blood plasma and PBS, and the lower layer is mainly red blood cells and granulocytes. There is a narrow band of white cloud layer mainly containing mononuclear cells at the interface of the upper and lower layers, and the layer of cells is sucked into another clean 15ml centrifuge tube by a pipette.
(4) PBS was added in a volume of 5 times or more, centrifuged at 800 g.times.5 minutes at room temperature, and the cells were washed twice.
(5) After resuspending the cells, erythrocyte lysate was added, and the mixture was left at room temperature for 3 minutes and centrifuged at 800 g.times.5 minutes.
(6) After centrifugation, the supernatant was discarded, and the cells were resuspended by adding serum-free RPMI1640 medium. Centrifuge at room temperature for 800 g.times.5 min and wash the cells twice with serum free RPMI 1640. Finally, the cells were resuspended by adding RPMI1640 medium containing 10% calf serum.
(7) A drop of cell suspension was mixed with a drop of 0.2% trypan blue stain, counted on a hemocytometer, and cell viability was measured. The percentage of viable cells above 95% allows for further experiments.
2. Total RNA extraction from cells (TRIzol method)
(1) Harvesting of cells 1-5X 10 7 The cells were transferred to a 1.5ml centrifuge tube, 1ml Trizol was added thereto, mixed well, and allowed to stand at room temperature for 5 min.
(2) 0.2ml of chloroform was added thereto, and the mixture was shaken for 15 seconds and allowed to stand for 2 min.
(3) Centrifuging at 4 deg.C, 12000g × 15min, and collecting supernatant.
(4) 0.5ml of isopropanol was added, and the liquid in the tube was gently mixed and left to stand at room temperature for 10 min.
(5) Centrifugation is carried out at 4 ℃, 12000g multiplied by 10min, and supernatant is discarded.
(6) 1ml of 75% ethanol was added and the precipitate was washed gently. At 4 deg.C, 7500g × 5min, discard the supernatant.
(7) Air-drying the specimen, and adding an appropriate amount of DEPC water to dissolve (promoting dissolution at 65 ℃ for 10-15 min). And determining OD260 and OD280 values to preliminarily judge the RNA quality.
(8) Total RNA extraction is processed by DNase I without RNase, total RNA is purified by QIAGEN RNeasy kit, and detailed operation principle and method are shown in kit instructions.
And 3. qRT-PCR detecting the expression level of miR-125a-3p in the cells.
The total RNA of human monocytes is used as a template, the miR-125a-3p gene is amplified by using the primers in the kit, and the expression quantity of the miR-125a-3p in the cells is quantitatively detected, and the detailed operation principle and the method are shown in the kit specification.
4. Statistical treatment
All data are expressed as means ± standard deviation, the comparison between the samples is performed by analysis of variance, the comparison between the two samples is performed by Kruskal-Wallis test, p<0.05 was considered statistically significant.
The results show that the expression level of miR-125a-3p in peripheral blood mononuclear cells of patients with hyperlipidemia and atherosclerosis is obviously reduced compared with that of normal healthy people (p<0.01) has statistical significance. The result indicates that the expression level of miR-125a-3p in the mononuclear cells in the peripheral blood can be used as a marker for diagnosing hyperlipidemia and atherosclerosis, and the specific result is shown in figure 1.
Example 2: WT mice and ApoE -/- And (3) the difference of miR-125a-3p expression quantity of different tissues of the mice.
1. Conventional methods for isolation of WT mice and ApoE -/- Heart and aorta of mice.
2. Tissue total RNA extraction (TRIzol method)
50-100mg of the tissue was placed in a 1.5ml centrifuge tube, 1ml of Trizol was added and homogenized well, and the mixture was allowed to stand at room temperature for 5 min. The subsequent steps refer to b to h in step (2) in example 1.
qRT-PCR detection of expression level of miR-125a-3p in different tissues
The qRT-PCR assay was performed using total RNA of mouse heart and aortic vessels as templates (the procedure was the same as in (3) of example 1).
4. Statistical treatment
Results were statistically analyzed and plotted using Prism graphics software, all data are expressed as mean ± sd, and comparisons between samples were statistically significant with p <0.05 using the t-test.
The results show that ApoE is a model for the study of hyperlipidemia and arteriosclerosis -/- The expression quantity of miR-125a-3p of aortic blood vessels of the mouse is obviously lower than that of a normal wild type WT mouse (a)p<0.01), having statistical significance; whileThere is no obvious difference in miR-125a-3p expression quantity of heart tissue (p>0.05), the result shows that certain tissue specificity exists in the expression and regulation of miR-125a-3p, namely, the reduction degree of hyperlipidemia and arteriosclerosis vascular tissues is more obvious than that of heart tissues, and the specific result is shown in figure 2.
Example 3: detecting the expression quantity of miR-125a-3p in RAW264.7 cells and the expression level of cell inflammatory factors.
1. Detection of high lipid status WT and ApoE cultures -/- Expression level of miR-125a-3p in mouse RAW264.7 cell
(1) Cells were seeded in 24-well plates with 4X 10 cells per well 4 。
(2) ox-LDL was added to a lipid-free DMEM medium to a concentration of 50 ug/ml.
(3) It was incubated in a standard cell incubator for 72 hours.
(4) Collecting total RNA of the cells, and detecting the expression quantity of miR-125a-3p in the cells by qRT-PCR. The specific procedure is as in example 2.
2. MiR-125a-3p mimic is used for regulating expression of miR-125a-3p in cells
(1) Cells were seeded in 24-well plates with 4X 10 cells per well 4 。
(2) 1ul of the transfection reagent siPORT NeoFX (AM4510, Ambion) and 25ul of OPTI-MEM I medium (Invitrogen) were mixed and incubated at room temperature for 10 minutes.
(3) miR-125a-3pmimic was diluted with OPTI-MEM I medium, mixed and incubated at room temperature for 10 minutes. Cells were treated with non-functional miRNA sequences (scrimbed miRNA, irrelevant sequence miRNA) as a control.
(4) And uniformly mixing the diluted miR-125a-3p mimic and the transfection reagent, and placing the mixture at room temperature for incubation for 10 minutes to form a transfection compound.
(5) The medium containing the transfection complex was added to the cell culture plate and incubated with the cells for 72 hours.
(6) Collecting total RNA of the cells, and carrying out qRT-PCR to detect the expression quantity of miR-125a-3p in the cells, wherein the specific method refers to example 2.
(7) The total RNA of the cells was collected and qRT-PCR was performed to detect the expression levels of inflammatory factors TNF-a, IL-10, IL-6, and iNOS in the cells, and the specific method was as described in example 2.
3. Statistical treatment
All data are expressed as means ± standard deviation, the comparison between groups of samples is performed by analysis of variance, the comparison between groups is performed by Kruskal-Wallis test,p<0.05 was considered statistically significant.
The results showed that the expression level of miR-125a-3p in mononuclear macrophage (RAW 264.7) cultured in high-lipid state (50 ug/ml ox-LDL) was reducedp<0.01, statistically significant compared to a negative control group; the miR-125a-3p imic can increase the expression level of miR-125a-3p in cellsp<0.01, statistically significant, #, compared to the negative control groupp<0.01, statistically significant compared to the ox-LDL control (FIG. 3). miR-125a-3p imic can reduce the inflammation level in high-fat state (50 ug/ml ox-LDL) culture mononuclear macrophage (RAW 264.7)p<0.01, statistically significant, #compared to the normal control group (control)p<0.01, statistically significant compared to the model control (ox-LDL), see FIG. 4.
SEQUENCE LISTING
<110> Liaoning university of traditional Chinese medicine
Application of <120> miR-125a-3p in preparation of cardiovascular disease diagnosis kit
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> unknown
<400> 1
acaggugagg uucuugggag cc 22
<210> 2
<211> 68
<212> DNA
<213> unknown
<400> 2
cugggucccu gagacccuuu aaccugugag gacguccagg gucacaggug agguucuugg 60
gagccugg 68
Claims (4)
- The application of miR-125a-3p in preparing a hyperlipidemia diagnostic kit, wherein the nucleotide sequence of miR-125a-3p is shown in SEQ ID NO. 1.
- 2. The use of claim 1, wherein the diagnostic kit is used for detecting the expression level of the miR-125a-3p protein in plasma by using the miR-125a-3p protein as a marker, and performing early diagnosis on the hyperlipidemia.
- 3. The use according to claim 1, wherein the diagnostic kit comprises the following components: 10 XmiRNA reverse transcription primer, 20 XmiRNA upstream primer, 20 XmiRNA universal primer, 4 Xone step miRNA RT Solution, 5 XSYBR Green qPCR Mix and 50 XROX Reference Dye aiming at miR-125a-3 p.
- 4. The use of the miR-125a-3p in the preparation of the hyperlipidemia diagnostic kit according to claim 1, wherein the miR-125a-3p reduces the level of inflammation in cultured mononuclear macrophages in a high-lipid state by increasing the expression of the miR-125a-3p mimic through miR-125a-3p mimic, and the nucleotide sequence of the miR-125a-3p mimic is shown in SEQ ID NO. 2.
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| CN102301002A (en) * | 2008-11-12 | 2011-12-28 | 卡里斯生命科学卢森堡控股有限责任公司 | Methods and systems of using exosomes for determining phenotypes |
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| CN107254538A (en) * | 2008-11-12 | 2017-10-17 | 卡里斯生命科学瑞士控股有限责任公司 | The method and system of phenotype is determined using allochthon |
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