miRNA-based lipoprotein a regulation method and application of miR-653-5p

Abstract

The present invention relates to the field of molecular biology and provides a novel method for regulating lipoprotein (a) levels. By utilizing a specific miRNA molecule, miR-653-5p, to target the LPA gene, the method effectively reduces lipoprotein (a) levels in the body, thereby enabling the cardiovascular disease inhibitor prepared by miR-653-5p to reduce the risk of cardiovascular disease.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a miRNA-based lipoprotein A regulation method and the application of miR-653-5p. Background Art

[0002] Lipoprotein (a) is a cholesterol-like particle composed of low-density lipoprotein (LDL), apolipoprotein (a), and apolipoprotein (b100). As an independent risk factor for cardiovascular disease, high levels of Lp(a) can increase the incidence of cardiovascular disease. Growing evidence indicates that Lp(a) plays a significant role in the development of atherosclerosis, leading to conditions such as stroke, aortic stenosis, myocardial infarction, and even death. Therefore, controlling Lp(a) levels in the human body is a key measure for preventing and mitigating cardiovascular risk. Unlike other lipoproteins, Lp(a) is largely genetically controlled and unaffected by environmental factors and lifestyle, making drug therapy the primary treatment option. However, current treatments are generally ineffective in lowering plasma Lp(a) concentrations, and long-term use can lead to serious adverse reactions, preventing these treatments from achieving the desired therapeutic effect and reducing cardiovascular risk. Therefore, the development of new drugs that can lower Lp(a) levels is crucial.

[0003] Since the level of lipoprotein a, or Lp(a), is mainly determined by the LPA gene that encodes ApoA and is not affected by age, gender, exercise, diet and nutrition (Journal of the American College of Cardiology - Cook NR, Mora S, Ridker PM. Lipoprotein(a)and Cardiovascular Risk Prediction Among Women[J]. J Am Coll Cardiol, 2018), targeting the LPA gene is a new strategy to effectively reduce lipoprotein a. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a miRNA-based LPA regulation method to effectively regulate LPA by downregulating the expression of the LPA gene.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solution: a miRNA-based lipoprotein a regulation method, which targets the LPA gene through miRNA molecules to downregulate its expression, thereby reducing the level of lipoprotein a, and the miRNA molecule is miR-653-5p.

[0006] The specific application of the above miR-653-5p is: to prepare cardiovascular disease inhibitors.

[0007] The cardiovascular disease inhibitor includes mir-653-5p agomir. The method for using the cardiovascular disease inhibitor is to synthesize mir-653-5p agomir and then directly inject it into the body via intravenous injection. The cardiovascular disease inhibitor reduces lipoprotein (A) levels by downregulating LPA gene expression.

[0008] The miR-653-5p used in the present invention is an existing miRNA molecule having an existing sequence. The synthesis of mir-653-5p agomir can be achieved by conventional chemical synthesis to obtain a double-stranded RNA molecule.

[0009] The beneficial effect of the present invention is that it provides a new method for regulating lipoprotein a levels. By utilizing the specific miRNA molecule miR-653-5p to target the LPA gene, the regulation of lipoprotein a is achieved, which can effectively reduce the level of lipoprotein a in the body, thereby enabling the cardiovascular disease inhibitor prepared by miR-653-5p to reduce the risk of cardiovascular disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The array images obtained by analyzing the experimental process in the embodiment are analyzed using Agilent feature extraction software;

[0011] Figure 2 Schematic diagram of LPA gene expression results in Hep G2 cells transfected with mir-653-5p mimic in the examples;

[0012] Figure 3 Schematic diagram of LPA gene expression results in Hep G2 cells transfected with miR-653-5p inhibitor in the Example;

[0013] Figure 4 Schematic diagram of the LPA gene expression results in the livers of the experimental group of mice injected with mir-653-5p agomir and the livers of the control group of mice not injected in the example. DETAILED DESCRIPTION

[0014] In order to facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments and drawings. The contents mentioned in the embodiments are not intended to limit the present invention.

[0015] miRNA differential analysis:

[0016] (1) Use Blood RNA was extracted using RT-PCR Reagent (Invitrogen life technologies), and total RNA was extracted using RNasey Mini Kit (Qiagen p / n 74104).

[0017] (2) Total RNA was labeled using the miRNA Complete Labeling and Hyb Kit (Agilent p / n 5190-0456), and the labeled total RNA was hybridized to the miRNA Complete Labeling and Hyb Kit (Agilent p / n 5190-0456).

[0018] (3) Clean the slide.

[0019] (4) Scanning was performed using an Agilent Microarray Scanner (Agilent p / n G2505C).

[0020] (5) Analyze the obtained array image using Agilent feature extraction software. Figure 1 As shown, individuals with high lipoprotein a were the Test group, and individuals with normal lipoprotein a were the Control group.

[0021] Preparation of cell model:

[0022] (1) Hep G2 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 6-well plate.

[0023] (2) Preparation of miR-653-5p mimic and miR-653-5p inhibitor working solutions: dilute miR-653-5p mimic and miR-653-5p inhibitor (stock concentration is 20 μM) with 250 μl serum-free DMEM medium (the final concentration of transfected cells is 100 nM), and mix thoroughly by pipetting 3-5 times.

[0024] (3) Gently invert to mix the transfection reagent, dilute 1.2ul Lipofectamine 2000 with 50ul serum-free DMEM medium, gently pipette 3-5 times to mix, and let it stand at room temperature for 5 minutes.

[0025] (4) Mix the transfection reagent and miR-653-5p mimic and miR-653-5p inhibitor dilutions to form a transfection complex. Gently pipette 3-5 times to mix thoroughly and let it stand at room temperature for 20 minutes.

[0026] (5) When the Hep G2 cells reach a density of 50%, discard the old culture medium and wash the cells with PBS solution, then discard the PBS solution. Finally, add 1500 μl of serum-free DMEM medium and 500 μl of transfection complex to each well. Gently shake the cell culture plate back and forth to mix thoroughly. Place the cell culture plate in a 37°C, 5% CO2 incubator for 4 h.

[0027] (6) After 4 hours, the culture medium was discarded, and the cells were washed with PBS solution and then discarded. 2000 μl of DMEM culture medium containing 10% fetal bovine serum was added to each well, and the cell culture plate was placed in a 37°C, 5% CO2 incubator for 20 hours.

[0028] RT-qPCR detection:

[0029] (1) Wash the cells with PBS solution and discard the PBS solution.

[0030] (2) Total RNA of Hep G2 cells in each group was extracted using RNA East Fast Animal Tissue / Cell Total RNA Extraction Kit (DP451) from TIANGEN. The RNA was dissolved in DEPC water and the OD260 / OD280 ratio was determined by UV spectrophotometer to be between 1.8 and 2.1. 2ul of total RNA of each group of cells was reverse transcribed into cDNA using TIANGEN Reverse Transcription Kit (KR118). 2ul of reverse transcription product was then amplified in PCR using TIANGEN qPCR Kit (FP217). The amplification system volume was 25ul. The Ct value was calculated, and β-actin was used as the internal reference. 2 -ΔΔCt It indicates the fold difference in the original copy number of β-actin in the experimental group LPA compared with that in the control group.

[0031] (3) 24 hours after Hep G2 cells were transfected with mir-653-5p mimic, it was found that the LPA gene in Hep G2 cells transfected with mir-653-5pmimic was significantly decreased compared with the control group. Figure 2 As shown. The LPA gene in Hep G2 cells transfected with miR-653-5pin inhibitor increased significantly compared with the control group, as shown Figure 3 This indicates that mir-653-5p can reduce the expression of LPA gene, thereby regulating lipoprotein a.

[0032] Injection experiment:

[0033] Animal model: B6-hLPA(CKI) / Alb-cre mice (Product No. C001522, Saiye Biotechnology). Three mice were set up in the experimental group and three mice were set up in the control group, all placed in the same environment.

[0034] The experimental group used mir-653-5p agomir to directly inject mice with tail vein. 80mg / kg, once a day, for 3 days. 7 days after the first injection, liver samples were taken for LPA gene detection, and the average value of 3 mice was taken. The control group did not receive injection, and liver samples were taken for LPA gene detection at the same time point, and the average value of 3 mice was taken. The results are shown in the figure below. Figure 4 As shown in the figure, it can be seen that the expression of LPA gene in the liver of mice injected with mir-653-5pagomir was significantly downregulated compared with the control group.

[0035] The present invention proposes an innovative miRNA-based method for regulating lipoprotein (a), which uses a specific miR-653-5p molecule to target the LPA gene and effectively reduce lipoprotein (a) levels in the body by downregulating its expression. This method not only provides a new strategy for the prevention and treatment of cardiovascular diseases, especially for patients whose lipoprotein (a) levels are difficult to reduce with traditional treatments, but also has high specificity, low side effects, simplicity, and broad application prospects. This is because miRNA-based treatments have lower side effects than traditional small molecule drugs or biomacromolecule drugs because miRNA is an endogenous molecule that is more easily accepted and processed by the body. By administering the drug through intravenous injection, the present invention can provide patients with a more accurate and convenient treatment plan and is expected to play an important role in the field of modern medicine.

[0036] In order to make it easier for ordinary technicians in this field to understand the improvements of the present invention over the prior art, some drawings and descriptions of the present invention have been simplified, and the above-mentioned embodiments are preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious replacement without departing from the concept of the present technical solution is within the scope of protection of the present invention.

Claims

1. Application of miR-653-5p in the preparation of drugs for reducing the risk of cardiovascular disease.

2. The use of miR-653-5p according to claim 1, characterized in that: The miR-653-5p is mir-653-5p agomir.

3. The use of miR-653-5p according to claim 2, characterized in that The method for using the miR-653-5p is as follows: after synthesizing the mir-653-5p agomir, it is directly injected into the body through intravenous injection.

4. The use of miR-653-5p according to claim 1, characterized in that: The miR-653-5p reduces the level of lipoprotein a by downregulating the expression of the LPA gene.

Images