Application of miR-30a in enhancing I-type IFN (interferon) antiviral immune response of immune cells

Abstract

The invention provides an application of miR-30a in enhancing I-type IFN (interferon) antiviral immune response of immune cells, the miR-30a can enhance RIG-I K63 ubiquitination by down-regulating the expression of USP14, and further enhance I-type IFN antiviral signals, thereby playing a broad-spectrum antiviral role. The invention provides a new prevention and treatment strategy for virus infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of miR-30a in antiviral treatment. Background Art

[0002] Viruses are small infectious bodies that replicate only inside the living cells of an organism. Symptoms of viral infections can range from mild to severe. If left untreated, viral infections can result in death. Currently, the main antiviral treatment strategies include preventive vaccination and therapeutic drug intervention. However, since it accelerates the generation of viruses and the emergence of drug-resistant strains, and most antiviral therapies are used for specific viral infections, there is an urgent need to develop new broad-spectrum antiviral strategies.

[0003] Viral infection can cause a variety of diseases, and innate immunity is the first natural line of defense of the host's immune system against pathogenic microorganisms. The antiviral innate immune response induces host cells to produce interferon to inhibit the reproduction and proliferation of viruses. Viruses will escape the innate immune response during evolution. Therefore, inducing the innate immune response to produce interferon provides a new idea for antiviral treatment. Summary of the invention

[0004] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed.

[0005] The first aspect of the present invention provides any of the following applications:

[0006] (1) Application of miR-30a promoter in the preparation of drugs for enhancing the antiviral immune response of immune cells;

[0007] (2) Application of miR-30a promoter in antiviral activities or in the preparation of drugs for treating diseases caused by viruses.

[0008] In the present invention, the promoter refers to any substance that can promote the activity of miR-30a, promote the expression level of miR-30a, promote the stability of the miR-30a gene, or promote the effective action time of miR-30a.

[0009] In the present invention, the effect achieved by the promoter can also be achieved by introducing an expression vector containing miR-30a into the host cell. Methods well known to those skilled in the art can be used to construct the expression vector required for the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells, such as kanamycin, gentamicin, hygromycin, and ampicillin resistance. In the present invention, the expression vector is a variety of vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, etc. The expression vector generally also contains a promoter, a replication origin, and / or a marker gene, etc. The introduction of the expression vector into the host cell can use well-known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, liposome transfection, and combination with cell membrane permeable peptides.

[0010] In the present invention, the effect achieved by the promoter can also be achieved by knocking in. Knock-in refers to the targeted insertion of a transgene into the host cell genome, resulting in the expression of the transgene and / or the expression of an altered endogenous target gene (e.g., elevated (including ectopic) or reduced expression), such as by introducing an extra copy of the target gene or by operatively inserting a regulatory sequence that provides enhanced expression of the target gene of the endogenous copy. Knock-in transgenes may include heterozygous knock-ins of transgenes or homozygous knock-ins of transgenes. Knock-in also encompasses conditional knock-ins, in which, for example, animals are exposed to substances that promote such expression, by introducing enzymes that promote recombination at the targeted insertion site, or by some other methods for changing the targeted insertion site, expression of the transgene and / or the expression of the altered endogenous target gene may occur.

[0011] In the present invention, miRNA mimic technology can also be used to synthesize miR-30a mimics by chemical methods to simulate the high-level expression of mature miR-30a in cells to enhance the effect of endogenous miR-30a.

[0012] Furthermore, the miR-30a promoter includes miR-30a itself, miR-30a mimics or other agents that promote miR-30a.

[0013] Furthermore, the other agents that promote miR-30a include but are not limited to nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, peptides, proteins or agents used for gene editing.

[0014] In the present invention, the term nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As equivalents, nucleic acids also include DNA or RNA analogs generated from nucleotide analogs and single-stranded (sense strand or antisense strand) and double-stranded polynucleotides when applicable. Liposomes refer to small vesicles composed of various types of lipids, phospholipids and / or surfactants that can be used to deliver drugs to mammals. Antibodies cover complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed by at least two complete antibodies, and antibody fragments, as long as they exhibit the desired antigen binding activity, they all fall within the scope of protection of the present invention.

[0015] Furthermore, the miR-30a promoter is selected from miR-30a mimics.

[0016] Furthermore, the sequence of the miR-30a mimetic is shown in SEQ ID NO:3 and SEQ ID NO:4.

[0017] In the present invention, treatment refers to the improvement, prevention, or reversal of a disease or condition or at least one distinguishable symptom thereof. Further, the treatment refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated, and the parameter is not necessarily identifiable in mammals or recognized by mammals. Further, the treatment refers to the inhibition or slowing down of a disease or disease course, and this inhibition or slowing down can be physical, such as certain distinguishable adverse symptoms. The "treatment" used in the present invention covers diseases of mammals, especially humans, including: (a) preventing the occurrence of diseases or conditions in individuals who are susceptible to the disease but have not yet been diagnosed with the disease. (b) Inhibiting the disease, such as blocking the development of the disease. Or (c) Alleviating the disease, such as alleviating the symptoms associated with the disease.

[0018] In the present invention, the antiviral means that the infectivity or pathogenicity or reproductive capacity of the virus is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., absent) relative to the reference value. The reference value may refer to the infectivity or pathogenicity or reproductive capacity of the virus before the antiviral operation is performed. The reference value may be an absolute value, a relative value, a value with an upper limit and / or a lower limit, a series of values, an average value, a median, a mean, or a value represented by a reference control or a reference value.

[0019] In the present invention, enhancement refers to allowing a subject or virus to improve its ability to respond to the treatment disclosed herein. For example, the response of the enhancement may include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 98% of the response capacity. As used herein, "enhancement" may also refer to increasing the number of subjects who respond to treatment, such as comprising a broad-spectrum antiviral drug, an anti-influenza virus drug, an anti-herpes virus drug or an anti-hepatitis virus drug. For example, the response of the enhancement may refer to the total percentage of subjects who respond to treatment, wherein the percentage is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 98%.

[0020] In the present invention, subjects include humans, mammals (e.g., cats, dogs, horses, etc.), living cells, and other living organisms. Living organisms can be as simple as, for example, a single eukaryotic cell or as complex as a mammal. Typical patients are mammals, particularly primates, and especially humans. For veterinary applications, a variety of subjects will be suitable, such as livestock such as cattle, sheep, goats, cows, pigs, etc.; poultry such as chickens, ducks, geese, turkeys, etc.; and domestic animals, particularly pets such as dogs and cats. For research applications, suitable subjects will be a variety of mammals, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs, such as inbred pigs, etc.

[0021] Furthermore, the immune cells include but are not limited to macrophages, T cells, B cells, NK cells, iNKT cells, NK92 cells, CTL cells, dendritic cells, myeloid cells, monocytes or neutrophils.

[0022] Furthermore, the immune cells are selected from macrophages.

[0023] Furthermore, the virus includes DNA virus and RNA virus.

[0024] Furthermore, the virus is selected from RNA viruses.

[0025] Furthermore, the RNA virus includes but is not limited to Sendai virus, vesicular stomatitis virus, influenza virus, coronavirus, polio virus, dengue virus, and rotavirus.

[0026] Furthermore, the virus is selected from Sendai virus and vesicular stomatitis virus.

[0027] Furthermore, the macrophages exert antiviral immune response through type I IFN.

[0028] In the present invention, the diseases caused by the virus include but are not limited to respiratory diseases, vesicular stomatitis, influenza, poliomyelitis, dengue fever, and diarrhea.

[0029] A second aspect of the present invention provides a pharmaceutical composition, comprising a miR-30a promoter and / or a USP14 inhibitor.

[0030] Furthermore, the miR-30a promoter includes miR-30a itself, miR-30a mimics or other drugs that promote miR-30a.

[0031] Furthermore, the miR-30a promoter is selected from miR-30a mimics.

[0032] Furthermore, the sequence of the miR-30a mimetic is shown in SEQ ID NO:3 and SEQ ID NO:4.

[0033] Furthermore, the USP14 inhibitors include but are not limited to reagents used in gene editing, nucleic acid inhibitors, protein inhibitors or compounds.

[0034] Furthermore, the nucleic acid inhibitor includes siRNA, shRNA or miRNA.

[0035] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0036] Furthermore, the sequence of the siRNA is shown in SEQ ID NO:7 and SEQ ID NO:8.

[0037] Furthermore, the pharmaceutical composition also includes other antiviral drugs.

[0038] Furthermore, other antiviral drugs include, but are not limited to, broad-spectrum antiviral drugs, anti-influenza virus drugs, anti-herpes virus drugs or anti-hepatitis virus drugs.

[0039] Furthermore, the broad-spectrum antiviral drugs include but are not limited to ribavirin, the anti-influenza virus drugs include but are not limited to oseltamivir and zanamivir, the anti-herpes virus drugs include but are not limited to acyclovir and valacyclovir, and the anti-hepatitis virus drugs include but are not limited to anti-hepatitis B virus drugs and anti-hepatitis C virus drugs.

[0040] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable carrier and / or excipient.

[0041] The pharmaceutically acceptable carrier and / or adjuvant described in the present invention include any substance suitable for human and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reaction), that is, with a reasonable benefit / risk ratio. The pharmaceutically acceptable carrier and / or adjuvant are used to help the stability of the formulation or to help improve its activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration as needed. The pharmaceutical composition thus prepared can be administered by any appropriate administration method known to those skilled in the art as needed. When using the pharmaceutical composition, the pharmaceutical composition described in the present invention is administered to humans at a safe and suitable dosage.

[0042] Furthermore, the pharmaceutically acceptable carrier and / or excipient includes, but is not limited to, a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant and / or a disintegrant. Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, and water; adhesives include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose; surfactants include but are not limited to polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid, and hexadecanol; humectants include but are not limited to glycerol and starch; adsorption carriers include but are not limited to starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants include but are not limited to zinc stearate, glyceride monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.

[0043] Furthermore, the pharmaceutical composition is a single compound preparation or a combination of multiple separate single preparations.

[0044] Furthermore, the multiple single-ingredient preparations in the combination of single-ingredient preparations are administered simultaneously or sequentially.

[0045] Furthermore, the dosage form of the single compound preparation or the combination of two separate single preparations includes a dosage form for enteral administration and a dosage form for parenteral administration.

[0046] Furthermore, the gastrointestinal dosage form includes solutions, drops, tablets, capsules, granules, films, gels, powders, emulsions, suspensions, pills, suppositories, aerosols, sprays, powder mists, patches, ointments or creams.

[0047] Furthermore, the non-intestinal administration dosage forms include injection dosage forms, respiratory tract administration dosage forms, cavity administration dosage forms, mucosal administration dosage forms, and skin administration dosage forms.

[0048] Furthermore, the injectable dosage forms include but are not limited to intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections and intracavitary injections; the respiratory tract administration dosage forms include but are not limited to sprays, aerosols, powder aerosols, etc.; the cavity administration dosage forms include but are not limited to suppositories, aerosols, effervescent tablets, drops, pills, etc., which are used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.; the mucosal administration dosage forms include but are not limited to eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, etc.; the skin administration dosage forms include but are not limited to external solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0049] The third aspect of the present invention provides any of the following methods:

[0050] (1) A method for regulating USP14 / RIG-I K63 ubiquitination / type I IFN, wherein the method regulates USP14 / RIG-I K63 ubiquitination / type I IFN by administering a miR-30a regulator.

[0051] In the present invention, the term "regulate" includes adjusting or controlling.

[0052] Furthermore, the miR-30a is negatively correlated with the USP14. When the miR-30a level increases, the USP14 level decreases, and when the miR-30a level decreases, the USP14 level increases.

[0053] Furthermore, the miR-30a is positively correlated with the RIG-I K63 ubiquitination. When the miR-30a level increases, the RIG-I K63 ubiquitination level increases, and when the miR-30a level decreases, the RIG-I K63 ubiquitination level decreases.

[0054] Furthermore, the miR-30a is positively correlated with the type I IFN. When the miR-30a level increases, the type I IFN level increases, and when the miR-30a level decreases, the type I IFN level decreases.

[0055] Furthermore, the miR-30a inhibits USP14 by binding to the 3'UTR of the USP14 mRNA.

[0056] Furthermore, the K63 ubiquitination includes endogenous K63 ubiquitination and exogenous K63 ubiquitination.

[0057] Furthermore, the type I IFN has a negative feedback regulatory effect on miR-30a, that is, the activation of type I IFN will in turn damage the expression of miR-30a.

[0058] Furthermore, the type I IFN includes IFN-α, IFN-β, IFN-τ, and IFN-ω.

[0059] Furthermore, the type I IFN is selected from IFN-β.

[0060] (2) A method for regulating TBK1 phosphorylation / IRF3 phosphorylation, wherein the method regulates TBK1 phosphorylation / IRF3 phosphorylation by administering a miR-30a regulator.

[0061] Furthermore, the miR-30a is positively correlated with the TBK1 phosphorylation / IRF3 phosphorylation. When the miR-30a level increases, the TBK1 phosphorylation / IRF3 phosphorylation level increases, and when the miR-30a level decreases, the TBK1 phosphorylation / IRF3 phosphorylation level decreases.

[0062] (3) A method for regulating ISGs, wherein the method regulates ISGs by administering a miR-30a regulator.

[0063] Furthermore, the miR-30a is positively correlated with the ISGs. When the miR-30a level increases, the ISGs level increases, and when the miR-30a level decreases, the ISGs level decreases.

[0064] In the present invention, the regulator includes a promoter and an inhibitor.

[0065] Furthermore, the promoter includes miR-30a itself, miR-30a mimics or other agents that promote miR-30a.

[0066] Furthermore, the other agents that promote miR-30a include but are not limited to nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, peptides, proteins, and agents used for gene editing.

[0067] Furthermore, the promoter is selected from miR-30a mimics.

[0068] Furthermore, the sequence of the miR-30a mimetic is shown in SEQ ID NO:3 and SEQ ID NO:4.

[0069] In the present invention, the inhibitor refers to any substance that can inhibit the activity of miR-30a, inhibit the stability of the miR-30a gene, inhibit the expression level of miR-30a, or inhibit the effective action time of miR-30a.

[0070] Furthermore, the inhibitors include but are not limited to reagents used in gene editing, nucleic acid inhibitors, protein inhibitors or compounds.

[0071] Furthermore, the reagents used for the gene editing include but are not limited to adeno-associated virus vectors, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS or reagents used for phiC31.

[0072] Furthermore, the nucleic acid inhibitor includes siRNA, shRNA or miRNA.

[0073] Furthermore, the nucleic acid inhibitor is shown in SEQ ID NO:6.

[0074] A fifth aspect of the present invention provides the use of a reagent for detecting miR-30a in the preparation of a product for diagnosing viral infection or a disease caused by a virus.

[0075] Furthermore, the reagents include but are not limited to oligonucleotide probes that specifically recognize miR-30a, primers that specifically amplify miR-30a, or chips that specifically analyze miR-30a.

[0076] Furthermore, the reagent is selected from primers that specifically amplify miR-30a.

[0077] A sixth aspect of the present invention provides a product for diagnosing viral infection or a disease caused by a virus, the product comprising a reagent capable of detecting the expression level of miR-30a.

[0078] Furthermore, the product also includes but is not limited to a chip, a kit or a nucleic acid membrane strip.

[0079] Furthermore, the kit includes but is not limited to reagents for detecting the expression level of miR-30a gene by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, and mass spectrometry.

[0080] Furthermore, the kit includes instruments or reagents for processing samples.

[0081] Furthermore, the sample includes but is not limited to cells, tissues, blood, urine, saliva or mucus.

[0082] Further, the sample is selected from blood.

[0083] The seventh aspect of the present invention provides the use of USP14 inhibitors in antiviral activities or in the preparation of drugs for treating diseases caused by viruses.

[0084] Furthermore, the inhibitors include but are not limited to reagents used in gene editing, nucleic acid inhibitors, protein inhibitors or compounds.

[0085] Furthermore, the reagents used for the gene editing include but are not limited to adeno-associated virus vectors, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS or reagents used for phiC31.

[0086] Furthermore, the nucleic acid inhibitor includes siRNA, shRNA or miRNA.

[0087] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0088] Furthermore, the sequence of the siRNA is shown in SEQ ID NO:7 and SEQ ID NO:8.

[0089] The eighth aspect of the present invention provides any one of the following applications:

[0090] (1) Application of miR-30a in regulating USP14 / RIG-I K63 ubiquitination / type I IFN.

[0091] (2) Application of miR-30a in regulating TBK1 phosphorylation / IRF3 phosphorylation.

[0092] (3) Application of miR-30a in regulating ISGs.

[0093] The present invention has the following advantages and beneficial effects:

[0094] The present invention provides the use of miR-30a in enhancing the type I IFN antiviral immune response of immune cells, wherein miR-30a enhances RIG-I K63 ubiquitination by downregulating the expression of USP14, thereby enhancing the type I IFN antiviral signal, thereby exerting a broad-spectrum antiviral effect. The present invention provides a new prevention and treatment strategy for viral infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 These are the results of miR-30a expression level after viral infection, wherein, Figure A is the result of the expression of multiple miRNAs after THP-1 cells were infected with VSV virus; Figure B is the result of the expression of miR-30a after THP-1 cells were infected with VSV; Figure C is the result of the expression of miR-30a after THP-1 cells were infected with SeV; Figure D is the result of the expression of miR-30a after THP-1 cells were transfected with poly (I:C)-LMW.

[0096] Figure 2 These are the result graphs of the effect of miR-30a on the replication of VSV or SeV, wherein, Figure A is the result graph of the RNA level and titer of SeV after overexpression of miR-30a; Figure B is the result graph of the RNA level and titer of SeV after knocking down miR-30a; Figure C is the result graph of the RNA level and titer of VSV after overexpression of miR-30a; Figure D is the result graph of the RNA level and titer of VSV after knocking down miR-30a; Figure E is the graph of the VSV-GFP intensity after overexpression of miR-30a; Figure F is the graph of the VSV-GFP intensity after knocking down miR-30a.

[0097] Figure 3 These are the result graphs of the effect of miR-30a on the type I IFN antiviral response triggered by SeV or VSV, wherein Figure A is the result graph of SeV-induced IFN-β, ISG15 and Viperin expression after overexpression of miR-30a; Figure B is the result graph of SeV-induced IFN-β, ISG15 and Viperin expression after knockdown of miR-30a; Figure C is the result graph of VSV-induced IFN-β, ISG15 and Viperin expression after overexpression of miR-30a; Figure D is the result graph of VSV-induced IFN-β, ISG15 and Viperins expression after knockdown of miR-30a.

[0098] Figure 4 Figure 1 is a graph showing the effect of miR-30a on RIG-I K63 ubiquitination and RIG-I signaling, wherein Figure A is a graph showing the phosphorylation of TBK1 and IRF3 triggered by SeV after overexpression of miR-30a; Figure B is a graph showing the phosphorylation of TBK1 and IRF3 after knockdown of miR-30a; Figures C, D, and E are graphs showing the effect of miR-30a on RIG-I ubiquitination caused by exogenous SeV stimulation; Figure F is a graph showing the effect of overexpression of miR-30a on endogenous RIG-I K63 ubiquitination; Figure G is a graph showing the effect of knockdown of miR-30a on RIG-I ubiquitination; Figure H is a graph showing the effect of miR-30a on the type I IFN signaling pathway activated by overexpression of RIG-I; and Figure I is a graph showing the effect of miR-30a on the type I IFN signaling pathway activated by overexpression of TBK1.

[0099] Figure 5Figure 1 is a graph showing the effect of specific binding of miR-30a to USP14 3'UTR on the expression level of USP14, wherein Figure A is a predicted graph of the miR-30a seed region and 3'UTR binding sites; Figure B is a graph showing the relative luciferase activity after the 3'UTR sequence was transfected into HEK293T cells; Figure C is a graph showing the binding sites between the miR-30a seed region and the wild-type (WT) or mutant (Mut) 3'UTR of USP14; Figure D is a graph showing the effect of miR-30a on the 3'-UTR luciferase activity of USP14; Figure E is a graph showing the effect of miR-30a on the 3'UTR luciferase activity of USP14; Figure F is a graph showing the effect of miR-30a on the 3'UTR luciferase activity of USP14. Figure 1 shows the effect of miR-30a on USP14 activity when the UTR binding site to miR-30a mutates; Figure F shows the effect of overexpression of miR-30a on USP14 expression level; Figure G shows the effect of knocking down miR-30a on USP14 expression level; Figure H shows the effect of VSV infection on USP14 expression level in THP-1 cells; Figure I shows the effect of SeV infection on USP14 expression level in THP-1 cells.

[0100] Figure 6 Figures A and B show the effect of USP14 on RIG-I-mediated type I IFN antiviral response. Figure C shows the effect of overexpression of USP14 on SeV-induced IFN-β expression. Figure D and E show the effect of siRNAs targeting USP14 (siUSP14#1 and siUSP14#2) on the mRNA and protein levels of USP14. Figure F shows the effect of knocking down USP14 on the cellular phosphorylation of TBK1 and IRF3 induced by SeV infection. Figure G shows the effect of knocking down USP14 on the ubiquitination of RIG-I.

[0101] Figure 7 Figure 1 is a study on the mechanism of antiviral effect of miR-30a; Figure A is a result diagram showing the effect of miR-30a and miR-30a in the presence of USP14 on the ubiquitination modification of endogenous RIG-I K63; Figure B is a result diagram showing the effect of miR-30a and miR-30a in the presence of USP14 on the ubiquitination modification of exogenous RIG-I K63; Figure C is a result diagram showing the effect of miR-30a and miR-30a in the presence of USP14 on the production of IFN-β; Figure D is a result diagram showing the effect of miR-30a and miR-30a in the presence of USP14 on the production of ISG15 and Viperin; Figure E is a result diagram showing the inhibitory effect of miR-30a and miR-30a on viruses in the presence of USP14. DETAILED DESCRIPTION

[0102] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0103] Example

[0104] 1. Experimental Materials

[0105] The antibodies used in the present invention are shown in Table 1, the cell lines are shown in Table 2, and the oligonucleotide sequences are shown in Table 3. The RIG-I plasmid with a Flag tag, the wild-type ubiquitin, K48-ubiquitin and K63-ubiquitin plasmids with an HA tag (all lysines except K48 or K63 sites in ubiquitin are mutated to alanine) used in the present invention are stored in this laboratory. The USP14 plasmid with a myc tag used in the present invention was provided by Professor Zhong Bo of Wuhan University. The USP14 inhibitor IU1 (HY-13817) was purchased from MCE. The double-stranded RNA mimic poly (I: C) -LMW (tlrl-picw) was purchased from InvivoGen.

[0106] Table 1 Antibody materials

[0107]

[0108] Table 2 Cell lines

[0109]

[0110] Table 3 Oligonucleotides

[0111]

[0112] 2. Experimental Methods

[0113] 1. Cell culture

[0114] THP-1 cells were cultured in 1640 medium (Bio-Channel) containing 10% fetal bovine serum (FBS, Bio-Channel). PMA (200 ng / mL, MedChemExpress, MCE) was added to THP-1 cells and cultured for 24 hours to induce differentiation into macrophages. HEK293T cells were cultured in DMEM medium (Bio-Channel) containing 10% FBS (Bio-Channel). All cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0115] 2. Viral infection and viral titration

[0116] THP-1 and HEK293T cells were infected with Sendai virus (SeV) or vesicular stomatitis virus (VSV). The final infection concentration of SeV was 100 hemagglutinin units / mL (HAU / mL), and the MOI of VSV was 0.1. The cell culture supernatant was collected and measured by TCID 50 The virus titer was determined in Vero cells using the following method: Vero cells were plated at 1×10 4 The cells were inoculated in a 96-well plate at a density of 10 cells / mL. The virus samples were diluted 10-fold and added to the 96-well plate. The culture was repeated three times for 3 days. The virus titer was calculated and expressed as log using the Reed-Muench method. 10 TCID 50 / mL.

[0117] 3. Plasmid construction

[0118] In order to explore the regulatory mechanism of miR-30a on RIG-I-mediated signal transduction, considering the effect of miR-30a on the ubiquitination modification of RIG-I, several genes related to ubiquitin regulation were selected, including ubiquitin-specific peptidase (USP) family members (USP48, USP45, USP22, USP2, USP14 and CYLD) and E3 ubiquitin ligases (TRIM13, TRIM27 and NEDD4). The 3' UTRs of these genes that can complementarily bind to the miR-30a seed region were predicted using online software (https: / / www.targetscan.org / vert_80 / ). Figure 5 Figure A in Figure 1). The 3'UTR sequences of the above genes and miR-30a binding were cloned into the pmiRGLO vector (a dual luciferase expression vector that simultaneously expresses firefly luciferase and Renilla luciferase) to construct a wild-type plasmid. Using the pmiRGLO-USP14-WT plasmid as a template, the 3'UTR of USP14 was mutated, and the 3'UTR mutant plasmid of human USP14 was constructed by fusion PCR technology ( Figure 5 Figure C in the figure).

[0119] 4. Transfection of siRNAs or miRNAs

[0120] siRNAs (60nM), miRNA mimics (80nM) or inhibitors (160nM) were mixed with jetPRIME in opti-DMEM for 15 minutes, transfected into cells, and cultured for 48 hours. Among them, the group transfected with miR-30a mimics was the miR-30a-M group, and the control group was the NC-M group; the group transfected with miR-30a inhibitors was the miR-30a-I group, and the control group was the NC-I group.

[0121] 5. Luciferase reporter gene assay

[0122] (1) 3'UTR luciferase reporter gene assay

[0123] The wild-type or mutant plasmids constructed in step 3 were co-transfected with miR-30a mimics or inhibitors into cells and cultured for 48 hours. The relative luciferase activities of the 3'-UTRs of ubiquitin-specific peptidase (USP) family members (USP48, USP45, USP22, USP2, USP14, and CYLD) and E3 ubiquitin ligases (TRIM13, TRIM27, and NEDD4) were then determined using the Dual-Luciferase Reporter Gene Assay System (Vazyme, DL101-01) by calculating the ratio of firefly luciferase to Renilla luciferase activities.

[0124] (2) IFN-β promoter luciferase reporter gene assay

[0125] HEK293T cells were co-transfected with the IFN-β luciferase (IFN-β-Luc) promoter and pRL-TK plasmid (expressing Renilla luciferase as an internal control) and USP14 expression plasmid or empty vector, cultured for 36 h, and then infected with SeV virus for 12 h. The activation degree of the IFN-β promoter was detected by calculating the ratio of firefly luciferase to Renilla luciferase activity.

[0126] 6. Real-time quantitative PCR (qRT-PCR)

[0127] (1) Quantitative analysis of cellular gene or viral copy number

[0128] Total RNA was extracted from cells using Trizol reagent. First-strand cDNA was synthesized by reverse transcription using random primers in HiScupiptII Q RT SuperMix (Vazyme, R223-01). qPCR was performed using ChamQSYBR Gareen MasterMix (Vazyme, Q311-02) with the LighetCycler480 system (Roche). Relative quantification was performed using GAPDH as an internal reference and 2 -△△CTThe threshold method was used to analyze the expression levels of SeV, VSV, IFN-β, ISG15, Viperin, and USP14 gene mRNA.

[0129] (2) Quantitative analysis of miRNAs

[0130] Total RNA was extracted from cells using Trizol reagent, and then reverse transcribed using the miRNA 1st Strand cDNA Synthesis Kit (Vazyme, MR101-01) using a stem-loop RT primer and a U6 reverse primer. Subsequently, qPCR was performed according to the method for quantitative analysis of cellular genes or viral copies. The relative quantification method was used, with U6 as the internal reference and 2 -△△CT The threshold method was used to analyze the expression levels of miRNAs.

[0131] 7. Immunoblotting analysis

[0132] Cells were lysed using RIPA or NP-40 lysis buffer (Beyotime) supplemented with a protease inhibitor cocktail (VIC MED, China). Cell lysates were sonicated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then electrotransferred to PVDF membranes (Millipore) and blocked with 5% skim milk for 1 hour. After blocking, the membranes were incubated with protein-specific primary antibodies at 4°C overnight, followed by incubation with IRDye® 680CW or 800CW secondary antibodies (LI-COR). Protein bands were finally visualized using Odyssey CLx Image Studio (LI-COR).

[0133] 8. Overexpression of USP14 by lentiviral infection

[0134] In order to overexpress USP14 in THP-1 cells, a lentivirus overexpressing USP14 was obtained by a three-plasmid packaging system. The method is as follows: the recombinant lentiviral vector expressing USP14 was co-transfected into HEK293T cells with pCMVR8.74 and pMD2G plasmids, the cell culture supernatant was collected after 72 hours of culture and concentrated by ultracentrifugation, and finally the concentrated lentivirus was infected into THP-1 cells using polybrene.

[0135] 9. Ubiquitination detection

[0136] Before collecting cells, cells were treated with MG132 (HY-13259, MCE) for 4 hours, then lysed with NP-40 lysis buffer and total protein was extracted. Anti-RIG-I antibody or anti-Flag antibody was added to the total protein sample, incubated at 4°C overnight, and pre-washed magnetic beads were added and incubated at 4°C for 8 hours. The magnetic beads were washed 5 times with PBS buffer. After the last wash, the PBS buffer was aspirated as much as possible and resuspended with 2× loading buffer. The ubiquitination of endogenous or exogenous RIG-I was detected by immunoblotting analysis.

[0137] 10. Statistical analysis

[0138] All experiments were repeated at least twice. GraphPad Prism 8.0 software was used for statistical analysis, and the experimental data were expressed as mean ± standard error. Two-tailed t-test was used to evaluate statistical significance, and the significance level was set as: NS (no significant difference) P>0.05, *P<0.05, **P<0.01, ***P<0.001.

[0139] 3. Experimental Results

[0140] 1. Type I IFN signaling triggered by viral infection downregulates the expression of miR-30a in macrophages

[0141] To identify miRNAs associated with RIG-I-mediated innate immune regulation, we selected several miRNAs and analyzed their expression in THP-1 cells infected with VSV, an RNA virus recognized by RIG-I. Figure 1 As shown, the expression of both miR-122 and miR-30a was significantly reduced after VSV infection (Figure A). Previous studies have shown that miR-122 can enhance the expression of type I IFN by targeting the negative regulator suppressor of cytokine signaling 3 (SOCS3). After VSV infection of THP-1 cells, the expression of miR-30a was significantly downregulated 4 hours after infection and reached the lowest level 24 hours after infection (Figure B). Infection with Sendai virus (another RNA virus recognized by RIG-I) also led to a decrease in miR-30a 12 or 24 hours after infection (Figure C). In addition, poly (I:C) treatment also reduced the expression of miR-30a in a dose-dependent manner (Figure D). In summary, the activation of the type I IFN antiviral signaling pathway can significantly downregulate the expression of miR-30a.

[0142] 2. miR-30a inhibits viral replication in macrophages by enhancing VSV- or SeV-induced type I IFN-mediated antiviral responses

[0143] To investigate whether changes in miR-30a expression levels in macrophages affect the host's antiviral response, we transfected THP-1 cells with miRNA mimics or inhibitors to overexpress or knockdown miR-30a, and then infected these cells with VSV or SeV, respectively. Figure 2 As shown, qRT-PCR and TCID 50 The results showed that overexpression of miR-30a significantly inhibited the RNA level of SeV in cells and the viral load in the cell supernatant (Figure A), while knockdown of miR-30a promoted SeV replication in cells and in the supernatant (Figure B). We observed that miR-30a also had a similar effect on VSV proliferation (Figure C, Figure D). In addition, the changes in GFP intensity observed by fluorescence microscopy further confirmed that overexpression of miR-30a and knockdown of miR-30a had opposite effects on VSV replication (Figure E, Figure F). These results suggest that miR-30a plays a broad-spectrum antiviral role in macrophages.

[0144] To verify whether the antiviral effect of miR-30a is achieved by regulating the antiviral response of type I IFN, we further studied the regulatory effect of miR-30a on the type I IFN signaling pathway in macrophages. Figure 3 As shown, overexpression of miR-30a significantly enhanced SeV-induced IFN-β and the expression of representative ISGs (ISG15 and Viperin) (Figure A), while knockdown of miR-30a inhibited the expression of IFN-β and representative ISGs (ISG15 and Viperin) (Figure B). Similar results were observed in VSV-infected cells as in SeV-infected cells (Figure C, Figure D). Taken together, these results indicate that miR-30a exerts antiviral effects by positively regulating type I IFN-mediated antiviral responses.

[0145] 3. miR-30a positively regulates the type I IFN signaling pathway by enhancing RIG-I K63 ubiquitination

[0146] Activation of the RIG-I-mediated signaling pathway triggers a series of downstream signaling cascades, including phosphorylation of TANK-binding kinase 1 (TBK1) and subsequent phosphorylation and dimerization of interferon regulatory factor 3 (IRF3), leading to the production of type I IFN. Therefore, we investigated the effect of miR-30a on the RIG-I signaling pathway in THP-1 cells. The results of immunoblot analysis are shown in Figure 2. Figure 4As shown, overexpression of miR-30a can promote SeV-triggered phosphorylation of TBK1 and IRF3 (Figure A); conversely, knockdown of miR-30a blocks TBK1 and IRF3 phosphorylation (Figure B). These results indicate that miR-30a can upregulate RIG-I activation in macrophages, thereby enhancing its mediated signal transduction. RIG-I binding to K63 polyubiquitin chains is essential for the activation of the RIG-I signaling pathway, so we further explored the effect of miR-30a on RIG-I ubiquitination. Consistent with the activation effect of TBK1 and IRF3 phosphorylation, miR-30a significantly promoted RIG-I ubiquitination induced by exogenous SeV stimulation, especially K63 ubiquitination, but not K48 ubiquitination (Figures C-E). Similarly, overexpression of miR-30a also enhanced endogenous K63 ubiquitination, while RIG-I ubiquitination was reduced in miR-30a knockdown THP-1 cells (Figure F, Figure G). In addition, miR-30a significantly enhanced the type I IFN signaling pathway activated by RIG-I overexpression, while having little effect on the downstream type I IFN signaling pathway activated by TBK1 overexpression (Figure H, Figure I), indicating that miR-30a directly regulates RIG-I-mediated signal transduction. In summary, our results indicate that miR-30a positively regulates the antiviral response of type I IFN by promoting RIG-I K63 ubiquitination.

[0147] 4. miR-30a targets USP14 3' UTR to inhibit USP14 expression

[0148] The experimental results are as follows Figure 5 As shown, Figure B shows that miR-30a significantly reduced the 3'-UTR luciferase activity of NEDD4. Among these candidate genes, overexpression of miR-30a significantly reduced the 3'-UTR luciferase activity of USP14, while knockdown of miR-30a increased the 3'-UTR luciferase activity of USP14 (Figure D). However, when the site of USP14 3' UTR binding to miR-30a was mutated, miR-30a mimics or inhibitors had no effect on luciferase activity (Figure E). Further studies showed that overexpression of miR-30a reduced the expression level of USP14 protein in THP-1 cells, while knockdown of miR-30a increased the expression level of USP14 protein (Figure F, Figure G).

[0149] To further investigate the association between miR-30a and USP14, we also detected the protein level of USP14 after virus infection. VSV or SeV infection significantly downregulated the expression of miR-30a in THP-1 cells ( Figure 1However, VSV or SeV infection significantly upregulated the expression of USP14 in THP-1 cells (Figure H, Figure I). Taken together, these results further demonstrate that miR-30a inhibits USP14 protein expression by specifically targeting USP14 3'UTR mRNA.

[0150] 5. USP14 weakens type I IFN antiviral response by deubiquitinating RIG-I K63

[0151] Since miR-30a targets USP14, we further explored how USP14 affects the regulation of type I IFN antiviral signaling pathway. Figure 6 As shown in Figures A-C, overexpression of USP14 can inhibit the production of IFN-β and its downstream ISGs (ISG15 and Viperin) induced by SeV virus, suggesting that USP14 is a negative regulator of the type I IFN antiviral signaling pathway. To further verify the role of USP14, we used siRNA to knock down USP14 in cells. Figure 6 The results showed that siRNA#2 had a better effect in knocking down USP14 (Figure D, Figure E). Knockdown of USP14 significantly enhanced the phosphorylation of TBK1 and IRF3 induced by SeV infection (Figure F). At the same time, knockdown of USP14 significantly increased the ubiquitination of RIG-I, especially K63 ubiquitination, but not K48 ubiquitination (Figure G). In summary, we confirmed that USP14 inhibits type I IFN antiviral response by deubiquitinating RIG-I K63.

[0152] 6. miR-30a exerts antiviral effects by downregulating USP14 and enhancing RIG-I K63 ubiquitination

[0153] Since miR-30a can target and inhibit the expression of USP14, and USP14 can lead to RIG-I K63 deubiquitination, we further explored whether miR-30a regulates the RIG-I-mediated antiviral signaling pathway by directly regulating the expression of USP14. Figure 7As shown, miR-30a can enhance both endogenous RIG-I K63 ubiquitination (Figure A, lane 2) and exogenous RIG-I K63 ubiquitination (Figure B, lane 2). As a negative regulator of IFN antiviral signaling, overexpression of USP14 significantly inhibited the increase in endogenous (Figure A, lanes 3 and 4) and exogenous (Figure B, lane 4) RIG-I K63 ubiquitination induced by miR-30a. In addition, similar to the effect of overexpression of miR-30a on USP14, the USP14 inhibitor IU1 enhanced the K63 ubiquitination of RIG-I by inhibiting the enzymatic activity of USP14 (Figure B, lane 3). Accordingly, miR-30a promoted the production of IFN-β and its downstream ISGs (ISG15 and Viperin) (Figure C, Figure D, lane 2), thereby inhibiting the replication of SeV in cells (Figure E, lane 2). However, in the presence of USP14, the increase in IFN-β and ISGs (ISG15 and Viperin) production induced by miR-30a was suppressed (Figure C, Figure D, lane 4), resulting in an increase in SeV replication level compared with the miR-30a-M group (Figure E, lane 4). In summary, we concluded that miR-30a strongly activated the immune response by downregulating USP14 expression and enhancing RIG-I K63 ubiquitination, ultimately exerting an antiviral effect.

[0154] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Any of the following applications: (1) Application of miR-30a promoter in the preparation of drugs for enhancing the antiviral immune response of immune cells; (2) Application of miR-30a promoter in antiviral or preparation of drugs for treating diseases caused by viruses; Preferably, the miR-30a promoter includes miR-30a itself, miR-30a mimics or other agents that promote miR-30a; Preferably, the other agents that promote miR-30a include nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, peptides, proteins or agents used in gene editing; Preferably, the miR-30a promoter is selected from miR-30a mimics; Preferably, the sequence of the miR-30a mimetic is shown in SEQ ID NO:3 and SEQ ID NO:

4.

2. The use according to claim 1, characterized in that: The immune cells include macrophages, T cells, B cells, NK cells, iNKT cells, NK92 cells, CTL cells, dendritic cells, myeloid cells, monocytes, and neutrophils; Preferably, the immune cells are selected from macrophages.

3. The use according to claim 1, characterized in that: The viruses include DNA viruses and RNA viruses; Preferably, the virus is selected from RNA viruses; Preferably, the RNA virus includes Sendai virus, vesicular stomatitis virus, influenza virus, coronavirus, polio virus, dengue virus, rotavirus; Preferably, the virus is selected from Sendai virus, vesicular stomatitis virus; Preferably, the macrophages exert antiviral immune response via type I IFN.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a miR-30a promoter and / or a USP14 inhibitor; Preferably, the miR-30a promoter includes miR-30a itself, miR-30a mimics or other drugs that promote miR-30a; Preferably, the miR-30a promoter is selected from miR-30a mimics; Preferably, the sequence of the miR-30a mimetic is shown in SEQ ID NO: 3 and SEQ ID NO: 4; Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the USP14 inhibitor includes reagents, nucleic acid inhibitors, protein inhibitors or compounds used in gene editing; Preferably, the nucleic acid inhibitor comprises siRNA, shRNA or miRNA; Preferably, the nucleic acid inhibitor is selected from siRNA; Preferably, the sequence of the siRNA is shown in SEQ ID NO:7 or SEQ ID NO:

8.

5. Any of the following methods: (1) A method for regulating USP14 / RIG-I K63 ubiquitination / type I IFN, characterized in that: The method regulates USP14 / RIG-I K63 ubiquitination / type I IFN by administering a miR-30a regulator; Preferably, the miR-30a is negatively correlated with the USP14; Preferably, the miR-30a is positively correlated with the RIG-I K63 ubiquitination; Preferably, the miR-30a is positively correlated with the type I IFN; Preferably, the miR-30a inhibits USP14 by binding to the 3'UTR of the USP14 mRNA; Preferably, the K63 ubiquitination includes endogenous K63 ubiquitination and exogenous K63 ubiquitination; Preferably, the type I IFN has a negative feedback regulatory effect on miR-30a; Preferably, the type I IFN includes IFN-α, IFN-β, IFN-τ, and IFN-ω; Preferably, the type I IFN is selected from IFN-β; (2) A method for regulating TBK1 phosphorylation / IRF3 phosphorylation, characterized in that the method regulates TBK1 phosphorylation / IRF3 phosphorylation by administering a miR-30a regulator; Preferably, the miR-30a is positively correlated with the TBK1 phosphorylation / IRF3 phosphorylation; (3) A method for regulating ISGs, characterized in that the method regulates ISGs by administering a miR-30a regulator; Preferably, the miR-30a is positively correlated with the ISGs.

6. The method according to claim 5, characterized in that The regulators include promoters and inhibitors; Preferably, the promoter includes miR-30a itself, miR-30a mimics or other agents that promote miR-30a; Preferably, the other agents that promote miR-30a include nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, peptides, proteins or agents used in gene editing; Preferably, the promoter is selected from miR-30a mimics; Preferably, the sequence of the miR-30a mimetic is shown in SEQ ID NO: 3 and SEQ ID NO: 4; Preferably, the inhibitor includes reagents, nucleic acid inhibitors, protein inhibitors or compounds used in gene editing; Preferably, the reagents used for gene editing include adeno-associated virus vectors, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS or reagents used for phiC31; Preferably, the nucleic acid inhibitor comprises siRNA, shRNA or miRNA; Preferably, the nucleic acid inhibitor is as shown in SEQ ID NO:

6.

7. Use of reagents for detecting miR-30a in the preparation of products for diagnosing viral infections or diseases caused by viruses; Preferably, the reagent comprises an oligonucleotide probe that specifically recognizes miR-30a, a primer that specifically amplifies miR-30a, or a chip that specifically analyzes miR-30a; Preferably, the reagent is selected from primers that specifically amplify miR-30a.

8. A product for diagnosing viral infection or a disease caused by a virus, characterized in that: The product includes a reagent capable of detecting the expression level of miR-30a; Preferably, the product further comprises a chip, a kit or a nucleic acid membrane strip; Preferably, the kit includes reagents for detecting the expression level of miR-30a gene by RT-PCR method, qRT-PCR method, biochip detection method, Southern blotting method, in situ hybridization method, and mass spectrometry; Preferably, the kit includes an instrument or reagent for processing a sample; Preferably, the sample comprises cells, tissue, blood, urine, saliva or mucus; Preferably, the sample is selected from blood.

9. Use of USP14 inhibitors in antiviral or preparation of drugs for treating diseases caused by viruses; Preferably, the inhibitor includes reagents, nucleic acid inhibitors, protein inhibitors or compounds used in gene editing; Preferably, the nucleic acid inhibitor comprises siRNA, shRNA or miRNA; Preferably, the nucleic acid inhibitor is selected from siRNA; Preferably, the sequence of the siRNA is shown in SEQ ID NO:7 or SEQ ID NO:

8.

10. Any of the following applications: (1) Application of miR-30a in regulating USP14 / RIG-I K63 ubiquitination / type I IFN; (2) Application of miR-30a in regulating TBK1 phosphorylation / IRF3 phosphorylation; (3) Application of miR-30a in regulating ISGs.

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