Application of a glucagon-like peptide-1 receptor-specific nucleic acid aptamer in the intervention of metabolic diseases

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to an application of a glucagon-like peptide-1 receptor-specific nucleic acid aptamer in the intervention of metabolic diseases. The present invention successfully improves cell proliferation ability, increases intracellular calcium ion concentration and insulin secretion level by using a glucagon-like peptide-1 receptor-specific aptamer, activates the glucagon-like peptide-1 (GLP-1) pathway, and increases the mRNA expression level of related genes, providing a stable and low-cost molecular tool for the intervention of metabolic diseases such as diabetes and obesity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an application of a glucagon-like peptide-1 receptor-specific nucleic acid aptamer in the intervention of metabolic diseases. Background Art

[0002] Glucagon-like peptide 1 (GLP-1) can intervene in the occurrence and development of diseases such as obesity by interacting with the GLP-1 receptor (GLP-1R). Since GLP-1 has a short half-life and is easily degraded and inactivated in the body, GLP-1 receptor agonists (GLP-1RA) have become a new type of drug that can maintain the pharmacological effects of GLP-1 while having a longer half-life. Science magazine listed the development of GLP-1RA and the discovery of drugs that can alleviate obesity-related health problems as the top ten scientific breakthroughs of 2023, which shows the importance of the GLP-1 / GLP-1R disease intervention pathway and GLP-1RA drugs in the field of clinical treatment.

[0003] At present, a variety of GLP-1RAs have been approved by the FDA for marketing, and some of them have also been approved to enter the list of medical insurance drugs in my country. GLP-1RA can be divided into short-acting preparations, long-acting preparations and weekly preparations according to their pharmacokinetic characteristics. The half-life of short-acting GLP-1RA in the body is usually 2~5 hours, while the half-life of long-acting GLP-1RA can reach 12~24 hours. The two also have different physiological regulatory effects on weight loss, gastric emptying rate, blood sugar level, and insulin secretion. Except for semaglutide, which has oral preparations, other drugs are administered by subcutaneous injection. The approved GLP-1RAs are basically peptide compounds, and their stability in the body is improved compared to GLP-1. However, the common GLP-1RA drugs are mostly protein drugs, which have high drug costs, poor batch stability, and strict conditions control in transportation. Therefore, GLP-1RAs with stable properties and low costs need to be developed.

[0004] Based on this, the present invention provides a DNA nucleic acid aptamer-type GLP-1RA, which can effectively overcome the current barriers in the field and provide a stable and low-cost molecular tool for the intervention of metabolic diseases such as diabetes and obesity. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to propose an application of a glucagon-like peptide-1 receptor-specific nucleic acid aptamer in the intervention of metabolic diseases. By adding specific nucleic acid aptamers, the problem of molecular intervention in metabolic diseases is solved.

[0006] To achieve the above objectives, on the one hand, the present invention provides an application of a glucagon-like peptide-1 receptor-specific nucleic acid aptamer in the intervention of metabolic diseases, characterized in that the nucleotide sequence of the glucagon-like peptide-1 receptor-specific nucleic acid aptamer is: 5'-AAGCAGGAAATTCTAGAAGACACGTTACAAATCAATCAGAAGAACGAG-3', as shown in SEQ ID NO: 1.

[0007] The nucleic acid aptamer can enhance the proliferation ability of pancreatic islet cells, increase the intracellular calcium ion concentration and insulin secretion level, and regulate the glucagon-like peptide-1 metabolic pathway.

[0008] Furthermore, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the above-mentioned nucleic acid aptamer.

[0009] Furthermore, the present invention provides a use of the above-mentioned nucleic acid aptamer or the above-mentioned pharmaceutical composition in the preparation of a glucagon-like peptide-1 receptor agonist drug.

[0010] Furthermore, the present invention provides a use of the above-mentioned nucleic acid aptamer or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating diabetes.

[0011] Furthermore, the present invention provides a use of the above-mentioned nucleic acid aptamer or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating obesity.

[0012] Furthermore, the present invention provides a use of the above-mentioned nucleic acid aptamer or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The proliferation rate of MIN6 cells after being treated with different concentrations of R2 aptamers.

[0014] Figure 2 Calcium ion concentration signal of MIN6 cells under fluorescence microscope.

[0015] Figure 3 Mouse Insulin ELISA Kit standard curve.

[0016] Figure 4 Insulin concentration in the culture medium of MIN6 cells in different treatment groups.

[0017] Figure 5 Relative quantification of gene expression levels in MIN6 cells under different treatment groups. DETAILED DESCRIPTION

[0018] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0019] Example 1: Effect of nucleic acid aptamer R2 on cell proliferation ability

[0020] (1) MIN6 cells were seeded into a 96-well plate and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h until the cells adhered to the wall and the density was about 60-70%;

[0021] (2) Then, the culture medium was aspirated and 100 μL of nucleic acid aptamer R2 solution diluted with complete culture medium at different concentrations (0.5, 1, 2, 5, 10, 15, 20 nmol / L) was added to set up the CK group (concentration was initially explored in advance), and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h;

[0022] Wherein, the nucleotide sequence of nucleic acid aptamer R2 is: 5'-AAGCAGGAAATTCTAGAAGACACGTTACAAATCAATCAGAAGAACGAG-3', as shown in SEQ ID NO: 1;

[0023] The nucleotide sequence of the CK sequence is: 5′-CCATCCCGGTCCCCCATCCCGAATTCCCATGCGCCTTACCCTACCTCC-3′, as shown in SEQ ID NO: 2;

[0024] (3) Add 10 μL of CCK-8 solution to each well and gently pipette to mix the solution. Incubate the wells in a 37°C, 5% CO2 cell culture incubator for 2 h. Finally, use an ELISA reader to measure the absorbance of the liquid in each well at 450 nm. Set up 5 parallel wells for each group and take the average value. Repeat the experiment three times.

[0025] After initial screening of concentration gradient, the concentration range of 0-20 nM was selected to determine the effect of aptamer R2 on the proliferation activity of MIN6 cells. The results showed that after treatment with low concentration of aptamer R2, the proliferation ability of MIN6 cells can be significantly improved. When MIN6 cells were treated with 15 nM R2 aptamer for 24 h, the proliferation rate increased by about 19% compared with the untreated group (e.g. Figure 1 As shown in Figure 3 ), it proves that aptamer R2 has significant effects in inhibiting pancreatic β-cell apoptosis and promoting pancreatic β-cell proliferation.

[0026] Example 2: Effect of nucleic acid aptamer R2 on intracellular calcium ion concentration

[0027] Subsequently, the intracellular calcium concentration was detected using a fluorescent probe.

[0028] (1) Dilute the Fluo-3 AM solution to 1 µM using PBS buffer.

[0029] (2) When the density of MIN6 cells in the six-well plate reaches about 70%, discard the culture medium in each well and rinse with PBS buffer three times;

[0030] (3) Add 1 mL of diluted Fluo-3 AM solution to each well and incubate at 37°C for 30 minutes to complete the fluorescent probe loading.

[0031] (4) After the probe is loaded, aspirate the liquid in the well and rinse twice with PBS buffer;

[0032] (5) The cells were incubated in PBS buffer for another 20 minutes at 37°C to ensure that MIN6 cells could completely convert Fluo-3 AM into Fluo-3.

[0033] (6) Place the six-well plate under a fluorescence microscope and observe and take photos to compare the fluorescence intensity of different groups.

[0034] Fluo-3 AM was used to characterize the intracellular calcium ion concentration of MIN6 cells treated with aptamer R2 and untreated. Fluo-3 AM was converted into Fluo-3 in the cells and showed green fluorescence at an excitation wavelength of 488 nm. The results are shown in Figure 2. Figure 2 As shown, under a fluorescence microscope, it can be observed that the green fluorescence in the MIN6 cells treated with aptamer R2 is very obvious, while the green fluorescence in the MIN6 cells treated with only PBS buffer is very weak, indicating that aptamer R2 can enhance the absorption of calcium ions by MIN6 cells, which helps MIN6 cells secrete insulin.

[0035] Example 3: Effect of nucleic acid aptamer R2 on cell insulin secretion ability

[0036] Furthermore, the double antibody sandwich ELISA method was used to determine the insulin secretion capacity of cells.

[0037] 1. Preparation

[0038] (1) Bring the refrigerated test kit to room temperature for about 20 minutes to equilibrate the liquid in the test kit;

[0039] (2) Dilute the washing solution in the kit to the working concentration with deionized water;

[0040] (3) Add the volume of standard diluent specified on the label of the standard bottle to the standard bottle and place it at room temperature for 15 minutes. Gently shake and pipette the mixture to completely dissolve the standard. The concentration of the standard in each bottle is 5.5 ng / mL.

[0041] (4) Take five clean 1.5 mL centrifuge tubes and add 250 μL of the standard diluent into each tube. Then dilute the standard sample by multiples to obtain seven concentrations: 0, 5.5, 2.75, 1.375, 0.687, 0.344, and 0.172 ng / mL (the standard diluent is 0 ng / mL).

[0042] 2. Sample processing and testing

[0043] (1) MIN6 cells seeded in six-well plates were divided into three groups: one group was treated with normal DMEM complete medium, one group was treated with 5 nM random library, and one group was treated with 5 nM aptamer R2. All groups were treated for 24 h.

[0044] The nucleotide sequence of the random library is: 5'-GGAATCGAGGATTCGTTANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGACCTCTAACTCTGACAT-3', as shown in SEQ ID NO: 3, wherein N is a random nucleotide;

[0045] (2) After 24 h of treatment, take out the cell culture medium samples according to the groups, centrifuge at 500 × g for 5 minutes, and take the supernatant for later use;

[0046] (3) Take out four pre-coated strips and place them in a 96-well frame;

[0047] (4) Add 10 μL of sample and different concentrations of standard into the corresponding wells;

[0048] (5) Then add 100 μL of horseradish peroxidase-labeled mouse insulin antibody to each well, cover the well with the transparent sealing film provided in the kit, and react at room temperature for 2 h;

[0049] (6) After the incubation, add 300 μL of washing solution to each well. Wash each well for 15 to 30 seconds for a total of five times. Finally, turn the plate upside down on absorbent paper and pat dry.

[0050] (7) Add 100 μL of the colorimetric reagent TMB solution to each well, cover the well with the white sealing film provided in the kit, and react at room temperature in the dark for 10 minutes;

[0051] (8) Add 50 μL of stop solution to each well and mix gently. Then use an ELISA reader to directly measure the absorbance of the liquid in each well at a wavelength of 450 nm.

[0052] The absorbance values ​​of insulin standards of different concentrations at a wavelength of 450 nm were measured using an enzyme reader, and then the standard curve of the mouse insulin ELISA Kit was drawn using GraphPad Prism software. The standard curve equation y=0.1970 x+0.02797, R 2 =0.993 (e.g. Figure 3 The corresponding insulin concentrations of different samples were calculated based on the absorbance values ​​of the culture medium samples at a wavelength of 450 nm measured by the microplate reader and the standard curve equation (as shown in Figure 4 As shown). The insulin concentration in the untreated cell culture medium was 7.533 ng / mL, the insulin concentration in the cell culture medium treated with the random library group was 7.630 ng / mL, and the insulin concentration in the cell culture medium treated with aptamer R2 was 9.366 ng / mL. By comparison, it can be seen that the insulin content in the MIN6 cell culture medium treated with aptamer R2 is significantly higher than that of the other two groups, indicating that the ability of MIN6 cells to secrete insulin is significantly improved after treatment with aptamer R2. The increase in insulin concentration in the culture medium is greater than 24%, which is higher than the increase in the proliferation rate of MIN6 cells in the CCK-8 experimental results. This shows that the aptamer R2 obtained by the experimental screening has a significant effect in promoting the secretion of insulin by MIN6 cells, and may have certain application potential in the prevention / treatment of diabetes, obesity or hyperlipidemia.

[0053] Example 4: Regulation of Cellular Pathways by Aptamer R2

[0054] Subsequently, the expression levels of GLP-1 pathway-related genes were determined by RT-PCR.

[0055] 1. Total RNA extraction from cells in each group

[0056] (1) After aspirating the culture medium in the six-well plate, add 600 μL of lysis buffer BB4 to each well, gently pipette 5-10 times to mix the suspension after cell lysis, and transfer them to RNase-free centrifuge tubes according to groups;

[0057] (2) Centrifuge at 12,000 × g for 5 min at room temperature, aspirate 500 μL of the supernatant in the centrifuge tube and transfer it to a new centrifuge tube;

[0058] (3) Add 500 μL of 70% ethanol to each centrifuge tube and shake gently to mix well. Sometimes precipitation will appear in the centrifuge tube;

[0059] (4) Vortex the liquid and precipitate in the centrifuge tube, then transfer the entire mixture to the centrifuge column provided in the kit, centrifuge at 12,000 × g for 30 s, and discard the effluent in the collection tube;

[0060] (5) Add 500 μL of CB4 solution to the centrifuge column, centrifuge at 12,000 × g for 30 s at room temperature, and discard the effluent in the collection tube;

[0061] (6) Add 500 μL of WB4 solution to the centrifuge column, centrifuge at 12,000 × g for 30 s at room temperature, and discard the effluent in the collection tube;

[0062] (7) Repeat step (6);

[0063] (8) Centrifuge at 12,000 × g for 2 minutes at room temperature to completely remove the liquid remaining in the centrifuge column;

[0064] (9) Place the centrifuge column in a new centrifuge tube, add 50 μL of RNase-free water onto the membrane at the bottom of the centrifuge column, let it stand at room temperature for 1-2 min, and centrifuge it at 12,000 × g in a high-speed centrifuge for 2 min. The solution in the centrifuge tube is the total RNA extracted from MIN6 cells and can be stored in a -80°C refrigerator.

[0065] 2. qRT-PCR

[0066] (1) Reverse transcription of mRNA in the extracted total RNA into cDNA Take out the frozen extracted total RNA, 5×FastKing-RT SuperMix and RNase-Free ddH2O, place the total RNA sample on ice and slowly thaw it, place the two reagents on ice after thawing at room temperature. Before use, shake and mix all three solutions, and quickly centrifuge them to allow the liquid on the tube wall to gather at the bottom of the tube. Prepare the reverse transcription reaction system required for the experiment as shown in Table 1.

[0067] Table 1 Reverse transcription reaction system

[0068]

[0069] The reverse transcription reaction was performed as shown in Table 2.

[0070] Table 2 Reverse transcription reaction procedure

[0071]

[0072] (2) qPCR determination of target gene mRNA expression level

[0073] According to the GLP-1R signaling pathway, the expression levels of genes related to promoting insulin secretion, inhibiting cell apoptosis, and inducing cell proliferation in the GLP-1R signaling pathway of pancreatic cells were verified, and the primer sequences were designed as shown in Table 3.

[0074] Table 3 qPCR primer sequences of target genes

[0075]

[0076] The total RNA of cells in different treatment groups was extracted using a kit, and then the total RNA extracted from each group of cells was reverse transcribed. Finally, qPCR was performed to obtain the gene amplification results extracted from each treatment group. -△△Ct Methods The expression levels of several genes in each treatment group were normalized and relatively quantified. Figure 5 As shown, aptamer R2 can increase the Akt1 , Bcl2 , NFκB1 , PDX1 Gene expression level. Akt1 Mainly regulates the volume of pancreatic islet cells and the ability to secrete insulin. Akt1 When the expression is upregulated, the size of pancreatic islet cells increases and the ability to secrete insulin improves; Bcl2 Mainly regulates cell apoptosis. Bcl2 When the expression is upregulated, it will inhibit cell apoptosis; NFκB1 It can maintain normal cell growth and inhibit cell apoptosis; PDX1 Regulate pancreatic development and differentiation and proliferation of pancreatic islet cells. The above genes regulate the quantity and quality of pancreatic β cells and the secretion capacity of insulin. In summary, after treatment with aptamer R2, these genes in MIN6 cells increased to a certain extent, indicating that aptamer R2 has a certain promoting effect on the increase of the quantity and quality of pancreatic β cells and the improvement of insulin secretion capacity.

[0077] In summary, the present invention successfully utilizes the glucagon-like peptide-1 receptor-specific aptamer R2 to improve cell proliferation ability, increase intracellular calcium ion concentration and insulin secretion level, activate the GLP-1 pathway, and increase the mRNA expression level of related genes, providing a stable and low-cost molecular tool for the intervention of metabolic diseases such as diabetes, obesity, and hyperlipidemia.

[0078] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. Use of a glucagon-like peptide-1 receptor-specific nucleic acid aptamer in the preparation of a drug for preventing and / or treating diabetes, characterized in that: The nucleotide sequence of the glucagon-like peptide-1 receptor-specific nucleic acid aptamer is: 5'-AAGCAGGAAATTCTAGAAGACACGTTACAAATCAATCAGAAGAACGAG-3'.

2. The use according to claim 1, characterized in that: The nucleic acid aptamer can enhance the proliferation ability of pancreatic islet cells, increase the intracellular calcium ion concentration and insulin secretion level, and regulate the glucagon-like peptide-1 metabolic pathway.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nucleic acid aptamer according to claim 1.

4. Use of the nucleic acid aptamer according to claim 1 or the pharmaceutical composition according to claim 3 in the preparation of a drug for preventing and / or treating diabetes.

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