Application of annexin A2 (ANXA2) and inhibitor thereof in diagnosis, treatment and / or prevention of pulmonary arterial hypertension

Abstract

The invention discloses annexin A2 (ANXA2) and application of an inhibitor thereof in diagnosis, treatment and / or prevention of pulmonary arterial hypertension. In particular discloses application of an ANXA2 inhibitor (including siRNA for silencing an ANXA2 gene, an ANXA2 antibody and a phosphorylation inhibitor) in preparation of a product for preventing and / or treating pulmonary arterial hypertension. Experiments prove that the ANXA2 inhibitor can significantly inhibit proliferation and migration of pulmonary artery smooth muscle cells, and significantly improve pulmonary arterial hypertension, pulmonary artery vascular remodeling and right ventricular hypertrophy. The ANXA2 or the Thr208 phosphorylation site of the ANXA2 protein can be used for diagnosis or auxiliary diagnosis of pulmonary arterial hypertension, or used for screening drugs for pulmonary arterial hypertension and developing new diagnosis and treatment methods and drugs. The therapeutic target and the ANXA2 inhibitor developed by the invention have wide clinical application value in the fields of diagnosis, prevention and treatment of pulmonary arterial hypertension.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of Annexin A2 (ANXA2) and its inhibitors in the diagnosis, treatment and / or prevention of pulmonary hypertension. Background Art

[0002] Pulmonary Artery Hypertension (PAH) is characterized by abnormally elevated pulmonary artery pressure. It is a chronic progressive disease. Its latest hemodynamic diagnostic criteria are: at rest, right heart catheter-detected pulmonary artery mean pressure ≥ 20 mmHg and pulmonary vascular resistance > 2.0 Wood units (WU). Pathological remodeling and vasoconstriction of pulmonary vessels can lead to progressive dyspnea, exercise intolerance, right heart failure and death. Its incidence is second only to coronary heart disease and hypertension among cardiovascular diseases. Since the clinical symptoms of early PAH are not obvious and the diagnosis of the disease requires right ventricular floating catheter detection, the actual incidence is much higher than the clinical detection rate, resulting in very high disability and mortality rates.

[0003] With the change of living environment, chronic lung diseases caused by industrial waste and dust have increased significantly. Chronic lung diseases such as chronic bronchitis, chronic obstructive pulmonary disease, and sleep apnea syndrome lead to hypoxia and pulmonary vascular lesions, which lead to pulmonary capillary preload PAH. The incidence of this type of PAH has increased year by year. In addition, studies have shown that the pulmonary blood vessels are thickened, suggesting that viral infection may be another new cause of PAH. Therefore, the pathogenesis and diagnosis and treatment of PAH have become a hot topic in current research.

[0004] The pathological characteristics of PAH are mainly early endothelial cell dysfunction leading to vasoconstriction, followed by endothelial damage and vascular smooth muscle cell proliferation, migration, and hypertrophy, leading to arteriolar muscularization, thickening of the pulmonary artery intima and media, and deposition of extracellular matrix, resulting in vascular sclerosis and even stenosis and obstruction, that is, pulmonary vascular remodeling. The clinical treatment of PAH currently mainly uses vasodilator drugs such as prostacyclin, endothelin receptor blockers, phosphodiesterase inhibitors, inhaled nitric oxide (NO) or its donor L-arginine, and calcium channel blockers to relieve symptoms, but there is no improvement in pathological changes such as vascular remodeling, so the efficacy of these drugs is not ideal. Therefore, it is particularly important to find new PAH treatment targets and explore new and more effective PAH diagnosis and treatment strategies. Summary of the invention

[0005] One of the purposes of the present invention is to provide the use of annexin A2 (ANXA2) and its inhibitors in the diagnosis, treatment and / or prevention of pulmonary hypertension. The technical problems to be solved are not limited to the described technical subjects, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] In order to achieve the above object, the present invention first provides any of the following uses of ANXA2 inhibitors:

[0007] A1) Use in the preparation of a product for preventing and / or treating pulmonary hypertension;

[0008] A2) Use in the preparation of a product for preventing and / or treating pulmonary artery vascular remodeling;

[0009] A3) Use in the preparation of a product for preventing and / or treating right ventricular hypertrophy;

[0010] A4) Use in the preparation of a product for inhibiting the proliferation and / or migration of pulmonary artery smooth muscle cells;

[0011] The ANXA2 inhibitor includes any of the following:

[0012] B1) substances that inhibit the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the ANXA2 gene;

[0013] B2) Substances that inhibit or reduce the content, activity and / or function of ANXA2 protein.

[0014] The substance may be any substance that inhibits the activity of ANXA2 protein and / or reduces the content of ANXA2 protein by regulating expression at the gene level or regulating the protein level.

[0015] The expression regulation at the gene level may include expression regulation at the chromatin level (such as histone modification, chromatin remodeling), transcription level (such as regulation of promoters, transcription factors, co-regulators), post-transcriptional level (such as RNA splicing, microRNA regulation) and post-translational level (such as ubiquitination, SUMOylation, acetylation, glycosylation, phosphorylation, methylation, NEDD8 modification, etc.), etc.

[0016] The regulation of protein levels may include regulating the activity and / or content of the protein by protein degradation, protein interaction or other methods capable of regulating protein activity.

[0017] Furthermore, the substance includes a substance that causes the ANXA2 gene to be deleted or inactivated by site-directed mutagenesis technology, gene knockdown technology, gene editing technology and / or gene knockout technology, or a substance that specifically binds to the ANXA2 protein to reduce its content or inactivate its function.

[0018] It is well known to those skilled in the art to inhibit gene expression, silence or knock out genes by using site-directed mutagenesis technology (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis and cassette mutagenesis, etc.), gene knockdown technology (including RNA interference technology, Morpholino interference technology, antisense nucleic acid technology and ribozyme technology, etc.), gene editing technology (including zinc finger ribozyme gene editing technology, TALEN gene editing technology and CRISPR gene editing technology, etc.) or gene knockout technology (including complete gene knockout and conditional gene knockout). For example, shRNA, siRNA or miRNA targeting the ANXA2 gene can be used to inactivate gene expression or silence genes at the post-transcriptional level or translation level. The ANXA2 gene can also be knocked out by using a CRISPR-Cas system containing sgRNA and Cas protein. Alternatively, the ANXA2 gene is mutated by using site-directed mutagenesis technology to produce a frameshift mutation or premature translation termination, thereby inactivating the ANXA2 gene or weakening its function. In some embodiments of the present invention, RNA interference technology is used to silence the ANXA2 gene.

[0019] Furthermore, the substance includes nucleic acid molecules, carbohydrates, lipids, compounds (including small molecule compounds), antibodies, peptides, proteins, recombinant vectors (such as gene editing vectors), recombinant cells (including extracellular vesicles, such as exosomes, etc.) or viral vectors (such as lentivirus and adeno-associated virus vectors).

[0020] Furthermore, the nucleic acid molecules may include (1) double-stranded RNA (dsRNA), small interfering RNA (siRNA), micro RNA (miRNA) and short hairpin RNA (shRNA) used in RNA interference technology; (2) antisense RNA (asRNA) and antisense oligonucleotide (AON) used in antisense nucleic acid technology; (3) gRNA and sgRNA used in gene editing technology; (4) aptamer and ribozyme, etc.

[0021] Those skilled in the art know that nucleic acid molecules such as siRNA, miRNA, shRNA or dsRNA can be designed by selecting target sequences based on the sequence of the ANXA2 gene or its transcribed mRNA. The nucleic acid molecules can interfere with the transcription, translation or post-transcription and translation modification of the gene, thereby affecting the expression of the protein.

[0022] Those skilled in the art know that in order to prolong the inhibitory effect of siRNA on target gene expression, a pair of specific oligonucleotide sequences can be designed and cloned into a vector after annealing. The transcription product of the recombinant vector is short hairpin RNA (shRNA), which can fold itself into a stem-loop structure with a stem length of 19-21 bases, wherein the 19-21 bases are derived from a specific sequence of the target gene mRNA, and the precursor of this stem-loop structure is quickly cut in the cell to form a functional siRNA. The shRNA expressed by this vector is sheared to form a siRNA with a stable expression amount and a long duration, thereby causing long-term inhibition of target gene expression.

[0023] The antibodies may include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, Fab, Fab′, F(ab′)2, antibody variable region (Fv), disulfide bond-stabilized Fv (dsFv), single-chain antibodies (ScFv), single-domain antibodies (sdAb, i.e., nanobodies), minibodies, and minimum recognition units (MRU).

[0024] It is well known to those skilled in the art that antibodies are commonly used protein inhibitors that can inhibit the activity of their ligands. It is also known to those skilled in the art that ANXA2 protein can be used to prepare ANXA2 antibodies, and the activity of ANXA2 protein can be inhibited by binding of the antibody to the protein.

[0025] In the above application, the ANXA2 inhibitor includes any one of the following:

[0026] C1) Nucleic acid molecules used to silence or knock out the ANXA2 gene (such as siRNA targeted to interfere with the expression of the ANXA2 gene or sgRNA targeted to knock out the ANXA2 gene, etc.);

[0027] C2) Antibodies that bind to ANXA2 protein (ANXA2 antibodies).

[0028] In the above application, the ANXA2 inhibitor includes any one of the following:

[0029] D1) siRNA, the nucleotide sequence of the sense strand of the siRNA may be 1 to 19 of SEQ ID No. 1 or SEQ ID No. 1; the nucleotide sequence of the antisense strand of the siRNA may be 1 to 19 of SEQ ID No. 2 or SEQ ID No. 2;

[0030] D2) a DNA molecule encoding the siRNA described in D1);

[0031] D3) An expression cassette, a recombinant vector or a recombinant host cell containing the siRNA described in D1) or the DNA molecule described in D2).

[0032] It is known to those skilled in the art that the end of one or both chains of siRNA may have an overhang (at least one unpaired nucleotide extending from the end of the RNA chain), and the overhang of siRNA may be present at the 5' end or the 3' end of siRNA, and the number of nucleotides constituting the overhang may be 1, 2, 3, 4, 5 or 6. The lengths of the overhangs of the two chains may be the same or different. The overhang may be TT, TU, UT or UU but is not limited thereto. In one or more embodiments of the present invention, both chains of siRNA include a 3' overhang. Specifically, adding a "TT" overhang at the 3' end of siRNA is used to stabilize siRNA, which does not affect the specificity and function of siRNA, and the "TT" portion of the 3' overhang is optional and non-restrictive.

[0033] The siRNA described herein or the DNA encoding the siRNA can be obtained using a variety of techniques known to those skilled in the art. For example, the siRNA or the DNA encoding the siRNA can be chemically synthesized or recombinantly produced using methods known in the art.

[0034] D3) The recombinant vector (including recombinant plasmid and recombinant viral vector) contains a DNA molecule encoding the siRNA described herein and any suitable promoter for expressing the siRNA. Any suitable promoter can be used to express siRNA from a recombinant plasmid or a recombinant viral vector. Suitable promoters for expressing siRNA described herein include, for example, U6 promoter, H1 promoter and CMV promoter. The selection of other suitable promoters is within the technical scope of the art.

[0035] In the above application, the ANXA2 inhibitor includes an inhibitor of phosphorylation of ANXA2 protein Thr208 site.

[0036] Furthermore, the Thr208 site of the ANXA2 protein may be the 208th threonine site of the ANXA2 protein.

[0037] Furthermore, the phosphorylation inhibitor includes a substance that inhibits the phosphorylation level of the Thr208 site of the ANXA2 protein.

[0038] Furthermore, the phosphorylation inhibitor includes a substance for mutating the threonine at position 208 of the ANXA2 protein into an amino acid that cannot be phosphorylated.

[0039] In the above application, the phosphorylation inhibitor may be an ANXA2 mutant protein, and the ANXA2 mutant protein may be obtained by mutating the threonine at position 208 of the amino acid sequence of the ANXA2 protein to an amino acid that cannot be phosphorylated.

[0040] The present invention also provides the use of ANXA2 protein, ANXA2 gene or Thr208 phosphorylation site of ANXA2 protein as a target in screening or developing products having any of the following functions:

[0041] E1) Prevention and / or treatment of pulmonary hypertension;

[0042] E2) prevention and / or treatment of pulmonary artery vascular remodeling;

[0043] E3) prevention and / or treatment of right ventricular hypertrophy;

[0044] E4) inhibits the proliferation and / or migration of pulmonary artery smooth muscle cells.

[0045] The screening method may include any of the following: (1) using ANXA2 protein and / or ANXA2 gene as a target to screen drugs or agents, and using drugs or agents that can reduce the expression level of ANXA2 gene and / or reduce the content or activity of ANXA2 protein as candidate drugs for preventing and / or treating pulmonary hypertension, pulmonary artery vascular remodeling or right ventricular hypertrophy, or as candidate drugs for inhibiting the proliferation and / or migration of pulmonary artery smooth muscle cells; (2) using the Thr208 phosphorylation site of ANXA2 protein as a target to screen drugs or agents, and using drugs or agents that can reduce or inhibit the phosphorylation level of the Thr208 site of ANXA2 protein as candidate drugs for preventing and / or treating pulmonary hypertension, pulmonary artery vascular remodeling or right ventricular hypertrophy, or as candidate drugs for inhibiting the proliferation and / or migration of pulmonary artery smooth muscle cells.

[0046] Pathological experiments, clinical trials or treatments of the candidate drugs can be used to further develop drugs related to pulmonary hypertension, such as drugs for preventing and / or treating pulmonary hypertension, pulmonary artery vascular remodeling or right ventricular hypertrophy, or drugs for inhibiting the proliferation and / or migration of pulmonary artery smooth muscle cells.

[0047] The present invention also provides a mutant protein, wherein the amino acid sequence of the mutant protein may be a sequence obtained by mutating the threonine at position 208 of SEQ ID No. 3 to an amino acid that cannot be phosphorylated.

[0048] The amino acids that cannot be phosphorylated herein include any amino acids other than threonine, serine and tyrosine.

[0049] The amino acids described herein that cannot be phosphorylated include, but are not limited to, alanine, cysteine, aspartic acid, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, arginine, valine, or tryptophan.

[0050] Furthermore, the amino acid that cannot be phosphorylated may be alanine.

[0051] The present invention also provides a biomaterial, which may be any of the following:

[0052] F1) siRNA as described herein;

[0053] F2) a DNA molecule encoding the siRNA described herein;

[0054] F3) a DNA molecule encoding a mutant protein as described herein;

[0055] F4) an expression cassette containing the DNA molecule described in F2) or F3);

[0056] F5) a recombinant vector containing the DNA molecule described in F2) or F3), or a recombinant vector containing the expression cassette described in F4);

[0057] F6) a recombinant microorganism containing the DNA molecule described in F2) or F3), or a recombinant microorganism containing the expression cassette described in F4), or a recombinant microorganism containing the recombinant vector described in F5);

[0058] F7) a recombinant host cell containing the DNA molecule described in F2) or F3), or a recombinant host cell containing the expression cassette described in F4), or a recombinant host cell containing the recombinant vector described in F5);

[0059] F8) Extracellular vesicles (such as exosomes, etc.) containing the DNA molecule described in F2) or F3), or extracellular vesicles (such as exosomes, etc.) containing the expression cassette described in F4), or extracellular vesicles (such as exosomes, etc.) containing the recombinant vector described in F5).

[0060] Furthermore, the recombinant vector includes a recombinant DNA molecule constructed by connecting a DNA molecule encoding the siRNA to a vector in vitro. The recombinant vector can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the siRNA into the recipient cell or extracellular vesicle and can express the siRNA so that it can perform the inhibitory function on the target gene.

[0061] Furthermore, the vector may be a plasmid or a virus.

[0062] Further, the virus may include baculovirus, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus) but is not limited thereto. The virus is preferably a retrovirus or adenovirus.

[0063] Furthermore, the recombinant vector may be a recombinant viral vector (such as a recombinant adenoviral vector or a recombinant adeno-associated viral vector, etc.) expressing the siRNA described herein. It is well known to those skilled in the art to suppress the expression of the ANXA2 gene by using the siRNA expressed by the viral vector to silence the ANXA2 gene.

[0064] Furthermore, the recombinant vector may also include a gene editing vector or a homologous recombination vector for mutating the threonine at position 208 of the ANXA2 protein into an amino acid that cannot be phosphorylated (such as alanine, etc.).

[0065] The method of constructing a gene editing vector is well known to those skilled in the art. The Cas protein (such as Cas9 protein) that matches (or binds to) the designed guide RNA (sgRNA) can be selected as long as the experimental purpose (such as gene mutation) can be achieved. For example, the sgRNA and the Cas protein can be connected to the same vector and started with a dual promoter, or the sgRNA and the Cas protein can be connected to different vectors, or a backbone vector (such as PX459 vector, PX458 vector, PX461 vector, PX462 vector, PX551 vector, PX552 vector, pGK1.1 vector, PX330 vector, PX335 vector, PX165 vector, eSpCas9 (1.1) vector, etc.) containing the Cas protein gene can be selected as an expression vector of the sgRNA, and the DNA molecule encoding the sgRNA is cloned into the backbone vector to construct a gene editing vector targeting the target gene. In order to achieve the purpose of gene mutation, a repair template plasmid (donor DNA molecule) can be provided, and the donor DNA molecule can contain the ANXA2 gene mutation site (e.g., Thr208Ala) and homologous arms located upstream and downstream of the mutation site, which can introduce site-directed mutations during the repair process to achieve the mutation of the threonine at position 208 of the ANXA2 protein to an amino acid that cannot be phosphorylated (e.g., alanine, etc.). The CRISPR-Cas system composed of the Cas protein and sgRNA expression plasmid and the donor DNA plasmid can be packaged into a virus or extracellular vesicle and delivered to the patient, and the purpose of preventing and / or treating pulmonary hypertension is achieved by inhibiting the phosphorylation of the Thr208 site of the ANXA2 protein.

[0066] It is known to those skilled in the art that Cas protein, Cas protein mRNA, Cas expression vector (a vector containing and expressing a DNA molecule encoding Cas protein), sgRNA, sgRNA expression vector (a vector containing and expressing a DNA molecule encoding sgRNA), donor DNA and / or donor DNA plasmid can be transferred into recipient cells by various methods known in the art, not limited to viral vector methods, and can also be achieved by non-viral vector introduction systems, including but not limited to physical and mechanical methods (such as direct injection, electroporation, ultrasound, and gene gun methods, etc.), cationic liposomes (such as DOTMA, DOTAP, DOTMAC, and DMG-PEG2000, etc.), cationic polymers (such as polyethyleneimine (PEI), polylysine (PLL), and polyamidoamine dendrimers (PAMAM), etc.), polysaccharide polymers (such as chitosan, cyclodextrin, and glucose, etc.), and inorganic nanomaterials (such as magnetic nanoparticles, carbon nanotubes, quantum dots, nanogold, and silica, etc.).

[0067] By constructing a homologous recombination vector, a method for performing site-directed mutagenesis on the ANXA2 gene based on homologous recombination technology is also well known to those skilled in the art. For example, a homologous recombination vector containing an upstream homologous arm of the ANXA2 gene mutation site, a fragment of the ANXA2 gene mutation site, and a downstream homologous arm of the ANXA2 gene mutation site can be constructed, and then homologous recombination occurs in the recipient cell under the action of a recombinase (such as Cre, Flp, Dre, VCre or SCre) or a site-specific recombinase system (such as Bxb1, Cre-LoxP, FLP-Frt, Dre-Rox, Cre-ERT2, Cre-ERT or ER-Cre-ER system). After homologous recombination occurs, the original site and its upstream and downstream fragments are replaced by the mutation site and its upstream and downstream fragments, thereby introducing a point mutation into the ANXA2 gene, and then achieving a mutation of the threonine at position 208 of the ANXA2 protein.

[0068] The present invention also provides a pharmaceutical composition comprising the biomaterial described herein.

[0069] Furthermore, the pharmaceutical composition may include the siRNA described herein, a DNA molecule encoding the siRNA described herein, or a recombinant vector (such as a recombinant viral vector) containing a DNA molecule encoding the siRNA described herein.

[0070] Furthermore, the pharmaceutical composition may include a DNA molecule encoding the mutant protein described herein or a recombinant vector (such as a recombinant viral vector) containing a DNA molecule encoding the mutant protein described herein.

[0071] The pharmaceutical composition has at least one of the following uses:

[0072] G1) Prevention and / or treatment of pulmonary hypertension;

[0073] G2) prevention and / or treatment of pulmonary artery vascular remodeling;

[0074] G3) prevention and / or treatment of right ventricular hypertrophy;

[0075] G4) inhibits the proliferation and / or migration of pulmonary artery smooth muscle cells.

[0076] Furthermore, the pharmaceutical composition also includes one or more pharmaceutically acceptable carriers.

[0077] The pharmaceutically acceptable carrier is selected from excipients, preservatives, protective agents, cosolvents, diluents (such as water, saline, PBS (phosphate buffer), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc.), wetting agents, disintegrants (such as dry starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, etc.), lubricants (such as sorbitan trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, etc.), fillers (such as starch, dextrin, etc.), adhesives (such as gelatin, pectin, gum arabic, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), penetration enhancers (such as Brij-78), pH adjusters, stabilizers (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactants (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium lauryl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption accelerators (such as chitosan), thickeners (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidants (such as sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, etc.), plasticizers (such as glycerol, sorbitol, phthalates, etc.), propellants (such as hydrofluoroalkanes, dimethyl ether, etc.), aerosolizers, suspending agents, dispersants, colorants (such as TiO2, pigments, etc.) and flavoring agents.

[0078] Excipients are usually used in medicines to shape the medicines, change the physical state of the medicines, and play a supporting role. Excipients include, but are not limited to: (1) Excipients for injections: such as solvent water, alcohols, ethers, amides, sulfoxides, esters, etc.; (2) Excipients for injection powders: such as sucrose, lactose, mannitol, etc.; (3) Excipients for sprays: such as soy lecithin, propylene glycol, borneol, ethanol, phenol, etc.; (4) Excipients for tablets: such as starch, sucrose, dextrin, methyl cellulose, gelatin, polyethylene glycol, tartaric acid, boric acid, etc.; (5) Excipients for eye drops: such as sodium hyaluronate, disodium ethylenediaminetetraacetic acid (EDTA-Na2), etc.; (6) Excipients for suppositories: such as cocoa butter, semi-synthetic or fully synthetic fatty acid glycerides, glycerol gelatin, polyethylene glycol, etc.; (7) Excipients for granules: such as corn flour, bentonite, zeolite powder, etc.; (8) Excipients for capsules: such as gelatin, etc.; (9) Excipients for gels: such as gelatin, pectin, gum arabic, etc.; (10) Excipients for ointments: such as vaseline, paraffin, liquid paraffin, lanolin, lanolin alcohol, beeswax, lard, vegetable oil, silicone oil, silicone, soaps, higher fatty alcohols, fatty alcohol sulfates, polyols, polyethylene glycol, FAPG, etc.; (11) Excipients for patches: such as ethylene-vinyl acetate copolymer (EVA), pressure-sensitive adhesive (PSA), etc.; (12) Excipients for films: such as gelatin, shellac, gum arabic, polyvinyl alcohol compounds, acrylic acid copolymers, etc.

[0079] Biological drug preparations are easily contaminated by exogenous microorganisms during production, transportation and storage, which may affect the quality of the drug. Therefore, preservatives are usually added to inhibit the growth and reproduction of microorganisms. The preservatives include but are not limited to thimerosal, formaldehyde, phenol and m-cresol.

[0080] Protective agents generally refer to any substance that can protect the activity of drugs, including freeze-drying protective agents. Freeze-drying protective agents can be used to change the physical and chemical environment of biological samples during freeze-drying, reduce cell damage, maintain original biological activity, improve the stability of proteins and cells, etc. The freeze-drying protective agents include but are not limited to sugars / polyols (such as sucrose, trehalose, lactose, glucose, maltose, maltodextrin, mannitol, sorbitol, etc.), polymers (such as HES, PVP, PEG, dextran, albumin, etc.), anhydrous solvents (such as polyethylene glycol, ethylene glycol, glycerol, DMSO, DMF, etc.), surfactants (such as Tween-80, etc.), amino acids (such as L-serine, sodium glutamate, alanine, glycine, sarcosine, acetyltryptophan, etc.), and salts and amines (such as phosphates, acetates, citrates, etc.).

[0081] The dosage form of the pharmaceutical composition includes, but is not limited to, injections (including injection solutions and powder injections for injection), gels, eye drops (including eye drops and intraocular injection solutions), oral solutions, suppositories, effervescent tablets, capsules, ointments, creams, sprays, aerosols, external solutions, tablets, powders, pills, granules, drops, paints, patches and long-acting sustained-release preparations.

[0082] Injections generally refer to solutions and emulsions for injection into the body made from extracted and purified medicinal materials, as well as sterile powders prepared into solutions before use, including injection solutions and powder injections for injection. The preparation method of injections is well known to those skilled in the art, for example, powder injections for injection can be prepared by vacuum freeze drying technology, spray drying technology, spray freeze drying technology. Injections can be prepared by preparing a solution with a drug and a solvent by a concentrated or diluted method, and then by filtering (such as surface filtration and / or deep filtration), encapsulation, sterilization and other steps.

[0083] In order to prepare the pharmaceutical composition into an injection preparation, such as a solution, emulsion, lyophilized powder injection and suspension, a commonly used diluent in the art, such as water, physiological saline, PBS (phosphate buffered saline), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc., can be used as a solvent to prepare a solution. In addition, in order to prepare an isotonic injection, an appropriate amount of a carrier such as sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, a conventional cosolvent, buffer, pH adjuster and other carriers can also be added.

[0084] The administration methods of the pharmaceutical composition include, but are not limited to, injection (such as administration in the form of injection), mucosal administration (such as administration in the form of sprays, aerosols, tablets, eye drops, suppositories, granules, capsules, etc.) and transdermal administration (such as administration in the form of gels, ointments, patches, films, etc.).

[0085] The present invention also provides the use of ANXA2 antibodies or ANXA2 Thr208 phosphorylated antibodies in the preparation of products (including reagents, kits, chips, test strips, test cards and immunosensors, etc.) for screening or diagnosing pulmonary hypertension.

[0086] The ANXA2 antibody may be a monoclonal antibody or a polyclonal antibody, and the ANXA2 antibody may be prepared by techniques well known to those skilled in the art. For example, polyclonal antibodies may be produced by immunizing mice, rabbits or rats with purified ANXA2 protein, or monoclonal antibodies may be produced by removing spleens from immunized mice and fusing spleen cells with myeloma cells to form hybridomas. ANXA2 antibodies may also be commercially available.

[0087] The ANXA2 Thr208 phosphorylation antibody can be an antibody that can recognize the phosphorylation site 208 of the ANXA2 protein, and the antibody can be prepared by immunizing an animal (such as a mouse, rabbit or rat) with an antigenic peptide (a polypeptide containing Thr 208 of the ANXA2 protein). In one or more embodiments of the present invention, the amino acid sequence of the antigenic peptide is shown in SEQ ID No. 4 (DAGVKRKGTDVPKW).

[0088] In one or more embodiments of the present invention, the ANXA2 Thr208 phosphorylation antibody is a polyclonal antibody, which is obtained by coupling the antigen polypeptide (SEQ ID No. 4) with the carrier protein hemocyanin (KLH) to immunize rabbits, and selecting rabbit serum with high titer for antigen affinity purification after three immunizations to obtain purified polyclonal antibodies. It is known to those skilled in the art that phosphorylation antibodies can recognize specific protein sites and can be used to detect the phosphorylation level of proteins.

[0089] The products described herein may be reagents, preparations, drugs, pharmaceutical compositions, kits, etc. but are not limited thereto.

[0090] The ANXA2 gene described herein may be endogenous or exogenous to the organism.

[0091] The amino acid sequence of the ANXA2 protein described herein may be the protein encoded by the following gene: Gene ID: 302, updated on 12-Nov-2024 (human), Gene ID: 56611, updated on 14-Nov-2024 (rat) or Gene ID: 12306, updated on 2-Nov-2024 (mouse).

[0092] The annexin family consists of 12 evolutionarily conserved and structurally related Ca 2+ANXA2 is composed of 12 family members, including annexin A1-A11 and A13, which have been identified in mammals. Among them, ANXA2 is one of the most widely studied members of the annexin family. ANXA2 is highly expressed in tissues such as the lung, pancreas, colon, and ileum, and consists of a variable N-terminal domain and a conserved C-terminal core domain. The N-terminal region of varying lengths contains binding sites for tissue plasminogen activator (tPA) and S100A10 (also known as p11). The N-terminal region of ANXA2 has three major phosphorylation sites, Tyr 23, Ser 11, and Ser 25, which can be phosphorylated by tyrosine kinases and serine kinases, respectively. The core region of ANXA2 contains specific sites that can bind to calcium and phospholipids. ANXA2 exists in two forms: ANXA2 monomer and heterotetrameric complex (ANXA2-S100A10). The heterotetrameric complex consists of two copies of ANXA2 and two copies of protein S100A10. After the heterotetrameric complex is phosphorylated at tyrosine 23 of ANXA2 mediated by Src kinase, the intracellular ANXA2 / S100A10 complex is translocated to the outer membrane leaflet. On the cell surface, the heterotetrameric ANXA2 / S100A10 complex acts as a scaffold to regulate the presentation of cell surface receptors and ion channels. ANXA2 is expressed at high levels in vascular cells and is involved in the occurrence and development of diseases such as atherosclerosis. At present, there are no reports on the study of ANXA2 in pulmonary hypertension.

[0093] After a large number of experimental studies, the inventors first discovered that the expression of ANXA2 in the pulmonary artery and serum of an animal model of pulmonary hypertension was significantly increased, and further confirmed through experiments that ANXA2 gene knockout can: (1) inhibit the increase of pulmonary artery pressure and significantly improve pulmonary hypertension; (2) inhibit the proliferation of pulmonary artery smooth muscle cells and significantly improve pulmonary artery vascular remodeling; (3) significantly improve right ventricular hypertrophy during pulmonary hypertension. In addition, overexpression of the ANXA2 gene can promote the proliferation and migration of pulmonary artery smooth muscle cells, thereby promoting pulmonary artery vascular remodeling and accelerating the occurrence and progression of pulmonary hypertension. The results show that the ANXA2 gene and / or ANXA2 protein can be used as a therapeutic target for the prevention and / or treatment of pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy, and has potential value as a marker of pulmonary hypertension, and can be used for pathological section diagnosis and serological diagnosis indicators of pulmonary hypertension.

[0094] In order to further verify that the ANXA2 gene and / or ANXA2 protein can be used as a therapeutic target, the inventors further designed and developed different types of ANXA2 inhibitors, including siRNA that silences the ANXA2 gene and inhibitors that inhibit the post-translational phosphorylation modification of the ANXA2 gene. The experimental results showed that different types of ANXA2 inhibitors can significantly (P < 0.001) inhibit the proliferation and migration of pulmonary artery smooth muscle cells, thereby effectively slowing down the process of pulmonary artery vascular remodeling and achieving the prevention and treatment of pulmonary hypertension.

[0095] The inventors also discovered for the first time a therapeutic target for ANXA2. Phosphorylation at the Thr208 site of ANXA2 plays an important role in the proliferation and migration of pulmonary arterial smooth muscle cells. Inhibiting phosphorylation at the Thr208 site of ANXA2 can significantly inhibit the proliferation and migration of pulmonary arterial smooth muscle cells, thereby playing a role in preventing and treating pulmonary hypertension.

[0096] The art has not conceived of using the ANXA2 gene and / or ANXA2 protein, or the Thr208 phosphorylation site of the ANXA2 protein as a target for preventing and / or treating pulmonary hypertension, pulmonary artery vascular remodeling, and right ventricular hypertrophy. Unexpectedly, the inventors of the present application have demonstrated that ANXA2 inhibitors can significantly improve pulmonary hypertension, pulmonary artery vascular remodeling, and right ventricular hypertrophy through different means including in vivo and in vitro. In addition, the ANXA2 gene and / or ANXA2 protein, or the Thr208 phosphorylation site of the present invention can also be used for the diagnosis or auxiliary diagnosis of pulmonary hypertension, or for screening pulmonary hypertension drugs, developing new diagnostic and therapeutic methods and drugs, and have a wide range of clinical application value in the prevention and treatment of pulmonary hypertension.

[0097] Definition of terms

[0098] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0099] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a target gene. A typical expression cassette comprises a promoter, an MCS (multiple cloning site) and a terminator. The expression cassette may also include a target gene, a marker gene (such as a TK gene, a DHFR gene, a CAT gene and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly (A) signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette may be directly connected or indirectly connected via a linker.

[0100] The term "vector" generally refers to a vector that can carry foreign DNA or target gene into host cells for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into host cells by transformation, transduction or transfection, so that the genetic material elements it carries are amplified and / or expressed in the host cells. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the properties of the recipient cells. The vector includes, but is not limited to, plasmid, phage (e.g., lambda phage or M13 phage), cosmid, phagemid, shuttle vector (e.g., yeast expression vector), Ti plasmid, artificial chromosome (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC), or Ti plasmid artificial chromosome (TAC)), viral vector (e.g., baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (e.g., SV40), herpesvirus (e.g., herpes simplex virus)). A vector may contain a variety of elements for controlling expression, including, but not limited to, promoter sequence, transcription initiation sequence, enhancer sequence, selection element, and reporter gene. In addition, the vector may also contain a replication initiation site.

[0101] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria may be from Corynebacterium sp. (e.g., Corynebacterium glutamicum, Corynebacterium pekinensis, Corynebacterium dentata, etc.), Brevibacterium sp. (e.g., Brevibacterium lactoferment, Brevibacterium flavum, Brevibacterium ammoniaphagoides, etc.), Escherichia sp. (e.g., Escherichia coli), Erwinia sp., Agrobacterium sp. (e.g., Agrobacterium tumefaciens), Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., and Bacillus sp. (e.g., Bacillus sp.), etc. The virus may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus), etc. The fungus may be from Saccharomyces sp. (such as saccharomyces cerevisiae, Candida, methanol yeast, Pichia pastoris), Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp. and Cephalosporium sp., etc. The actinomycete may be from Streptomyces sp. (such as Streptomyces). The algae may be from Cyanophyta (such as Cyanobacteria), Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., and the like.

[0102] The term "host cell" is also referred to as a recipient cell, and generally refers to any type of cell that can be used to introduce a vector, such as a plant cell and an animal cell. The host cell can be understood to refer not only to a specific recipient cell, but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell. Suitable host cells are known in the art, wherein: the plant cell may be Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like, but is not limited thereto; the animal cell may be a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), Chinese hamster ovary cell substrain (CHO-K1 cell), African green monkey kidney cell (Vero cell), SV40-transformed African green monkey kidney cell (COS cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human embryonic kidney cell (HEK293 cell), human HeLa cell, fibroblast, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., African clawed frog (Xenopus laevis) cell or giant salamander (Andrias davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5), etc., but are not limited thereto.

[0103] The term "extracellular vesicles (EVs)" generally refers to any type of vesicles that can be obtained from cells in any form, such as microvesicles, exosomes, apoptotic bodies, microparticles, ectosomes, etc. Extracellular vesicles can be derived from any cell type in vivo, ex vivo, and in vitro.

[0104] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0105] The term "recombinant microorganism" generally refers to a recombinant microorganism with changed functions obtained by manipulating and modifying the genes of a target microorganism, such as introducing an exogenous target gene or a recombinant vector into the target microorganism, or directly editing the endogenous genes of the target microorganism.

[0106] The term "recombinant host cell" generally refers to a recombinant host cell whose function has been changed by manipulating and modifying the genes of the host cell, such as introducing an exogenous target gene or a recombinant vector into the host cell, or directly editing the endogenous genes of the host cell.

[0107] The term "antibody" generally refers to an immunoglobulin that can specifically bind to a target antigen or target protein. When referring to the term "antibody", unless the context clearly indicates, it includes not only complete antibodies, but also antigen-binding fragments that can specifically bind to a target antigen or target protein.

[0108] The term "introduction" generally refers to the transfer of foreign genes into recipient cells such as eukaryotic recipient cells or prokaryotic recipient cells. The method of introduction is not particularly limited, and any known transformation method can be used as long as the target gene can be transferred into the recipient cell. The method of introduction may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria by chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria by phage transduction. (3) transferring the target gene into plant recipient cells by physical or chemical methods, such as gene gun method (also known as microprojectile bombardment method or biological missile method), chemical stimulation method, electric shock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasonic method, air gun method, and vortex method. (4) Using vectors as media to transfer target genes into plant recipient cells, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system)-mediated method (Agrobacterium-mediated method), plant virus vector-mediated transformation method, etc. (5) Introducing target genes into ex vivo animal cells (transfection) by calcium phosphate coprecipitation method, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e. electrotransfection method), microinjection, gene gun method or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method), etc. (6) Introducing target genes into in vivo animal cells by microinjection method, retrovirus vector method, somatic cell nuclear transfer method, sperm vector method or embryonic stem cell method, etc., and further preparing transgenic animals.

[0109] The term "mutation" generally refers to changes in amino acid sequences or nucleotide sequences, which may include changes in the base pair composition or arrangement order of the gene structure, such as point mutations caused by changes in a single base, or deletions, duplications and insertions of multiple bases, etc. It may also include replacement, deletion and insertion (addition) of one or more amino acid residues of a protein.

[0110] The term "inhibitor (also called antagonist)" has a well-known meaning in the art and may refer to any substance that reduces (downregulates) the level and / or activity of a target protein or gene. The inhibitor may also refer to any substance (including phosphorylation inhibitors) that inhibits post-translational modification of a target gene.

[0111] The term "phosphorylation inhibitor" generally refers to any substance that blocks, inhibits or reverses protein phosphorylation. Herein, "ANXA2 protein Thr208 site phosphorylation inhibitor" refers to any substance that blocks, inhibits or reverses the phosphorylation of Threonine 208 of ANXA2 protein.

[0112] The term "ANXA2 inhibitor" in the present application may be a substance that inhibits the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the ANXA2 gene, or a substance that inhibits or reduces the content, activity and / or function of the ANXA2 protein. The ANXA2 inhibitor includes a substance that deletes or inactivates the ANXA2 gene by techniques such as site-directed mutagenesis, gene knock-down, gene editing, and gene knock-out. The ANXA2 inhibitor also includes a substance that can target and bind to the ANXA2 protein, inhibit the activity of the ANXA2 protein, or prevent the ANXA2 protein from performing its function.

[0113] The term "site-directed mutagenesis" generally refers to changing one or several bases in a gene through site-directed mutagenesis, resulting in a change in the amino acid composition of the corresponding protein. Site-directed mutagenesis methods include oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis.

[0114] The term "gene knock-down" is also called gene knockdown, which generally refers to the technology of inactivating gene expression or silencing gene at the post-transcriptional level or translation level without changing the DNA sequence of the gene. Gene knock-down includes RNA interference technology, Morpholino interference technology, antisense nucleic acid technology and ribozyme technology.

[0115] The term "gene editing" generally refers to the technology that can change a specific gene sequence in any cell, including somatic cells, and can cause base deletion, duplication, insertion, frameshift mutation, replacement and knockout of target genes, and achieve replacement, deletion, shearing and single base change of genomic sequences, that is, the technology of "editing" the genome or the sequence of a specific gene at will. Gene editing includes zinc finger nuclease gene knockout technology, TALEN gene editing technology and CRISPR gene editing technology.

[0116] The term "gene knock-out" generally refers to a technique in which an exogenous mutated gene is used to replace an endogenous normal homologous gene through homologous recombination, thereby inactivating the endogenous gene, including complete gene knockout (e.g., complete mutation of the target gene based on a replacement targeting vector or an insertion targeting vector) and conditional gene knockout (e.g., tissue-specific knockout based on the Cre-LoxP recombinase system or the FLP-FRT recombinase system).

[0117] The term "RNA interference (RNAi)" generally refers to a technique that uses double-stranded RNA (dsRNA) to induce the degradation of mRNA of a target gene that is homologous and complementary to it, so that the expression of the gene is silenced and shut down, thereby inducing post-transcription gene silencing (PTGS) to achieve the purpose of preventing gene expression. Double-stranded RNA (dsRNA) is the trigger of RNAi, which triggers the degradation of single-stranded RNA (ssRNA) that is complementary to it. Long-chain dsRNA can be cut into small fragments of dsRNA, i.e., small interfering RNA (siRNA), by the Dicer enzyme in cells, and siRNA mediates mRNA cutting. Therefore, RNAi can also be achieved by introducing siRNA corresponding to the target gene. In addition, RNAi induced by introducing dsRNA into mammalian cells is transient. A gene encoding RNA with an inverted repeat sequence that can form a hairpin (i.e., short hairpin RNA, shRNA) can be introduced. This gene continuously provides dsRNA in the form of a hairpin structure, so that RNAi can continue. RNA interference in a broad sense also includes gene silencing at the transcriptional level (pre-transcriptional gene silencing) triggered in the gene regulatory region. This silencing process involves DNA methylation rather than degradation of mRNA. The siRNA used to silence genes directly acts on the regulatory region of the gene rather than the coding region. RNA interference in a broad sense can also include gene silencing at the translational level (translational silencing). For example, microRNA (microRNA, miRNA, a single-stranded RNA molecule) mainly silences gene expression by preventing the translation of mRNA and interfering with the accumulation of target mRNA protein products.

[0118] The term "Morpholino interference technology" usually refers to the use of morpholino to replace the pentose ring on traditional nucleotides, and the original phosphate group is also changed, so that the molecule as a whole does not carry any charge, cannot be recognized and degraded by RNase and DNase, and has extremely strong stability. Its principle is the same as that of antisense nucleic acid technology, which binds to the mRNA molecule by complementing the homologous sequence of the target gene mRNA, thereby hindering the binding of other molecules and proteins to the specific mRNA nucleic acid sequence, and ultimately preventing the target gene mRNA from being translated into protein.

[0119] The term "antisense nucleic acid technology" generally refers to the technology of inhibiting the expression of specific genes by artificially synthesizing antisense RNA or introducing its gene into cells, based on the principle that antisense RNA can complementarily bind to specific mRNA molecules with homologous sequences, thereby inhibiting the processing and translation of the mRNA. Antisense nucleic acid technology mainly includes antisense RNA (antisense RNA, asRNA) and antisense oligonucleotide (antisense oligonucleotide, AON).

[0120] The term "ribozyme technology" generally refers to the technology of using ribozymes to cut and degrade target RNA molecules. Ribozymes are a type of RNA molecules with biocatalytic activity that can specifically bind to and cut target RNA molecules, and can achieve degradation of specific RNA molecules, thereby inhibiting the expression of target genes. Ribozymes include hammerhead ribozymes, hairpin ribozymes, hepatitis D virus ribozymes, VS (Varkud satellite) ribozymes, and class I intron ribozymes.

[0121] The term "frameshift mutation" generally refers to the insertion or deletion of one, two or several pairs of bases (not 3 or multiples of 3) at a certain point in the DNA sequence, resulting in the shift of the amino acid triplet codon during transcription, a complete change in the base sequence starting from the damaged point, and translation into different amino acids.

[0122] The term "premature translation termination" generally refers to the mutation of the codon encoding the amino acid into a premature termination codon (PTC), resulting in premature termination of protein translation.

[0123] The term "siRNA (small interfering RNA)" generally refers to a type of double-stranded RNA molecule that can mediate the cleavage, degradation or silencing of a target RNA (such as mRNA, tRNA and viral RNA, etc.) that is complementary thereto. siRNA is generally double-stranded, comprising an antisense strand complementary to the target RNA, and a sense strand complementary to the antisense strand. Methods for preparing siRNA are well known to those skilled in the art, such as chemical synthesis, in vitro transcription, in vitro preparation of long fragment dsRNAs by degradation of RNaseIII (or Dicer), and methods for preparing by transfecting host cells with siRNA expression vectors for expression.

[0124] The term "DNA encoding siRNA" generally refers to a DNA molecule that can be transcribed to generate the siRNA. It is well known to those skilled in the art that, if the sequence of a siRNA is given, the U in the siRNA sequence can be replaced by T, while other nucleotides remain unchanged to obtain a DNA sequence encoding the siRNA.

[0125] The term "Cas protein (also called Cas endonuclease)" generally refers to a series of endonucleases with multiple activities that can cut the target site under the guidance of guide RNA (gRNA). The Cas protein may include 2 types of CRISPR-Cas systems, such as Cas9, Cas12a, Cas12b, Cas12i3, Cas13a, Cas13b and Cas13c.

[0126] The term "endogenous" generally refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0127] The term "exogenous" generally refers to any substance introduced from outside of or generated externally to an organism, cell, tissue, or system.

[0128] The term "pharmaceutically acceptable carrier" generally refers to a substantially chemically inert and non-toxic component of a pharmaceutical composition or drug that does not inhibit the effectiveness and / or safety of the drug. Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0129] The term "prevention" generally refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom in a subject.

[0130] The term "treatment" generally refers to a method implemented to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, reduction in the extent of the disease, stabilization of the disease (i.e., no longer worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. In addition, treatment may also refer to prolonging the survival of the subject compared to the expected survival if the subject had not received treatment.

[0131] The term "comprising" is not intended to be limiting, but is intended to be inclusive and means that there may be other elements besides the listed elements, which can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 Schematic diagram of increased expression of ANXA2 in pulmonary artery vessels and serum in animal models of pulmonary hypertension; A. Representative images of immunofluorescence staining of ANXA2 in pulmonary arteries in lungs of rats in the control group (Con) and rats with pulmonary hypertension induced by monocrotaline (MCT); B. Statistics of ANXA2 positive staining areas in pulmonary arteries. There were 6 rats in each group. One lung tissue section was taken from each rat and all pulmonary arteries were counted. There were 30 vessels in each group. The results were expressed as mean ± standard error. *** indicates p < 0.001; C. Pulmonary artery of mice in the control group (Con) and hypoxia combined with Su5 Representative images of immunofluorescence staining of ANXA2 in pulmonary arteries of mice with 416 (HySu)-induced pulmonary hypertension; D. Statistics of ANXA2-positive staining areas in pulmonary arteries. There were 6 rats in each group. One lung tissue section was taken from each rat and all pulmonary arteries were counted. There were 26 and 31 blood vessels in each group, respectively. The results are expressed as mean ± standard error, *** indicates p < 0.001; E. ANXA2 content in the lungs of control group (Con) rats and in the serum of rats with monocrotaline (MCT)-induced pulmonary hypertension. There were 7 and 6 samples in each group, respectively. The results are expressed as mean ± standard error, ** indicates p < 0.01.

[0133] Figure 2Schematic diagram of knocking out ANXA2 to improve pulmonary hypertension; AC. Identification results of ANXA2 knockout mice (ANXA2- / -), the mRNA expression level in the mouse lung was detected by real-time quantitative PCR (A), and the ANXA2 protein expression level was detected by western blot and immunohistochemistry (B and C); DG. After HySu-induced pulmonary hypertension in wild-type control mice (ANXA2+ / +) and knockout mice (ANXA2- / -), the right ventricular pressure was detected by catheterization and statistically analyzed (D and E), and the pulmonary artery blood flow spectrum was detected by blood flow Doppler ultrasound and its PAT / PET ratio was analyzed (F and G). The results are expressed as mean ± standard error, *** indicates p < 0.001).

[0134] Figure 3 Schematic diagram of knocking out ANXA2 to improve pulmonary artery remodeling; A and B. H&E staining of lung sections of ANXA2+ / + mice and ANXA2- / - mice after HySu-induced pulmonary hypertension, and the thickness of pulmonary artery vessels greater than 50 microns and less than 50 microns were counted respectively. C and D. α-SMA and PCNA immunofluorescence co-staining of lung sections of ANXA2+ / + mice and ANXA2- / - mice after HySu-induced pulmonary hypertension, and statistical analysis of PCNA-positive cells, DAPI staining to show cell nuclei.

[0135] Figure 4 Schematic diagram of knocking out ANXA2 to improve right ventricular hypertrophy; A and B. ANXA2+ / + mice and ANXA2- / - mice were subjected to echocardiography to measure the right ventricular thickness after HySu-induced pulmonary hypertension, and statistical analysis was performed; C and D. ANXA2+ / + mice and ANXA2- / - mice were subjected to right ventricular weight after HySu-induced pulmonary hypertension, and the ratio of right ventricle to left ventricle plus ventricular septum weight or body weight was calculated, and statistical analysis was performed. The results are expressed as mean ± standard error, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0136] Figure 5Schematic diagram of knocking down ANXA2 to slow down the proliferation of pulmonary artery smooth muscle cells; A-C. After transfection of pulmonary artery smooth muscle cells with ANXA2 small interfering RNA, the mRNA level of ANXA2 was detected by real-time quantitative PCR (A), and the protein expression level of ANXA2 was detected by Western blot and statistical analysis was performed (B and C); D and E. After transfection of pulmonary artery smooth muscle cells with ANXA2 small interfering RNA, the number of cells was observed and statistical analysis was performed; F and G. After transfection of pulmonary artery smooth muscle cells with ANXA2 small interfering RNA, the EdU incorporation experiment was used to detect cell proliferation and the EdU positive cells were counted. The results are expressed as mean ± standard error, ** indicates p < 0.01, *** indicates p < 0.001.

[0137] Figure 6 Schematic diagram of knocking down ANXA2 to slow down the migration of pulmonary artery smooth muscle cells; A. Transwell assay of pulmonary artery smooth muscle cells transfected with ANXA2 small interfering RNA; B. Statistical analysis of the number of migrated cells. The results are expressed as mean ± standard error, *** indicates p < 0.001.

[0138] Figure 7 Schematic diagram of overexpression of ANXA2 promoting the proliferation of pulmonary artery smooth muscle cells; A. After transfection of pulmonary artery smooth muscle cells with ANXA2 overexpression plasmid, the mRNA level of ANXA2 was detected; B and C. After transfection of pulmonary artery smooth muscle cells with ANXA2 overexpression plasmid, the protein expression levels of ANXA2, PCNA and Cyclin D1 were detected and quantitatively analyzed; D. After transfection of pulmonary artery smooth muscle cells with ANXA2 overexpression plasmid, EdU incorporation experiment was used to detect cell proliferation and EdU positive cells were counted. The results are expressed as mean ± standard error, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

[0139] Figure 8 Schematic diagram of overexpression of ANXA2 promoting the migration of pulmonary artery smooth muscle cells; A. Representative diagram of cell migration after transfection of ANXA2 overexpression plasmid in pulmonary artery smooth muscle cells by Transwell assay; B. Statistical analysis of the number of migrated cells. The results are expressed as mean ± standard error, *** indicates p < 0.001.

[0140] Fig. 9 Schematic diagram of the inhibition of pulmonary artery smooth muscle cell proliferation after mutating the threonine (T) at position 208 of ANXA2 to alanine (A); A. Analysis of PKA phosphorylation sites of ANXA2 proteins from different species; B. Schematic diagram showing the mutation of the threonine (T) at position 208 of rat ANXA2 protein to alanine (A), and the mutant plasmid is represented as ANXA2 T208A;CE. Overexpression of ANXA2 in pulmonary artery smooth muscle cells T208A Then the mRNA expression level and protein expression level of ANXA2 were detected, and the protein expression level was quantitatively analyzed; F and G. Pulmonary artery smooth muscle cells were transfected with ANXA2 T208A Plasmid was used to detect the expression levels of ANXA2, p-ANXA2(T208), PCNA and Cyclin D1 proteins by western blot and quantitative analysis; H. Pulmonary artery smooth muscle cells transfected with ANXA2 T208A Plasmids were then used to detect cell proliferation using EdU incorporation assays, and EdU-positive cells were statistically analyzed. The results are expressed as mean ± standard error, ** indicates p < 0.01, *** indicates p < 0.001.

[0141] Fig.10 Schematic diagram of the inhibition of pulmonary artery smooth muscle cell migration by the mutation of T at position 208 of ANXA2; A and B. Pulmonary artery smooth muscle cells transfected with ANXA2 T208A After plasmid expression, cell migration was detected by Transwell assay and statistical analysis was performed. The results are expressed as mean ± standard error, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. DETAILED DESCRIPTION

[0142] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0143] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0144] The following examples were processed using Graphpad Prism 10 statistical software, and the experimental results were expressed as mean ± standard error. The Student's t-test method was used for comparison between two groups, and the ANVOA test method was used for comparison between multiple groups. P < 0.05 (*) indicates that there is a statistical difference, P < 0.01 (**) indicates that the statistical difference is significant, and P < 0.001 (***) indicates that the statistical difference is extremely significant. The quantitative experiments in the following examples, unless otherwise specified, were set up for three or more biological replicates, and the results were averaged.

[0145] The siRNAs in the following examples were all synthesized by Genentech.

[0146] The pulmonary artery smooth muscle cells (PASMC) in the following examples are cultured primary rat pulmonary artery smooth muscle cells.

[0147] Example 1: ANXA2 expression in pulmonary artery vessels and serum of animal models of pulmonary hypertension is significantly increased

[0148] Pulmonary hypertension refers to a chronic progressive disease in which the pulmonary artery pressure is increased due to various reasons and exceeds a certain threshold (its hemodynamic diagnostic criteria are: at sea level resting state, the right cardiac catheter detects the mean pulmonary artery pressure> 20 mmHg). Its pathological characteristics are the thickening of the pulmonary artery wall, that is, pulmonary artery remodeling. At present, there are two common methods for constructing a pulmonary hypertension model: one is to give rats a single subcutaneous injection of monocrotaline (MCT, 60 mg / kg) to construct a pulmonary hypertension rat model (MCT model); the other is to give mice 10% hypoxia combined with Su5416 injection (20 mg / kg, once a week, a total of 3 injections) to construct a pulmonary hypertension mouse model (HySu mouse model). The animal models used in this embodiment are all constructed by the inventor according to the classic method.

[0149] The lung tissue sections of the successfully constructed MCT rat pulmonary hypertension model and the HySu mouse pulmonary hypertension model were stained, and the serum samples of the successfully constructed MCT rat pulmonary hypertension model were used for detection. The lung tissue sections of the pulmonary hypertension model were subjected to immunofluorescence staining, and the serum samples of the MCT rat pulmonary hypertension model were subjected to ELISA to detect the expression level of ANXA2 protein.

[0150] Test results such as Figure 1 As shown in Figure 2, the expression level of ANXA2 protein was significantly increased in the pulmonary artery of the MCT rat pulmonary hypertension model ( Figure 1 A and B); Similarly, the expression level of ANXA2 protein was significantly increased in the pulmonary artery of the HySu mouse pulmonary hypertension model ( Figure 1 C and D). Serum test results showed that the ANXA2 protein content in the serum of MCT rat pulmonary hypertension model was significantly increased ( Figure 1 E).

[0151] The above analysis results suggest that ANXA2 protein (NCBI Gene ID: 302 (human), 12306 (mouse), 56611 (rat)) may have potential value as a marker of pulmonary hypertension and can be used for pathological section diagnosis and serological diagnostic indicators of pulmonary hypertension.

[0152] Example 2: Application of ANXA2 knockout in improving pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy

[0153] This example uses ANXA2 gene knockout mice to verify that ANXA2 protein deficiency can improve pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy. The specific experimental steps are as follows:

[0154] 1. ANXA2 gene knockout can improve pulmonary arterial hypertension

[0155] Using wild-type mice (ANXA2 + / + , purchased from Saiye Biotechnology Co., Ltd.) and ANXA2 whole-body knockout mice (ANXA2 - / - , purchased from Saiye Biotechnology Co., Ltd.), and the HySu mouse pulmonary hypertension model was induced by hypoxia combined with Su5416.

[0156] After the pulmonary hypertension model of HySu mice was established, the following analyses were performed: 1) Body weight was measured; 2) Ultrasound analysis of pulmonary artery blood flow velocity was used to indirectly measure pulmonary artery pressure: two-dimensional and pulsed Doppler ultrasound was performed using ultra-high resolution ultrasound, and the PWD of pulmonary blood flow was obtained at the level of the aortic valve in the parasternal aortic valve right ventricular outflow view, and PAT (time from the start of blood flow to peak velocity) and PET (time from the start to the end of pulmonary blood flow) were measured. Pulmonary artery pressure was evaluated based on the PAT / PET ratio; 3) Direct measurement of pulmonary artery pressure: Under tribromoethanol anesthesia, a catheter was inserted into the right ventricle, and the right ventricular pressure (RVSP) was measured using a multi-conductor physiograph; 4) After detecting RVSP, the right ventricle, left ventricle + ventricular septum and lung tissues of the mice were obtained, and gene and protein expression in the lung tissues was detected.

[0157] mRNA and protein were extracted from lung tissue to verify whether ANXA2 was successfully knocked out. Real-time quantitative PCR results showed that ANXA2 - / - The mRNA level of ANXA2 in the lung tissue of mice was significantly lower than that of wild-type mice ( Figure 2 A); Meanwhile, Western blot results showed that ANXA2 - / - There is no ANXA2 protein expression in mouse lung tissue ( Figure 2 B); In addition, immunohistochemical staining results showed that ANXA2 - / - ANXA2 is not expressed in mouse lung tissue ( Figure 2 C). The above results indicate that ANXA2 - / - There is no ANXA2 expression in mouse lungs.

[0158] The results of intubation pressure testing showed that ANXA2 - / - The right ventricular pressure (RVSP) of mice was significantly lower than that of wild-type mice ( Figure 2D and E). In addition, ultrasound data showed that ANXA2 - / - The PAT / PET ratio of mice was significantly higher than that of wild-type mice, indicating that the acceleration time of pulmonary artery blood flow was longer than that of wild-type mice. It was also confirmed that knocking out ANXA2 could inhibit the increase of pulmonary artery pressure ( Figure 2 F and G). The above experimental results show that ANXA2 gene knockout can significantly improve HySu-induced pulmonary hypertension.

[0159] 2. ANXA2 gene knockout can improve pulmonary artery vascular remodeling

[0160] We further used the above-mentioned HySu-induced ANXA2 + / + and ANXA2 - / - The lung tissue of the mouse model of pulmonary hypertension was paraffin-embedded and stained with hematoxylin-eosin (H&E) to observe the pulmonary artery vascular remodeling. The results showed that whether the pulmonary artery was larger than 50 μm in diameter or smaller than 50 μm in diameter, ANXA2 + / + The mice all underwent significant vascular remodeling (i.e., the pulmonary artery wall was significantly thickened), and in the case of ANXA2 - / - In mice, pulmonary artery thickness was significantly reduced ( Figure 3 In addition, we performed immunofluorescence staining on mouse lung tissue sections, using vascular smooth muscle cell-specific antibody α-SMA and cell proliferation indicator PCNA for staining. The results showed that the pulmonary artery smooth muscle cells of wild-type mice were very abundant and contained a large number of PCNA-positive cells, while in ANXA2 - / - In the pulmonary artery, smooth muscle cells were fewer and PCNA-positive cells were significantly reduced ( Figure 3 C and D), indicating that knockout of ANXA2 inhibited the proliferation of pulmonary artery smooth muscle cells in the HySu-induced pulmonary hypertension model. The above results prove that knockout of ANXA2 can significantly improve pulmonary artery vascular remodeling.

[0161] 3. ANXA2 gene knockout can improve right ventricular hypertrophy

[0162] By analyzing the ultrasound results, we found that ANXA - / - The thickness of the right ventricular free wall of mice was significantly reduced compared with that of wild-type mice ( Figure 4 A and B). We further weighed the right ventricle (RV), left ventricle + ventricular septum (LV + S) and body weight (BW) of mice, and calculated the right ventricle / (left ventricle + ventricular septum) ratio (RV / LV + S, i.e., Fulton index) and right ventricle / body weight ratio (RV / BW). The results showed that ANXA - / - The two ratios of mice were significantly lower than those of wild-type mice ( Figure 4C and D). The above results indicate that knocking out ANXA2 gene can significantly improve right ventricular hypertrophy in the process of pulmonary hypertension.

[0163] The above results indicate that ANXA2 can be used as a therapeutic target to prevent, treat, alleviate and / or improve pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy.

[0164] Example 3: Use of ANXA2 inhibitors in the prevention and / or treatment of pulmonary arterial hypertension

[0165] ANXA2 inhibitors can be substances that inhibit the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the ANXA2 gene, or substances that inhibit or reduce the content, activity and / or function of the ANXA2 protein. This example exemplarily uses RNA interference (RNAi) technology to verify the use of ANXA2 inhibitors in the prevention and / or treatment of pulmonary hypertension, and the specific steps are as follows:

[0166] 1. Experimental groups:

[0167] ANXA2 was knocked down in pulmonary artery smooth muscle cells by ANXA2-specific small interfering RNA (siRNA), and the experimental groups were as follows:

[0168] Experimental group (ANXA2-siRNA): transfected with ANXA2-siRNA designed by the present invention;

[0169] Negative control (NC-siRNA): transfected with NC-siRNA.

[0170] 2. Design and sequence of siRNA

[0171] The sequence of the negative control group NC-siRNA is as follows:

[0172] NC-siRNA sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3',

[0173] NC-siRNA antisense strand (antisense): 5'-ACGUGACACGUUCGGAGAATT-3'.

[0174] The sequence of ANXA2-siRNA in the experimental group is as follows:

[0175] ANXA2-siRNA sense strand: 5'-GCAAAUCCCUGUACUACUUTT-3' (SEQ ID No. 1),

[0176] ANXA2-siRNA antisense strand (antisense): 5′-AAGUAGUACAGGGAUUUGCTT-3′ (SEQ ID No. 2).

[0177] 3. Transfection

[0178] The company synthesized siRNA and used Lipofectamine TM Pulmonary artery smooth muscle cells were transfected with Lipofectamine 3000 transfection reagent (purchased from Thermo Fisher Scientific, catalog number L3000015). The transfection steps were as follows: Pulmonary artery smooth muscle cells were cultured in 6-well plates until the confluence reached 70-80%. TM 3000 transfection reagent instructions, use Opti-MEM medium to add control siRNA and ANXA2-siRNA to different wells, and replace normal medium after 6 hours. Perform subsequent detection 48 hours after transfection.

[0179] 4. Real-time quantitative PCR detection of ANXA2 mRNA level

[0180] After collecting the treated pulmonary artery smooth muscle cells, mRNA was extracted using an RNAiso kit (Takara, Catalog No. 9108) and then reversed to cDNA using a cDNA reverse transcription kit (Thermo Fisher Scientific, Catalog No. K1622). Real-time quantitative PCR was then used to detect the expression level of ANXA2. The primer sequences for ANXA2 are as follows: F: 5'-CAGCTTGGAGGGTGATCATT-3', R: 5'-CATTGCTGCGGTTAGTCAGA-3'. The real-time quantitative PCR reaction was performed using Power Green PCR MasterMix Kit (ABI, Cat. No. 4367659) was used and the reaction system was carried out according to its instruction manual. The PCR reaction conditions were: 95°C for 30 seconds, (95°C for 10 seconds, 60°C for 30 seconds, 40 cycles), and finally the melting curve was detected.

[0181] 5. Western blot detection of ANXA2 protein expression level

[0182] After the treatment, the pulmonary artery smooth muscle cells were collected and the total protein was extracted, and the expression of ANXA2 was detected by Western blot. The ANXA2 antibody was purchased from Santa Cruz Company with the catalog number of sc-28385.

[0183] 6. EdU incorporation experiment

[0184] Using BeyoClick TMEdU cell proliferation kit and Alexa Fluor 488 in vitro imaging kit (Biyuntian, Cat. No. C0071L) were used for EdU incorporation assay. PASMCs growing in logarithmic numbers were plated at 5 × 10 4 The number of cells was plated on a 12-well plate. After 24 hours of the designated treatment, each well was incubated with 1 ml of 10 μM EdU medium for 2 hours. The cells were fixed with 4% paraformaldehyde for 30 minutes. After washing 3 times with PBS, the cells were washed with PBS containing 0.5% TritonX-100 for 10 minutes. Then, the reaction solution was prepared according to the kit instructions and added to each well and incubated in the dark at room temperature for 30 minutes. The staining solution was discarded and the cells were washed with PBS for 10 minutes. For nuclear staining, 1× Hoechest-33342 was added and incubated at room temperature for 30 minutes. After washing with PBS, positive cells were observed by fluorescence microscopy.

[0185] 7. Cell migration assay

[0186] Transwell assays were performed using 24-well plates with polycarbonate membrane filters (Corning) containing 8 μM pores. 5 Starved pulmonary artery smooth muscle cells were seeded into the upper chamber, and serum-free medium or 2.5% FBS medium containing PDGF-BB was added to the lower chamber. After 24 hours, cells on the upper or lower surface of the transwell insert were fixed with methanol for 15 minutes and stained with crystal violet for 30 minutes at room temperature. Cells on the upper surface of the filter were scraped off. Cells that migrated to the lower surface were counted in five random fields (20x magnification) using an Olympus inverted microscope.

[0187] 8. Results and Analysis

[0188] The results are as follows Figure 5 and Figure 6 As shown. ANXA2-specific small interfering RNA can effectively knock down the mRNA and protein expression levels of ANXA2 ( Figure 5 AC). Subsequently, we found that knocking down ANXA2 could significantly inhibit the proliferation of pulmonary artery smooth muscle cells ( Figure 5 D and E). At the same time, knockdown of ANXA2 can also significantly inhibit EdU incorporation in pulmonary artery smooth muscle cells, further proving that knockdown of ANXA2 inhibits the proliferation of pulmonary artery smooth muscle cells ( Figure 5 F and G). Transwell assay showed that knockdown of ANXA2 could significantly inhibit the migration of pulmonary artery smooth muscle cells ( Figure 6 A and B).

[0189] The above analysis shows that ANXA2 inhibitors (such as siRNA for knocking down ANXA2) can significantly inhibit the proliferation and migration of pulmonary artery smooth muscle cells, which are important factors leading to pulmonary artery vascular remodeling. By inhibiting the proliferation and migration of pulmonary artery smooth muscle cells, the process of pulmonary artery vascular remodeling can be effectively slowed down (references: PMID: 39206778; 24134901), thereby preventing and treating pulmonary hypertension.

[0190] Example 4: Overexpression of ANXA2 can promote proliferation and migration of pulmonary artery smooth muscle cells

[0191] Using pcDNA3.1 as the basic vector plasmid, the coding region (CDS) of rat ANXA2 (GenBank Accession No. NM_019905 (VERSION: NM_019905.1) from position 43 to 1062) was constructed into the pcDNA3.1 vector plasmid to construct a rat ANXA2 overexpression plasmid. TM The cells were transfected with 3000 transfection reagent, and the overexpression was detected after 36 hours. The effects of overexpression of ANXA2 on cell proliferation and migration were studied according to the EdU staining and Transwell method mentioned above. The results showed that after transfection of ANXA2 overexpression plasmid, the gene and protein expression levels of ANXA2 were significantly increased, accompanied by upregulation of protein expression of cell proliferation indicators PCNA and Cyclin D1 ( Figure 7 AC). Furthermore, we found that overexpression of ANXA2 increased EdU incorporation, demonstrating increased cell proliferation ( Figure 7 D). In addition, we used transwell experiments to demonstrate that overexpression of ANXA2 can significantly promote the proliferation of pulmonary artery smooth muscle cells ( Figure 8 A and B).

[0192] The above results indicate that ANXA2 can promote the proliferation and migration of pulmonary artery smooth muscle cells, and its up-regulated expression may become a marker for the diagnosis of pulmonary hypertension.

[0193] Example 5. Inhibition of ANXA2 Thr208 phosphorylation can inhibit the proliferation and migration of pulmonary artery smooth muscle cells. Analysis of ANXA2 protein sequences in different species (human, rat, mouse) revealed the presence of a potential PKA phosphorylation site (RKGT) ( Fig. 9A), a plasmid (i.e., ANXA2) in which the Thr208 site of rat ANXA2 (amino acid sequence as shown in SEQ ID No. 3, GenBank Accession No. NP_063970 (VERSION: NP_063970.1)) was mutated to alanine (an amino acid that cannot be phosphorylated) was constructed by point mutation. T208A )( Fig. 9 B), and then the plasmid was transfected into pulmonary artery smooth muscle cells in the same manner as the ANXA2 overexpression plasmid described above. Cells were collected 36 hours after transfection, and the mRNA of ANXA2 was detected by real-time quantitative PCR ( Fig. 9 C), the total protein expression of ANXA2 was detected by western blot ( Fig. 9 D and E), the results showed that overexpression of mutant ANXA2 T208A Furthermore, we compared the expression of wild-type ANXA2 plasmid and ANXA2 T208A The expression levels of ANXA2 protein, phosphorylated ANXA2(T208) protein, PCNA and Cyclin D1 in pulmonary artery smooth muscle cells were investigated. T208A Although the total protein of ANXA2 could be overexpressed after plasmid transfection, its phosphorylation level was significantly reduced, and the corresponding expression levels of PCNA and Cyclin D1 were significantly inhibited ( Fig. 9 F and G), indicating that mutation of ANXA2 Thr208 site can inhibit the proliferation of pulmonary artery smooth muscle cells. Further EdU staining showed that, consistent with the above results, overexpression of ANXA2 promoted EdU incorporation, while mutation of ANXA2 Thr208 to alanine significantly reduced EdU incorporation ( Fig. 9 H). In addition, transwell experiments showed that mutation of ANXA2 Thr208 to alanine significantly inhibited wild-type ANXA2-induced pulmonary artery smooth muscle cell migration ( Fig.10 A and B).

[0194] The above results indicate that phosphorylation of ANXA2 Thr208 plays an important role in the proliferation and migration of pulmonary artery smooth muscle cells. Inhibiting phosphorylation of ANXA2 Thr208 can inhibit the proliferation and migration of pulmonary artery smooth muscle cells, thereby playing a role in preventing and treating pulmonary hypertension.

[0195] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Any of the following uses of ANXA2 inhibitors: A1) Use in the preparation of a product for preventing and / or treating pulmonary hypertension; A2) Use in the preparation of a product for preventing and / or treating pulmonary artery vascular remodeling; A3) Use in the preparation of a product for preventing and / or treating right ventricular hypertrophy; A4) Use in the preparation of a product for inhibiting the proliferation and / or migration of pulmonary artery smooth muscle cells; The ANXA2 inhibitor includes any of the following: B1) substances that inhibit the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the ANXA2 gene; B2) Substances that inhibit or reduce the content, activity and / or function of ANXA2 protein.

2. The use according to claim 1, characterized in that: The ANXA2 inhibitor includes any of the following: C1) Nucleic acid molecules for silencing or knocking out the ANXA2 gene; C2) Antibodies that bind to ANXA2 protein.

3. The use according to claim 1 or 2, characterized in that: The ANXA2 inhibitor includes any of the following: D1) siRNA, wherein the nucleotide sequence of the sense strand of the siRNA is nucleotides 1 to 19 of SEQ ID No. 1 or SEQ ID No. 1; the nucleotide sequence of the antisense strand of the siRNA is nucleotides 1 to 19 of SEQ ID No. 2 or SEQ ID No. 2; D2) a DNA molecule encoding the siRNA described in D1); D3) An expression cassette, a recombinant vector or a recombinant host cell containing the siRNA described in D1) or the DNA molecule described in D2).

4. The use according to claim 1, characterized in that: The ANXA2 inhibitor includes an ANXA2 protein Thr208 site phosphorylation inhibitor.

5. The use according to claim 4, characterized in that: The phosphorylation inhibitor is an ANXA2 mutant protein, which is obtained by mutating the threonine at position 208 of the amino acid sequence of the ANXA2 protein to an amino acid that cannot be phosphorylated.

6. Use of ANXA2 protein, ANXA2 gene or Thr208 phosphorylation site of ANXA2 protein as a target in screening or developing products having any of the following functions: E1) Prevention and / or treatment of pulmonary hypertension; E2) prevention and / or treatment of pulmonary artery vascular remodeling; E3) prevention and / or treatment of right ventricular hypertrophy; E4) inhibits the proliferation and / or migration of pulmonary artery smooth muscle cells.

7. A mutant protein, characterized in that The amino acid sequence of the mutant protein is a sequence obtained by mutating the threonine at position 208 of SEQ ID No. 3 to an amino acid that cannot be phosphorylated.

8. Biomaterial, characterized in that The biological material is any of the following: F1) The siRNA according to claim 3; F2) a DNA molecule encoding the siRNA according to claim 3; F3) a DNA molecule encoding the mutant protein according to claim 7; F4) an expression cassette containing the DNA molecule described in F2) or F3); F5) a recombinant vector containing the DNA molecule described in F2) or F3), or a recombinant vector containing the expression cassette described in F4); F6) a recombinant microorganism containing the DNA molecule described in F2) or F3), or a recombinant microorganism containing the expression cassette described in F4), or a recombinant microorganism containing the recombinant vector described in F5); F7) a recombinant host cell containing the DNA molecule described in F2) or F3), or a recombinant host cell containing the expression cassette described in F4), or a recombinant host cell containing the recombinant vector described in F5); F8) An extracellular vesicle containing the DNA molecule described in F2) or F3), or an extracellular vesicle containing the expression cassette described in F4), or an extracellular vesicle containing the recombinant vector described in F5).

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the biomaterial according to claim 8.

10. Use of ANXA2 antibody or ANXA2 Thr208 phosphorylated antibody in the preparation of a product for screening or diagnosing pulmonary arterial hypertension.

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