CN111979311A

CN111979311A - Application of myosin 1b as atherosclerosis diagnosis and treatment target

Info

Publication number: CN111979311A
Application number: CN202010894303.8A
Authority: CN
Inventors: 熊裕焱; 闫媛; 牛芳林; 于怡; 王迓君
Original assignee: Northwestern University
Current assignee: Northwestern University
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2020-11-24

Abstract

The invention provides application of myosin 1b as an atherosclerosis diagnosis and treatment target. Judging whether the subject suffers from atherosclerosis by detecting the expression level of myosin 1b in the blood of the subject, wherein if the expression level of myosin 1b mRNA in the blood sample is up-regulated (high expression), the blood sample is mainly atherosclerosis patients; the detection result is accurate and reliable, and the application value of clinical diagnosis is realized; in addition, the atherosclerotic plaque can be reduced by targeting myosin 1b gene or protein, a foundation is laid for developing a novel drug for treating atherosclerosis based on the target, and the method has potential application value.

Description

Application of myosin 1b as atherosclerosis diagnosis and treatment target

Technical Field

The application relates to the field of biomedicine, in particular to a biomarker and a therapeutic target for atherosclerosis.

Technical Field

Atherosclerosis (AS) is a chronic inflammatory disease that seriously compromises human health and is characterized by intravascular plaques composed of fat, cholesterol, calcium, and fibrin. When plaque is accumulated, the channel in the artery is narrowed, the plaque can partially or completely prevent blood from flowing through the large or medium-sized artery of the heart, brain, pelvis, legs, arms or kidney, the main factor causing various cardiovascular and cerebrovascular diseases such as coronary heart disease, angina pectoris, stroke, carotid artery disease, peripheral artery disease and chronic nephropathy, and the diagnosis of atherosclerosis and the development and research of drug targets have become hot spots in the cardiovascular field.

With the rapid increase of the global aging population, aging-related diseases, especially cardiovascular and cerebrovascular diseases, present increasingly serious epidemic situations, and the main disease-causing population is the middle-aged and old people, which has the characteristics of high morbidity, high disability rate and high mortality rate. By 2030, about 20% of the world's population will reach over 65 years of age, and at this age, cardiovascular and cerebrovascular diseases will account for about 40% of all death factors, and therefore, the development of atherosclerosis diagnosis and new drug targets is of great importance.

The vascular endothelial cells are continuous monolayer flat cells which are attached to the innermost layer of the blood vessel, not only are a barrier of blood, blood vessel and surrounding tissues, but also can secrete a plurality of active substances for regulating the functions of the blood vessel, and have important function in maintaining the steady state of the blood vessel. Vascular endothelial cell aging, dysfunction and inflammatory factor release are major causes of atherosclerosis, and with increasing age or stimulation by stress conditions, vascular endothelial cells undergo aging, resulting in vascular endothelial dysfunction, showing marked endothelial dysfunction characteristics such as increased Reactive Oxygen Species (ROS), decreased NO, increased levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, accompanied by increased levels of pro-inflammatory factors such as TNF- α, MCP-1, IL-6 and IL-8. Endothelial cell-derived NO is a vasodilator of vascular and tissue perfusion, and can regulate endothelial cell division and survival, while having effects of inhibiting inflammatory cell adhesion to endothelial cells, disrupting proinflammatory cytokine-induced signaling, and protecting blood vessels and heart. In addition, endothelial cell senescence causes increased ROS levels, leading to endothelial nitric oxide anabolic coupling (eNOS-uncovered) and NO inactivation, resulting in vasomotor dysfunction, and ultimately vascular aging and dysfunction. The expression of adhesion molecules (VCAM-1, ICAM-1) on endothelial cell membranes is increased and proinflammatory factors are released, so that more monocytes/macrophages, dendritic cells, natural killer cells and B and T lymphocytes participate in the inflammatory reaction of blood vessel walls, the blood vessel dysfunction is aggravated, and the development of atherosclerosis is accelerated. Therefore, delaying aging of vascular endothelial cells, improving vascular endothelial dysfunction and reducing inflammatory factor release are becoming hot spots for development of novel drugs for atherosclerosis.

At present, the identification and diagnosis of atherosclerosis mainly depends on imaging examination, such as CT and MRI, because blood vessels move continuously, the imaging quality is unstable, the accuracy of the diagnosis result is affected, and in addition, certain radiation of the imaging examination makes many patients unwilling to do CT and MRI for excessive times. With the continuous acceleration of the aging process of the global population, the number of patients with atherosclerosis will also increase rapidly, and the simple, rapid and accurate diagnosis of atherosclerosis becomes an urgent problem to be solved. In addition, the pathogenesis and the mechanism of occurrence and development of atherosclerosis are still unclear, and the prevention and treatment measures and medicines for thoroughly and efficiently diagnosing and radically treating the disease are still lacked. Therefore, the search and development of novel atherosclerosis diagnosis biomarkers and drug therapy targets have great significance, and are problems to be solved urgently in the field of atherosclerosis at present.

Myosin constitutes a multigenic family of large actin-based molecular motors that are essential for eukaryotic homeostasis throughout the phylogenetic spectrum. Myosin 1b (Myosin 1b, Myo1b) belongs to type I Myosin, a widely existing motor protein in cells. Myosin class I is a single-headed member of the large family of myosins, capable of binding to the fiber filaments and producing kinetic energy by hydrolysis of ATP, and plays a role in cellular material transport, formation of cell membrane processes, cell migration and nuclear transcription. Myo1b is widely expressed in vertebrate tissues such as brain, heart, lung, kidney, and liver, and it can cross-link actin-loaded filaments, which is beneficial for better maintenance and control of cortical tone. At present, no report about the clinical diagnosis of atherosclerosis and the development of therapeutic drugs on myosin 1b is found.

Disclosure of Invention

The application provides a biomarker and a target for diagnosing and treating atherosclerosis, and researches show that: whether the test subject suffers from atherosclerosis or not is judged by detecting the expression level of myosin 1b in blood of the test subject, the result is accurate and reliable, and the test subject has an application value of clinical diagnosis; in addition, the biomarker is targeted, and the biomarker can be used as a target for treating atherosclerosis in vivo and in vitro, so that a foundation is laid for developing a novel medicament for treating atherosclerosis based on the target, and the biomarker has potential application value.

The following practical schemes in industry are specifically defined:

in a first aspect, the use of myosin 1b as a biomarker in the manufacture of a product for use in the diagnosis/detection of atherosclerosis.

Furthermore, the product for diagnosing/detecting atherosclerosis is used for detecting whether the mRNA of myosin 1b in the blood sample is highly expressed or not as a basis for distinguishing. For example, the kit and the test strip for colloidal gold or gold magnetic chromatography detection are suitable for real-time quantitative fluorescent PCR detection.

The product can be a product which only detects the mRNA expression level of myosin 1b, and the subsequent steps are used for judging whether the expression level is high or not; or a product which can directly give whether or not the mRNA of myosin 1b is highly expressed. In addition, the product can also record the mRNA expression level of myosin 1b in healthy human blood in advance so as to judge whether the expression level is high or not.

The product can also prompt a judgment basis, namely: the blood sample is a subject with atherosclerosis if the mRNA expression level of myosin-1 b is up-regulated (high expression) compared to normal healthy persons. Here, the specific form of the "prompt detection basis" is not limited, and for example: the detection basis is recorded in the attached product specification; if software is involved, the detection basis can also be embodied by a corresponding algorithm.

In a second aspect, a kit for diagnosis/detection of atherosclerosis, comprising reagents for detecting the mRNA expression level of myosin 1 b.

Further, the kit is suitable for real-time quantitative fluorescent PCR detection, the reagent for detecting the expression level of myosin 1b is an amplification primer pair, and the nucleotide sequence of the amplification primer pair is as follows:

forward amplification primer sequence: 5'-AACATGGCCTCATTGGAAAG-3' (SEQ ID NO. 2);

reverse amplification primer sequence: 5'-CGTTGCTTCCTCAGGTCTTC-3' (SEQ ID NO. 3).

The specific method of real-time quantitative fluorescent PCR is as follows: extracting total RNA from a sample of a subject with a blood RNA extraction kit, reverse-transcribing the total RNA into cDNA using a reverse transcription kit, and then subjecting the cDNA to reverse transcription using a reverse transcription kit

qPCR was performed on qPCR Master mix of Green I dye, relative quantification was calculated using the Ct value comparison method, and relative standards were normalized to internal reference.

In a third aspect, the use of myosin 1b as a biomarker in the manufacture of a product for use in the treatment of atherosclerosis by targeting myosin 1b gene or protein to achieve atherosclerotic plaque reduction.

Further, the product for treating atherosclerosis is a medicine and/or a treatment device, wherein the medicine comprises a chemical medicine and/or a traditional Chinese medicine for inhibiting the expression of the myosin 1b gene or protein, and the treatment device comprises a heart scaffold for treating atherosclerosis, which precisely targets the myosin 1b gene or protein.

Furthermore, the chemical medicine and/or the Chinese medicine can be prepared into injection, tablets, pills, granules, capsules, nanocapsules and the like.

In a fourth aspect, an interfering adenovirus for the treatment of atherosclerosis, targeting the human myosin 1b gene as a target, the interfering sequence being: 5'-GGGCTTTATGGATCATGAAGC-3' (SEQ ID NO. 4).

In a fifth aspect, an intelligent terminal includes a processor and a memory, where the memory stores a program, and the program implements the following steps when being loaded and executed by the processor:

obtaining the expression level of mRNA of myosin 1b in a blood sample;

judging whether the expression level is up-regulated (high expression);

if the blood sample is up-regulated, prompt information indicating that the blood sample is an atherosclerosis patient is output.

Here, the smart terminal may communicate with a corresponding mRNA expression level detection device, may be connected to a removable storage medium, or the like, or may directly input detection data by a user, thereby conveniently detecting the expression level of mRNA of myosin 1b in a stored blood sample; the intelligent terminal local memory can also record the mRNA expression level of myosin 1b in healthy human blood in advance so as to rapidly judge whether the expression is high or not and obtain a diagnosis/detection result.

The output prompt information can be in a form of directly displaying the diagnosis/detection result on a display screen, forming a report sheet by connecting printing equipment, or summarizing the report sheet as diagnosis reference information.

Drawings

FIG. 1 is a graph showing the expression levels of myosin 1b in the blood of a normal healthy group and an atherosclerotic patient;

FIG. 2 shows that myosin 1b knockdown retards vascular endothelial cell senescence;

FIG. 3 is a graph showing that myosin 1b knockdown improves the function of vascular endothelial cells;

FIG. 4 is a graph showing the effect of myosin 1b knockdown significantly reducing inflammatory factor release from vascular endothelial cells;

FIG. 5 is a graph showing that myosin 1b knockdown significantly reduces the size of vascular atherosclerotic plaques resulting from ligation of a portion of the mouse carotid artery;

FIG. 6 shows that myosin 1b knockdown significantly reduces ApoE^-/-Mouse vascular atherosclerotic plaque size.

Detailed Description

The following detailed description will be made of the relevant validation experiments and analyses of the present application with reference to the accompanying drawings, and the specific development process of the inventors is not limited thereto.

Experiment 1: real-time fluorescent quantitative PCR detection for comparing mRNA expression level of myosin 1b in blood samples of atherosclerotic and normal healthy persons

Whole blood samples were collected from 20 patients with confirmed atherosclerosis and 20 normal healthy persons as controls.

PureLink using Saimer Feishale technologies, Inc^TMTotal RNA was extracted from each Blood sample using Total RNA Blood kit (K156001) according to the protocol. The mRNA concentration in each sample was measured using Thermo Scientific NanoDrop 2000, and then GoScript from Promega^TMSynthesis of cDNA Using Reverse Transcriptase (A5001)

qPCR Master mix (A6001) with Green I dye quantitative analysis of mRNA levels of myosin 1b in blood of atherosclerotic and healthy persons using the Ct value comparison method

Calculating relative quantification, wherein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as the optimal reference;

the forward primer sequence for myosin 1b is: 5' -AACATGGCCTCATTGGAAAG-3(SEQ ID NO.2), and the reverse primer is: 5'-CGTTGCTTCCTCAGGTCTTC-3' (SEQ ID NO. 3);

the forward primer for GAPDH is: 5'-TGCACCACCAACTGCTTAGC-3' (SEQ ID NO.5), and the reverse primer is: 5'-GGCATGGACTGTGGTCATGAG-3' (SEQ ID NO. 6).

And (4) analyzing results: as shown in FIG. 1, the mRNA of myosin 1b in the blood of atherosclerotic patients was about 2.7 times higher than that of normal healthy people, so that myosin 1b gene could be used as a molecular marker for early detection of atherosclerotic patients. All data are expressed as ± standard deviation, indicating a significant difference p <0.01 in myosin 1b mRNA levels in atherosclerotic patients compared to normal healthy groups.

Experiment 2: beta-galactosidase staining method for detecting influence of myosin 1b gene knock-down on vascular endothelial cell senescence

Vascular endothelial cell senescence is one of the important causes of atherosclerosis, and a large amount of clinical data indicate that a large number of senescent vascular endothelial cells are present in the blood vessel of atherosclerotic patients. Therefore, targeting vascular endothelial cell aging also becomes an important way for developing drugs for treating atherosclerosis. To detect vascular endothelial senescence, we used a β -galactosidase staining method. Aged vascular endothelial cells, HUVECs, were first washed twice with PBS 72 hours after infection with adenovirus targeting interfering myosin 1b, and then fixed with 3.7% formaldehyde PBS for 10-15 minutes. After washing twice again with PBS, the cells were stained with a beta-galactosidase staining solution (1 mg/ml X-gal, 40 mM Na)₃C₆H₅O₇·2H₂O、5 mM K₄Fe(CN)₆·3H₂O、5 mM K₃Fe(CN)₆150 mM NaCl and 2 mM MgCl₂Dissolved in phosphate buffer at pH 6.0) CO-free at 37 deg.C₂Was incubated overnight in the incubator of (1). The blue senescent cells were examined by conventional light microscopy, the percentage of positive senescent cells for each group was calculated, and significance analysis was performed.

And (4) analyzing results: as shown in figure 2, Western Blot (Western immunoblot) detection shows that the myosin 1b interference virus can obviously reduce the expression level of HUVEC myosin 1b of aged vascular endothelial cells, and a beta-galactosidase staining method also shows that the knocking-down of myosin 1b can obviously delay the aging of the vascular endothelial cells. Vascular endothelial senescence is an important cause of atherosclerosis, and thus targeting myosin 1b is likely to be a drug target for treating atherosclerosis. All data are expressed as ± sd, which represents a significant difference p <0.001 in the myosin 1b knock-down group compared to the control group.

Experiment 3: detection of the Effect of myosin 1b Gene knockdown on the function of senescent vascular endothelial cells

Dysfunction of vascular endothelial cells is also an important factor causing atherosclerosis, and vascular endothelial cells, namely Nitric Oxide (NO) and Reactive Oxygen Species (ROS), are two important indexes for evaluating the function of vascular endothelium, wherein the Nitric Oxide (NO) can promote the relaxation of blood vessels, but the excessive generation of the reactive oxygen species can promote vascular aging and inflammatory reaction, so that the process of atherosclerosis is accelerated. Aged vascular endothelial cells HUVEC were infected with adenovirus targeting interfering myosin 1b 72 hours later, and in order to measure cellular ROS, the cells were incubated with 5 μ M DHE (Dihydroethidium) working solution for 20 minutes, and fluorescence signal images were collected with a fluorescence microscope to detect the intracellular ROS production. For detection of NO, HUVEC was used without Ca²⁺Was gently washed twice with PBS, and then the cells were mixed with Krebs-Ringer bicarbonate solution (NaCl 118mM, KCl 4.7mM, CaCl) containing 5. mu.M DAF-2DA₂ 2.5mM、MgSO₄ 1.2mM、KH₂PO₄ 1.2mM、NaHCO₃25mM, EDTA 0.026mM and glucose 5.5mM) for 30 minutes. The cells were then washed 3 times and fluorescence signal images were collected with a fluorescence microscope and the fluorescence intensity was quantified by Image J software (NIH). All data are expressed as ± sd, which represents a significant difference p between myosin 1b knock-down groups compared to control groups<0.001。

And (4) analyzing results: as shown in fig. 3, in aging vascular endothelial cells, the knocking-down of myosin 1b can significantly increase the nitric oxide content of aging vascular endothelial cells while reducing the generation of reactive oxygen species, so that targeting myosin 1b can significantly improve the function of vascular endothelial cells, thereby facilitating the treatment of atherosclerosis.

Experiment 4: real-time fluorescent quantitative PCR detection for detecting influence of myosin 1b gene knock-down on release of vascular endothelial cell inflammatory factor

Atherosclerosis is an inflammatory disease in which the vascular endothelium releases many cytokines and adhesion molecules such as VCAM-1, ICAM-1, TNF- α, MCP1, IL-6, and IL-8, etc. to recruit innate inflammatory cells and then recruit cells of the adaptive immune system into the vessel wall, resulting in the further development of atherosclerosis. The real-time fluorescent quantitative PCR detection is adopted to detect the influence of the knocking-down of the myosin 1b gene on the release of the vascular endothelial cell inflammatory factor. Aged vascular endothelial cells HUVEC, infected with adenovirus targeting interfering myosin 1b for 72 hours, extracted total RNA from cell and tissue samples by Trizol method, reverse transcribed into cDNA using reverse transcription kit, and then reverse transcribed into cDNA using reverse transcription kit

qPCR Master mix of Green I dye qPCR was performed using the Ct value comparison method

Relative quantification was calculated and the relative standard was normalized to GAPDH (internal reference). All data are expressed as ± sd, which represents a significant difference p between myosin 1b knock-down groups compared to control groups<0.01; significant differences p in myosin 1b knock-down groups compared to control groups<0.001。

The specific primer sequences are as follows:

GAPDH：

an upstream primer: 5'-TGCACCACCAACTGCTTAGC-3' (SEQ ID NO. 5);

a downstream primer: 5'-GGCATGGACTGTGGTCATGAG-3' (SEQ ID NO. 6);

VCAM-1:

an upstream primer: 5'-CAGACAGGAAGTCCCTGGAA-3' (SEQ ID NO. 7);

a downstream primer: 5'-TTCTTGCAGCTTTGTGGATG-3' (SEQ ID NO. 8);

ICAM-1:

an upstream primer: 5'-GGCTGGAGCTGTTTGAGAAC-3' (SEQ ID NO. 9);

a downstream primer: 5'-ACTGTGGGGTTCAACCTCTG-3' (SEQ ID NO. 10);

TNF-α:

an upstream primer: 5'-CCCAGGGACCTCTCTCTAATCA-3' (SEQ ID NO. 11);

a downstream primer: 5'-GCTACAGGCTTGTCACTCGG-3' (SEQ ID NO. 12);

MCP1:

an upstream primer: 5'-GATCTCAGTGCAGAGGCTCG-3' (SEQ ID NO. 13);

a downstream primer: 5'-TGCTTGTCCAGGTGGTCCAT-3' (SEQ ID NO. 14);

IL-6:

an upstream primer: 5'-GGCACTGGCAGAAAACAACC-3' (SEQ ID NO. 15);

a downstream primer: 5'-GCAAGTCTCCTCATTGAATCC-3' (SEQ ID NO. 16);

IL-8:

an upstream primer: 5'-GTGCAGTTTTGCCAAGGAGT-3' (SEQ ID NO. 17);

a downstream primer: 5'-CTCTGCACCCAGTTTTCCTT-3' (SEQ ID NO. 18);

and (4) analyzing results: as shown in FIG. 4, myosin 1b knockdown significantly reduced mRNA levels of the inflammatory factors VCAM-1, ICAM-1, TNF- α, MCP 1L-6, and IL-8 released by vascular endothelial cells that lead to atherosclerosis. Therefore, targeting myosin 1b can significantly reduce the release of vascular endothelial inflammatory factors, thereby facilitating the treatment of atherosclerosis.

Experiment 5: study at animal level, in vivo myosin 1b Gene knockdown in mice improves carotid Atherosclerosis

Ligation of the carotid artery in mice is a rapid model for the study of atherosclerosis, and after 3 weeks of ligation, 200. mu.L of the virus was injected via the caudal vein into mice with a titer of 3.3X 10⁷～2×10⁸Adenovirus specifically targeting interfering myosin 1b for 2 weeks, once weekly injection, followed by euthanasia of mice, isolation of ligated vessels for fluorescent quantitative PCR to detect myosin 1b knock-down efficiency, paraffin sectioning of part of vessels, followed by EVG (elastic Van Giesen) staining, evaluation of myosin 1b knock-down on arteriesEffects of atherosclerosis. All data are expressed as ± sd, which represents a significant difference p between myosin 1b knock-down groups compared to control groups<0.001。

And (4) analyzing results: as shown in figure 5, the expression of the gene can be effectively reduced by injecting adenovirus targeting knocking down myosin 1b through tail vein of a mouse, and meanwhile, EVG staining results show that the knocking down myosin 1b can effectively improve vascular blockage caused by atherosclerosis, reduce the size of atherosclerotic plaques, and further verify that myosin 1b can be used as a target for treating atherosclerosis from in vivo level.

Experiment 6: studies at animal level of myosin 1b Gene knockdown in mice to reduce atherosclerotic plaque area

ApoE^-/-Mice are another important model of atherosclerosis, and symptoms of atherosclerosis develop after 12 weeks of high fat feeding. The titer of the virus was 3.3X 10 at 200. mu.L by tail vein injection in mice⁷～2×10⁸Adenovirus specifically targeting interfering myosin 1b was injected once a week for 2 weeks, and then mice were euthanized. Separating aorta, performing fluorescence quantitative PCR (polymerase chain reaction) detection on myosin 1b knocking efficiency, taking part of blood vessel as paraffin section, and then performing H&E (haemotoxylin and eosin) staining, to assess the effect of myosin 1b knockdown on atherosclerotic plaque size. All data are expressed as ± sd, which represents a significant difference p between myosin 1b knock-down groups compared to control groups<0.001。

And (4) analyzing results: as shown in figure 6, the expression of the gene can be effectively reduced by injecting adenovirus targeting knocking down myosin 1b through tail vein of a mouse, and meanwhile, H & E staining results show that the knocking down of myosin 1b can effectively improve vascular blockage caused by atherosclerosis, reduce the size of atherosclerotic plaques, and further verify that myosin 1b can be used as a target for treating atherosclerosis from in vivo level.

<110> northwest university

Application of <120> myosin 1b as diagnosis and treatment target of atherosclerosis

<160> 18

<210> 1

<211> 5129

<212> DNA

<213> human (Homo sapiens)

<400> 1

GAGCACTCCGCAGCTGGGTGCGCACGGGAGCCTCAACCGCGGCGCGTGGAGGCAGTACCA 60

GAGCGCGCGGCGCGCAGTCGGCCGGCAGCCGCGGGACAGCCTTGGCAGAACAGCCGCGGC 120

GGGAGCCCCGCAGCCCCGGGCTGGGCCCGCGTCCCGGAGCGCCACCGGAGAGCGAGGACG 180

ACGTGGAGGCGGAGTGGCGCCCGGCGAGGTAGCGCCAGGCGAGCTGGAGACCATGGCCAA 240

AATGGAGGTGAAAACCTCACTTCTGGACAATATGATTGGAGTTGGGGATATGGTTCTTTT 300

AGAACCTCTCAATGAGGAGACCTTCATCAACAACCTCAAGAAGCGCTTTGACCACAGTGA 360

AATATACACATACATTGGAAGTGTGGTTATATCTGTTAACCCATACCGGTCTTTACCCAT 420

TTATTCACCAGAGAAAGTGGAAGAATACAGGAACAGAAATTTTTATGAACTGAGCCCTCA 480

CATCTTTGCCCTTTCGGATGAAGCATACAGATCCCTACGAGATCAAGATAAGGACCAATG 540

TATTCTCATTACTGGGGAAAGTGGAGCAGGAAAAACAGAGGCCAGTAAGCTTGTCATGTC 600

CTATGTGGCAGCTGTTTGTGGAAAAGGAGCAGAAGTTAATCAAGTTAAAGAACAGCTTTT 660

ACAGTCCAACCCGGTCCTGGAAGCTTTTGGAAATGCCAAAACTGTAAGGAATGACAACTC 720

CTCTAGATTTGGCAAATATATGGATATTGAATTTGACTTTAAAGGCGATCCACTAGGAGG 780

AGTAATAAGTAACTATCTTTTAGAGAAATCTCGGGTTGTTAAACAGCCAAGAGGTGAAAG 840

AAACTTCCATGTGTTCTATCAGCTGCTCTCTGGTGCCTCTGAAGAGCTCCTCAATAAACT 900

TAAGCTTGAGAGGGATTTCAGCAGGTATAACTACCTGAGTCTGGATTCGGCCAAAGTGAA 960

TGGAGTGGATGATGCAGCAAATTTTAGAACCGTGCGGAATGCCATGCAGATTGTGGGCTT 1020

TATGGATCATGAAGCTGAGTCTGTCTTGGCGGTGGTGGCAGCAGTGTTGAAACTGGGGAA 1080

CATTGAGTTCAAGCCCGAATCTCGAGTGAATGGTCTAGATGAAAGCAAAATCAAAGATAA 1140

AAATGAGTTAAAAGAAATTTGTGAATTGACCGGCATTGATCAATCAGTTCTAGAACGAGC 1200

ATTCAGTTTCCGAACAGTTGAGGCCAAACAGGAGAAAGTTTCAACTACACTGAATGTGGC 1260

TCAGGCTTATTATGCCCGTGATGCTCTGGCTAAAAACCTCTACAGCAGGTTGTTTTCATG 1320

GTTGGTAAATCGAATCAATGAAAGCATTAAGGCACAAACAAAAGTGAGAAAGAAGGTCAT 1380

GGGTGTTCTGGACATTTATGGCTTTGAGATTTTCGAGGACAACAGCTTTGAGCAGTTCAT 1440

TATTAATTATTGTAACGAAAAGCTGCAACAAATCTTCATTGAACTTACTCTTAAAGAAGA 1500

GCAGGAGGAGTATATACGGGAGGATATAGAATGGACTCACATTGACTACTTCAATAATGC 1560

TATCATTTGTGACCTAATAGAAAATAACACAAATGGAATCCTGGCCATGCTGGATGAAGA 1620

GTGCCTCAGACCTGGCACAGTCACTGATGAGACCTTCTTAGAAAAGCTGAACCAAGTATG 1680

TGCCACCCACCAGCATTTTGAGAGCAGGATGAGCAAGTGCTCTCGGTTCCTCAATGACAC 1740

GTCTCTGCCTCACAGCTGCTTCAGGATCCAGCATTATGCTGGAAAGGTGCTGTACCAGGT 1800

GGAAGGATTCGTTGACAAAAACAATGACCTTCTCTATCGAGACCTGTCCCAAGCCATGTG 1860

GAAGGCCAGCCATGCCCTCATCAAGTCTTTGTTCCCCGAAGGGAATCCCGCCAAGATCAA 1920

CCTGAAAAGGCCTCCTACAGCAGGCTCACAGTTCAAGGCATCCGTGGCCACTCTGATGAA 1980

AAACCTACAGACCAAGAACCCAAACTATATTAGGTGTATCAAACCGAATGATAAAAAAGC 2040

AGCACACATCTTCAACGAGGCTCTAGTGTGTCATCAGATCAGGTACCTGGGGCTTTTGGA 2100

GAACGTCCGAGTGCGGAGGGCAGGCTACGCCTTCAGGCAGGCCTATGAACCTTGCCTAGA 2160

AAGATACAAAATGCTTTGTAAACAAACATGGCCTCATTGGAAAGGACCAGCCAGGTCTGG 2220

TGTGGAGGTCCTATTTAATGAATTAGAAATTCCCGTGGAAGAATACTCCTTTGGTAGATC 2280

AAAGATATTCATCCGAAACCCAAGAACATTATTCAAATTAGAAGACCTGAGGAAGCAACG 2340

CCTGGAGGACTTGGCCACTCTCATTCAGAAGATATATCGGGGGTGGAAATGCCGCACACA 2400

CTTCCTGCTAATGAAAAAAAGCCAAATTGTGATTGCCGCCTGGTACAGGAGATATGCGCA 2460

ACAAAAGAGGTACCAGCAGACAAAGAGTTCCGCCTTAGTAATTCAGTCTTATATCCGGGG 2520

TTGGAAGGCTCGAAAAATTCTGCGGGAACTGAAGCATCAAAAGCGCTGTAAGGAAGCAGT 2580

CACGACCATTGCTGCATATTGGCATGGGACCCAGGCACGAAGGGAACTGAGACGGCTGAA 2640

GGAGGAGGCTAGGAATAAACATGCTATTGCAGTTATTTGGGCTTACTGGCTTGGATCTAA 2700

GGCTCGAAGGGAATTGAAACGCTTGAAGGAGGAGGCTAGGCGTAAGCATGCAGTTGCTGT 2760

CATTTGGGCTTACTGGCTTGGACTGAAGGTACGTAGAGAATACAGGAAATTCTTCAGAGC 2820

CAATGCTGGAAAGAAAATCTATGAGTTTACGCTTCAGAGAATTGTGCAAAAATACTTCTT 2880

GGAAATGAAAAATAAGATGCCTTCCTTATCTCCAATAGACAAGAATTGGCCCTCAAGACC 2940

TTACTTATTCTTGGATTCTACTCACAAGGAGCTAAAAAGGATTTTCCACTTGTGGAGGTG 3000

TAAAAAATACAGGGACCAATTCACAGACCAGCAGAAACTTATTTATGAAGAGAAACTAGA 3060

AGCCAGTGAACTCTTCAAAGACAAGAAGGCTTTATACCCATCTAGTGTTGGGCAACCATT 3120

CCAAGGGGCTTACCTGGAAATCAACAAGAACCCCAAGTATAAGAAACTCAAAGATGCCAT 3180

TGAAGAAAAGATCATCATTGCTGAAGTCGTGAACAAAATTAACCGTGCTAATGGGAAGAG 3240

TACATCTCGGATTTTCCTCTTAACAAACAATAATCTCCTTCTTGCTGACCAAAAGTCTGG 3300

ACAAATCAAGTCAGAGGTTCCATTGGTGGATGTGACCAAGGTATCAATGAGCTCACAAAA 3360

TGATGGCTTCTTCGCCGTCCACCTCAAAGAGGGCTCAGAAGCAGCTAGTAAAGGAGACTT 3420

TCTCTTCAGCAGTGATCACCTGATTGAAATGGCCACCAAGCTCTATCGCACAACTCTCAG 3480

CCAAACCAAACAGAAGCTCAATATTGAGATTTCCGATGAGTTCCTGGTACAGTTCAGACA 3540

GGACAAAGTATGTGTGAAGTTTATTCAGGGAAACCAGAAAAATGGGAGTGTCCCAACATG 3660

TAAACGAAAAAACAACCGTCTCCTTGAAGTTGCTGTCCCTTAACTGGCGCCTCCTCTCTA 3720

CTTTCATGGACTTGTTCCTTTGTAATAGTGCAATTTGGTTTTGTTTTATTTGGGGTTCAT 3780

TGTATGTTTGGGAATCACCAAAGGCTTTTAGAGTTCTTTGGCAAAATAAAAATATTTGAC 3840

TAATCAATTTTTATTATTGGAATAGTTTTAACCTTTCAAATACATGTTCTGTCCTGGAGC 3900

AGGATTGTAGAAACTAACAGTGTCTATTTTCATGTCTGATGTGTTCTTCCTTTAGTCATC 3960

ATGTTAGGTCTGTGTACCCTAAATCAGCATATTACTCATAAATCATTAATTAATATAAGC 4020

ATAGGAAATGGTCTTAAAAGATACTGCATTCATTCATCAGATATTTATTCCATGCCTACT 4080

CTATGCTAGGCACTGTGCTAGATGGTATGAAAACTTATTAGGAACCTTTTTGTTTTTGAG 4140

ACCATTGCATTCTGGCTGGTTTGTGCTGGTTTAACGACATCTAAGAAGGTTTAGAAATGG 4200

TGAGACCAAAACAATAACTGTTAATGATGGACAGCATTATTAGGAACCCTGTAGTATGAT 4260

ATTTAACAATATAGGCTTCAAGAAGGGCTGGTCCTAAGAGGGGGCAGAAATGAATGACCA 4320

GGTTAAATCCCTCTACATGTGGTTTCTGTTTGAAAAAAAGAAAACTGACATTTGAACAGG 4380

ACTTTTAATTTGTTTAAAACTCTGGTAATTACTTGTAACAGTAGAAAATAGAAGTCATTC 4440

TTATTTTAGAAAAAGTGACAGAAGCAGTCCAGTAAGATTATATGTTTCTGTTTCTGGTAA 4500

ATACCATATATGATCCTCGAAATGATAATATCTCCAGAATATTGTTTTCACCCAAATTTG 4560

AGTAGATATTTTAAACACCTAACAAAGTAAAGGGCTAAAAGCCATTCAGATAGCAGTAAA 4620

ACATTCTGTATGATGTGCAATAAAACATCCAAGATCTTTTTTGAAAGTTTTATTTATAAT 4680

ATACATTTTTGTATGAGAAAGGTGATTGGTACAGGGTGCCTATTTTAGTCATGGATCAAA 4740

ATTTGTGTAACTTGCAGGGCTTTCTTTCTTTTTCTTCAAATTTACAAGGGTTCATTTTGG 4800

AAACTACATTTTAAACTTTGGAATCAAATTGTTTCTTATTTGGGAGGATAATGTATATAC 4860

ATTGGTATTATGTTAAATAATAAAATTGTTCTAATTTGGTGCCATTTCCTGAATCACAAC 4920

TGTATTTTTGTATCTCAAGCTATTTTCATATGTTGTGTGTCAATGTATCATCTCTCAGAA 4980

AGGTTTTACAATCCAAACATTATATGTTCTCTGTGTAACTGAATTTCACTTATCTTTTAT 5040

AAACCAGAAACATTAATTGAAAATATTTTCTGGGGATTTTCTCTTGACTTGTATTTTTAA 5100

AAATTGCTCACATAAAGAAGTTCTCAGAA 5129

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

AACATGGCCTCATTGGAAAG 20

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

CGTTGCTTCCTCAGGTCTTC 20

<210>4

<211> 21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

GGGCTTTATGGATCATGAAGC 21

<210>5

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

TGCACCACCAACTGCTTAGC 20

<210>6

<211> 21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

GGCATGGACTGTGGTCATGAG 21

<210>7

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

CAGACAGGAAGTCCCTGGAA 20

<210>8

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

TTCTTGCAGCTTTGTGGATG 20

<210>9

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

GGCTGGAGCTGTTTGAGAAC 20

<210>10

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ACTGTGGGGTTCAACCTCTG 20

<210>11

<211> 22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

CCCAGGGACCTCTCTCTAATCA 22

<210>12

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

GCTACAGGCTTGTCACTCGG 20

<210>13

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

GATCTCAGTGCAGAGGCTCG 20

<210>14

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

TGCTTGTCCAGGTGGTCCAT 20

<210>15

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

GGCACTGGCAGAAAACAACC 20

<210>16

<211> 21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

GCAAGTCTCCTCATTGAATCC 21

<210>17

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

GTGCAGTTTTGCCAAGGAGT 20

<210>18

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

CTCTGCACCCAGTTTTCCTT 20

Claims

1. Use of myosin 1b as a biomarker in the manufacture of a product for the diagnosis/detection of atherosclerosis.

2. The use according to claim 1, wherein said product for diagnosing/detecting atherosclerosis is used as a basis for determining whether or not mRNA of myosin-1 b is highly expressed in a blood sample.

3. The use according to claim 1, wherein the product for atherosclerosis diagnosis/detection is a kit suitable for real-time quantitative fluorescence PCR detection, colloidal gold or gold magnetic chromatography detection test strip.

4. A kit for diagnosis/detection of atherosclerosis, comprising a reagent for detecting the mRNA expression level of myosin 1 b.

5. The kit for diagnosing/detecting atherosclerosis according to claim 4, wherein the kit is a kit suitable for real-time quantitative fluorescent PCR detection, and the reagent for detecting the expression level of myosin 1b is an amplification primer pair, and the nucleotide sequence of the amplification primer pair is as follows:

forward amplification primer: 5'-AACATGGCCTCATTGGAAAG-3' (SEQ ID NO. 2);

reverse amplification primer: 5'-CGTTGCTTCCTCAGGTCTTC-3' (SEQ ID NO. 3).

6. The kit for diagnosing/detecting atherosclerosis according to claim 5, wherein the kit further comprises an amplification primer pair of GAPDH as an internal reference, and the nucleotide sequence of the amplification primer pair is as follows:

forward amplification primer: 5'-TGCACCACCAACTGCTTAGC-3' (SEQ ID NO. 5);

reverse amplification primer: 5'-GGCATGGACTGTGGTCATGAG-3' (SEQ ID NO. 6).

7. Use of myosin 1b as a biomarker in the manufacture of a product for use in the treatment of atherosclerosis by targeting myosin 1b gene or protein to achieve atherosclerotic plaque reduction.

8. The use according to claim 7, wherein the product for the treatment of atherosclerosis is a medicament and/or a therapeutic device, wherein the medicament comprises a chemical and/or a Chinese medicine that inhibits the expression of the myosin 1b gene or protein, and the therapeutic device comprises a heart scaffold for the treatment of atherosclerosis that precisely targets the myosin 1b gene or protein.

9. An interfering adenovirus for treating atherosclerosis, wherein the interfering targeting sequence is directed to a human myosin 1b gene target: 5'-GGGCTTTATGGATCATGAAGC-3' (SEQ ID NO. 4).

10. An intelligent terminal comprising a processor and a memory, the memory storing a program, characterized in that: the program realizes the following steps when being loaded and run by a processor:

obtaining the expression level of mRNA of myosin 1b in a blood sample;

determining whether the expression level is up-regulated;