Nucleic acid molecule combinations and their use in medicaments for the treatment of liver diseases

Abstract

The application discloses a nucleic acid molecule combination and application thereof in a medicine for treating liver diseases. The nucleic acid molecule combination comprises a first nucleic acid molecule, for example, miR-451a, or a first analog or derivative thereof. The nucleic acid molecule combination can be used for preparing a medicine for treating acute liver failure and other liver diseases through in-vitro and in-vivo experiments, a brand-new nucleic acid medicine strategy is provided for clinical treatment of liver diseases, and the nucleic acid molecule combination has application potential in development into various dosage forms such as tablets, capsules and injections.

Description

Technical Field

[0001] This application relates to the fields of biotechnology and medical technology, specifically to nucleic acid molecular combinations and their application in drugs for treating liver diseases. Background Technology

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding small RNA molecules, approximately 20-24 bases in length. They originate from pre-miRNAs of about 70-90 bases in length, which are cleaved into mature miRNAs by the enzyme Dicer, forming secondary structures with hairpin structures. The seed sequence of a miRNA, especially the 2nd to 8th nucleotides at the 5' end, is the most conserved region during evolution, determining the specific binding of the miRNA to its target gene mRNA. miRNAs exert a negative regulatory effect by precisely pairing their seed sequence with the 3' untranslated region (3' UTR) of the target gene mRNA, leading to mRNA degradation or translational repression, thereby regulating gene expression. A single miRNA can regulate the expression of multiple target genes by binding to their mRNAs, while multiple miRNAs may collectively regulate a single gene, forming a complex regulatory network. The liver, as a vital organ for metabolism and synthesis, is one of the organs with the richest miRNA expression profile and the most in-depth functional studies. Under normal physiological conditions, liver-specific miRNAs (such as miR-122) maintain hepatocyte differentiation and metabolic homeostasis. However, under pathological conditions, dysregulation of miRNA expression is closely related to the development and progression of various liver diseases. Studies have shown that the expression profiles of miRNAs are significantly altered in the pathological processes of acute liver failure, liver fibrosis, non-alcoholic fatty liver disease, and hepatocellular carcinoma. Their abnormal expression not only serves as a molecular marker of disease progression but also directly participates in the regulation of hepatocyte damage, inflammatory responses, oxidative stress, and regeneration and repair. For example, in the serum of patients with acute liver failure, the expression of liver-specific miRNAs such as miR-122 is significantly elevated, with changes even preceding traditional liver function indicators (such as ALT and AST), suggesting their unique value as early diagnostic markers. Furthermore, in recent years, intervention strategies targeting miRNAs have shown great promise in the treatment of liver diseases. By exogenously supplementing protective miRNAs or inhibiting the expression of pathogenic miRNAs, it is possible to effectively regulate hepatocyte survival, reduce inflammatory damage, and promote liver regeneration, providing new directions for precision treatment and targeted drug development for critical illnesses such as acute liver failure. With the continuous advancement of delivery technology and clinical translation, miRNA will undoubtedly become an important force in the future diagnosis and treatment of liver diseases and biomedical research.

[0003] In view of the above, this application is hereby submitted. Summary of the Invention

[0004] Based on this, this application provides at least one nucleic acid molecule combination and its application in drugs for treating liver diseases. The main purpose is to provide new ideas for the clinical treatment of liver diseases by using nucleic acid molecule combinations, such as miR-mix and related nucleic acid molecules, as examples to prepare drugs for treating liver diseases.

[0005] In a first aspect of this application, the use of nucleic acid molecular combinations in a drug for treating liver diseases is provided;

[0006] The nucleic acid molecule assembly includes a first nucleic acid molecule having one or more of the items shown in (a) to (e):

[0007] (a) The nucleic acid molecule assembly includes miR-451a;

[0008] (b) The nucleic acid molecule combination includes a first mimic or derivative having the same or similar function as miR-451a;

[0009] (c) The nucleic acid molecule combination includes a first miRNA precursor, which can be processed in the host to form at least one of the miR-451a and the first mimic or derivative;

[0010] (d) The nucleic acid molecule combination includes a first polynucleotide fragment that can be transcribed in the host to form the first miRNA precursor;

[0011] (e) The nucleic acid molecule combination includes a first expression vector containing the first polynucleotide fragment.

[0012] In a second aspect of this application, the use of a second nucleic acid molecule and a third nucleic acid molecule in a drug for treating liver diseases is provided;

[0013] (d') A second polynucleotide fragment that can be transcribed within the host to form the second miRNA precursor;

[0014] (e') A second expression vector containing the second polynucleotide fragment;

[0015] The third nucleic acid molecule has one or more of the items shown in (a'') to (e''):

[0016] (a'')miR-302c-3p;

[0017] (b'') A third analogue or derivative having the same or similar function as miR-302c-3p;

[0018] (c'') A third miRNA precursor, which can be processed in the host to form at least one of the miR-302c-3p and the third mimic or derivative;

[0019] (d'') is a third polynucleotide fragment that can be transcribed within the host to form the third miRNA precursor;

[0020] (e'') A third expression vector containing the third polynucleotide fragment.

[0021] In a third aspect of this application, a nucleic acid molecule combination is provided, which is composed of at least two of the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule as defined above.

[0022] In a fourth aspect of this application, a medicament for treating liver diseases is provided, comprising a combination of nucleic acid molecules as described in the third aspect.

[0023] In a fifth aspect of this application, a method for in vitro protection of hepatocytes is provided, the method comprising culturing hepatocytes in a culture medium supplemented with at least two of a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule as defined above.

[0024] Compared to traditional technologies, the beneficial effects of this application include:

[0025] This application reveals that nucleic acid molecule combinations, exemplified by miR-mix, can enhance the viability of damaged hepatocytes and inhibit their apoptosis in vitro; in vivo, they can significantly improve the survival rate of mice with acute liver failure, reduce serum ALT / AST levels, improve liver function, and alleviate pathological damage to liver tissue. Transcriptome analysis shows that nucleic acid molecule combinations, exemplified by miR-mix, can restore the normal gene expression program of hepatocytes, enhance liver metabolism and detoxification functions, and inhibit inflammation and apoptosis-related pathways. In summary, miR-mix can block the activation of mitochondrial apoptosis signaling pathways in hepatocytes, inhibit early hepatocyte apoptosis in acute liver failure, reduce local inflammatory responses, thereby preventing massive hepatocyte death, ultimately improving liver function in mice with liver failure, alleviating liver tissue damage, and increasing survival rate. Based on this, this application proposes the use of nucleic acid molecule combinations, exemplified by miR-mix, and related nucleic acid molecules in the preparation of drugs for treating liver diseases, providing new insights for the clinical treatment of liver diseases. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0027] Figure 1 This is a schematic diagram illustrating the cell viability test results of miRNA mimics in vitro, demonstrating their protective effect against hepatocyte damage, according to one embodiment of this application.

[0028] Figure 2 This is a schematic diagram illustrating the cell viability test results of miR-mix in vitro, demonstrating its protective effect against liver cell damage, according to one embodiment of this application.

[0029] Figure 3A and Figure 3B This is a schematic diagram illustrating the inhibitory effect of miR-mix and miR-451a on D-GalN-induced apoptosis in one embodiment of this application.

[0030] Figure 4 This is a survival curve diagram of the control group and the miR-mix and miR-451a treatment group in an in vivo experiment to protect mice with acute liver failure according to one embodiment of this application.

[0031] Figure 5 This diagram illustrates the serum ALT and AST levels in the control group and the miR-mix and miR-451a treatment group during an in vivo experiment demonstrating the protective effect of miR-mix and miR-451a against acute liver failure in mice, as described in one embodiment of this application.

[0032] Figure 6A and Figure 6B The images show the histopathological (H&E staining) images of the control group and the miR-mix and miR-451a treatment group in a mouse experiment for treating acute liver failure according to one embodiment of this application.

[0033] Figure 7A and Figure 7B This diagram illustrates the results of Western blot analysis (TUNEL staining) of liver tissue proteins in the miR-mix and miR-451a treatment groups in a mouse modeling control group (Control) and miR-mix and miR-451a treatment groups in an embodiment of this application.

[0034] Figure 8 This is a schematic diagram of the transcriptome sequencing analysis results of primary hepatocytes from mice with acute liver failure treated with miR-mix in one embodiment of this application.

[0035] Figure 9 and Figure 10 This is a schematic diagram of the transcriptome sequencing results of liver tissue from mice with acute liver failure treated with miR-mix in one embodiment of this application. Detailed Implementation

[0036] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0037] 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0038] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0039] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0040] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0041] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0042] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0043] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0044] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0045] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0046] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0047] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0048] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0049] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0050] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0051] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0052] miR-mix (miR-302b, miR-302c, miR-451a) are miRNAs highly expressed in human embryonic stem cells. miR-302b-3p can participate in cell damage regulation by targeting PTEN and activating the AKT signaling pathway; miR-302b-3p and miR-302c-3p can also inhibit cell proliferation and promote apoptosis by targeting cyclins. miR-451a, as an important member of the miR-144 / miR-451a cluster, plays a key regulatory role in various liver diseases. It can regulate tumor-associated macrophage polarization by targeting hepatocyte growth factor and macrophage migration inhibitory factor, regulate hepatic lipid metabolism by targeting glycerol kinase, and alleviate liver inflammation and hepatocyte damage by targeting histone deacetylase 8. However, the function of miR-302b, miR-302c, and miR-451a in combination in hepatocytes is unclear, and there are no reports on their therapeutic effects on acute liver failure. This application aims to provide the application of miR-mix and its related nucleic acid molecules in the preparation of drugs for treating liver diseases. The miR-mix can be actively taken up by hepatocytes, inhibit the activation of hepatocyte apoptosis signaling pathways, reduce liver tissue damage, improve liver function, and provide a new strategy for the clinical treatment of liver diseases such as acute liver failure.

[0053] In one aspect of this application, the use of a combination of nucleic acid molecules in a drug for treating liver diseases is provided;

[0054] The nucleic acid molecule assembly includes a first nucleic acid molecule having one or more of the items shown in (a) to (e):

[0055] (a) The nucleic acid molecule assembly includes miR-451a;

[0056] (b) The nucleic acid molecule combination includes a first mimic or derivative having the same or similar function as miR-451a;

[0057] (c) The nucleic acid molecule combination includes a first miRNA precursor, which can be processed in the host to form at least one of the miR-451a and the first mimic or derivative;

[0058] (d) The nucleic acid molecule combination includes a first polynucleotide fragment that can be transcribed in the host to form the first miRNA precursor;

[0059] (e) The nucleic acid molecule combination includes a first expression vector containing the first polynucleotide fragment.

[0060] In some embodiments, the sequence of miR-451a is as shown in SEQ ID NO: 4.

[0061] In this application, "drug" includes any agent, compound, composition, or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects produced by a "drug" in vivo is not particularly limited; it may have systemic effects or only local effects. The activity of the "drug" is not particularly limited; it may be an active substance that can interact with other substances or an inert substance that does not interact with other substances.

[0062] In one example, the drug may be a liquid formulation or a solid formulation. Liquid formulations refer to formulations containing a liquid phase, and non-limiting examples include solutions, suspensions, emulsions, etc. Non-limiting examples of solid formulations include tablets, capsules, granules, pills, etc.

[0063] In one example, depending on the method of administration, it could be an oral preparation, injection, drops, patch, tube feeding preparation, etc.

[0064] In one example, the excipients chosen may differ depending on the dosage form.

[0065] In this application, "excipients" include, but are not limited to, mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, sodium calcium EDTA, carbonates, acetates, phosphates or aqueous solutions of monovalent alkali metals, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipids, kaolin, talc, calcium stearate, and magnesium stearate.

[0066] In this application, "pharmaceutically acceptable" means those ligands, materials, compositions, and / or dosage forms that are suitable for administration to patients within the bounds of reasonable medical judgment and that are commensurate with a reasonable benefit / risk ratio.

[0067] In this application, "pharmaceuticalally acceptable excipient" means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. As used herein, the term "pharmaceuticalally acceptable carrier" includes buffers compatible with drug administration, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, and the like. Each carrier must be "pharmaceuticalally acceptable" in the sense of compatibility with other components in the formulation and harmlessness to the patient. Suitable examples include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn... Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0068] In one example, the drug contains an effective amount of the combination of nucleic acid molecules.

[0069] In this application, "effective amount" refers to the dose by which the component corresponding to the term achieves treatment, prevention, reduction and / or relief of a specific disease, condition and / or symptom in a subject. In this application, unless otherwise specified, it refers to the dose by which liver disease, condition and / or symptom are treated, prevented, reduced and / or relieved.

[0070] In this application, "subject" refers to an animal, preferably a mammal, and more preferably a human. Subjects include, but are not limited to, consumers of health products and patients with diseases, conditions, and / or symptoms. The subject in this application is preferably a mammal. The term "mammal" primarily refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats and mice), pigs, cattle, sheep, horses, and humans, preferably primates, and more preferably humans.

[0071] In one of the examples, the subjects were mammals.

[0072] In one example, the subjects were either humans or mice.

[0073] In this application, "patient" refers to an animal, preferably a mammal, and more preferably a human. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats, pigs, cattle, sheep, horses, and humans, preferably primates, and more preferably humans.

[0074] In some embodiments, the nucleic acid molecule combination further includes a second nucleic acid molecule and / or a third nucleic acid molecule.

[0075] This application has been verified through in vitro and in vivo experiments that the nucleic acid molecule combination can be used to prepare drugs for treating liver diseases such as acute liver failure, providing a novel nucleic acid drug strategy for the clinical treatment of liver diseases, and has the potential to be developed into various dosage forms such as tablets, capsules, and injections.

[0076] For example, the nucleic acid molecule combination further includes a second nucleic acid molecule, that is: the nucleic acid molecules include at least a first nucleic acid molecule and a second nucleic acid molecule.

[0077] For example, the nucleic acid molecule combination further includes a third nucleic acid molecule, that is: the nucleic acid molecule includes at least a first nucleic acid molecule and a third nucleic acid molecule.

[0078] For example, the nucleic acid molecule combination further includes a second nucleic acid molecule and a third nucleic acid molecule, that is: the nucleic acid molecule includes at least a first nucleic acid molecule, a second nucleic acid molecule and a third nucleic acid molecule.

[0079] In some embodiments, the second nucleic acid molecule has one or more of the items shown in (a') to (e'):

[0080] (a')miR-302b-3p;

[0081] (b') A second analogue or derivative having the same or similar function as miR-302b-3p;

[0082] (c') A second miRNA precursor, which can be processed in the host to form at least one of the miR-302b-3p and the second mimic or derivative;

[0083] (d') A second polynucleotide fragment that can be transcribed within the host to form the second miRNA precursor;

[0084] (e') A second expression vector containing the second polynucleotide fragment.

[0085] In some embodiments, the sequence of miR-302b-3p is as shown in SEQ ID NO: 2.

[0086] In some embodiments, the third nucleic acid molecule has one or more of the items shown (a'') to (e''):

[0087] (a'')miR-302c-3p;

[0088] (b'') A third analogue or derivative having the same or similar function as miR-302c-3p;

[0089] (c'') A third miRNA precursor, which can be processed in the host to form at least one of the miR-302c-3p and the third mimic or derivative;

[0090] (d'') is a third polynucleotide fragment that can be transcribed within the host to form the third miRNA precursor;

[0091] (e'') A third expression vector containing the third polynucleotide fragment.

[0092] In some embodiments, the sequence of miR-302c-3p is as shown in SEQ ID NO: 3.

[0093] In some embodiments, the composition of the nucleic acid combination is as follows:

[0094] The first nucleic acid molecule, and:

[0095] The second nucleic acid molecule and / or the third nucleic acid molecule.

[0096] For example, the nucleic acid combination may consist of a first nucleic acid molecule and a second nucleic acid molecule, or the nucleic acid combination may consist of a first nucleic acid molecule and a third nucleic acid molecule, or the nucleic acid combination may consist of a first nucleic acid molecule to a third nucleic acid molecule.

[0097] In some embodiments, the molar ratio of the first nucleic acid molecule to the second nucleic acid molecule in the nucleic acid combination is (0.5~2.0):(0.5~2.0).

[0098] In some embodiments, the molar ratio of the first nucleic acid molecule to the third nucleic acid molecule in the nucleic acid combination is (0.5~2.0):(0.5~2.0).

[0099] In some embodiments, the molar ratio of the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule in the nucleic acid combination is (0.5~2.0):(0.5~2.0):(0.5~2.0).

[0100] The first nucleic acid molecule may be miR-451a, the second nucleic acid molecule may be miR-302b-3p, and the third nucleic acid molecule may be miR-302c-3p.

[0101] In another aspect of this application, the use of the second and third nucleic acid molecules as defined above in a medicament for treating liver diseases is provided.

[0102] In some embodiments, the molar ratio of the second nucleic acid molecule to the third nucleic acid molecule is (0.5~2.0):(0.5~2.0).

[0103] In some embodiments, the second nucleic acid molecule is miR-302b-3p, and the third nucleic acid molecule is miR-302c-3p.

[0104] In another aspect of this application, a combination of nucleic acid molecules is provided, which consists of at least two of the first to third nucleic acid molecules as defined above.

[0105] In some embodiments, the nucleic acid combination includes at least the second nucleic acid molecule.

[0106] For example, the nucleic acid combination may consist of the second nucleic acid molecule and the first nucleic acid molecule, or the second nucleic acid molecule and the third nucleic acid molecule, or the first nucleic acid molecule and the third nucleic acid molecule, or the first nucleic acid molecule to the third nucleic acid molecule.

[0107] In some embodiments, the nucleic acid molecule combination consists of a second nucleic acid molecule and a first / third nucleic acid molecule; for example, in the nucleic acid combination, the molar ratio of the second nucleic acid molecule to the first / third nucleic acid molecule is (0.5~2.0):(0.5~2.0).

[0108] In some embodiments, the nucleic acid combination consists of the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule; for example, the molar ratio of the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule in the nucleic acid combination is (0.5~2.0):(0.5~2.0):(0.5~2.0).

[0109] In some embodiments, the nucleic acid molecule combination consists of a and b; wherein: a is miR-302b-3p, and b is miR-451a and / or miR-302c-3p. For example:

[0110] The nucleic acid molecule combination consists of miR-302b-3p and miR-451a, or miR-451a, miR-302b-3p and miR-302c-3p, or miR-302b-3p and miR-302c-3p.

[0111] Another aspect of this application provides a medicament for treating liver diseases, comprising a combination of nucleic acid molecules as described above.

[0112] Another aspect of this application provides a method for in vitro protection of hepatocytes, the method comprising culturing hepatocytes in a culture medium supplemented with at least two of the first to third nucleic acid molecules as described above.

[0113] In one example, the hepatocytes are D-gal-induced damaged hepatocytes.

[0114] In one example, the hepatocytes are mPH.

[0115] In one example, the total concentration of nucleic acid molecules is 10 nM to 100 nM, for example, such as 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or any value or range between two values.

[0116] In another aspect of this application, a method for treating liver disease is provided, the method comprising administering to a subject in need an effective amount of at least two of the first to third nucleic acid molecules as described above.

[0117] The following are some examples.

[0118] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0119] Example 1: In vitro functional identification of miR-mix

[0120] 1. miRNA mimics

[0121] We purchased cell mimics miR-302b-3p, miR-302c-3p, miR-451a, miR-92a-3p, miR-127-3p, and miR-26a-5p, along with their control mimic NC, from Guangzhou Ruibo Biotechnology Co., Ltd.

[0122] mimic NC (SEQ ID NO: 1): UUUGUACUACACAAAAGUACUG.

[0123] miR-302b-3p (SEQ ID NO: 2):UAAGUGCUUCCAUGUUUUGGUGA.

[0124] miR-302c-3p (SEQ ID NO: 3):UAAGUGCUUCCAUGUUUUAGUAG.

[0125] miR-451a (SEQ ID NO: 4): AAACCGUUACCAUUACUGAGUU.

[0126] miR-92a-3p (SEQ ID NO: 5): UAUUGCACUUGUCCCGGCCUGU.

[0127] miR-127-3p (SEQ ID NO: 6):UCGGAUCCGUCUGAGCUUGGCU.

[0128] miR-26a-5p (SEQ ID NO: 7): UUCAAGUAAUCCAGGAUAGGCU.

[0129] 2. miRNAs have a protective effect on hepatocytes in vitro.

[0130] mPH obtained by in situ perfusion was cultured in William's Medium E medium containing 10% (v / v) fetal bovine serum, 1% (v / v) penicillin and streptomycin, and 1 wt% glutamine. D-gal stimulation of mPH was used to construct an in vitro cell model simulating in vivo liver injury. For nucleic acid transfection, the medium was not supplemented with penicillin and streptomycin; instead, different miRNA mimics or their control mimic NCs were added. After 24 hours of culture, cell viability was assessed using a lactate dehydrogenase cytotoxicity assay kit to determine the in vitro effectiveness of the miRNA mimics. The results were expressed by the following formula: Cell viability (%) = (Absorbance of treated sample - Absorbance of sample control well) / (Absorbance of enzyme activity in untreated cells - Absorbance of sample control well).

[0131] The results showed that miR-302b-3p and miR-302c-3p exhibited the most significant protective effects, while miR-451a also showed significant cell-protective activity. Figure 1 As shown.

[0132] 3. miR-mix has a protective effect on hepatocytes in vitro

[0133] On the basis of determining the optimal single-drug concentration of each miRNA mimic, it was further investigated whether combined delivery could produce a synergistic enhanced cytoprotective effect superior to single-drug therapy. miR-451a has a significant protective effect on primary hepatocytes (mPH), while the ternary combination composed of miR-302b-3p, miR-302c-3p and miR-451a (miR-mix) has a more excellent protective effect on mPH, which can significantly improve the cell viability of mPH, and the results are shown in Figure 2 .

[0134] 4. miR-mix has an inhibitory effect on the apoptosis of mPHs

[0135] An Annexin V / propidium iodide staining kit was used to determine the protective effect of miR-mix on hepatocytes. The results show that miR-mix has an inhibitory effect on D-GalN-induced apoptosis, and the results are shown in Figure 3A and Figure 3B .

[0136] Example 2 Modeling and treatment of acute liver failure in mice

[0137] 1. Experimental animals: SPF-grade male Kunming mice, weighing 18-22g, were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Production License: SCXK(Xiang)2021-0002). The mice were fed with normal mouse chow and had free access to filtered tap water. The room temperature was 22°C±1°C, the humidity was 65%±5%, with a 12h light / dark cycle. The animal experimental protocol was reviewed and approved by the Animal Ethics Committee of Central South University. The experimental process was strictly implemented in accordance with the National Standard of the People's Republic of China (GB / T 35892-2018) Guidelines for Ethical Review of Laboratory Animal Welfare.

[0138] 2. miR-mix mimics: The miR-mix mimics for animal use, namely miR-302b-3p agomir, miR-302c-3p agomir and miR-451a agomir, were purchased from Guangzhou RiboBio Co., Ltd., and normal saline was used as the control for miR-302b-3p agomir, miR-302c-3p agomir and miR-451a agomir. miRNA agomir is a specially chemically modified miRNA agonist.

[0139] 3. Modeling and Treatment: An acute liver failure mouse model was established by intraperitoneal injection of D-gal and lipopolysaccharide (LPS), followed by tail vein injection of 10 nmol miR-mix agomir. The results showed that miR-451a and miR-mix had a certain protective effect on mice with acute liver failure, significantly improving their survival rate and slowing their mortality. (See results below.) Figure 4 As shown.

[0140] 4. Analysis of Treatment Results

[0141] (1) Observation of serum ALT / AST levels and tissue structure

[0142] Serum ALT and AST levels were measured using a standard automated clinical analyzer in the clinical laboratory of Hunan Guangxiu Hospital. Results are as follows: Figure 5 As shown, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicators of liver damage in mice, increased sharply 6 hours after modeling, and decreased slightly at 24 hours. Compared with the control group, the ALT and AST values ​​of the miR-451a and miR-mix groups were significantly lower at all time points than the control group, indicating that the liver damage in the treatment group was significantly less and liver function was improved.

[0143] (2) Histopathological analysis

[0144] After liver harvesting, fresh liver tissue was fixed in 4% paraformaldehyde and embedded in paraffin. The embedded tissue was then sectioned into 5 μm pieces and stained with hematoxylin and eosin (H&E). The results are as follows: Figure 6A and Figure 6B As shown, the liver tissue of the control group exhibited extensive diffuse hemorrhage and large-scale necrosis, with severe damage to the liver lobe structure. The liver damage in the miR-451a and miR-mix treatment groups was significantly reduced, and the liver lobe structure was more intact, suggesting that miR-451a and miR-mix have therapeutic efficacy for liver injury. The results of TUNEL staining are shown below. Figure 7A and Figure 7BAs shown, at 6 hours after induced injury in the control group, a large number of apoptotic cells with dark brown positive staining were observed in the liver tissue; at 12 hours, punctate brown-stained positive cells and large areas of brown necrosis were also observed; by 24 hours, the predominance was in large areas of brown staining with few brown positive staining nuclei. This is because severe liver damage and extensive hepatocyte necrosis in the later stages of ALF lead to cell lysis and nucleus disappearance. After miR treatment, only a small number of scattered apoptotic cells were observed in the liver tissue of ALF mice at 6 hours, and the number of positive staining cells was significantly lower than that in the control group. At 12 hours, the number of apoptotic cells increased slightly, and by 24 hours, few positive staining nuclei were observed, with only small areas of necrosis. This indicates that miR-451a and miR-mix significantly inhibited hepatocyte apoptosis and necrosis.

[0145] Example 3 Transcriptome Analysis

[0146] 1. Samples from each group (in vitro experiments: normal control group, mPH control group, miR-mix treatment group; in vivo experiments: normal control group, ALF model group, miR-mix treatment group at 6h and 12h) were sent to BGI Genomics Co., Ltd. in Shenzhen for cDNA library construction and high-throughput sequencing.

[0147] 2. Transcriptome analysis results

[0148] (1) Transcriptome sequencing and analysis of primary hepatocytes

[0149] Total RNA from hepatocytes in the normal group, control group, and miR-mix treatment group was analyzed by in vitro transcriptome sequencing. PCA analysis results are as follows: Figure 8 As shown in Figure A, the three groups of samples were clearly separated, with the first principal component (PC1) explaining 67.2% of the variance and the second principal component (PC2) explaining 19.3% of the variance, indicating significant transcriptomic differences among the groups. The miR-mix treatment group showed significant cluster separation from the control group and exhibited a trend towards the normal group, suggesting that miR-mix can induce significant changes in the transcriptional level of hepatocytes in vitro.

[0150] The results of the differential expression analysis are as follows: Figure 8 As shown in Figure B, the volcano plot reveals extensive transcriptomic alterations in the miR-mix treatment group compared to the control group, identifying a total of 709 significantly upregulated genes and 594 significantly downregulated genes. The hierarchical clustering analysis heatmap is shown below. Figure 8 As shown in Figure C, there are significant differences in gene expression patterns among the three groups of samples. The miR-mix treatment group exhibits an intermediate expression profile between the normal group and the control group on multiple gene clusters, suggesting that miR-mix treatment can partially restore the normal gene expression program of hepatocytes.

[0151] The results of the KEGG pathway enrichment analysis are as follows: Figure 8 As shown in Figure D, the upregulated pathways mainly include: chemical carcinogenesis-reactive oxygen species pathway, prions, chemical carcinogenesis-DNA adducts, retinol metabolism, steroid hormone biosynthesis, drug metabolism-other enzymes, PPAR signaling pathway, cytochrome P450-mediated metabolism of exogenous substances, cytochrome P450-mediated drug metabolism, bile secretion, arachidonic acid metabolism, and glutathione metabolism, indicating that miR-mix treatment can enhance the detoxification capacity, antioxidant defense, and metabolic function of hepatocytes. The downregulated pathways mainly include: human papillomavirus infection, herpes simplex virus type 1 infection, Epstein-Barr virus infection, influenza A, Kaposi's sarcoma-associated herpesvirus infection, NOD-like receptor signaling pathway, hepatitis C, cytotoxicity, measles, TNF signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and cytoplasmic DNA sensing pathway, indicating that miR-mix exerts an anti-inflammatory effect in hepatocytes and inhibits pathological stress responses.

[0152] To further validate the RNA sequencing results, qPCR was used to quantify key genes, and the results are as follows: Figure 8 As shown in Figure E, compared with the control group, the expression levels of Cyp2d9, Cyp2e1, Cyp12a, Fapp1, and Sirt1 were significantly upregulated in the miR-mix treatment group, while the expression level of Pla2g15 was significantly downregulated, and the expression level of Hacd1 showed no significant difference. The above qPCR validation results are generally consistent with the RNA sequencing data, confirming the upregulation effect of miR-mix on cytochrome P450 enzymes and Sirt1, supporting the conclusion that miR-mix can activate protective cellular programs and enhance liver metabolism and detoxification functions.

[0153] (2) Transcriptome sequencing and analysis of mouse liver tissue

[0154] Transcriptome sequencing analysis was performed on liver tissue from mice with acute liver failure. Samples were collected at 6 h (early recovery phase) and 12 h (mid-recovery phase) after miR-mix treatment. PCA analysis results are as follows: Figure 9 As shown in Figure A, the clusters of the normal group and the acute liver failure group were clearly separated, indicating that acute liver failure induced significant transcriptomic changes. Both the miR-mix treatment groups (6 h and 12 h) showed a gradual shift towards the normal group, suggesting that miR-mix treatment initiated the liver recovery process at the transcriptional level. Untreated acute liver failure mice (ALF_6h and ALF_12h) showed overlapping clusters, indicating the persistence of their pathological gene expression patterns, while the miR-mix treatment group showed a time-dependent recovery towards a normal transcriptional state.

[0155] Results of differential gene expression analysis as follows Figure 9China B and Figure 9 As shown in C. 6 hours after treatment ( Figure 9 (B) suggests that miR-mix can promote hepatocyte regeneration and regulate apoptosis in the early recovery phase. 12 h after treatment ( Figure 9 The results showed that liver metabolic function gradually recovered; at the same time, Fst and Tifa were significantly downregulated, suggesting that inflammation and growth factor signaling pathways were regulated.

[0156] Hierarchical clustering analysis of differentially expressed genes, such as Figure 9 As shown in Figure D, the heatmap clearly separates the samples in each group, and the three biological replicates within each group are highly consistent. Analysis identified several gene clusters with different expression patterns: genes upregulated in acute liver failure but downregulated by miR-mix treatment (pro-inflammatory and apoptosis-related genes), and genes downregulated in acute liver failure but restored by miR-mix (liver protection and metabolic function-related genes).

[0157] The results of the KEGG pathway enrichment analysis are as follows: Figure 9 China E and Figure 9 As shown in Figure F. 6 hours after treatment ( Figure 9 Middle E), downregulated pathways include primary bile acid biosynthesis and PPAR signaling pathways, upregulated pathways include signaling pathways regulating stem cell pluripotency and apoptosis pathways. 12 h after treatment ( Figure 9 The significantly upregulated pathways included steroid hormone biosynthesis, retinol metabolism, drug metabolism-cytochrome P450, cortisol synthesis and secretion, and aldosterone synthesis and secretion, indicating that miR-mix treatment gradually restored liver metabolic function, particularly steroid hormone metabolism and drug detoxification-related functions. The significantly downregulated TGF-β signaling pathway suggests that miR-mix may exert its anti-fibrotic effect by regulating this key pathway.

[0158] To further validate the RNA sequencing results, qPCR was used to quantify key differentially expressed genes. The results are as follows: Figure 10 As shown. Compared with the acute liver failure group, the miR-mix treatment group showed significantly downregulated expression levels of Cyp8b1 and Sec14l2, and significantly upregulated expression levels of Dffb and Onecut1; the expression levels of Fst and Tifa were significantly downregulated, while the expression levels of Elovl3 and Gpt were significantly upregulated. Figure 10 The qPCR validation results were largely consistent with the RNA sequencing data, confirming the regulatory role of miR-mix in liver regeneration, metabolic function, and inflammation-related genes.

[0159] in conclusion:

[0160] miR-mix is ​​a miRNA specifically expressed in embryonic stem cells, typically playing a crucial role in early development, with its expression level rapidly decreasing after differentiation. While miR-mix has been reported to have effects in other somatic cells, it is almost not expressed in hepatocytes, and no studies have been found regarding its role in hepatocytes or liver diseases. This application, however, experimentally demonstrates that miR-mix can inhibit hepatocyte apoptosis, thereby exerting a protective effect against acute liver failure in mice, providing a new approach for the clinical treatment of liver failure or other liver-related diseases.

[0161] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0162] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. The application of nucleic acid molecular combinations in drugs for treating liver diseases; characterized in that, The nucleic acid molecule assembly includes a first nucleic acid molecule having one or more of the items shown in (a) to (e): (a) The nucleic acid molecule assembly includes miR-451a; (b) The nucleic acid molecule combination includes a first mimic or derivative having the same or similar function as miR-451a; (c) The nucleic acid molecule combination includes a first miRNA precursor, which can be processed in the host to form at least one of miR-451a and the first mimic or derivative; (d) The nucleic acid molecule combination includes a first polynucleotide fragment that can be transcribed in the host to form the first miRNA precursor; (e) The nucleic acid molecule combination includes a first expression vector containing the first polynucleotide fragment.

2. The application as described in claim 1, characterized in that, The nucleic acid molecule combination also includes a second nucleic acid molecule and / or a third nucleic acid molecule; The second nucleic acid molecule has one or more of the items shown in (a') to (e'): (a')miR-302b-3p; (b') A second analogue or derivative having the same or similar function as miR-302b-3p; (c') A second miRNA precursor, which can be processed in the host to form at least one of the miR-302b-3p and the second mimic or derivative; (d') A second polynucleotide fragment that can be transcribed within the host to form the second miRNA precursor; (e') A second expression vector containing the second polynucleotide fragment; The third nucleic acid molecule has one or more of the items shown in (a'') to (e''): (a'')miR-302c-3p; (b'') A third analogue or derivative having the same or similar function as miR-302c-3p; (c'') A third miRNA precursor, which can be processed in the host to form at least one of the miR-302c-3p and the third mimic or derivative; (d'') is a third polynucleotide fragment that can be transcribed within the host to form the third miRNA precursor; (e'') A third expression vector containing the third polynucleotide fragment; Optionally, the composition of the nucleic acid combination is as follows: The first nucleic acid molecule, and, The second nucleic acid molecule and / or the third nucleic acid molecule; Optionally, in the nucleic acid combination, the molar ratio of the first nucleic acid molecule to the second nucleic acid molecule or the third nucleic acid molecule is (0.5~2.0):(0.5~2.0). Optionally, in the nucleic acid combination, the molar ratio of the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule is (0.5~2.0):(0.5~2.0):(0.5~2.0). Optionally, the first nucleic acid molecule is miR-451a, the second nucleic acid molecule is miR-302b-3p, and the third nucleic acid molecule is miR-302c-3p.

3. The use of the second and third nucleic acid molecules as defined in claim 2 in a medicament for treating liver diseases; Optionally, the molar ratio of the second nucleic acid molecule to the third nucleic acid molecule is (0.5~2.0):(0.5~2.0). Optionally, the second nucleic acid molecule is miR-302b-3p, and the third nucleic acid molecule is miR-302c-3p.

4. The application as described in any one of claims 1 to 3, characterized in that, The drug meets one or more of the following conditions: (A1) also includes pharmaceutically acceptable excipients; (A2) The dosage form of the drug is tablets, capsules or granules; (A3) The dosage form of the drug is a suspension, oral solution or injection; (A4) The liver disease described is acute liver failure.

5. A nucleic acid molecule assembly, characterized in that, It is composed of at least two of the first nucleic acid molecule as defined in claim 1, the second nucleic acid molecule as defined in claim 2, and the third nucleic acid molecule; Optionally, the nucleic acid combination consists of the second nucleic acid molecule and the first / third nucleic acid molecule; more preferably, in the nucleic acid combination, the molar ratio of the second nucleic acid molecule to the first / third nucleic acid molecule is (0.5~2.0):(0.5~2.0). Optionally, the nucleic acid combination consists of the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule; more preferably, in the nucleic acid combination, the molar ratio of the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule is (0.5~2.0):(0.5~2.0):(0.5~2.0).

6. The nucleic acid molecule combination as described in claim 5, characterized in that, The nucleic acid molecule combination consists of a and b; wherein: a is miR-302b-3p, and b is miR-451a and / or miR-302c-3p.

7. A medicine for treating liver diseases, characterized in that, It includes the nucleic acid molecule combinations as described in claim 5 or 6; Optionally, the drug meets one or more of the following conditions: (B1) The drug also includes pharmaceutically acceptable excipients; (B2) The dosage form of the drug is tablets, capsules or granules; (B3) The dosage form of the drug is a suspension, oral solution, or injection; and, (B4) The liver disease described is acute liver failure.

8. A method for in vitro protection of hepatocytes, characterized in that, The method includes culturing hepatocytes in a culture medium containing at least two of the first nucleic acid molecule as defined in claim 1 and the second and third nucleic acid molecules as defined in claim 2.

9. The method for in vitro protection of hepatocytes as described in claim 8, characterized in that, The hepatocytes are hepatocytes that have suffered D-galactosamine-induced damage; and / or, The hepatocytes were primary mouse hepatocytes.

10. The method for in vitro protection of hepatocytes as described in claim 8 or 9, characterized in that, The concentration of the combination of at least two nucleic acid molecules, including the first nucleic acid molecule to the third nucleic acid molecule, is 10 nM to 100 nM.

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