Use of miR-1246-like miRNAs in the treatment of type ii diabetes and its complications

Abstract

The application belongs to the field of biological medicine, and more particularly relates to the use of miR-1246 type miRNAs in treating type II diabetes and type II diabetes complications. The application discloses the use of a miRNA, or a precursor sequence, an expression vector thereof, or an agonist thereof, the miRNA being miR-1246 type miRNAs selected from the group consisting of miR-1246 or a miR-1246 derivative sequence or a modified derivative thereof, for use in the preparation of a medicine for preventing and treating type II diabetes and other metabolism related diseases. The application first finds that miR-1246 type miRNAs can reduce body weight and improve body shape, effectively reduce the concentration of blood sugar and blood lipid, and significantly improve insulin tolerance, glucose tolerance related indexes, improve the body insulin sensitivity, repair the secretion function of islets and islet beta cells, thereby improve the metabolic disorder state, maintain the body metabolic homeostasis, and finally achieve the purpose of preventing or treating type II diabetes and its complications.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically relates to the use of miR-1246 class miRNAs in drugs for the treatment of type II diabetes and its complications. Background Technology

[0002] Diabetes mellitus is a complex chronic systemic disease characterized by metabolic and endocrine disorders, with typical manifestations including hyperglycemia, hyperinsulinemia, and hypertriglyceridemia. Approximately 90-95% of patients have type 2 diabetes mellitus (T2DM), which is primarily caused by insulin resistance and insufficient insulin secretion, ultimately leading to persistently elevated blood glucose levels. Statistics show that in 2021, there were 463 million people with T2DM worldwide, and this number is projected to rise to 700 million by 2045. Obesity, a sedentary lifestyle, and poor dietary habits are all significant contributing factors to T2DM. This disease can cause multi-organ damage and various serious complications, posing a major global public health challenge.

[0003] Insulin resistance is a core initiating factor and early pathological feature in the development of type 2 diabetes mellitus (T2DM). It manifests as a significant decrease in the sensitivity of peripheral tissues to insulin, leading to reduced efficiency in insulin-mediated glucose uptake and utilization. To maintain glycemic stability, pancreatic β-cells compensate by secreting more insulin, resulting in hyperinsulinemia. Under the combined effects of prolonged high workload, glucose and lipid toxicity, and inflammatory stress, β-cells gradually suffer damage, including a decrease in number and impaired secretory function. Insulin resistance and pancreatic β-cell dysfunction interact, forming a vicious cycle that ultimately leads to an imbalance in blood glucose homeostasis, driving the occurrence and progression of T2DM.

[0004] Currently, clinical treatment for type 2 diabetes mellitus (T2DM) and its comorbidities mainly relies on lifestyle interventions and first-line medications, such as metformin, thiazolidinediones (TZDs, such as pioglitazone), and GLP-1 receptor agonists (such as liraglutide, smegglutide, and dulaglutide). However, existing drugs all have certain limitations, potentially causing weight gain, edema, increased risk of heart failure, gastrointestinal adverse reactions, increased risk of genitourinary tract infections, and increased risk of fractures in some female patients. These potential side effects limit their long-term widespread use. Therefore, exploring safer and more effective new treatment strategies and drugs remains an urgent need and core direction in the field of diabetes research. Summary of the Invention

[0005] The purpose of this invention is to develop the use of miR-1246 class miRNAs to provide new drugs for the prevention and treatment of type II diabetes and its complications.

[0006] This invention discloses the use of a miRNA, or its precursor sequence, expression vector, or agonist thereof, wherein the miRNA is a miR-1246 class miRNA, and the miR-1246 class miRNA is selected from the group consisting of miR-1246 or miR-1246-derived sequences, or modified derivatives thereof, for use in the preparation of drugs for the prevention and treatment of metabolic-related diseases such as type II diabetes.

[0007] In a preferred embodiment, the miR-1246 is preferably human has-miR-1246, the sequence of which is shown in SEQ ID NO:1.

[0008] In a preferred embodiment, the miR-1246 derived sequence is derived from the sequence shown in SEQ ID NO:1, including derived sequences formed by single base deletions, insertions, and / or substitutions.

[0009] In another preferred embodiment, the miR-1246 derived sequence is shown in SEQ ID NO:2 to 21.

[0010] In a preferred embodiment, the precursor sequence is shown in SEQ ID NO:22.

[0011] In another preferred embodiment, the precursor sequence is derived from the sequence shown in SEQ ID NO:22, including derived sequences formed by single-base deletions, insertions, and / or substitutions.

[0012] In another preferred embodiment, the miR-1246 class of miRNAs includes isolated or artificially synthesized ones.

[0013] In another preferred embodiment, the modified derivative includes miR-1246 or a modified derivative of a miR-1246-derived sequence, wherein the modification is selected from one or more of the following: glycosyl modification of nucleotides, modification of the linkage between nucleotides, cholesterol modification, locked nucleotide modification, peptide modification, lipid modification, halogen modification, hydrocarbon modification, and nucleic acid modification.

[0014] In another preferred embodiment, the glycosyl modification of the nucleotide includes 2-O-methyl glycosyl modification, 2-O-methoxyethyl glycosyl modification, 2-O-alkyl glycosyl modification, 2-fluoro glycosyl modification, sugar ring modification, and locked nucleotide modification; and / or the modification of the linkage between the nucleotides includes thiophosphate modification and phosphorylation modification; and / or the nucleic acid modification includes "TT" modification.

[0015] In another preferred embodiment, the modified miRNA derivative is a monomer or polymer of a compound having the structure shown in Formula I: Equation I: (X)n-(Y)m In equation I, Each X represents the miRNA described above; Each Y is an independent modifier that promotes the stability of miRNA administration; Y connects to the left, right, or middle of X; n is a positive integer from 1 to 100 (preferably 1 to 20) (preferably n is 1, 2, 3, 4 or 5); m is a positive integer from 1 to 1000 (preferably from 1 to 200); Each "-" indicates a linker, chemical bond, or covalent bond.

[0016] In another preferred embodiment, the adapter is a nucleic acid sequence of 1-10 bases in length.

[0017] In another preferred embodiment, Y includes (but is not limited to) cholesterol, steroids, sterols, alcohols, organic acids, fatty acids, esters, monosaccharides, polysaccharides, amino acids, polypeptides, mononucleotides, and polynucleotides.

[0018] In another preferred embodiment, the precursor sequence is selected from the group consisting of: (a) a precursor miRNA capable of being processed into the miRNA described herein; or (b) Can be transcribed by the host to form the precursor miRNA and processed to form the polynucleotide of the miRNA.

[0019] In another preferred embodiment, the nucleotide sequence of the precursor miRNA is shown in SEQ ID NO:23.

[0020] In another preferred embodiment, the polynucleotide has the structure shown in Formula II: Seq 正向 -X-Seq 反向 Formula II, In formula II, Seq 正向 In order to be processed into the miRNA nucleotide sequence described above in the host; Seq 反向 These are nucleotide sequences that are substantially or completely complementary to the forward direction of the Seq sequence; X is located at Seq 正向 and Seq 反向 The interval sequence between, and the interval sequence with Seq 正向 and Seq 反向 Not complementary; Furthermore, the structure shown in Formula II, after being transferred into the host cell, forms the secondary structure shown in Formula III:

[0021] In Equation III, Seq 正向 Seq 反向 The definitions of X and X are as described above. "||" indicates that in Seq 正向 and Seq 反向 The complementary base pairing relationship formed between them.

[0022] In another preferred embodiment, the expression vector is used to express the miR-1246 class miRNAs or their precursor miRNAs.

[0023] In another preferred embodiment, the expression vector contains the miR-1246 class of miRNAs, or their precursor miRNAs, or polynucleotides that can be transcribed by the host to form the precursor miRNAs.

[0024] In another preferred embodiment, the expression vector includes: a viral vector and a non-viral vector.

[0025] In another preferred embodiment, the expression vector is a plasmid.

[0026] In another preferred embodiment, the miRNA agonist is selected from the group consisting of substances that promote the expression of miR-1246 class miRNAs and substances that enhance the activity of miR-1246 class miRNAs.

[0027] In another preferred embodiment, the aforementioned type II diabetes and other metabolic-related diseases refer to a class of metabolic syndromes caused by glucose metabolism disorders. Type II diabetes and other metabolic-related diseases refer to a class of diseases closely related to abnormal glucose metabolism processes in the human body, including but not limited to type II diabetes, diabetic nephropathy, hyperlipidemia, obesity, atherosclerosis, or combinations thereof.

[0028] In another preferred embodiment, the pharmaceutical composition comprises (a) has-miR-1246 class miRNAs, or their precursor sequences, expression vectors, or agonists thereof; and (b) pharmaceutically acceptable vectors.

[0029] In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof.

[0030] In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient, wherein the second active ingredient is selected from the group consisting of drugs for treating type II diabetes, non-alcoholic fatty liver disease, obesity, hyperlipidemia, hypertension, atherosclerosis, or combinations thereof.

[0031] In another preferred embodiment, the drug for treating type II diabetes, non-alcoholic fatty liver disease, obesity, hyperlipidemia, hypertension, and atherosclerosis is selected from the group consisting of: insulin, biguanides, insulin secretagogues, insulin sensitizers, incretins, α-glucosidase inhibitors, sodium-glucose cotransporter 2 inhibitors, statins, fibrates, choline sequestrants, niacin and its derivatives, cholesterol absorption inhibitors, bile acids, PCSK9 inhibitors, or combinations thereof.

[0032] A second aspect of the present invention provides a pharmaceutical composition comprising: The first active ingredient is a has-miR-1246 class miRNA, or its precursor sequence, expression vector, or agonist: wherein the has-miR-1246 class miRNA is selected from the group consisting of miR-1246 or miR-1246-derived sequences, or their modified derivatives. A second active ingredient, which differs from the first active ingredient, and is selected from the group consisting of: drugs for treating type II diabetes, non-alcoholic fatty liver disease, obesity, hyperlipidemia, hypertension, atherosclerosis, or combinations thereof; and Pharmaceutically acceptable carrier.

[0033] In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof.

[0034] In another preferred embodiment, the dosage form of the pharmaceutical composition is a liquid dosage form, preferably an injection, and more preferably an intravenous injection or an intraperitoneal injection.

[0035] In another preferred embodiment, the method of administration of the pharmaceutical composition includes direct injection of miR-1246 class miRNAs or plasmids containing such miRNAs.

[0036] The beneficial effects of this invention are as follows: This invention is the first to discover that miR-1246 class miRNAs can effectively reduce blood glucose and blood lipid concentrations; they can also significantly improve insulin tolerance and glucose tolerance-related indicators, enhance insulin sensitivity, repair pancreatic β cells and pancreatic β cell secretion, thereby improving metabolic disorders, maintaining metabolic homeostasis, and ultimately achieving the goal of preventing or treating type II diabetes and related metabolic diseases. Attached Figure Description

[0037] Figure 1miR-1246 reduced body weight and improved body shape in DB / DB (homozygous leptin receptor Lepr gene mutation) type II diabetic mice. The mice were divided into different treatment groups: DB / DM+NS group (heterozygous Lepr gene mutation mice + saline group), DB / DB+NS group (homozygous Lepr gene mutation mice + saline group), DB / DB+agomir-1246 group (homozygous Lepr gene mutation mice + chemically modified miR-1246, agomir-1246 group), and DB / DB+antagomir-1246 group (homozygous Lepr gene mutation mice + an inhibitor of miR-1246, antagomir-1246). (A) Representative body shape of mice in different treatment groups. agomir-1246 significantly improved obesity in DB / DB type II diabetic mice, while antagomir-1246 did not have this effect. (B) Body weight of mice in different treatment groups. agomir-1246 significantly reduced the body weight of DB / DB type II diabetic mice, while antagomir-1246 did not have this effect. p < 0.05 was considered statistically significant (compared to the control group). ### p < 0.001 indicates a difference; compared with the model group, ** p < 0.01, *** p < 0.001 indicates a difference. Figure 2 Agomir-1246 reduced blood glucose and lipid concentrations in DB / DB type II diabetic mice. (A, B) Changes in blood glucose and fasting blood glucose concentration in mice under different treatment groups. Agomir-1246 significantly reduced blood glucose in DB / DB diabetic mice, while antagomir-1246 did not have this effect. (C, D, E) Lipid concentrations in mice under different treatment groups. TG: Total triglyceride concentration; TC: Total cholesterol concentration; LDL-c: Total low-density lipoprotein cholesterol. Agomir-1246 effectively reduced TG, TC, and LDL levels in DB / DB diabetic mice and improved blood lipids, while antagomir-1246 did not have this effect. p < 0.05 was considered statistically significant (compared to the control group). ## p<0.01, ### p < 0.001 indicates a difference; compared with the model group, * p < 0.05, ** p < 0.01, *** p < 0.001 indicate a difference. Figure 3Agomir-1246 improved glucose tolerance (GTT) and insulin tolerance (ITT) in DB / DB type II diabetic mice. (A, B) GTT detection in mice of different treatment groups. Agomir-1246 significantly improved GTT in DB / DB diabetic mice, while antagomir-1246 did not have this effect. (C, D) ITT detection in mice of different treatment groups. Agomir-1246 significantly improved ITT in DB / DB diabetic mice, with a significantly increased reduction in blood glucose, while antagomir-1246 did not have this effect. p < 0.05 was considered statistically significant (compared to the control group). ### p < 0.001 indicates a difference; compared with the model group, * p < 0.05, ** p < 0.01, *** p < 0.001 indicate a difference. Figure 4 Anagomir-1246 effectively improved insulin resistance and repaired pancreatic β-cell function in DB / DB type II diabetic mice. (A, B) Insulin levels and insulin resistance (IR) in mice treated with different methods. Anagomir-1246 significantly reduced serum insulin levels and improved the insulin resistance index (HOMR-IR) in type II diabetic mice, while anagomir-1246 did not have this effect. (C) Histopathological examination of pancreatic tissue in mice treated with different methods. Anagomir-1246 significantly repaired the volume of pancreatic β-cells, contributing to insulin secretion function, while anagomir-1246 did not have this effect. p < 0.05 was considered statistically significant (compared to the control group). ### p < 0.001 indicates a difference; compared with the model group, ** p < 0.01, *** p < 0.001 indicates a difference. Detailed Implementation

[0038] Through extensive and in-depth research, the inventors have discovered for the first time that miR-1246-type miRNAs can prevent or treat type II diabetes and other related chronic metabolic diseases. Based on this, the inventors completed this invention.

[0039] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0041] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0042] As used herein, the terms “host,” “subject,” and “required object” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, and non-human primates such as cattle, horses, dogs, cats, pigs, sheep, goats, giraffes, deer, camels, sheep, rats, mice, hares, and rabbits.

[0043] miRNA and its precursors microRNAs (miRNAs) are non-coding RNAs, approximately 21-23 nucleotides in length, widely found in eukaryotes. They primarily participate in the negative regulation of mRNA levels after gene transcription and can regulate the expression of other genes. miRNAs can participate in physiological processes such as cell communication, cell signal transduction, and changes in cell or tissue metabolism, either short-term or long-term. miRNAs play a significant role in cell growth, differentiation, development, and the occurrence and development of diseases.

[0044] miR-1246 was first discovered using high-throughput sequencing technology in human embryonic stem cells. Subsequent studies have located the human miR-1246 encoding gene, located on chromosome 2q31.1a. miR-1246 regulates the RAF / MEK / ERK, GSK3β, Wnt / β-catenin, JAK / STAT, PI3K / AKT, THBS2 / / MMP, and NOTCH2 signaling pathways.

[0045] The mature sequence of miR-1246 (AAUGGAUUUUUGGAGCAGG, SEQ ID NO:1) shows that mature miR-1246 usually negatively regulates target genes through two sequence complementarity-dependent mechanisms with its complementary mRNA. The mechanism involves base pairing of the seed sequence (2nd-7th nucleotides) at the 5' apos; end of miR-1246 with the 3' UTR region of the target gene mRNA. The tightness of the pairing determines the degree of inhibition of the target gene protein by the miRNA. Pairing forms include: (1) 6mer site: the 2nd-7th nucleotides of the miRNA are completely paired with the target gene. This method has higher sensitivity to target gene recognition, lower specificity, incomplete inhibition, and more protein accumulation. (2) 7mer-1A site: the 2nd-7th nucleotides are complementary to the target gene, and the first nucleotide of the miRNA corresponding to the target gene is A. This method has high specificity, moderate inhibitory effect, and is relatively universal. (3) 7mer-m8 site: that is, the 2nd to 8th nucleotides of the miRNA are completely paired with the target gene. This method has the second highest specificity, strong conservation, and general inhibitory effect; (4) 8mer site: that is, the 2nd to 8th nucleotides of the miRNA are completely paired with the target gene, and the first nucleotide of the miRNA corresponding to the target gene is A. This method has the highest specificity, complete inhibition, and the least protein accumulation. miR-1246 is related to cancer, tumors, Ebola virus infectious diseases, immune diseases, etc. In these diseases, miR-1246 mainly acts as an inhibitor. However, in some cancers and cells, miR-1246 also shows a promoting effect. This may be because its role is closely related to the tissue and cell environment in which it is located.

[0046] Human-derived miRNAs can be isolated from human cells. As used herein, "isolated" means that the substance has been isolated from its native environment (or, in the case of a native substance, the native environment). Polynucleotides and polypeptides in their native state within living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in their native state.

[0047] The miRNA can be processed from a precursor miRNA (Pre-miRNA), which can fold into a stable stem-loop (hairpin) structure, typically 50-100 bp in length. The precursor miRNA folds into a stable stem-loop structure, with two substantially complementary sequences on either side of the stem. The precursor miRNA can be natural or synthetic. The miR-1246 precursor miRNA sequence of this invention is shown in SEQ ID NO:22.

[0048] Precursor miRNAs can be cleaved to generate miRNAs that are substantially complementary to at least a portion of the sequence of the mRNA encoding the gene. As used herein, “substantially complementary” means that the nucleotide sequences are sufficiently complementary to interact in a predictable manner, such as forming secondary structures (e.g., stem-loop structures). Typically, two “substantially complementary” nucleotide sequences have at least 70% complementary nucleotides to each other; preferably, at least 80%; more preferably, at least 90%; and even more preferably, at least 95%; such as 98%, 99%, or 100%. Generally, two sufficiently complementary molecules may have up to 40 mismatched nucleotides; preferably, up to 30; more preferably, up to 20; and even more preferably, up to 10, such as 1, 2, 3, 4, 5, 8, or 11 mismatched nucleotides.

[0049] As used herein, a "stem-loop" structure, also known as a "hairpin" structure, refers to a nucleotide molecule that can form a secondary structure including a double-stranded region (stem) formed by two regions of the nucleotide molecule (located on the same molecule), positioned on either side of the double-stranded portion; it also includes at least one "loop" structure, comprising a non-complementary nucleotide molecule, i.e., a single-stranded region. Even if the two regions of the nucleotide molecule are not perfectly complementary, the double-stranded portion of the nucleotide can remain double-stranded. For example, insertions, deletions, substitutions, etc., can lead to a small region becoming non-complementary or that small region itself forming a stem-loop structure or other forms of secondary structure; however, the two regions can still be substantially complementary and interact in a predictable manner to form a double-stranded region of a stem-loop structure. Stem-loop structures are well known to those skilled in the art, and typically, after obtaining a nucleic acid with a nucleotide sequence having a primary structure, those skilled in the art can determine whether the nucleic acid can form a stem-loop structure.

[0050] The miRNAs mentioned in this invention refer to miR-1246 class miRNAs, which are selected from the following group: miR-1246 or miR-1246-derived sequences, or their modified derivatives.

[0051] In another preferred embodiment, the miRNA is derived from a human or a non-human mammal; preferably, the non-human mammal is a monkey (such as a macaque) or an orangutan (such as a chimpanzee), and the miR-1246 sequence of chimpanzees and humans is completely identical, while the mature sequence of miR-1246 of macaques and humans differs by one base.

[0052] This invention also includes miRNA variants and derivatives. Furthermore, miRNA derivatives in a broader sense may also include miRNA variants. Those skilled in the art can modify the miR-1246 cluster using common methods, including (but not limited to): methylation modification, hydrocarbon modification, glycosylation modification (such as 2-methoxy-glycosylation, hydrocarbon-glycosylation, glycan ring modification, etc.), nucleic acid modification, peptide modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification), etc.

[0053] In another preferred embodiment, the miR-1246 class of miRNAs includes isolated or artificially synthesized ones.

[0054] In another preferred embodiment, the miR-1246 has the sequence shown in SEQ ID NO:1.

[0055] In another preferred embodiment, the nucleotide sequence of the precursor miRNA is shown in SEQ ID NO:22.

[0056] Polynucleotide constructs Based on the miRNA sequence provided by this invention, polynucleotide constructs that, upon introduction, can be designed to process miRNAs into miRNAs that can affect the expression of the corresponding mRNAs, i.e., the polynucleotide constructs can upregulate the amount of the corresponding miRNAs in vivo. Therefore, this invention provides an isolated polynucleotide (construct), which can be transcribed into a precursor miRNA by human cells, and the precursor miRNA can be cleaved and expressed into the miRNA by human cells.

[0057] In a preferred embodiment of the present invention, the polynucleotide construct contains the structure shown in Formula II: Seq 正向 -X-Seq 反向 Formula II, In Equation II, Seq 正向 In order to be processed into the miRNA nucleotide sequence described above in the host; Seq 反向 To be with Seq 正向 Nucleotide sequences that are essentially complementary or completely complementary; X is located at Seq 正向 and Seq 反向 The interval sequence between, and the interval sequence with Seq 正向 and Seq 反向 Not complementary; Furthermore, the structure shown in Formula II, after being transferred into the host cell, forms the secondary structure shown in Formula III:

[0058] In Equation III, Seq 正向 Seq 反向 The definitions of X and X are as described above. "||" indicates that in Seq 正向 and Seq 反向 The complementary base pairing relationship formed between them.

[0059] In another preferred embodiment, the expression vector is used to express the miR-1246 class miRNAs or their precursor miRNAs.

[0060] In another preferred embodiment, the expression vector contains the miR-1246 class of miRNAs, or their precursor miRNAs, or polynucleotides that can be transcribed by the host to form the precursor miRNAs.

[0061] Typically, the polynucleotide construct is located on an expression vector. Therefore, the present invention also includes a vector containing the miRNA or the polynucleotide construct. The expression vector typically also contains a promoter, a replication origin, and / or a marker gene. Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells, such as resistance to kanamycin, gentamicin, hygromycin, and ampicillin.

[0062] Pharmaceutical Composition and Administration This invention provides a pharmaceutical composition comprising: (i) A first active ingredient, wherein the first active ingredient is a miR-1246 class miRNA, or its precursor sequence, expression vector, or agonist: wherein the miR-1246 class miRNA is selected from the group consisting of miR-1246 or miR-1246-derived sequences, or their modified derivatives. (ii) a second active ingredient, wherein the second active ingredient is different from the first active ingredient, and wherein the second active ingredient is selected from the group consisting of: hypoglycemic agents, lipid-lowering agents, drugs for treating fatty liver and other metabolic disorders of glucose and lipid metabolism, or combinations thereof; and (iii) Pharmaceutically acceptable carriers.

[0063] In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof.

[0064] In another preferred embodiment, the dosage form of the pharmaceutical composition is a liquid dosage form, preferably an injection, and more preferably an intravenous injection or an intraperitoneal injection.

[0065] In another preferred embodiment, the method of administration of the pharmaceutical composition includes direct injection of miR-1246 class miRNAs or plasmids expressing them.

[0066] In another preferred embodiment, the second active ingredient is a therapeutic agent selected from the group consisting of: type II diabetes, non-alcoholic fatty liver disease, obesity, hyperlipidemia, hypertension, atherosclerosis, or a combination thereof.

[0067] In another preferred embodiment, the second active ingredient is selected from the group consisting of: insulin, biguanides, insulin secretagogues, insulin sensitizers, incretins, α-glucosidase inhibitors, sodium-glucose cotransporter 2 inhibitors, statins, fibrates, choline sequestrants, niacin and its derivatives, cholesterol absorption inhibitors, bile acids, PCSK9 inhibitors, or combinations thereof.

[0068] As used herein, the term "first active ingredient" refers to miR-1246 class miRNAs, or their precursor sequences, or expression vectors containing them, or their agonists, that can be used in this invention. Preferably, the active ingredient is selected from the group consisting of: (a) miR-1246 class miRNAs, wherein the miR-1246 class miRNAs are selected from the group consisting of miR-1246 or miR-1246-derived sequences or modified derivatives thereof, wherein the miR-1246-derived sequences have any of the sequences described in (b)-(e); (b) a precursor miRNA, wherein the precursor miRNA can be processed in the host into the miR-1246 class miRNA described in (a); (c) A polynucleotide, wherein the polynucleotide can be transcribed by the host to form the precursor miRNA described in (b) and processed to form the miRNA described in (a); (d) An expression vector containing the miR-1246 class miRNA described in (a), the precursor miRNA described in (b), or the polynucleotide described in (c); Agonists of miRNAs as described in (e)(a).

[0069] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0070] As used herein, the term "pharmaceuticalally acceptable" refers to a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.

[0071] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. They are prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0072] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the active ingredient of this invention is administered every 3 days at a dose of approximately 0.00001 mg to 50 mg / kg animal body weight (preferably 0.00001 mg to 50 mg / kg animal body weight). For example, due to the urgency of the treatment condition, separate daily doses can be administered, or the dose can be reduced proportionally.

[0073] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0074] In this invention, miRNA can be used to prepare pharmaceutical compositions for treating type II diabetes and other related chronic metabolic diseases.

[0075] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described by Sambrook et al., or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0076] Unless otherwise specified, all materials used in the embodiments of this invention are commercially available products, and all methods used in this invention are conventional methods in the art.

[0077] The following nucleic acid molecules were synthesized by Guangzhou Ruibo Company:

[0078] agomir-1246 is a human-derived double-stranded modified miRNA molecule of has-miR-1246 (SEQ ID NO:1). Modifications are made to the antisense strand, including a cholesterol modification at the 3' end, two thiocarbamate modifications at the 5' end, four thiocarbamate modifications at the 3' end, and a full-strand methoxy modification. This chemically modified miRNA-1246 exhibits good tissue compatibility, single-base specificity, efficacy, and stability. agomir-1246 was synthesized by Guangzhou Ruibo Company.

[0079] Antagomir-1246 is the antisense strand of miRNA-1246 (SEQ ID NO:1), with cholesterol modification at the 3' end, two thiocyanate modifications at the 5' end, four thiocyanate modifications at the 3' end, and a full-chain methoxyl modification. This inhibitor interferes with the function of endogenous miRNA-1246. Chemically modified miRNA-1246 inhibitors exhibit good tissue compatibility, single-base specificity, efficacy, and stability. Antagomir-1246 was synthesized by Guangzhou Ruibo Company.

[0080] Example 1. The therapeutic effect of miR-1246 on DB / DB type II diabetes. agomir-1246 reduced the weight of type II diabetic mice with DB / DB (leptin receptor Lepr point mutation) and also improved their body shape.

[0081] agomir-1246 reduced blood glucose and blood lipid levels in DB / DB type II diabetic mice.

[0082] agomir-1246 can improve insulin tolerance and glucose tolerance-related indicators in DB / DB mice.

[0083] agomir-1246 can improve insulin sensitivity and repair pancreatic β cells and their secretory regulation function.

[0084] 1.1 Experimental Materials and Methods DB / DB type II diabetes mouse model.

[0085] Method: Six-week-old male DB / DB mice were used to establish a type II diabetes model after one week of acclimatization by measuring fasting blood glucose.

[0086] Normal diet control group (heterozygous mice with Lepr gene mutation DB / DM+NS): Mice were fed a normal diet, and after 8 weeks of normal diet, they were injected with saline via the tail vein weekly.

[0087] DB / DB control group (homozygous mice with Lepr gene mutation DB / DB+NS): Mice were fed a normal diet and received weekly intravenous injections of saline.

[0088] agomir-1246 group (homozygous mice with Lepr gene mutation DB / DB+ agomir-1246): Mice were fed a normal diet and injected 10-50 nM agomir-1246 0.1ml / time / mice via tail vein once a week.

[0089] The antagomir-1246 group (homozygous mice with Lepr gene mutation DB / DB+ antagomir-1246): Mice were fed a normal diet and injected 0.1 ml of 10-50 nM antagomir-1246 into the tail vein once a week per mouse.

[0090] 1.2 Experimental Results See results Figure 1 A, 1B, observed and monitored the body size and weight of DB / DB mice. agomir-1246 reduced the weight and improved the body size of DB / DB mice with type II diabetes.

[0091] like Figure 2 Figures A and 2B show that peripheral blood was collected from each group of mice, and blood glucose concentration was measured. Agomir-1246 reduced blood glucose levels in DB / DB type II diabetic mice. Figure 2 In C-2E, peripheral blood samples were collected and blood lipid concentrations were measured. Agomir-1246 significantly reduced the levels of TG, TC, and LDL in Db / DB diabetic mice, effectively improving blood lipids.

[0092] like Figure 3 A-3D visualization showed that glucose tolerance (GTT) and insulin sensitivity (ITT) were measured in each group of mice. agomir-1246 improved glucose tolerance and insulin sensitivity in DB / DB type II diabetic mice.

[0093] like Figure 4 A-4C assays showed that peripheral blood samples were collected to detect insulin levels in each group of mice, and the pathological condition of pancreatic tissue was observed. Agomir-1246 effectively reduced insulin levels, improved insulin resistance, and played a crucial role in repairing the secretory regulatory function of pancreatic β-cells.

[0094] However, antagomir-1246 does not have the above effects.

[0095] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The uses of miR-1246 class miRNAs in drugs for the treatment of type 2 diabetes and its complications, including: The use of a miRNA, or its precursor sequence, expression vector, or agonist thereof, characterized in that the miRNA is a miR-1246 class miRNA, wherein the miR-1246 class miRNA is selected from the group consisting of miR-1246 or miR-1246-derived sequences, or modified derivatives thereof, for use in the preparation of drugs for the prevention and treatment of type II diabetes and type II diabetes complications.

2. The use as described in claim 1, characterized in that, The miR-1246 is a human-derived has-miR-1246, the sequence of which is shown in SEQ ID NO:

1.

3. The use as described in claim 1, characterized in that, The miR-1246 derived sequence is derived from the sequence shown in SEQ ID NO:1, including derived sequences formed by single base deletions, insertions, and / or substitutions.

4. The use as described in claim 3, characterized in that, The miR-1246 derived sequence is shown in SEQ ID NO:2~21.

5. The use as described in claim 1, characterized in that, The precursor sequence is shown in SEQ ID NO:

22.

6. The use as described in claim 1, characterized in that, The modified derivatives include miR-1246 or miR-1246-derived sequences, wherein the modification is selected from one or more of the following: glycosyl modification of nucleotides, modification of the linkage between nucleotides, cholesterol modification, locked nucleotide modification, peptide modification, lipid modification, halogen modification, hydrocarbon modification, and nucleic acid modification.

7. The use as described in claim 1, characterized in that, The expression vector contains the miR-1246 class of miRNAs, or their precursor miRNAs, or polynucleotides that can be transcribed by the host to form the precursor miRNAs.

8. The use as described in claim 1, characterized in that, The agonist is selected from the group consisting of substances that promote the expression of miR-1246 class miRNAs and substances that enhance the activity of miR-1246 class miRNAs.

9. The use as described in claim 1, characterized in that, The type 2 diabetes-related diseases refer to the syndromes caused by type 2 diabetes.

10. The use as described in claims 1-9, further comprising a pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: 1) The first active ingredient is a has-miR-1246 class miRNA, or its precursor sequence, expression vector, or agonist: wherein the has-miR-1246 class miRNA is selected from the group consisting of miR-1246 or miR-1246-derived sequences, or their modified derivatives. 2) A second active ingredient, which is different from the first active ingredient, and the second active ingredient is selected from the group consisting of: treatment of type II diabetes, non-alcoholic fatty liver disease, hyperlipidemia, or combinations thereof; 3) Pharmaceutically acceptable carrier.

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