CN109055544B

CN109055544B - Molecular marker of atherosclerosis and application thereof

Info

Publication number: CN109055544B
Application number: CN201811140906.8A
Authority: CN
Inventors: 朱鑫星; 林俊堂; 于金金; 杨芬; 钟根深; 郭睿; 杜江
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2018-09-28
Filing date: 2018-09-28
Publication date: 2021-12-28
Anticipated expiration: 2038-09-28
Also published as: CN109055544A

Abstract

The invention relates to an atherosclerosis molecular marker and application thereof. The atherosclerosis molecular marker can better reflect the progression status of atherosclerosis, so that the atherosclerosis molecular marker is applied to the preparation of related products for diagnosing or detecting atherosclerosis, and enriches the means for diagnosing and detecting atherosclerosis. And the atherosclerosis molecular marker can inhibit the adhesion between monocyte THP-1 and vascular endothelial cells HUVECs and inhibit the expression of inflammatory factors such as adhesion factors and chemotactic factors, so that the overactivation of vascular endothelial inflammation can be effectively inhibited, and the atherosclerosis molecular marker can also be used as a molecular drug target for inhibiting the formation and development of atherosclerosis, thereby providing a new experimental theoretical basis and direction for further researching the pathogenesis of atherosclerosis and exploring the prevention and treatment drugs thereof.

Description

Molecular marker of atherosclerosis and application thereof

Technical Field

The invention relates to the field of atherosclerosis, in particular to an atherosclerosis molecular marker and application thereof.

Background

Atherosclerosis is a chronic inflammatory disease that is initiated primarily by a disturbed Low Density Lipoprotein (LDL) metabolism, with intimal deposition in the sub-endothelial layer of blood vessels. Clinically, atherosclerosis is closely related to the occurrence of common cardiovascular diseases such as myocardial infarction, stroke, ischemic heart pain, hypertension and the like. At present, cardiovascular and cerebrovascular diseases caused by atherosclerosis become one of the main diseases endangering human health. According to the latest data statistics of the world health organization WHO, the number of people WHO die from cardiovascular and cerebrovascular diseases accounts for 48 percent of the total number of non-infectious diseases, and is more than twice of the number of people WHO die from cancers. Therefore, the further research on the pathogenesis of atherosclerosis and the search for an important marker which can be used for the clinical diagnosis of atherosclerosis have important significance.

Disclosure of Invention

Based on the above, there is a need for providing an atherosclerosis molecular marker which can be used as a marker for clinical diagnosis of atherosclerosis.

An atherosclerosis molecular marker, which is a long-chain non-coding RNA and has a nucleotide sequence shown as SEQ ID NO. 1.

The invention also provides a PCR amplification primer, which comprises an upstream primer and a downstream primer for specifically amplifying the atherosclerosis molecular marker.

In one embodiment, the upstream primer has a nucleotide sequence shown as SEQ ID NO.2, and the downstream primer has a nucleotide sequence shown as SEQ ID NO. 3.

The invention also provides a small interfering RNA, which comprises a complementary sense strand and an antisense strand, and the small interfering RNA can specifically inhibit the expression of the atherosclerosis molecular marker.

In one embodiment, the sense strand has the nucleotide sequence shown in SEQ ID No.2 and the antisense strand has the nucleotide sequence shown in SEQ ID No. 3.

The invention also provides the application of the atherosclerosis molecular marker, the PCR amplification primer or the small interfering RNA in the preparation of products for diagnosing or detecting atherosclerosis.

In one embodiment, the product is a detection reagent, a kit, a gene chip or a test strip.

The invention also provides application of the atherosclerosis molecular marker, the PCR amplification primer or the small interfering RNA in preparation of drugs for preventing or treating atherosclerosis.

The invention also provides a kit for detecting atherosclerosis, which comprises the PCR amplification primer.

In one embodiment, the kit further comprises at least one of an RNA extraction reagent, a reverse transcription reagent and a PCR amplification reagent.

Two thirds of the human gene sequence of 30 billion base pairs are reverse transcribed and eventually less than 2% of the nucleic acid sequence is used to encode proteins, most genes do not express proteins, and this class of genes is called non-coding RNA. Non-coding RNA includes short non-coding RNA and long non-coding RNA, which may be involved in cell differentiation and ontogeny regulation at multiple levels, and are closely related to various diseases. The inventor of the application finds in research that the expression level of long-chain non-coding RNA NKILA (the sequence is shown as SEQ ID NO. 1) in peripheral blood mononuclear cells of an atherosclerotic patient is obviously lower than that of peripheral blood mononuclear cells of a normal person (the P value is less than 0.05), and the long-chain non-coding RNA NKILA can better reflect the progress state of atherosclerosis, so that the long-chain non-coding RNA NKILA can be used as a clinical diagnosis molecular marker of atherosclerosis and is applied to the preparation of related products for diagnosing or detecting atherosclerosis, thereby enriching the diagnosis and detection means of atherosclerosis.

The development of atherosclerosis is directly linked to the abnormality of vascular endothelial cells. In the early stage of atherosclerosis, due to metabolic disorder of low density lipoprotein, inflammatory reaction of vascular endothelial cells is initiated, thereby promoting the endothelial cells to secrete a large amount of inflammatory factors such as adhesion factors, chemotactic factors and the like. The secretion of these inflammatory factors promotes adhesion between monocytes and endothelial cells, allowing monocytes to cross the endothelial cell space into the endothelial lining of the subendothelial vasculature. After entering the intima, monocytes can be induced by oxidized low-density lipoprotein to become macrophages and phagocytose the lipoprotein, so that foam cells are gradually formed, and finally, the formation of hardened plaques is caused. Through further research, the inventor discovers that the atherosclerosis molecular marker can inhibit the adhesion between the monocyte THP-1 and vascular endothelial cells HUVECs and inhibit the expression of inflammatory factors such as adhesion factors, chemotactic factors and the like, so that the overactivation of vascular endothelial inflammation can be effectively inhibited, and the atherosclerosis molecular marker can also be used as a molecular drug target for inhibiting the formation and development of atherosclerosis, thereby providing a new experimental theoretical basis and direction for further researching the pathogenesis of atherosclerosis and exploring the prevention and treatment drugs.

Drawings

FIG. 1 is a comparison of the expression levels of atherosclerotic molecular markers in peripheral blood mononuclear cells of atherosclerotic patients and normal persons;

FIG. 2 is a comparison of the effect of siRNA on the inhibition of interference with the expression level of an atherosclerotic molecular marker;

FIG. 3 is a graph showing the effect of siRNA reducing the expression of atherosclerotic molecular markers on adhesion between monocytes and endothelial cells;

FIG. 4 is a graph showing the effect of siRNA reduction of expression of atherosclerotic molecular markers on the expression levels of adhesion factors VCAM1 and SELE and chemokines MCP1 and IL 8;

in FIG. 5, A is the detection result of the overexpression efficiency of the atherosclerosis molecular marker, and B is the influence of the overexpression of the atherosclerosis molecular marker on the adhesion between the monocyte and the endothelial cell in FIG. 5;

FIG. 6 shows the effect of over-expression of atherosclerotic molecular markers on the expression levels of adhesion factors VCAM1 and SELE and chemokines MCP1 and IL 8.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The molecular markers of atherosclerosis and their use are described in further detail below, primarily with reference to the detailed description and the accompanying drawings.

Detecting and comparing the expression level of atherosclerosis molecular marker in peripheral blood mononuclear cell of normal person and atherosclerosis patient

1.1 isolation and preparation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells were separated from lymphocyte separation solutions (purchased from tertiary ocean biology, Tianjin) of normal persons (10 as a control group) and atherosclerotic patients (10 as an experimental group), respectively, and the middle tunica albuginea layer was aspirated after centrifugation. Then, the cells were washed twice with 0.9% sodium chloride solution, resuspended in X-VIVO medium containing 10% PBS, and adjusted to 1X 10⁶Individual cells/mL.

1.2 extraction of RNA

In an EP tube containing 50. mu.L to 1.5mL of peripheral blood mononuclear cells, 500. mu.L of Trizol reagent was added. After blowing with a pipette until no more viscous, 100. mu.L of chloroform (one fifth of the volume of Trizol) was added to the EP tube and allowed to stand for 5min after shaking vigorously enough (becoming cloudy). Centrifuging at 12000rpm for 10min, and separating into three layers with RNA at the uppermost layer, protein at the middle layer, and organic phase at the lowermost layer. And (3) sucking the supernatant into a new EP tube, adding isopropanol with the same volume, shaking and uniformly mixing, and standing at-20 ℃ for 20min to ensure that the RNA is completely precipitated. Centrifugation was carried out at 12000rpm for 10min, and the supernatant was removed to leave a precipitate, which was washed with 500. mu.L of 75% ethanol. Then, the mixture was centrifuged at 12000rpm for 5min, and the supernatant was removed and the washing was repeated once. And (3) standing at room temperature for 5-10 min, airing, adding 20 mu L of DEPC water, and dissolving at room temperature for 30 min.

1.3 Spectrophotometer measurement of RNA concentration

Respectively taking 2 mu L of RNA, adding 198 mu L of DEPC water, uniformly mixing, respectively measuring OD230, OD260 and OD280 in a spectrophotometer to evaluate the nucleic acid quality, and obtaining the RNA concentration according to the following calculation formula.

1.4 digestion and removal of residual genomic DNA from RNA

For example, when 5. mu.g of RNA was digested, 0.4. mu.L of RNase inhibitor (40U/. mu.L), 1. mu.L of DNase, 2. mu.L of 10 XDNase buffer and 5. mu.g of RNA were added in this order, and DEPC water was added to make up a 20. mu.L system. Then put into a PCR instrument and the following procedures are carried out: 30min at 37 ℃ (DNA digested by DNase); and (3) at 68 ℃ for 10min (inactivating DNase).

1.5 reverse transcription

For example, reverse transcription of 1. mu.g RNA was performed by adding 4. mu.L RNA (1. mu.g), 2. mu.L random primer (25. mu.M) and 6. mu.L DEPC water to prepare a 12. mu.L system, mixing well, and placing into a PCR instrument, the procedure was as follows: 70 ℃, 10min (opening complex RNA structures); immediately afterwards, the RNA was placed on ice for 3-5 min (random primers were attached to the RNA template). mu.L dNTP (10mM), 4. mu.L 5 Xreverse transcription buffer, 0.5. mu.L reverse transcriptase (MLV) and 2.5. mu.L DEPC water were added to the above system, mixed well and placed in a PCR instrument for the following procedures: 30 ℃ for 10 min; 42 ℃ for 1 h; 70 ℃ for 15 min.

1.6qRT-PCR detection

Firstly, an RNA sequence of NKILA (RefSeq sequence number: NR _131157.1, the specific sequence is shown in SEQ ID NO. 1) is found in an NCBI database, primer design is carried out according to the sequence of the NKILA, synthesis is carried out, and the sequences of an upstream primer (primer-F and primer-R) and a downstream primer (primer-F and primer-R) are respectively shown in SEQ ID NO.2 and SEQ ID NO. 3. The cDNA obtained by reverse transcription was used as a template to prepare the following reaction system:

then put into a fluorescent quantitative PCR instrument to carry out the following amplification procedures: 95 ℃ for 7 min; 40 cycles: 95 ℃ for 10 s; 60 ℃ for 30 s. And (3) performing melting curve analysis after amplification is completed: 95 ℃ for 15 s; 60 ℃ for 15 s; 95 ℃ for 15 s. By use of 2^-ΔΔCTRelative quantification of NKILA expression in peripheral blood mononuclear cells of normal and atherosclerotic patientsAnd comparing and analyzing the amount of the obtained product.

As a result, as shown in fig. 1, the expression level of NKILA in peripheral blood mononuclear cells of an atherosclerotic patient was significantly lower than that of NKILA in peripheral blood mononuclear cells of a normal patient (represented by the value of P in the figure < 0.05).

Secondly, design of atherosclerosis molecular marker siRNA sequence and detection of interference effect thereof

RNA interference (RNAi) technology refers to a highly conserved phenomenon of highly efficient and specific degradation of homologous mrnas induced by double-stranded small RNA molecules during evolution, and RNA molecules inhibit the biological process of certain gene expression by destroying specific mrnas. It is a sequence-specific post-transcriptional gene silencing process that is widely found in animals and plants, and is a bioprotective mechanism for the resistance of biological genomes to the invasion of foreign genetic elements such as viruses. Since the expression of a specific gene can be specifically knocked out or turned off using the RNAi technology, the technology is rapidly becoming one of the most interesting research tools in the field of gene function research and gene therapy research, and has been widely used for exploring the biological functions of genes and the treatment of various diseases.

2.1 design of siRNA

According to the complete RNA sequence (shown as SEQ ID NO. 1) of NKILA, a siRNA sequence is designed and screened by using a design website (http:// sidirect2.rnai. jp /) of siRNA, Shanghai Biolabe company is entrusted to synthesize the siRNA, and specific sequences of a sense strand and an antisense strand of the siRNA are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5.

2.2 detection of the interfering Effect of the above siRNA Using umbilical vein endothelial cells (HUVECs)

HUVECs are inoculated on a six-hole plate, when the cell density reaches about 70%, control group siRNA (specific sequences are shown as SEQ ID NO.6 and SEQ ID NO. 7) and the experimental group siRNA are transfected into HUVECs respectively by using a Lipofectamine 3000 transfection reagent.

The specific method steps for transfecting siRNA are as follows: mu.L of serum-free medium was added to each of two 1.5mL EP tubes, 6. mu.L of Lipofectamine 3000 was added to one of the EP tubes, and 6. mu.L of siRNA at a concentration of 10. mu.M was added to the other of the EP tubes. The mixture of siRNA medium was added to the mixture of Lipofectamine 3000 medium, vortexed for 5 seconds to mix well, allowed to stand at room temperature for 10min, and added drop-wise to HUVECs cells. After 6 hours, the complete medium was replaced with fresh medium, and after 48 hours, total RNA was extracted by Trizol for reverse transcription and the amount of NKILA expressed by fluorescent quantitative PCR was measured as described above.

As a result, as shown in fig. 2, the expression level of NKILA in the cells of the experimental group was significantly lower than that of NKILA in the cells of the control group (P value < 0.001 in the figure), indicating that the siRNA designed specifically targets NKILA and decreases its expression level, and thus the siRNA can be used for further exploring the biological function of NKILA and the treatment of atherosclerosis.

Thirdly, reducing the influence of the expression level of the atherosclerosis molecular marker on the vascular dermatitis

The inflammatory reaction is a pathological reaction which is very common clinically, and refers to a basic pathological process which takes defense reaction as a main part and is generated by the stimulation of living tissues with a vascular system to various injury factors. Inflammation often manifests as redness, swelling, heat, pain, and dysfunction, as well as systemic reactions such as fever, altered peripheral blood leukocyte counts, and the like. Excessive activation of inflammation is closely related to the development and progression of atherosclerosis.

3.1 Effect of decreasing expression levels on adhesion between monocytes and endothelial cells

After the vascular endothelial inflammation is activated, endothelial cells can be promoted to secrete a plurality of related adhesion factors, so that the adhesion between monocytes and the endothelial cells is promoted, and the degree of the inflammation of the endothelial cells can be reflected by verifying the adhesion between the cells.

HUVECs of endothelial cells are inoculated on a 6-well plate overnight, and when the density reaches 80%, siRNA of a control group and siRNA of an experimental group are transfected into the HUVECs respectively by using a Lipofectamine 3000 transfection reagent (the specific transfection steps are as described above). The medium was changed to fresh 6 hours after transfection, the mononuclear cells THP-1 were labeled with a fluorophore by Tracker Green CMFDA 48 hours after transfection, HUVECs were changed to fresh medium 1 hour after transfection, and the THP-1 cells were washed 3 times with RPMI medium. THP-1 cells were added to HUVECs of the control group and the experimental group, respectively, and incubated in an incubator for 1 hour. The THP-1 cells not adhering to HUVECs were removed by washing 3 times with ECM medium for culturing HUVECs, and the THP-1 cells adhering to HUVECs were counted under a fluorescent microscope and photographed.

As shown in FIG. 3, it can be seen that the adhesion between THP-1 and HUVECs cells was significantly enhanced after the NKILA expression level was decreased as compared with the control group.

3.2 Effect of decreasing expression level on expression level of inflammatory factor in endothelial cells

HUVECs cells are inoculated on a six-well plate, and after the cell density reaches about 70%, control group siRNA and experimental group siRNA are transfected into HUVECs by using a Lipofectamine 3000 transfection reagent. After 48 hours, total RNA was extracted by Trizol, reverse transcription was performed, and the mRNA expression levels of four inflammatory factors, i.e., adhesion factors VCAM1 and SELE and chemokines MCP1 and IL8, were measured by fluorescent quantitative PCR.

As a result, as shown in fig. 4, when the expression level of NKILA decreased, the expression levels of inflammatory factors such as adhesion factors VCAM1 and SELE and chemokines MCP1 and IL8 were significantly increased (in the figure, the value of P is less than 0.01, and in the figure, the value of P is less than 0.001).

Fourth, the influence of the over-expression of the atherosclerosis molecular marker on the vascular dermatitis

4.1 construction of Lentiviral expression vectors for molecular markers of atherosclerosis

Based on the RNA sequence of NKILA, a double-stranded DNA sequence was synthesized, and two sites of BamH I and Xba I were attached to the both ends of the DNA sequence. The synthesized NKILA was ligated to the lentiviral empty vector of pLV CS2.0 by DNA T4 ligase (the lentiviral empty vector sequence is shown in SEQ ID NO. 8) by double cleavage at both BamH I and Xba I sites.

The enzyme digestion reaction system is as follows:

after the addition of water to a volume of 20. mu.L, the cells were incubated at 37 ℃ for 4 hours.

The T4 DNA ligase reaction system is as follows:

10 Xbuffer 1. mu.L

The NKILA fragment: pLV vector (mole number) (1-5): 1

T4 ligase 1. mu.L

After adding water to make up to 10. mu.L, the cells were incubated overnight at 16 ℃.

Respectively packaging lentiviruses of pLV CS2.0 empty vector and NKILA overexpression vector, and specifically comprising the following steps: the method comprises the following steps of inoculating HEK293T cells into a 6-well plate, and transfecting NKILA overexpression plasmids, empty control vectors and commercial lentiviral packaging plasmids pSPAX2 and pMD2G into HEK293T cells by using Lipofectamine 3000 when the cell density reaches 70%, wherein the plasmids are prepared in the following specific ratio:

pSPAX2 0.5μg

pMD2G 0.5μg

NKILA overexpression plasmid or empty vector 1. mu.g

After transfection for 48 hours, virus supernatants were collected and used directly for infecting cells or frozen in a minus 80 ℃ ultra low temperature freezer for future use.

4.2 testing the effect of overexpression on the adhesion between monocytes and endothelial cells.

HUVECs of umbilical vein endothelial cells are inoculated into a six-well plate, when the cell density reaches 80%, the original culture medium is discarded, 1mL of supernatant of the empty vector (vec) and NKILA overexpression virus (NKILA-flag) is taken respectively, added into the HUVECs, 1mL of fresh culture medium is added, and finally 2 μ L of Polybrene (Polybrene) with the concentration of 4 μ g/μ L is added. After 8 hours, the cells were replaced with fresh medium. 48 hours after infection, HUVECs cells were treated with inflammatory response inducer TNF α for 1 hour, while mononuclear cells THP-1 were labeled with a fluorophore using Tracker Green CMFDA and incubated at 37 ℃ for 1 hour. HUVECs were washed 3 times with ECM medium and replaced with fresh ECM medium, while THP-1 cells were washed 3 times with RPMI medium. THP-1 cells labeled with Tracker Green were added to HUVECs infected with empty vector and NKILA-overexpressing lentivirus, respectively, and incubated in an incubator for 1 hour. The THP-1 cells not adhering to HUVECs were removed by washing 3 times with ECM medium for culturing HUVECs, and the THP-1 cells adhering to HUVECs were counted under a fluorescent microscope and photographed.

As shown in fig. 5A, the expression level of NKILA was significantly increased in the cells of the experimental group after infection with the NKILA overexpressing lentivirus, compared to the control group infected with the empty vector (represented by the value of P < 0.001 in the figure). More importantly, as shown in FIG. 5B, the increased expression of NKILA significantly inhibited the adhesion between monocyte THP-1 and endothelial HUVECs.

4.3 testing the Effect of overexpression on inflammatory factors in vascular endothelial cells

HUVECs are inoculated on a 6-well plate, and are infected by packaged empty vectors and NKILA overexpression lentiviruses respectively when the cell density reaches 80%. HUVECs were infected 48 hours later, and treated with TNF α, an inducer of inflammatory response, for 1 hour. Extracting total RNA by using Trizol lysis cells, digesting by DNase, taking 1 mu g of RNA for reverse transcription, carrying out fluorescent quantitative PCR on the cDNA after the reverse transcription, and respectively detecting the expression quantity of related inflammatory factors after infecting empty vectors and NKILA over-expressing lentivirus.

As a result, as shown in fig. 6, the expression amounts of inflammatory factors such as adhesion factors VCAM1 and SELE and chemokines MCP1 and IL8 were significantly decreased in the cells of the experimental group infected with the NKILA-overexpressing lentivirus compared to the control group infected with the empty vector (in the figure, the P value is less than 0.05, and in the figure, the P value is less than 0.01), and the important inhibitory effect of the long non-coding RNA NKILA on the vascular dermatitis was confirmed again.

In conclusion, the expression level of the long-chain non-coding RNA NKILA (the sequence is shown as SEQ ID NO. 1) in peripheral blood mononuclear cells of an atherosclerotic patient is obviously lower than that of peripheral blood mononuclear cells of a normal person (the P value is less than 0.05), and the development condition of atherosclerosis can be better reflected, so that the long-chain non-coding RNA NKILA can be used as a clinical diagnosis molecular marker of atherosclerosis and applied to preparation of related products for diagnosing or detecting atherosclerosis, and the diagnosis and detection means of atherosclerosis are enriched. The atherosclerosis molecular marker can inhibit the adhesion between monocyte THP-1 and vascular endothelial cells HUVECs and inhibit the expression of inflammatory factors such as adhesion factors, chemotactic factors and the like, so that the overactivation of vascular endothelial inflammation can be effectively inhibited, and the atherosclerosis molecular marker can also be used as a molecular drug target for inhibiting the formation and development of atherosclerosis, thereby providing a new experimental theoretical basis and direction for further researching the pathogenesis of atherosclerosis and exploring the prevention and treatment drugs thereof.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

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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aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc 720

ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag 780

acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat 840

caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac 900

aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat 960

gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg 1020

agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat 1080

aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat 1140

gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 1200

gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 1260

gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat 1320

ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag 1380

taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat 1440

taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa 1500

gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat 1560

aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt 1620

aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt 1680

atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga 1740

agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta 1800

tcggttaact tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt 1860

agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaattcaa 1920

aattttatcg atgcctcccc gtcaccaccc cccccaaccc gccccgaccg gagctgagag 1980

taattcatac aaaaggactc gcccctgcct tggggaatcc cagggaccgt cgttaaactc 2040

ccactaacgt agaacccaga gatcgctgcg ttcccgcccc ctcacccgcc cgctctcgtc 2100

atcactgagg tggagaagag catgcgtgag gctccggtgc ccgtcagtgg gcagagcgca 2160

catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga 2220

gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg 2280

agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg 2340

ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta 2400

cgggttatgg cccttgcgtg ccttgaatta cttccacgcc cctggctgca gtacgtgatt 2460

cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga 2520

gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga 2580

atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat 2640

ttttgatgat atcctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa 2700

gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc 2760

cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg 2820

tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg 2880

ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt 2940

cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt 3000

gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc 3060

acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg 3120

tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag 3180

actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag 3240

tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt 3300

caggtgtcgt gaaaactacc cctgagctcc ttaaggttaa cgccaccatg gactacaaag 3360

acgatgacga caagtctaga gaattcggat ccaatattcc cgggctcgag ccatggaagc 3420

ttgatatcta actgactgaa ccggtggtac cgatccacgc gtctccggcc tagggataac 3480

agggtaatcc gctagcccct ctccctcccc cccccctaac gttactggcc gaagccgctt 3540

ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg 3600

caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc 3660

ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga 3720

agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc 3780

tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc 3840

acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc 3900

aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga 3960

tctggggcct cggtacacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg 4020

ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacac 4080

tagagatcca ccggtcgcca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg 4140

cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac 4200

gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt 4260

cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt 4320

ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc 4380

gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct 4440

ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga 4500

ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg 4560

cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg 4620

gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat 4680

gacccgcaag cccggtgcct gagcggccgc gtcgacaatc aacctctgga ttacaaaatt 4740

tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 4800

gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 4860

tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 4920

gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 4980

cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 5040

gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 5100

ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg 5160

cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 5220

ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 5280

atctcccttt gggccgcctc cccgcctgga attcgagctc ggtaccttta agaccaatga 5340

cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 5400

taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag 5460

accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat 5520

aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact 5580

agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc 5640

atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact 5700

tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 5760

aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 5820

atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 5880

ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 5940

cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac 6000

gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa 6060

cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 6120

ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 6180

agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 6240

gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 6300

ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 6360

cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 6420

gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 6480

tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 6540

gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag 6600

ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg 6660

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 6720

atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 6780

agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 6840

ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 6900

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 6960

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 7020

tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 7080

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 7140

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 7200

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 7260

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 7320

cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 7380

tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 7440

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 7500

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 7560

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 7620

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 7680

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 7740

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 7800

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 7860

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 7920

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 7980

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 8040

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 8100

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 8160

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 8220

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 8280

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 8340

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 8400

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 8460

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 8520

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 8580

cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 8640

gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 8700

gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 8760

gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 8820

agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgc 8865

Claims

1. The application of the atherosclerosis molecular marker in the preparation of products for diagnosing atherosclerosis is characterized in that the atherosclerosis molecular marker is long-chain non-coding RNA, and the nucleotide sequence of the atherosclerosis molecular marker is shown as SEQ ID NO. 1.

2. Use of a PCR amplification primer in the preparation of a product for diagnosing atherosclerosis, comprising an upstream primer and a downstream primer for specifically amplifying the molecular marker of atherosclerosis according to claim 1; the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.

3. The use according to any one of claims 1 to 2, wherein the product is a detection reagent.

4. The use according to any one of claims 1 to 2, wherein the product is a kit, a gene chip or a test strip.

5. The use of claim 4, wherein the kit further comprises at least one of an RNA extraction reagent, a reverse transcription reagent, and a PCR amplification reagent.

6. Use of a small interfering RNA in the preparation of a medicament for preventing and treating atherosclerosis, wherein the small interfering RNA comprises a complementary sense strand and an antisense strand, and the small interfering RNA is capable of specifically inhibiting the expression of the atherosclerotic molecular marker of claim 1; the nucleotide sequence of the sense strand is shown as SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 5.