Application of a gene detection reagent in the preparation of a kit for diagnosing diabetes mellitus complicated with coronary heart disease

Abstract

The present invention provides an application of a gene detection reagent in the preparation of a kit for diagnosing diabetes mellitus complicated with coronary heart disease, belonging to the technical field of biomedicine. The detection reagent provided by the present invention is a reagent for detecting the expression level of LncRNA-AC141930.2. By detecting the expression level of LncRNA-AC141930.2, it is possible to detect at an early stage whether a type 2 diabetes patient is complicated with coronary heart disease. At the same time, the siRNA provided by the present invention can effectively reduce the senescence of human microvascular endothelial cells caused by high glucose and can reduce the decline in the tube formation ability of human microvascular endothelial cells caused by high glucose. Therefore, it can be used in the preparation of drugs for treating diabetes mellitus complicated with coronary heart disease.

Description

[0001] This is a divisional application. The invention name of the original application is: A drug for treating type 2 diabetes mellitus complicated with coronary heart disease. The application date of the original application is: 2022-02-15, and the application number of the original application is: CN202210136598.1. Technical Field

[0002] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of a gene detection reagent in the preparation of a kit for diagnosing diabetes mellitus complicated with coronary heart disease. Background Art

[0003] Type 2 diabetes mellitus is a common metabolic disease, and its prevalence rate has been increasing year by year. Currently, type 2 diabetes mellitus is no longer just a disease of the wealthy in developed countries. Developing countries including China have also become hard-hit areas for type 2 diabetes mellitus. Type 2 diabetes mellitus generally causes damage to blood vessels throughout the body. Since the affected blood vessels are spread throughout the body and irreversible, type 2 diabetes mellitus has become an important killer among modern diseases.

[0004] The continuous elevation of blood glucose is a significant feature of patients with type 2 diabetes mellitus. Long-term hyperglycemia significantly increases the probability of patients with type 2 diabetes mellitus developing cardiovascular diseases compared with non-diabetic patients. Diabetes mellitus complicated with coronary heart disease is the main cause of death or disability due to cardiovascular complications. Having type 2 diabetes mellitus complicated with coronary heart disease is a type of diabetic coronary heart disease, which is a heart disease that causes atherosclerotic changes or functional changes in the coronary arteries on the basis of type 2 diabetes mellitus, resulting in stenosis or obstruction of the cardiac blood cavity, or local ischemia, hypoxia or even necrosis of the heart. Therefore, early diagnosis and early treatment of type 2 diabetes mellitus are required to improve the quality of life of patients.

[0005] Meanwhile, existing research has found that long-term abnormal glucose and lipid metabolism leads to endothelial dysfunction. Therefore, effectively reducing the endothelial damage caused by high glucose will contribute to the treatment of type 2 diabetes mellitus complicated with coronary heart disease. Summary of the Invention

[0006] The problem to be solved by the present invention is to provide a drug for treating type 2 diabetes mellitus complicated with coronary heart disease and to provide a biomarker for diagnosing type 2 diabetes mellitus complicated with coronary heart disease.

[0007] To solve the above problems, the present invention provides the following technical solutions:

[0008] A drug for treating type 2 diabetes mellitus complicated with coronary heart disease, wherein the drug for treating type 2 diabetes mellitus complicated with coronary heart disease contains siRNA of LncRNA-AC141930.2 and a drug carrier.

[0009] Further, the drug carrier is one or more of cholesterol, liposome, and nanoparticle;

[0010] Furthermore, the drug can be administered orally or parenterally. When administered parenterally, it can be administered by intravenous injection, intranasal injection, local injection, intraventricular injection, intraspinal injection, subcutaneous injection, intraperitoneal injection, transdermal administration, etc.

[0011] Furthermore, the transcript sequence of the LncRNA-AC141930.2 is as shown in SEQ ID NO.1, and the sequences of the siRNA are as shown in SEQ ID NO.4 and SEQ ID NO.5.

[0012] Use of siRNA of LncRNA-AC141930.2 in the preparation of a drug for treating diabetes mellitus combined with coronary heart disease.

[0013] Furthermore, the transcript sequence of the LncRNA-AC141930.2 is as shown in SEQ ID NO.1, and the sequences of the siRNA are as shown in SEQ ID NO.4 and SEQ ID NO.5.

[0014] Use of siRNA of LncRNA-AC141930.2 in the preparation of a biological agent for inhibiting cell senescence induced by high glucose.

[0015] Furthermore, the transcript sequence of the LncRNA-AC141930.2 is as shown in SEQ ID NO.1, the sequences of the siRNA are as shown in SEQ ID NO.4 and SEQ ID NO.5, and the cell is a human microvascular endothelial cell.

[0016] Use of siRNA of LncRNA-AC141930.2 in the preparation of a biological agent for inhibiting the reduction of cell angiogenesis ability induced by high glucose.

[0017] Furthermore, the transcript sequence of the LncRNA-AC141930.2 is as shown in SEQ ID NO.1, the sequences of the siRNA are as shown in SEQ ID NO.4 and SEQ ID NO.5, and the cell is a human microvascular endothelial cell.

[0018] Use of a reagent for detecting the expression level of LncRNA-AC141930.2 in the preparation of a kit for diagnosing diabetes mellitus combined with coronary heart disease.

[0019] Furthermore, the transcript sequence of the LncRNA-AC141930.2 is as described in SEQ ID NO.1, the reagent is a PCR primer for LncRNA-AC141930.2, and the sequences of the PCR primer are as shown in SEQ IDNO.2 and SEQ ID NO.3.

[0020] The beneficial effects of the present invention are as follows:

[0021] The siRNA of LncRNA-AC141930.2 provided by the present invention can reduce the senescence of human microvascular endothelial cells caused by high glucose and can reduce the decline in the tube formation ability of human microvascular endothelial cells caused by high glucose. Therefore, it can be used to prepare drugs for the treatment of type 2 diabetes complicated with coronary heart disease. At the same time, the LncRNA-AC141930.2 provided by the present invention can be used as a biomarker to diagnose patients with diabetes complicated with coronary heart disease. Brief Description of the Drawings

[0022] Figure 1 Expression differences of LncRNA-AC141930.2 in different groups;

[0023] Figure 2 ROC curves between the healthy control group and the group of type 2 diabetes complicated with coronary heart disease;

[0024] Figure 3 ROC curves between the type 2 diabetes group and the group of type 2 diabetes complicated with coronary heart disease;

[0025] Figure 4 ROC curves between the coronary heart disease group and the group of type 2 diabetes complicated with coronary heart disease;

[0026] Figure 5 Effect of high glucose on the RNA level of LncRNA-AC141930.2 in human microvascular endothelial cells;

[0027] Figure 6 Interference effect of the designed si-lnc of the present invention;

[0028] Figure 7 Effect of si-lnc on the cellular senescence of human microvascular endothelial cells caused by high glucose;

[0029] Figure 8 Effect of si-lnc on the tube formation ability of human microvascular endothelial cells caused by high glucose. Detailed Embodiments

[0030] To clearly illustrate the technical features of the present solution, the content of the present invention will be further described in detail below through specific embodiments. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. According to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution or change can be made. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0031] Example 1

[0032] (1) Collect peripheral blood from normal individuals (30 cases, healthy control group), patients with type 2 diabetes (30 cases, type 2 diabetes group), patients with coronary heart disease (30 cases, coronary heart disease group), and patients with type 2 diabetes complicated with coronary heart disease (30 cases, type 2 diabetes complicated with coronary heart disease group). There were no statistically significant differences in age and gender among the groups. The patients voluntarily participated in this study and filled out the informed consent form.

[0033] (2) Diagnostic criteria for diabetes: Fasting blood glucose ≥ 7.0 mmol·L －1 , or random blood glucose ≥ 11.1 mmol·L －1 ; Diagnostic criteria for coronary heart disease: Coronary angiography showed stenosis of at least one epicardial major blood vessel ≥ 50%;

[0034] (3) Diagnostic criteria for diabetes complicated with coronary heart disease: Fasting blood glucose ≥ 7.0 mmol·L−1, or random blood glucose ≥ 11.1 mmol·L−1 and coronary angiography showed stenosis of at least one epicardial major blood vessel ≥ 50%;

[0035] (4) Exclusion criteria: The diabetes group excluded patients with diabetic nephropathy, diabetic retinopathy, hypertension, peripheral arteriosclerosis, and those who were taking hypoglycemic, lipid-lowering, antihypertensive drugs or insulin;

[0036] History of acute and chronic infections, hepatic and renal insufficiency, severe cerebrovascular accidents, malignant tumors, rheumatism, immune system diseases, endocrine diseases such as hyperthyroidism, gout, and pituitary diseases;

[0037] Female patients needed to be excluded if they were pregnant or lactating;

[0038] Patients with mental diseases;

[0039] (5) Draw fasting morning venous blood from the selected subjects in the experiment. After centrifuging at 3000 r / min for 15 min using a centrifuge, take the supernatant to obtain the sample serum.

[0040] Example 2

[0041] Extract RNA from the sample

[0042] (1) Take 200 ul of the sample serum and add it to a centrifuge tube. Add 200 ul of RNA lysis buffer and mix well by shaking for 30 s;

[0043] (2) After standing at room temperature for 5 min, centrifuge at 12000 rpm for 10 min, and then aspirate the supernatant into a new centrifuge tube;

[0044] (3) Add 200 μl of chloroform, shake for 15 s, then leave it at room temperature for 5 min, centrifuge at 12,000 rpm for 15 min, and transfer the upper aqueous phase to a new centrifuge tube;

[0045] (4) Add an equal volume of isopropanol, mix well and leave it for 10 min, centrifuge at 12,000 rpm for 10 min, discard the supernatant and retain the precipitate;

[0046] (5) Dissolve the precipitate with 500 μl of 70% ethanol prepared with pre-cooled DEPC water, centrifuge at 8,000 rpm for 5 min, repeat once, discard the supernatant, dry at room temperature for 10 min, and then add DEPC water to obtain RNA.

[0047] Example 3

[0048] Reverse transcription reaction

[0049] (1) The reverse transcription process was carried out with reference to the PrimeScript TM RT reagent Kit of TaKaRa Company;

[0050] (2) Prepare the following reaction system:

[0051] Reagent Dosage 5×PrimeScript Buffer (for Real Time) 2.0 μl PrimeScript RT Enzyme Mix I 0.5 μl Oligo dT Primer (50 μM) *1 0.5 μl Random 6mers (100 μM) *1 0.5 μl Total RNA 500 ng RNase Free dH2O Up to 10 μl

[0052] (3) Carry out the reverse transcription reaction on a PCR instrument under the following conditions:

[0053] 37 °C for 15 min * 3; 85 °C for 5 s; hold at 4 °C for 5 min.

[0054] Example 4

[0055] PCR reaction

[0056] (1) The PCR reaction was carried out with reference to the steps of the TB Premix Ex Taq TM II Kit of TaKaRa Company;

[0057] (2) Prepare the following reaction system:

[0058]

[0059]

[0060] (3) Carry out the reaction under the following reaction conditions:

[0061] Step1 95 °C for 30 s, Step2 95 °C for 5 s, 60 °C for 30 s for 40 cycles;

[0062] (4) Design and synthesize the primer sequences of LncRNA-AC141930.2 to be detected in the present invention:

[0063]

[0064] (5) The results obtained are as Figure 1 shown.

[0065] Compared with the healthy control group, the expression level of LncRNA-AC141930.2 in the type 2 diabetes group was 0.93 ± 0.53, and the difference was not statistically significant. The expression level of LncRNA-AC141930.2 in the coronary heart disease group was 1.10 ± 0.54, and the difference was not statistically significant. The expression level of LncRNA-AC141930.2 in the type 2 diabetes complicated with coronary heart disease group was 2.40, and the difference was statistically significant. At the same time, the difference compared with the type 2 diabetes group and the coronary heart disease group was also statistically significant.

[0066] According to Figure 1 the results, draw the ROC curves of the type 2 diabetes complicated with coronary heart disease group and the healthy control group, draw the ROC curves of the type 2 diabetes complicated with coronary heart disease group and the type 2 diabetes group, draw the ROC curves of the type 2 diabetes complicated with coronary heart disease group and the coronary heart disease group. The curves obtained are respectively as Figure 2 , Figure 3 and Figure 4 shown. Among them, Figure 2 the AUC value in Figure 3 is 0.9180, Figure 4 the AUC value in

[0067] Example 5

[0068] Effect of high glucose on the expression of LncRNA-AC141930.2 in human microvascular endothelial cells (HMEC-1)

[0069] (1) Seed HMEC-1 cells in a 6-well plate and culture them in a cell culture incubator;

[0070] (2) When the cell density reaches more than 85%, add normal DMEM medium to the control group, add DMEM medium containing 30 mmol / L D-glucose to experimental group a, and add DMEM medium containing 25 mmol / L mannitol to experimental group b. Set 3 replicates for each group;

[0071] (3) After culturing the cells for 48 h, RNA was extracted to detect the expression of LncRNA-AC141930.2 in different groups.

[0072] The experimental results were as Figure 5 , in which the expression level of LncRNA-AC141930.2 in experimental group a was 3.68 ± 0.52, with a statistically significant difference. The expression level of LncRNA-AC141930.2 in experimental group b was 0.98 ± 0.10, without a statistically significant difference. The above results indicate that high glucose can cause an increase in the expression of LncRNA-AC141930.2 in human microvascular endothelial cells, and the increase in expression is not caused by osmotic pressure.

[0073] Example 6

[0074] Construct siRNA of LncRNA-AC141930.2

[0075] (1) Design and synthesize siRNA of LncRNA-AC141930.2. For convenience of representation, it was named si-lnc. The sequence of si-lnc is as follows:

[0076] Sense strand: AUUUCAUAAUUGCAUUUGC, SEQ ID NO.4;

[0077] Antisense strand: GCAAAUGCAAUUAUGAAAU, SEQ ID NO.5;

[0078] (2) Seed HMEC-1 cells in a 6-well culture plate. When the cell density reaches more than 85%, transfect si-nC and si-lnc into the cells;

[0079] (3) After 48 h of transfection, extract cellular RNA to detect the expression level of LncRNA-AC141930.2.

[0080] The experimental results were as Figure 6 , and the si-lnc provided by the present invention can effectively inhibit the expression level of LncRNA-AC141930.2 in HMEC-1 cells, with an inhibition rate of 82.3%.

[0081] Example 7

[0082] Effect of transfecting si-lnc on high glucose-induced cell senescence of HMEC-1

[0083] (1) Seed HMEC-1 cells in a 6-well culture plate. When the cell density reaches over 85%, for the control group: Transfect with si-NC and culture using normal DMEM medium; for experimental group a: Transfect with si-NC and culture using DMEM medium containing 30 mmol / L D-glucose; for experimental group b: Transfect with si-lnc and culture using DMEM medium containing 30 mmol / L D-glucose.

[0084] (2) After 48 hours of transfection, remove the medium and wash the cells 3 times with PBS.

[0085] (3) Add 1 ml of cell fixative to fix the cells and place them at room temperature for 20 minutes.

[0086] (4) After fixation, remove the fixative, wash the cells with PBS, then add β-galactosidase staining solution and stain overnight at 37°C.

[0087] (5) After staining, remove the staining solution, wash the cells 2 times with PBS, add 2 ml of PBS, and then take pictures under a microscope to detect the positive rate of the cells.

[0088] The experimental results are as Figure 7 shown. It can be seen that transfection with si-lnc can effectively inhibit cell senescence caused by high glucose.

[0089] Example 8

[0090] Effect of Transfecting si-lnc on the Decrease in Tube Formation Ability of HMEC-1 Induced by High Glucose

[0091] (1) One day before the experiment, place Matrigel in an ice box and put it in a 4°C refrigerator to allow the gel to slowly melt overnight.

[0092] (2) Mix ECM medium and Matrigel in a ratio of 1:1 to obtain a mixed solution.

[0093] (3) In a 48-well plate, add 180 μl of the mixed solution to each well, and place the 48-well plate at 37°C for 30 minutes to allow the gel to solidify.

[0094] (4) For the control group, use cells transfected with si-nc and cultured in normal medium; for experimental group a, use cells transfected with si-nc and cultured in DMEM medium containing 30 mmol / L D-glucose; for experimental group b, use cells transfected with si-lnc and cultured in DMEM medium containing 30 mmol / L D-glucose. Add 500 μl containing 4×10 4 cells into the 48-well plate and incubate at 37°C.

[0095] (5) After incubation for 4 h, photographs were taken under an inverted microscope, and the number of branches was calculated.

[0096] The experimental results are as Figure 8 shown. It can be seen that after transfection with si-lnc, the tube formation ability reduced by high glucose can be effectively restored.

Claims

1. Use of a reagent for detecting the expression level of LncRNA-AC141930.2 in the preparation of a kit for diagnosing diabetes mellitus complicated with coronary heart disease, characterized in that, The transcript sequence of the LncRNA-AC141930.2 is as described in SEQ ID NO.1, the reagent is the PCR primer for LncRNA-AC141930.2, and the sequences of the PCR primers are as shown in SEQ ID NO.2 and SEQ ID NO.3.

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