CN112159842A

CN112159842A - Molecular marker for early warning of atherosclerosis and application thereof

Info

Publication number: CN112159842A
Application number: CN202011080892.2A
Authority: CN
Inventors: 姜怡邓; 张慧萍; 张辉; 刘圆; 马芳; 杨安宁; 马胜超
Original assignee: Ningxia Medical University; General Hospital of Ningxia Medical University
Current assignee: Ningxia Medical University; General Hospital of Ningxia Medical University
Priority date: 2020-10-11
Filing date: 2020-10-11
Publication date: 2021-01-01

Abstract

A molecular marker for early warning of atherosclerosis, comprising: (1) detecting the influence of different concentrations of Hcy on the proliferation of endothelial cells by an MTT colorimetric method, and screening appropriate concentrations; (2) after the optimum concentration Hcy intervenes the cells, the cell viability is stained and detected; (3) after the optimum concentration Hcy intervenes the cells, detecting endothelial cell apoptosis by flow cytometry; (4) after the optimum concentration Hcy intervenes the cells, the expression of LncRNA DINO is detected by real-time fluorescence quantitative PCR; (5) transfecting LncRNA DINO siRNA, and after interfering the LncRNA DINO, carrying out cell viability staining detection; (6) constructing LncRNA DINO siRNA, and detecting endothelial cell apoptosis by flow cytometry after interfering the LncRNA DINO; (7) constructing LncRNA DINO siRNA, and detecting the expression of apoptosis-related protein by using Western immunoblotting after interfering LncRNA DINO. The invention provides a new molecular target for early diagnosis and early warning of atherosclerosis and a new direction for treatment.

Description

Molecular marker for early warning of atherosclerosis and application thereof

Technical Field

The invention belongs to the technical field of medicine, and particularly relates to a molecular marker for early warning of atherosclerosis and application thereof.

Background

Atherosclerosis (AS) is a chronic progressive disease that is well developed in the large and medium sized arteries, characterized primarily by endothelial cell apoptosis, smooth muscle cell proliferation, and foam cell formation, and is responsible for the high morbidity and mortality of cardiovascular and cerebrovascular diseases worldwide. In recent years, with the rapid development of social economy, the living standard of people is continuously improved, and under the influence of factors such AS dietary structure change, working pressure increase and the like, the incidence rate of AS (atherosclerosis) shows a trend of rising year by year and gradually develops towards the younger state, so that the early discovery, prevention and delay of the process of atherosclerosis is important in the current clinical science field. Therefore, the search for an early marker of atherosclerosis has important significance for preventing and early diagnosing atherosclerosis.

The mechanism of development of atherosclerosis is complex, and there are several hypotheses: inflammation hypothesis, lipid infiltration hypothesis (lipogenesis hypothesis), vascular smooth muscle cell monoclonal hypothesis, endothelial cell dysfunction hypothesis, and the like. The functional and structural integrity of vascular endothelial cells of the artery can maintain the normal functions of contraction, relaxation and endocrine of the blood vessel, and can prevent various cells (monocytes, neutrophils, erythrocytes and the like) and various substances (triglyceride, cholesterol, low-density lipoprotein and the like) in the blood from migrating and depositing to the subintimal space, thereby providing natural guarantee for the arterial blood vessel wall. Endothelial cells, which serve as the natural barrier of the vascular wall, have a series of subsequent chain reactions (release of chemokines, migration and proliferation of monocytes, phagocytosis of lipid into foam cells by macrophages and the like) caused by dysfunction, which are the initial and necessary steps of atherosclerosis, so that the apoptosis of the endothelial cells is closely related to the development of atherosclerosis.

Homocysteine (Hcy) is a sulfur-containing amino acid formed in the metabolic pathway of methionine in vivo, cannot be synthesized by the body per se, and belongs to an intermediate product of methionine cycle. Recent epidemiological studies have shown that hyperhomocysteinemia (heromoeysiteinemia, HHcy) is an independent risk factor for atherosclerotic cardiovascular disease. Epidemiological investigation shows that the risk of stroke is increased by 59 percent when Hcy (homocysteine) in blood plasma is increased by 5 mu mol/L; every 3 mu mol/L reduction of Hcy can reduce the risk of stroke by about 24%; the incidence rate of cardiovascular events of patients with hypertension and Hcy blood is 3 times higher than that of patients with simple hypertension and 12-25 times higher than that of normal people, so that the search for preventing and treating the effect of the Hcy blood on atherosclerotic cardiovascular diseases is particularly important.

In recent years, research on non-coding RNAs (ncRNAs) and cardiovascular diseases has become a hot spot. There is increasing evidence that various types of ncRNAs (non-coding ribonucleic acids) can regulate the expression of a large number of genes, play an important role in the physiological response of cells, and participate in many pathological changes including atherosclerosis. Long non-coding RNA (lncRNA) is a transcription product with the length of more than 200nt and without coding capacity. The long-chain non-coding RNA can regulate and control gene expression at multiple levels such as epigenetic modification level, transcription level, post-transcription level and the like, thereby playing an important role in multiple physiological processes such as cell differentiation, proliferation, apoptosis, migration, pluripotent stem cell regeneration and the like. DNA Damage Induced non-coding RNA (DINO), a member of lncRNA (long non-coding RNA) newly discovered in 2016, widely involved in cancer development mechanisms, and studies have shown that lncRNA DINO (DNA Damage Induced non-coding RNA) is essential for tumor suppressor p 53-dependent gene expression, cell cycle arrest, and apoptosis in DNA Damage response; when no DNA is damaged, the expression of IncRNA DINO can effectively activate a DNA damage pathway, promote the stability increase of p53 protein, regulate p53 to automatically enhance a loop and inhibit the development of oncogenes; it is also reported in literature that it can significantly inhibit the apoptosis rate of gastric cancer cells and play a tumor-inhibiting role in the progression of gastric cancer. lncRNA DINO (DNA damage-induced non-coding RNA), a member of lncRNA (long non-coding RNA), plays an important role in cancer cell migration and apoptosis, but its role in endothelial cell apoptosis by Hcy is not clear.

In the past, AS (atherosclerosis) has been studied, and the relationship between the AS and the gene expression and the signal pathway has been studied in many cases. We clarify the influence of long-chain non-coding RNA (ribonucleic acid) on HUVECs (human umbilical vein endothelial cells) apoptosis, and from the perspective of early prevention and diagnosis, search for a marker target capable of being used for AS clinical diagnosis, and further confirm the relationship between lncRNA DINO (DNA damage-induced non-coding RNA) and atherosclerotic diseases, which is helpful for better understanding of disease pathogenesis and provides theoretical basis and experimental basis for the diagnosis and treatment of AS.

Disclosure of Invention

The invention aims to provide a long-chain non-coding RNA for atherosclerosis diagnosis and application thereof.

A molecular marker for early warning of atherosclerosis, wherein the molecular marker for early warning of atherosclerosis is long-chain non-coding RNA, and the method comprises the following steps:

(1) MTT (tetrazolium salt) colorimetric method is used for detecting the influence of different concentrations of Hcy on endothelial cell proliferation, and proper concentrations are screened;

(2) after the optimum concentration Hcy (homocysteine) intervenes the cells, a control group (0 mu mol/L Hcy) and an experimental group (100 mu mol/L Hcy) are taken as groups, and the change of the cell activity is detected by cell activity staining;

(3) after the cells are intervened by the Hcy with the optimal concentration, a control group and an experimental group are taken as groups, and the change of the endothelial cell apoptosis level is detected by flow cytometry;

(4) after the cells are intervened by the optimal concentration of Hcy, grouping a control group and an experimental group, and detecting the expression of LncRNA DINO (non-coding RNA induced by DNA damage) by real-time fluorescent quantitative PCR (RT-qPCR);

(5) transfecting LncRNA DINO siRNA (interfering RNA induced by DNA damage) and detecting the change of cell viability by cell viability staining after interfering the LncRNA DINO;

(6) constructing LncRNA DINO siRNA, and detecting the apoptosis level change of endothelial cells by flow cytometry after interfering the LncRNA DINO;

(7) constructing LncRNA DINO siRNA, and detecting the expression of apoptosis-related proteins Bax and Bcl-2 by using Western Blot (Western Blot) after interfering LncRNA DINO.

Primers for detecting the long non-coding RNA (LncRNA DINO) are also within the scope of the present invention.

It is also within the scope of the invention that the agent for inhibiting the long non-coding RNA is a small interfering RNA (si RNA).

The invention relates to application of long-chain non-coding RNA in atherosclerosis in prevention and diagnosis of the atherosclerosis.

Compared with the prior art, the invention has the beneficial effects that:

experiments prove that the regulation and control effect of lncRNA DINO (non-coding RNA induced by DNA damage) on the apoptosis of endothelial cells induced by homocysteine participates in the process of regulating and controlling the disease in the initiating link of atherosclerosis. Through the verification of the target function, the early diagnosis and early warning function of early atherosclerosis can be realized, clinical timely intervention is facilitated, and the disease development is delayed. Therefore, the invention provides a new molecular target for early diagnosis and early warning of atherosclerosis and also provides a new direction for treating atherosclerosis.

Drawings

FIG. 1 is a schematic diagram showing the determination of the optimum concentration of Hcy interfering endothelial cells by MTT (tetrazolium salt) colorimetric method;

FIG. 2 is a schematic diagram showing the change of cell viability after detecting the intervention of Hcy in endothelial cells by cell viability staining;

FIG. 3 is a schematic diagram showing the apoptosis of endothelial cells after detecting Hcy by flow cytometry;

FIG. 4 is a schematic view showing the expression of LncRNA DINO detected by RT-qPCR (real-time fluorescent quantitative PCR);

FIG. 5 is a schematic view showing the detection of expression of LncRNA DINO (DNA damage-induced non-coding RNA) after transfection of an interference fragment;

FIG. 6 is a schematic diagram showing apoptosis detection by cell viability staining after interference of LncRNA DINO (DNA damage induced non-coding RNA);

FIG. 7 is a schematic diagram of detection of apoptosis by flow cytometry after interference of LncRNA DINO;

FIG. 8 is a diagram showing the Western Blot detection of the expression of Bax (Bcl-2-related X protein) and Bcl-2 (B-lymphocytoma-2) after the interference of LncRNA DINO.

In fig. 2, DIPA denotes the nucleus; cy3 represents dead cells; FITC stands for live cells; merge denotes a combined picture.

In fig. 3, the lower left quadrant (first quadrant) is live cells, the lower right quadrant (second quadrant) is early apoptotic cells, the upper right quadrant (third quadrant) is late apoptotic cells, and the upper left quadrant (fourth quadrant) is injured cells; ordinate: apoptosis ratio (%): rate of Apoptosis; compared with the control group, the compound of the formula,^** P< 0.01。

in fig. 4, ordinate: lncRNA DINO expression levels, expression of non-coding RNA induced by DNA damage; compared with the control group, the compound of the formula,^** P < 0.01。

in fig. 5, ordinate: lncRNA DINO expression levels, expression of non-coding RNA induced by DNA damage; si-NC (negative control for interfering fragments) or si-DINO (interfering fragments of DNA damage-induced non-coding RNA) transfected cells, compared to si-NC (negative control),^** P< 0.01。

in fig. 6, DIPA is blue indicating nuclei; cy3 is red indicating dead cells; FITC is green indicating viable cells; merge denotes a combined picture.

In fig. 7, ordinate: apoptosis ratio (%): rate of Apoptosis; + symbol: si-NC (negative control for interfering fragments) or si-DINO (interfering fragments of DNA damage-induced non-coding RNA) transfected cells, or Hcy interfering cells; -symbol: the intervention factor is not met; compared with the si-NC, the method has the advantages that,^** P < 0.01。

FIG. 8 is a striped graph of apoptosis-related proteins Bax (Bcl-2-related X protein), Bcl-2 (B-lymphoma-2); B. apoptosis-related protein Bax (Bcl-2-related X protein), Bcl-2 (B-lymphocytoma-2) statistical map; compared to si-NC (negative control for interfering fragments),^** P< 0.01。

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

A molecular marker for early warning of atherosclerosis comprises the following specific embodiments:

1 Material preparation

1.1 materials and reagents Human Umbilical Vein Endothelial Cells (HUVECs) (China, famous Biotechnology, Inc.); homocysteine (Sigma, usa); fetal bovine serum, DMEM medium (australia, Gibco); streptomycin, trypsin digest, NP-40 (ethylphenyl polyethylene glycol) (china, bi yun tian institute of biotechnology); cell viability fluorescence assay kit (switzerland, Roche); PMSF (china, beijing solibao technologies ltd); bax (Bcl-2-associated protein X) antibody, Bcl-2 (B-lymphoma-2) antibody (Abcam, UK), beta-actin antibody, and horseradish peroxidase-labeled secondary antibody (Chinese, Beijing gold bridge, Inc.); skimmed milk powder (us, BD corporation); a cDNA (complementary DNA) kit, an RT-qPCR (real-time fluorescent quantitative PCR) kit (Japan, Takara Co.); lipo2000 (Thermo Fisher corporation, usa); the primers were synthesized by Shanghai Biotech, Inc.; LncRNA DINO siRNA (DNA damage induced non-coding RNA interference RNA) by Huzhou river horse science and technology company Limited.

1.2 clean bench for instruments and equipment (Suzhou, Antai); CO 2₂Incubators (Heraeus, germany); model 5415D micro bench centrifuge (Eppendorf, Germany); ultramicrospectrophotometers (simplina, usa); vertical electrophoresis apparatus, membrane transfer apparatus, exposure apparatus (Bio-Rad, USA); precision balances of the BS110S type (Sartorius, germany); horizontal shaker (Thermo Fisher, usa) gradient RCR instrument (Biometra Tone, germany); fluorescent quantitative PCR instrument (Shanghai, Feng Ling); FACS Calibur flow cytometer (USA; BD); confocal laser microscopy (Germany; ZEISS).

2 preparation process and method of long-chain non-coding RNA marker

2.1 cell culture

Culturing HUVECs (human umbilical vein endothelial cells) in DMEM medium containing 10% fetal bovine serum at 37 deg.C under 5% CO₂In the incubator, when the cells grow to about 70%, the cells were intervened with Hcy (control group) at a final concentration of 0. mu. mol/L and Hcy (experimental group) at a final concentration of 100. mu. mol/L for 72 hours, and then the cells were collected for subsequent experiments.

2.2 MTT (tetrazolium salt) colorimetric method for detecting the optimum concentration of Hcy (homocysteine).

HUVECs (human umbilical vein endothelial cells) cells were seeded in 3 96-well cell culture plates, cultured for 24 hours, the supernatant was discarded, and 100ul cell culture medium containing different concentrations of Hcy (homocysteine) (0. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L, 200. mu. mol/L, 500. mu. mol/L) was added to the cells, 3 multiple wells were set for each concentration, 37 ℃, 5% CO₂After 72h of culture, cell viability was determined by MTT (tetrazolium salt) colorimetric assay (according to the instructions).

2.3 cell viability staining to detect apoptosis

The medium was discarded, the cells in the laser confocal dish were washed 3 times with PBS, 5 ml of PBS, 5. mu.l of nucleic acid Dye, 25. mu.l of Dead Dye and 3. mu.l of visible Dye were added to a 15 ml centrifuge tube, and after mixing the three dyes, 200. mu.l of mixed Dye was sequentially added to each dish, and after incubation at 37 ℃ for 30 min, the cells were removed and observed for apoptosis by laser confocal microscopy.

2.4 detection of apoptosis by flow cytometry

Endothelial cells were digested with pancreatin without EDTA (ethylenediaminetetraacetic acid), and the supernatant was discarded by centrifugation at 800rpm at 4 ℃. After washing the cells 2 times with cold PBS, suspending the cells with 400. mu.l of 1 × Annexin V (phospholipid binding protein) binding solution, adding 5. mu.l of Annexin V-FITC (phospholipid binding protein-fluorescein isothiocyanate) staining solution, gently mixing, incubating at 4 ℃ in the dark for 15 min, adding 10. mu.l of PI (propidium iodide) staining solution, mixing, incubating in the dark for 5min, and immediately placing in a flow cytometer to detect the change of apoptosis.

2.5 RT-qPCR (real-time fluorescent quantitative PCR) detection of IncRNA DINO (DNA damage-induced non-coding RNA) self-expression in each cell group.

Total RNA (ribonucleic acid) of each group of cells was extracted and reverse transcribed according to the instructions of the Takara RNA kit. PCR amplification procedure: 30 s at 95 ℃; 5 s at 95 ℃; 34 s at 60 ℃ for 40 cycles, and the same line was amplified with an internal control GADPH (glyceraldehyde-3-phosphate dehydrogenase). The relative amount of the target gene is 2^-△△CtAnd (4) showing.

2.6 LncRNA DINO (DNA damage induced non-coding RNA) interference fragment transfected cells

After HUVECs (human umbilical vein endothelial cells) cells are inoculated in a 6-well plate and cultured for 24 hours, the cell density is 60-70%, transfection operation is carried out strictly according to the operation instruction of a Lipofectamine 2000 (DNA transfection reagent) kit, 5 mu L of negative controls si-NC and si-DINO are respectively added into 250 mu L of serum-free culture medium, the mixture is gently and uniformly mixed, 6 mu L of Lipofectamine 2000 (DNA transfection reagent) liposome is uniformly mixed with 250 mu L of serum-free culture medium, and the mixture is placed at room temperature for 5 minutes. After 5min, mixing the two solutions, and standing at room temperature for 20 min. And (3) metering the volume of each hole in the six-hole plate to 2ml by using a serum-free culture medium, putting the six-hole plate into a constant-temperature incubator for continuous culture, replacing the complete culture medium after 6h of transfection, and continuously culturing for 72 h.

2.7 Western blot (immunoblot) assay of protein expression of Bax (Bcl-2-associated X protein), Bcl-2 (B-lymphoma-2)

Preparing a protein lysate by PMSF (phenylmethylsulfonyl fluoride) and NP-40 (ethylphenyl polyethylene glycol) lysate according to a ratio of 1:100, adding the lysate according to the cell amount, placing the lysate in a shaking table, centrifuging at 12000 rpm and 4 ℃ for 20min after 30 min at 4 ℃, transferring protein supernatant into a new EP tube, adding a sample buffer according to a ratio of 4:1 of protein to buffer, and boiling and denaturing at 99 ℃ for 5 min. Performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) electrophoresis on 300 mu g of protein sample per well, transferring the gel onto 0.22 mu m PVDF (polyvinylidene fluoride) membrane, sealing 5% skimmed milk at room temperature for 2h, washing the membrane with PBST (phosphate buffer solution containing Tween-20) for 10 min for 3 times, respectively adding beta-actin (beta-actin) antibody (1: 10000), Bax (Bcl-2-related X protein), Bcl-2 (B-lymphocytoma-2) antibody (1: 2000), standing overnight at 4 ℃, washing the membrane with PBST (phosphate buffer solution containing Tween-20) at room temperature for 10 min, repeating for 3 times, incubating with horseradish peroxidase-labeled secondary antibody (1: 5000) at room temperature for 2h, washing the membrane with PBST (phosphate buffer solution containing Tween-20) at room temperature for 10 min, repeating for 3 times, and (6) exposing. The ratio of the optical density values of Bax (Bcl-2 related X protein), Bcl-2 (B lymphomatosis-2) and beta-actin (beta-actin) is taken as the relative amount of Bax (Bcl-2 related X protein) and Bcl-2 (B lymphomatosis-2) protein expression.

2.8 statistical methods

The experimental results of the research are all measured data, the statistical analysis is carried out by prism7.0 statistical software, the results are expressed by X +/-S, and the comparison between the two groups is carried outtAnd (4) testing, wherein the One-way ANOVA test is adopted for the comparison among the average numbers of multiple samples, the Student-Newman-Keuls test is adopted for the comparison between every two groups, and P < 0.05 shows that the difference has statistical significance.

4 results

4.1 determination of the optimum concentration of Hcy (homocysteine) by MTT (tetrazolium salt) colorimetric method

MTT (tetrazolium salt) colorimetric method for detecting Hcy (homocysteine) with different concentrations of 0 mu mol/L, 50 mu mol/L, 100 mu mol/L, 200 mu mol/L and 500 mu mol/LEffect on endothelial cell survival, results show: the survival rate of endothelial cells is gradually reduced along with the increase of Hcy concentration and shows a dose-dependent relationship, the survival rate of the endothelial cells is obviously reduced when the concentration is 100 mu mol/L, 200 mu mol/L and 500 mu mol/L, and the survival rate of the endothelial cells is not obviously changed when the concentration is 50 mu mol/L Hcy (homocysteine) compared with that of a control group (the survival rate of the endothelial cells is not obviously changed when the concentration is 50 mu mol/L: (homocysteine) (P <0.001). See fig. 1.

4.2 cell viability staining to detect apoptosis

The influence of Hcy (homocysteine) on endothelial cell apoptosis is detected by cell viability staining, and the result is observed by a laser confocal microscope, and the result shows that: compared with the control group, the number of the apoptotic cells (the number of the red cells) of the experimental group is obviously increased, and the cell activity is obviously reduced. See fig. 2.

4.3 flow cytometry detection of apoptosis

Quantitative analysis of endothelial cell apoptosis level change after Hcy (homocysteine) intervention by flow cytometry shows that: increased endothelial apoptosis levels in the experimental group compared to the control group (P <0.01), suggesting that Hcy (homocysteine) is capable of promoting endothelial cell apoptosis. See fig. 3.

4.4 RT-qPCR detection of LncRNA DINO (DNA Damage-induced non-coding RNA) expression

RT-qPCR detects the expression of LncRNA DINO (DNA damage induced non-coding RNA), and the result shows that: increased expression of LncRNA DINO (DNA damage-induced non-coding RNA) in experimental group compared with control group: (P <0.01). See fig. 4.

4.5 detection of LncRNA DINO (DNA Damage-induced non-coding RNA) expression after transfection of interfering fragments

After transfection of the interference fragment, RT-qPCR detected LncRNA DINO (DNA damage induced non-coding RNA) expression, and the results showed: (iii) significantly reduced expression of LncRNA DINO compared to si-NC: (P <0.01). See fig. 5.

4.6 detection of apoptosis by staining for cell viability following interference with LncRNA DINO (DNA Damage-induced non-coding RNA)

To further clarify the effect of LncRNA DINO (DNA damage-induced non-coding RNA) on endothelial apoptosis, cell viability staining after interfering with LncRNA DINO (DNA damage-induced non-coding RNA) detected apoptosis, the results showed: after Hcy (homocysteine) intervention, the number of red cells in the si-DINO group is obviously reduced compared with that in the si-NC group. See fig. 6.

4.7 interfering LncRNA DINO (DNA damage induced non-coding RNA), flow cytometry detection of apoptosis

Flow cytometry after interference with LncRNA DINO (DNA damage-induced non-coding RNA) detected levels of endothelial apoptosis, showing: decreased endothelial apoptosis levels in the si-DINO group compared to the si-NC group after Hcy (homocysteine) intervention (II)P <0.01). See fig. 7.

4.8 interference of LncRNA DINO (DNA damage induced non-coding RNA), Western Blot detection of expression of Bax (Bcl-2 associated X protein), Bcl-2 (B-lymphocytoma-2)

Western Blot after interference with LncRNA DINO (DNA damage-induced non-coding RNA) detected the expression of Bax (Bcl-2-associated X protein), Bcl-2 (B-lymphocytoma-2), and the results show: (Bcl-2-associated X protein/B lymphoma-2) expression in si-DINO (interfering fragment of DINO) is reduced following Hcy (homocysteine) intervention compared to si-NC (negative control for interfering fragment) ((Bcl-2-associated X protein/B lymphoma-2))P <0.01). See fig. 8.

5 conclusion

In conclusion, the present project has focused on HUVECs (human umbilical vein endothelial cell) apoptosis, and aims to investigate the role of LncRNA DINO (DNA damage-induced non-coding RNA) in homocysteine-induced endothelial cell apoptosis, and elucidates that LncRNA DINO (DNA damage-induced non-coding RNA) can promote Hcy (homocysteine) -induced endothelial cell apoptosis. From the perspective of early diagnosis and early warning, a new target point is provided for prevention and treatment of AS (atherosclerosis).

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Sequence listing

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Claims

1. A molecular marker for early warning of atherosclerosis is characterized in that the molecular marker for early warning of atherosclerosis is long-chain non-coding RNA, and comprises the following steps:

(7) constructing LncRNA DINO siRNA long-chain non-coding RNA, and detecting the expression of apoptosis-related proteins Bax and Bcl-2 by using a protein immunoblot (Western Blot) after interfering LncRNA DINO.

2. The molecular marker for early warning of atherosclerosis according to claim 1, wherein homocysteine interferes with endothelial cell lncRNA DINO (DNA damage-induced non-coding RNA) to be increased significantly, and endothelial cell apoptosis is reduced significantly after the lncRNA DINO is interfered.

3. The molecular marker for early warning of atherosclerosis according to claim 1, wherein the primer for detecting the long non-coding RNA (LncRNA DINO) is also within the protection scope of the present invention.

4. The molecular marker for early warning of atherosclerosis according to claim 1, wherein the agent for inhibiting the long non-coding RNA is interfering RNA (si RNA).

5. The use of the molecular marker for early warning of atherosclerosis as claimed in claim 1, wherein the long non-coding RNA in atherosclerosis is used for the prevention and diagnosis of the disease.