Use of miR156a or preparation thereof in regulating triglyceride metabolism

Abstract

The application provides application of miR156a or a preparation thereof in triglyceride metabolism regulation. The application of miR156a in a triglyceride metabolism product or medicine, wherein the miR156a is an RNA sequence of SEQ ID No. 1 in the sequence listing. The application of miR156a in a very low density lipoprotein level product or medicine, wherein the miR156a is an RNA sequence of SEQ ID No. 1 in the sequence listing. The application of miR156a in a product or medicine for preventing or treating non-alcoholic fatty liver or hypertriglyceridemia, wherein the miR156a is an RNA sequence of SEQ ID No. 1 in the sequence listing. The application of a preparation of miR156a in triglyceride metabolism regulation, wherein the miR156a preparation comprises any one or more of the following: I, miR156a, II, a miR156a precursor, III, a modified miR156a molecule, IV, a recombinant vector of miR156a, V, a derivative with biological activity of miR156a, and VI, a mimic with biological activity of miR156a. The miR156a or the preparation thereof provided by the application can reduce the content of triglyceride in serum and liver in a targeted manner, and can be used in combination with the same kind of medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, specifically to the application of miR156a or its formulations in regulating triglyceride metabolism. Background Technology

[0002] The liver is the core organ for maintaining triglyceride metabolic homeostasis. Hepatocytes synthesize and secrete triglycerides (TG). Under normal physiological conditions, the liver processes a large amount of fatty acids, but stores only a small amount as triglycerides. In pathological conditions such as obesity, triglyceride accumulation in hepatocytes increases. When the liver fat content is greater than 5%, it is defined as nonalcoholic fatty liver (NAFL). Accompanied by necrotizing inflammation and damage to hepatocytes, NAFL further develops into nonalcoholic steatohepatitis (NASH), cirrhosis, and even hepatocellular carcinoma. It is estimated that by 2030, the overall prevalence of nonalcoholic fatty liver in adults (≥15 years) will be 33.5%, the median age will increase from 50 years to 55 years, the number of people with decompensated cirrhosis will increase to 105,430, the number of liver cancer cases will increase to 12,240, and the number of deaths from liver disease is estimated to reach 78,300. Therefore, non-alcoholic fatty liver disease has become a new challenge in the field of contemporary medicine, and its harm to human health is constantly increasing. At present, more effective means are needed to prevent and treat non-alcoholic fatty liver disease.

[0003] Very low-density lipoprotein (VLDL) is a triglyceride-rich particle produced by the liver, accounting for the vast majority of fasting plasma triglyceride levels. In patients with severe steatosis or NASH, VLDL-TG secretion is increased, while plasma VLDL clearance is reduced, leading to hypertriglyceridemia. Clinically, 80-90% of patients with hypertriglyceridemia have moderately elevated TG levels, ranging from 150 mg / dL (1.7 mmol / L) to 400 mg / dL (4.6 mmol / L). Epidemiological and genetic studies have shown that abnormally elevated serum TG is an independent risk factor for cardiovascular disease. Currently, fibrates are the first-line targeted therapy for hypertriglyceridemia, reducing TG levels by 20%-70%. However, their effect on low-density lipoprotein cholesterol differs between moderate hypertriglyceridemia (decreased or unchanged LDL cholesterol) and severe hypertriglyceridemia (elevated LDL cholesterol). Given the adverse reactions of drugs used to treat hypertriglyceridemia, there is an urgent need to design and develop novel drugs for the treatment of hypertriglyceridemia.

[0004] Minimal RNAs (miRNAs) are a class of single-stranded, non-coding small RNA molecules, approximately 19-23 nucleotides in length. They can regulate the expression of target genes through base pairing, thereby modulating cell growth, differentiation, apoptosis, cell cycle, and immune responses. Furthermore, miRNAs are not degraded by endogenous ribonucleases and can remain stably present in human blood. Recent research has found that plant miRNAs can be absorbed by the animal gastrointestinal tract and enter tissues through blood circulation to regulate the expression of endogenous genes and exert biological functions. Therefore, application strategies based on plant miRNAs have high practical value. Investigating the regulatory role of plant miRNAs in hepatic lipid metabolism is of great significance for the prevention and treatment of triglyceride metabolism disorders such as non-alcoholic fatty liver disease, hypertriglyceridemia, and cardiovascular diseases. Summary of the Invention

[0005] To address the above problems, this invention provides an application of miR156a or its formulation in regulating triglyceride metabolism. To achieve the above objective, the technical solution of this invention is as follows:

[0006] This invention relates, in one aspect, to the application of miR156a in products or drugs that regulate triglyceride metabolism. The miR156a is the RNA sequence of SEQ ID No. 1 in the sequence listing. miR156a inhibits the transcription and expression of the PGC-1β gene by targeting and binding to the transcriptional coactivator Pgc-1β, thereby inhibiting the expression of fatty acid synthesis-related genes: sterol regulatory element-binding transcription factor 1 (Srebf1), acetyl-CoA carboxylase 1 (Acc1), fatty acid synthase (Fasn), and stearoyl-CoA desaturase 1 (Scd1). Furthermore, miR156a also inhibits the synthesis of fatty acids into triglycerides by targeting and regulating 3-phosphoglyceryl acyltransferase 1 (Gpat1), thus achieving the effect of regulating triglyceride metabolism.

[0007] VLDL are triglyceride-rich particles produced by the liver, accounting for the vast majority of fasting plasma triglyceride levels. Another aspect of this invention relates to the use of miR156a in products or drugs for regulating very low-density lipoprotein levels, wherein miR156a is the RNA sequence of SEQ ID No. 1 in the sequence listing.

[0008] Another aspect of this invention relates to the use of miR156a in the preparation of products or medicaments for the prevention or treatment of non-alcoholic fatty liver disease or hypertriglyceridemia, wherein miR156a is the RNA sequence of SEQ ID No. 1 in the sequence listing. The miR156a of this invention can reduce triglyceride levels in serum and liver, and can be used in products or medicaments for the prevention or treatment of non-alcoholic fatty liver disease, hypertriglyceridemia, or cardiovascular disease. The medicament can be administered via intravenous injection, direct intra-organ injection, or oral administration, or other routes of administration well known to those skilled in the art, wherein the administration composition can be used in combination with other treatment methods. Specifically, the combined use of miR156a or its formulations with statins for the treatment of cardiovascular disease can simultaneously reduce plasma triglyceride and low-density lipoprotein cholesterol levels while avoiding the side effects of fibrates.

[0009] Another aspect of the present invention relates to a formulation of miR156a, wherein miR156a is the RNA sequence of SEQ ID No. 1 in the sequence listing. The miR156a formulation is a formulation containing the miR156a, specifically, the miR156a formulation comprises any one or more of the following substances:

[0010] I. The miR156a mentioned above

[0011] II. miR156a precursor

[0012] III. Modified miR156a molecule,

[0013] IV. The recombinant vector of miR156a,

[0014] V. Derivatives possessing the biological activity of miR156a,

[0015] VI. Mimics possessing the bioactivity of miR156a,

[0016] The miR156a formulation may be used in products or drugs for regulating triglyceride metabolism, or in the preparation of products or drugs for the prevention or treatment of non-alcoholic fatty liver disease or hypertriglyceridemia.

[0017] In this invention, miR156a is derived from plants.

[0018] The miR156a precursor described in this invention refers to a precursor that can be processed into miR156a in cells or in vivo.

[0019] The derivatives with miR156a bioactivity described in this invention include derivatives that still retain miR156a bioactivity after modification.

[0020] The miR156a biomimics described in this invention include synthetically produced or commercially available miR156a biomimics. For example, miR156amimics are synthetically produced according to the miR156a sequence of this invention.

[0021] According to any of the above applications, the miR156a formulation can also be used as a tool drug for studying the functional mechanisms of the aforementioned related diseases or related proteins.

[0022] The beneficial effects of this invention are that miR156a or its formulation provided by this invention can specifically reduce the triglyceride content in serum and liver, and can be used in conjunction with similar drugs for simultaneous administration, providing a method for preparing a product or drug for the prevention or treatment of triglyceride metabolism-related diseases.

[0023] Secondly, miR156a, as described in this invention, is widely found in grains, vegetables, and fruits, and has a positive effect on improving diseases related to abnormal triglyceride metabolism. Therefore, it can serve as a basis for dietary adjustments. Individuals who wish to improve their triglyceride metabolism can appropriately increase their intake of grains and fruits and vegetables high in miR156a.

[0024] Third, applying an effective amount of plant miR156a or its preparations to drugs for the prevention or treatment of non-alcoholic fatty liver, hypertriglyceridemia, or cardiovascular diseases can reduce the triglyceride content in human serum and liver, thus solving the problem of some adverse reactions of existing drugs. Attached Figure Description

[0025] Figure 1 The content of miR156a in grains, vegetables, and fruits.

[0026] Figure 2 Serum and liver triglyceride levels were abnormally elevated in miR156a Sponge transgenic mice that underwent obesity induced by a high-fat diet.

[0027] Figure 2 A represents the content of miR156a in the high-fat diet;

[0028] Figure 2 B represents the miR156a content in the liver of obese mice;

[0029] Figure 2 C represents the body weight of the obese mouse;

[0030] Figure 2 D represents the serum triglyceride level in obese mice;

[0031] Figure 2 E represents the liver triglyceride content in obese mice;

[0032] Figure 2 F shows the liver H&E staining and Oil Red O staining images of obese mice.

[0033] Figure 3 miR156a was administered via gavage to reduce serum and liver triglyceride levels in mice with high-fat diet-induced obesity.

[0034] Figure 3 A is the experimental flowchart of high-fat diet-induced obese mice by gavage administration of miR156a;

[0035] Figure 3 B represents the miR156a content in the liver of obese mice;

[0036] Figure 3 C represents the triglyceride content in the liver of obese mice;

[0037] Figure 3 D represents the body weight of the obese mouse;

[0038] Figure 3 E represents the serum triglyceride level in obese mice;

[0039] Figure 3 F shows the liver H&E staining and Oil Red O staining images of obese mice.

[0040] Figure 4 To reduce lipid droplet accumulation in hepatocytes, miR156a

[0041] Figure 4 A represents the efficiency of miR156a transfection into primary mouse liver cells;

[0042] Figure 4 B shows the Nile Red staining pattern of lipid droplets in primary liver cells;

[0043] Figure 4 C represents the efficiency of miR156a transfection into mouse hepatocellular carcinoma cells Hepa1-6;

[0044] Figure 4 D is a Nile Red staining image of lipid droplets in Hepa1-6 liver cancer cells. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified in the embodiments, conditions should be performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0046] Example 1

[0047] This embodiment demonstrates that miR156a is widely present in grains, vegetables, and fruits.

[0048] 1. miRNA extraction

[0049] Weigh 50 mg each of rice, wheat, potato, cabbage, lettuce, broccoli, spinach, apple, orange, grape, and banana, respectively, and grind them in liquid nitrogen. Transfer the ground samples to 1.5 mL centrifuge tubes. Extract miRNA using the BioTeke Universal Plant MicroRNA Rapid Extraction Kit. Add 1 mL of lysis buffer (PL), invert and mix well, incubate at 65°C for 10 minutes, inverting and mixing several times during incubation to fully lyse the ribosomes. Centrifuge at 12000 rpm at room temperature for 5 minutes. Transfer the supernatant to a 2 mL centrifuge tube, add an equal volume of pre-chilled isopropanol, invert and mix well, centrifuge at 12000 rpm at 4°C for 5 minutes. Discard the supernatant, add 100 μL of LEPC H2O to dissolve the precipitate. Add 900 μL of lysis buffer (MRL), mix well. Add 200 μL of chloroform, mix well, and let stand for 5 minutes. Centrifuge at 12000 rpm at 4°C for 10 minutes, and transfer 700 μL of the supernatant to a new centrifuge tube. Add 0.6 times the volume of 70% ethanol and mix by inversion. Transfer the solution to the RA adsorption column, passing it through the column twice. Centrifuge at 10000 rpm for 30 seconds, collect the filtrate, add 2 / 3 volume of anhydrous ethanol, and mix by inversion. Transfer the solution to the RB adsorption column and centrifuge at 10000 rpm for 30 seconds. Discard the filtrate, add 700 μL of RW wash buffer, centrifuge at 12000 rpm for 1 minute, and discard the filtrate. Centrifuge the RB adsorption column at 12000 rpm for 2 minutes. Transfer the adsorption column to a new EP tube, add 30-50 μL of DEPC H2O, let stand for 2 minutes, and centrifuge at 12000 rpm for 1 minute. The collected miRNA can be used for subsequent quantitative PCR (q-PCR) experiments or stored at -80℃.

[0050] 2. Reverse transcription

[0051] Based on the principle of stem-loop primer q-PCR, stem-loop primers were designed for reverse transcription. Reverse transcription of miRNA was performed using stem-loop primer probes from ABI (catalog number 000333). A mixture of 1 μL 10×RT buffer, 0.5 μL reverse transcriptase, 0.1 μL deoxyribonucleotide (dNTP) mixture, 0.12 μL RNase inhibitor, 0.5 μL Taqman-probe, 2 μL RNA, and 5.78 μL LDPPC H2O was gently mixed and placed in a PCR instrument for reverse transcription. The reaction program was 16℃ for 30 min; 42℃ for 30 min; 85℃ for 5 min; and stored at 4℃.

[0052] 3. Q-PCR detection

[0053] Quantitative detection was performed using an ABI Q-PCR kit, with three replicates per sample. 10 μL of universal reaction mixture, 0.33 μL of Taqman probe-TM, 7.17 μL of H2O, and 2.5 μL of cDNA were mixed and placed in an LC480 real-time PCR instrument for PCR. The reaction program was 95℃ for 5 min; (95℃, 15 s; 60℃, 1 min) for 45 cycles. The synthesized single-stranded miR156a of known concentration was serially diluted to 10... 5 fM, 10 4 fM, 10 3 fM, 10 2 fM, 10fM, 1fM, and the corresponding CT values ​​generated by Q-PCR were used to plot the miR156a standard curve, and the absolute content of miR156a in the sample to be tested was calculated from this curve.

[0054] 4. Results

[0055] like Figure 1 As shown, the abundance of miR156a varies in grains, vegetables, and fruits. Grains and vegetables have higher levels of miR156a, such as wheat, which has a high content of 12.01 fmol / g. Rice, broccoli, and bananas have miR156a contents of 6.47 fmol / g, 6.18 fmol / g, and 2.61 fmol / g, respectively. Apples, oranges, and grapes have relatively lower levels of miR156a.

[0056] Example 2

[0057] This embodiment demonstrates, using the miR156a Sponge transgenic mouse model, that the absence of miR156a in vivo leads to abnormally elevated serum and liver triglyceride levels in mice.

[0058] 1. Eight-week-old male miR156a Sponge transgenic mice and their littermates were fed a high-fat diet (60% fat content) for 16 weeks to induce obesity. The high-fat diet was found to contain a certain amount of miR156a, such as... Figure 2 As shown in Figure A. Mice were weighed weekly during the period, and their weight changes were recorded. After successful establishment of the obesity model, the mice were fasted overnight, and blood and liver tissue were collected. The triglyceride content in serum and liver was detected using a triglyceride detection kit provided by Nanjing Jiancheng Saihao Technology Co., Ltd. Additionally, the accumulation of lipid droplets in the liver was detected by H&E staining and Oil Red O staining.

[0059] 2. Results

[0060] like Figure 2As shown in Figure B, the liver miR156a content in obese miR156a Sponge transgenic mice was significantly lower than that in their littermates' wild-type mice, while their body weight was not different from that of wild-type mice (e.g., ...). Figure 2 (as shown in C), but serum triglyceride levels (as shown in C) Figure 2 (as shown in D) and liver triglyceride levels (e.g. Figure 2 The levels shown in E were significantly higher than in wild-type mice, and H&E and Oil Red O staining results of liver tissue showed increased accumulation of lipid droplets in the liver (as shown in E). Figure 2 (As shown in F). The above results indicate that the absence of miR156a leads to abnormally elevated serum and liver triglyceride levels in obese mice, and exacerbates hepatic steatosis.

[0061] Example 3

[0062] This embodiment demonstrates that oral administration of miR156a reduces serum and liver triglyceride levels in mice with high-fat diet-induced obesity.

[0063] 1. Eight-week-old male miR156a Sponge transgenic mice and their littermates were fed a high-fat diet (60% fat content) for 16 weeks to induce obesity. Obese mice were divided into four groups and administered miR156a RNA (SEQ ID No. 15′-UGACAGAAGAGAGUGAGCAC-3′) and NC RNA (SEQ ID No. 25′-UUGUACUACACAAAAGUACUG-3′) by gavage at a dose of 20 nnol / kg, respectively, at the same time daily for one week. Figure 3 As shown in Figure A. Mice were fasted overnight, and blood and liver samples were collected to detect triglyceride levels; liver lipid droplet accumulation was detected by H&E staining and Oil Red O staining.

[0064] 2. Results

[0065] like Figure 3 As shown in Figure B, after gavage administration of miR156a, the content of miR156a in the liver of wild-type obese mice was significantly increased, while no significant change was observed in miR156a Sponge transgenic mice. Figure 3 As shown in D, gavage administration of miR156a did not affect the body weight of mice, but wild-type mice showed the following effects after gavage administration of miR156a: Figure 3 E and Figure 3 As shown in Figure C, serum and liver triglyceride levels were significantly reduced, while... Figure 3As shown in Figure F, H&E and Oil Red O staining of liver tissue revealed reduced lipid droplet accumulation in the liver, while serum and liver triglyceride levels in miR156a Sponge transgenic mice showed no significant changes. These results indicate that miR156a can reduce serum and liver triglyceride levels in obese mice, decrease liver lipid accumulation, and improve hepatic steatosis and hypertriglyceridemia.

[0066] Example 4

[0067] This embodiment demonstrates that miR156a reduces the accumulation of lipid droplets in hepatocytes.

[0068] 1. Mouse primary liver cells and Hepa1-6 hepatocellular carcinoma cells were used to establish miR156a group and control group, respectively. Hepa1-6 hepatocellular carcinoma cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. In the experiment, mouse primary liver cells and Hepa1-6 hepatocellular carcinoma cells were seeded into 12-well cell culture plates and cultured in DMEM medium containing 10% fetal bovine serum. After the cells were fully adhered, the miR156a group was transfected with miR156a, and the control group was transfected with NC RNA. Six hours after transfection, 0.125% bovine serum albumin (BSA) and 0.1 mM palmitic acid (PA) were added to each group to stimulate the cells. Twenty-four hours later, the intracellular lipid droplet content was detected by Nile Red staining.

[0069] 2. Results

[0070] like Figure 4 A and Figure 4 As shown in Figure C, transfection with miR156a significantly increased the levels of miR156a in both primary liver cells and Hepa1-6 liver cancer cells. Nile red staining results showed that... Figure 4 B and Figure 4 As shown in Figure D, compared with the BSA-NC group, the BSA-miR156a group had fewer lipid droplets in hepatocytes. Under PA stimulation, the number of lipid droplets in hepatocytes of the NC group increased significantly, while the number of lipid droplets added in hepatocytes of the miR156a group was significantly less than that of the NC group. These results demonstrate in vitro that miR156a can reduce the accumulation of lipid droplets in hepatocytes.

[0071] It should be noted that the described embodiments are only some, not all, of the embodiments of the present invention. In the experiment, the miR156a Sponge transgenic mouse refers to a mouse obtained by integrating the plant miR156a Sponge sequence into the mouse genome and stably transmitting it to offspring.

[0072] Example 1 involves the study and verification of the distribution and content of miR156a in plants, and Examples 2-4 involve the study of the regulatory function of miR156a in animals or in vitro. Through a series of experimental studies, the inventors found that (1) miR156a is widely present in grains, vegetables and fruits. (2) The absence of miR156a leads to abnormally high levels of serum and liver triglycerides in obese mice, and exacerbates hepatic steatosis. (3) Gavage administration of miR156a reduces serum and liver triglyceride levels in obese mice induced by a high-fat diet. (4) miR156a reduces the accumulation of lipid droplets in hepatocytes.

[0073] In summary, miR156a is widely found in grains, vegetables, and fruits, and endogenous miR156a is currently absent in animals. This invention, through studies on the regulatory functions of miR156a in vivo and in vitro, has discovered that miR156a or its formulations play a crucial role in serum and liver triglyceride levels, and can reduce lipid accumulation in hepatocytes. This suggests its potential effectiveness in preventing or treating non-alcoholic fatty liver disease, hypertriglyceridemia, or cardiovascular diseases. Furthermore, the drug can be administered via intravenous injection, direct intra-organ injection, oral administration, or other routes of administration well-known to those skilled in the art.

Claims

1. The use of miR156a in the preparation of medicaments for the prevention or treatment of non-alcoholic fatty liver disease or hypertriglyceridemia, characterized in that, The miR156a is the RNA sequence shown in UGACAGAAGAGAGUGAGCAC.

Images