CN113913511A - Application of OTULIN in preparation of reagents for diagnosing and treating cilia-related diseases - Google Patents

Abstract

The invention discloses application of OTULIN in preparation of a reagent for diagnosing and treating cilia-related diseases, finds that OTULIN regulates and controls generation of primary cilia for the first time, and provides a new means for diagnosis and treatment of the primary cilia-related diseases.

Description

Application of OTULIN in preparation of reagents for diagnosing and treating cilia-related diseases

Technical Field

The invention belongs to the field of biomedicine, and relates to application of OTULIN in preparation of a reagent for diagnosing and treating cilia-related diseases.

Background

Cilia are membrane-bounded projections of the cell surface from centrosomes and comprise a microtubule cytoskeleton, the ciliary axon, surrounded by the ciliated membrane. The axon in mammalian epithelial multifilament hair cells is 9+2, has motor protein arms, and has motility. A single non-motile 9+0 primary cilium is found on epithelial cells, such as renal tubular epithelial cells, or non-epithelial cells, such as chondrocytes, fibroblasts and neurons. All fibrillar membranes contain specific receptors and ion channel proteins that initiate signaling pathways that regulate differentiation, migration and cell growth during development and adulthood (S W, A P-M, M B-D. Maintaining centrosomes and cilia [ J ]. Journal of cell science,2017,130(22): 3789. 3800.). The axons are attached to the matrix below the cell membrane. The matrix originates from the central granule, which is a barrel-shaped structure, and is critical for the formation of centrosomes, cilia and flagella, and structural or functional central granule aberrations cause ciliary lesions (Ea N, Jw R. centrioles, centrosomes, and cilia in health and disease [ J ]. Cell,2009,139(4): 663-.

The primary cilia play an irreplaceable role in the development of the embryo and also have an important function in the tissues of adults. Because cilia exist widely and play a very important role, mutations in cilia genes can cause many types of cilial diseases. The hereditary diseases caused by defects in cilia function or occurrence are called cilia-related diseases, the cilia-related diseases include polycystic kidney disease, nephtophitis disease (NPHP), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), Bardet Biedl Syndrome (BBS) and the like, the symptoms of the diseases mainly include visceral translocation, polydactyly (toes), reduced reproductive capacity, decreased vision, olfaction, hearing, respiratory system infection, polycystic hyperplasia of liver and kidney and the like, and the occurrence rates of the symptoms are different. The study of genes related to ciliogenesis is of great significance for the study of cilia-related diseases as well as developmental diseases.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a molecular marker related to ciliation, and provides a new target for diagnosis and treatment of cilia-related diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a diagnostic product comprising a reagent for detecting OTULIN.

Further, the reagent comprises a reagent for detecting the OTULIN expression level in the sample by a digital imaging technology, a protein immunization technology, a dye technology, a nucleic acid sequencing technology, a nucleic acid hybridization technology, a chromatographic technology and a mass spectrometry technology.

Further, the reagent comprises:

a probe that specifically recognizes OTULIN mRNA; or

Primers for specifically amplifying OTULIN mRNA; or

An antibody that specifically binds to an OTULIN protein.

Further, the product comprises a chip and a kit.

Further, the kit comprises a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry kit, an immunohistochemical detection kit, an ELISA kit and an electrochemiluminescence detection kit.

In a second aspect, the present invention provides a pharmaceutical composition comprising an enhancer of the functional expression of OTULIN.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

Further, the promoter is an agent comprising a nucleic acid encoding a functional OTULIN protein, an activator of OTULIN protein, or an agent comprising OTULIN protein.

Further, the promoter is an expression vector containing an OTULIN nucleic acid.

In a third aspect, the invention provides a pharmaceutical composition comprising an inhibitor of the functional expression of OTULIN.

Further, the inhibitor reduces OTULIN mRNA or egg levels, or reduces OTULIN activity.

Further, the inhibitor includes nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules.

Further, the inhibitor is a nucleic acid inhibitor.

Further, the nucleic acid inhibitor is selected from shRNA, small interfering RNA, dsRNA, micro RNA, antisense nucleic acid or a construct thereof.

Further, the nucleic acid inhibitor is selected from small interfering RNA or a construct thereof.

Further, the sequence of the small interfering RNA is shown as SEQ ID NO: 1 or 2.

In a fourth aspect, the present invention provides a method of screening a candidate drug for treating cilia-related disease, comprising the steps of:

treating a system expressing or containing an OTULIN gene or protein encoded thereby with a substance to be screened; and detecting the expression or activity of the OTULIN gene or protein encoded thereby in said system;

wherein, if the candidate substance can inhibit the expression or activity of OTULIN, the candidate substance is indicated to be a candidate drug for treating cilia-related diseases.

Further, the cilia-related disease is a primary cilia formation disorder disease.

Further, the system includes (but is not limited to): a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

Further, the steps also include: the obtained drug candidates are subjected to further cell experiments and/or animal experiments to further select substances from the drug candidates that can treat the cilia-related disease.

A fifth aspect of the invention provides a method of inhibiting cilia formation, characterized in that an effective amount of an accelerant for OTULIN is administered.

Further, the promoter is an agent comprising a nucleic acid encoding a functional OTULIN protein, an activator of OTULIN protein, an agent comprising OTULIN protein;

further, the promoter is an expression vector containing an OTULIN nucleic acid.

Further, the cilia are primary cilia.

In a sixth aspect, the invention provides a method of promoting cilia formation, comprising administering an effective amount of an inhibitor of OTULIN.

Further, the inhibitor reduces OTULIN mRNA or egg levels, or reduces OTULIN activity.

Further, the inhibitor includes nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules.

Further, the inhibitor is a nucleic acid inhibitor.

Further, the nucleic acid inhibitor is selected from shRNA, small interfering RNA, dsRNA, micro RNA, antisense nucleic acid or a construct thereof.

Further, the nucleic acid inhibitor is selected from small interfering RNA or a construct thereof.

Further, the sequence of the small interfering RNA is shown as SEQ ID NO: 1 or 2.

Further, the cilia are primary cilia.

A seventh aspect of the invention provides the use of any one of:

1) use of a diagnostic product according to the first aspect of the invention in the manufacture of a means for diagnosing a cilia-related disease;

2) use of a pharmaceutical composition according to the second aspect of the invention for the preparation of a model for constructing a cilium-associated disease;

3) use of a pharmaceutical composition according to the second aspect of the invention in the manufacture of a product for inhibiting cilia formation;

4) use of a pharmaceutical composition according to the third aspect of the invention for the manufacture of a medicament for the treatment of a cilia-related disease;

5) use of a pharmaceutical composition according to the third aspect of the invention in the manufacture of a product for promoting cilia formation;

6) use of a method according to the fourth aspect of the invention for screening a candidate drug for the treatment of a cilia-related disease;

7) use of a method according to the fifth aspect of the invention for constructing a model of a cilium-associated disease.

Further, the cilia-related disease includes polycystic kidney disease, nephronophhitis disease, Joubert syndrome, Meckel-Gruber syndrome, and Bardet Biedl syndrome.

Further, the cilia in 3) or 5) are primary cilia.

Further, the model in 2) or 7) is a cell model or an animal model.

Further, the cell model is an RPE-1 cell model.

The invention has the advantages and beneficial effects that:

the invention discovers that OTULIN is related to the generation of primary cilia for the first time, thereby providing a new technical scheme for diagnosing and treating cilia related diseases.

Drawings

FIG. 1 is a western blot of over-expressed OTULIN;

figure 2 is an immunofluorescence plot of the effect of over-expression of OTULIN on primary cilia;

figure 3 is a statistical plot of the effect of over-expression of OTULIN on primary cilia;

FIG. 4 is a western blot of knockdown OTULIN;

figure 5 is an immunofluorescence plot of knockdown OTULIN effects on primary cilia;

figure 6 is a statistical plot of the effect of knockdown of OTULIN on primary cilia.

Detailed Description

Through extensive and intensive research, the invention discovers that OTULIN is related to the generation of primary cilia, inhibits the generation of cilia by inhibiting OTULIN in cultured cells, and performs zebra fish experiments to discover that the inhibition of OTULIN influences the development of zebra fish, the bilateral symmetry defect caused by ciliary dysfunction and scoliosis occur, and the defect caused by the knocking-down of OTULIN can be compensated by overexpression. The present invention has been completed based on the finding that OTULIN may cause diseases associated with ciliary abnormalities, which can be used as a target for diagnosis and treatment of the related diseases.

In the present invention, HOIL-1L (OTU deubiquitinase with linear linkage specificity, gene ID: 90268); including wild type, mutant or fragments thereof. The term encompasses full-length, unprocessed biomarkers, as well as any form of biomarker that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of the biomarkers. The gene ID is available at https:// www.ncbi.nlm.nih.gov/gene/.

In the present invention, the expression of a gene or protein can be determined using any method known in the art. It will be appreciated by those skilled in the art that the means by which gene or protein expression is determined is not an important aspect of the present invention.

The gene markers of the present invention can be detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification techniques, and protein immunodetection techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

The present invention can amplify nucleic acids (e.g., ncRNA) prior to or simultaneously with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification commonly known as NASBA; amplification of the probe molecule itself using RNA replicase (commonly known as Q.beta.replicase), transcription-based amplification methods, and self-sustained sequence amplification.

Protein immunological techniques include, but are not limited to, sandwich immunoassays, such as sandwich ELISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.

Any direct (e.g., using a sensor chip) or indirect method may be used in the detection of the molecular markers of the invention.

In the present invention, the term "probe" refers to a molecule that is capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The term "primer" means an oligonucleotide, whether naturally occurring or synthetically produced in a purified restriction digest, that serves as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be single-stranded or double-stranded and must be long enough to prime synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer depends on many factors, including temperature, source of primer, and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. Factors involved in determining the appropriate length of a primer will be readily known to those skilled in the art.

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

"diabodies" (diabodies) refer to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) joined in the same polypeptide chain (VH-VL). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. Diabodies may be bivalent or bispecific.

In one aspect, the present invention discloses a pharmaceutical composition comprising an OTULIN promoter, wherein the OTULIN promoter refers to any substance that can improve the stability of OTULIN genes or expression products, up-regulate the expression of OTULIN, increase the effective duration of OTULIN action, or promote the transcription of OTULIN genes, and can be used in the present invention as a substance useful for up-regulating the expression of OTULIN, thereby being useful for the preparation of cilia-related disease models.

As a preferred mode of the present invention, the promoter of OTULIN is an expression vector containing OTULIN. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.

In yet another aspect, the present invention discloses a pharmaceutical composition comprising an inhibitor of the functional expression of OTULIN; the inhibitor is any substance which can reduce the stability of the OTULIN gene or an expression product, down-regulate the expression of the OTULIN, reduce the effective acting time of the OTULIN or inhibit the transcription of the OTULIN gene, and the substances can be used for the invention, can be used as substances which are useful for down-regulating the expression of the OTULIN, and can be used for treating cilia related diseases.

As an alternative of the invention, the inhibitor of OTULIN is an antibody that specifically binds to OTULIN. The specific antibody comprises a monoclonal antibody and a polyclonal antibody; the invention encompasses not only intact antibody molecules, but also any fragment or modification of an antibody, e.g., chimeric antibodies, scFv, Fab, F (ab')2, Fv, etc. As long as the fragment retains the ability to bind to the OTULIN protein. The preparation of antibodies for use at the protein level is well known to those skilled in the art and any method may be used in the present invention to prepare such antibodies.

As a preferred mode of the invention, the inhibitor of OTULIN is an OTULIN-specific small interfering RNA molecule. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, which is the RNA interference (RNA interference) process. Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

The nucleic acid inhibitor of the present invention, such as siRNA, can be chemically synthesized or can be prepared by transcribing an expression cassette in a recombinant nucleic acid construct into single-stranded RNA. Nucleic acid inhibitors, such as siRNA, can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells using a variety of techniques known in the art.

In an alternative embodiment of the present invention, the inhibitor of OTULIN may be a "Small hairpin RNA (shRNA)" which is a non-coding Small RNA molecule capable of forming a hairpin structure, and the Small hairpin RNA can inhibit gene expression via an RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.

As a preferred embodiment of the present invention, the inhibitor of OTULIN is an antisense oligonucleotide. The antisense oligonucleotide has a sequence complementary to a target sequence, and by the complementary sequence, the suppression of a target gene can be achieved, the antisense oligonucleotide being a ribonucleic acid or a DNA. As a preferred embodiment, the antisense oligonucleotide comprises at least one chemical modification. Antisense oligonucleotides can comprise one or more Locked Nucleic Acids (LNAs). Locked nucleic acids are modified ribonucleic acids that contain an additional bridge between the 2 'to 4' carbons of the ribose moiety to have a locked (locked) conformation, and thus oligonucleotides with locked nucleic acids have improved thermal stability. Alternatively, the antisense oligonucleotide may comprise Peptide Nucleic Acids (PNA), and the antisense oligonucleotide comprises a peptide-based backbone in place of the sugar-phosphate backbone. Other chemical modifications that the antisense oligonucleotides can comprise include: sugar modifications such as 2 ' -O-alkyl (e.g., 2 ' -O-methyl, 2 ' -O-methoxyethyl), 2 ' -fluoro, and 4 ' -thio-oxy modifications; backbone modifications such as phosphorothioate, morpholino, or phosphocarboxylic acid linkages. The antisense oligonucleotide is 7 to 50 nucleotides, preferably 10 to 40 nucleotides, more preferably 15 to 30 nucleotides, and most preferably 20 to 25 nucleotides in length.

The pharmaceutical composition of the invention comprises active ingredients and pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier includes (but is not limited to) diluents, binders, surfactants, humectants, adsorptive carriers, lubricants, fillers, disintegrants.

Wherein the diluent is lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powder), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrating agent such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonizing agents, chelating agents, pH control agents, and surfactants.

Wherein the stabilizer comprises human serum protein, L-amino acid, sugar and cellulose derivative. The L-amino acid may further include any one of glycine, cysteine and glutamic acid. Saccharides include monosaccharides such as glucose, mannose, galactose, fructose, and the like; sugar alcohols such as mannitol, cellosolve, xylitol, and the like; disaccharides such as sucrose, maltose, lactose, and the like; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, etc. and their derivatives. The cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and sodium hydroxymethylcellulose. Surfactants include ionic or non-ionic surfactants such as polyoxyethylene alkyl esters, sorbitan monoacyl esters, fatty acid glycerides. Additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid, and the corresponding salts (alkali metal or alkaline rare earth metal salts thereof, such as sodium, potassium, calcium, and magnesium salts). Isotonic agents include potassium chloride, sodium chloride, sugars and glycerol. The chelating agent comprises sodium ethylene diamine tetracetate and citric acid.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or injection administration is preferred. The pharmaceutical composition of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or excipients.

The dosage of the pharmaceutical composition of the present invention can be adjusted during the course of treatment depending on the severity of symptoms, the frequency of relapse, and the physiological response of the treatment regimen.

The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example Effect of OTULIN on Primary cilia development

The experimental principle is as follows: under the normal culture condition of serum, primary cilia do not occur, but when the mechanism for inhibiting the primary cilia in cells is defective, the primary cilia are abnormal, the invention uses RNA interference technology (RNAi) to knock down OTULIN endogenous to the cells for 48h, and detects the occurrence condition of the primary cilia of the cells under the normal condition of the serum through immunofluorescence staining.

Experimental cell lines: human retinal epithelial cells (RPE-1)

Experimental materials: ac-tubulin antibody: sigma, cat # T6793, gamma-tubulin antibody: sigma corporation, cat # T6557; CP110 antibody: proteitech, Inc., cat # 12780-1-AP.

The experimental method comprises the following steps:

first, OTULIN overexpression inhibits primary cilia,

to verify that OTULIN inhibits the development of primary cilia, the present inventors constructed wild-type OTULIN plasmids and CS mutants to transfect RPE-1 cells, followed by serum starvation to induce the development of primary cilia, which were detected by immunofluorescent staining.

Experimental procedure for overexpression:

1. cell culture: using advanced DMEM/F12 medium at 37 deg.C, 5% CO₂Culturing the RPE-1 cells in the culture chamber. The next experiment was carried out after the first generation.

2. The seed cell: 5X 10 in the evening before the day⁴cells/ml cell density were plated on 24-well and 6-well plates with slides, respectively, and the next experiment was continued after 12 hours as appropriate depending on the density.

3. The transfection test was carried out by changing the 24-well plate to 200. mu.l of the normal culture solution and the 6-well plate to 900. mu.l of the normal culture solutionThe agent is Lipofectamine^TMLTX Reagent with PLUS^TMReagent, ratio of transfectants: LTX, plus ═ 1: 2.5: 1. the system is as follows:

24-well plate: (0.1. mu.g of plasmids + 0.1. mu.l of plus + 25. mu.l of opti-MEM) + (0.25. mu.l of LTX + 25. mu.l of opti-MEM)

6, a pore plate: (0.25. mu.g of plasmids + 0.25. mu.l of plus + 50. mu.l of opti-MEM) + (0.625. mu.l of LTX + 50. mu.l of opti-MEM)

The system is well configured and then stands for 5min, and then is respectively added into cells. DMEM/F12 medium was changed after 6 hours.

4. After normal culture for 24 hours, the opti-MEM reduced serum culture medium is changed for culture for 48 hours, then the cells are collected for western blot detection and immunofluorescence detection, the western blot detection is the same as the above method, and the experimental steps of immunofluorescence are as follows:

(1) the 24-well plate was placed on ice for 10 minutes, after which the medium was discarded and then washed 3 times with PBS.

(2) Fixing and permeabilizing: 4% paraformaldehyde was fixed at room temperature for 3 minutes, followed by-20 ℃ cell permeabilization with ice methanol (300ul) for 20 minutes, and PBS washing was performed 3 times.

(3) And (3) sealing: blocking solution was prepared by 0.25% PBST + 3% NGS (sheep serum), 300. mu.l per well, and blocked for 1 h.

(4) Primary antibody incubation: the blocking solution was used as an antibody dilution, and the AC-tubulin antibody was diluted in the blocking solution at a ratio of 1:400 and the CP110 at a ratio of 1:500, and each slide was 30. mu.l. Incubate at room temperature for 1 h.

(5) PBS was washed three times for 10 minutes each.

(6) And (3) secondary antibody incubation: taking blocking solution as a secondary antibody diluent, and adding Alex-488 and Alex-647 secondary antibodies into a mixture of 1: the ratio of 400 was diluted into the blocking solution, 30. mu.l each. Incubate at room temperature for 1 h.

(7) PBS was washed 3 times. 10 minutes each time.

(8) Counterdyeing: hoechst for 10 minutes.

(9) Sealing: sigma mounting medium was used to seal the slide.

(10) And (5) placing under a fluoroscope for microscopic examination.

Di, OTULIN knockdown promotes primary cilia development

1. Cell culture: using advanced DMEM/F12 medium at 37 deg.C, 5% CO₂Culturing the RPE-1 cells in the culture chamber. The next experiment was carried out after the first generation.

2. The seed cell: 3 × 10 in the evening before⁴cells/ml cell density were plated on 24-well and 6-well plates with slides, respectively, and the next experiment was continued after 12 hours as appropriate depending on the density.

3. Interference

The interference sequence was designed for OTULIN, purchased from Thermo fisher, as follows:

1#:5′-GGAAGAAUGAGGACCUGGUUGAUAA-3′(SEQ ID NO：1)

2#:5′-GCGGAGGAAUAUAGCCUCUAUGAAG-3′(SEQ ID NO：2)。

preparing an interference system, wherein the final concentration of the interference sequence of the OTULIN is 50nM, the transfection reagent is RNAiMAX (thermo fisher), the total system is a 24-well plate which is 300ul, and the other well plates are half of the volume of the original culture medium. The system is as follows:

24-well plate: (0.75. mu.l siRNA + 50. mu.l opti-MEM) + (0.75. mu.l RNAImax + 50. mu.l opti-MEM)

6, a pore plate: the prepared system was left to stand for 15min (2.5. mu.l siRNA + 50. mu.l opti-MEM) + (2.5. mu.l RNAImax + 50. mu.l opti-MEM), added dropwise to a well plate, mixed well, and cultured in a normal medium after 6 hours.

4. Cells were harvested, 48 hours after interference, the target protein was knocked down by siRNAs, cells in 6-well plates were lysed on M2 buffer ice for 30 minutes (the usual protein inhibitor cocktail was added to M2 buffer), centrifuged at 12000rpm for 15min, protein concentration was measured, and the concentration of all protein lysates was normalized according to a protein concentration standard curve. And finally, detecting the knocking-down effect by immunoblotting.

5. Experimental procedure for immunoblotting

(1) Preparing glue: a10% SDS-PAGE protein gel was prepared. After the concentrated gel solidified, carefully pull off the comb.

(2) Loading: the gel was fixed to an electrophoresis apparatus, a sufficient amount of 1 × Tris-glycine electrophoresis buffer was added, and 40 μ L of each sample (40 μ g total protein) was added to each well.

(3) Electrophoresis: firstly, carrying out electrophoretic separation under the constant voltage of 80V, and adjusting the voltage to 120V after loading runs to the separation gel until the loading runs out of the gel bottom.

(4) Transfer printing: PVDF film transfer printing is carried out for 2.5h under 400 mA.

(5) And (3) sealing: 5% skimmed milk (TBST buffer as blocking diluent) is prepared and blocked for 1 h.

(6) Primary antibody incubation: anti-sheet-HOIP was incubated overnight at 4 ℃.

(7) And (3) secondary antibody incubation: incubation with sheet secondary antibody at room temperature for 1 h.

(8) And (3) developing: the method adopts a substrate catalysis mode. Mixing A, B solution at a ratio of 1:1, dripping on a transfer film, coating, and developing in a cassette.

6. After the cells in the 24-well plate are cultured for 48 hours under the condition of a normal culture medium, the creeping pictures in the 24-well plate are collected, and the occurrence condition of primary cilia is detected by immunofluorescence staining. The immunofluorescence assay procedure was as follows:

(1) the 24-well plate was placed on ice for 10 minutes, after which the medium was discarded and then washed 3 times with PBS.

(2) Fixing and permeabilizing: cells (300ul) were fixed in ice methanol at-20 ℃ for 5min and washed 3 times with PBS.

(3) And (3) sealing: blocking solution was prepared by 0.25% PBST + 3% NGS (sheep serum), 300. mu.l per well, and blocked for 1 h.

(4) Primary antibody incubation: the blocking solution was used as an antibody dilution, and the AC-tubulin antibody was diluted in the blocking solution at a ratio of 1:400 and the gamma-tubulin at a ratio of 1:200, at 30. mu.l per slide. Incubate at room temperature for 1 h.

(5) PBS was washed three times for 10 minutes each.

(6) And (3) secondary antibody incubation: taking blocking solution as a secondary antibody diluent, and mixing Alex-488 and Alex-546 secondary antibodies in a ratio of 1: the ratio of 400 was diluted into the blocking solution, 30. mu.l each. Incubate at room temperature for 1 h.

(7) PBS was washed 3 times. 10 minutes each time.

(8) Counterdyeing: hoechst for 10 minutes.

(9) Sealing: sigma mounting medium was used to seal the slide.

(10) And (5) placing under a fluoroscope for microscopic examination.

Experimental results and analysis:

the invention takes RPE-1 cells as a research model, and finds that the primary cilia generation induced by serum starvation can be inhibited when wild-type OTULIN protein is over-expressed in the RPE-1 cells (figure 1, figure 2 and figure 3), and the primary cilia generation can be effectively promoted by knocking down the OTULIN protein (figure 4, figure 5 and figure 6).

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> military medical research institute of military science institute of people's liberation force of China

Application of <120> OTULIN in preparation of reagents for diagnosing and treating cilia-related diseases

<141> 2021-11-19

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggaagaauga ggaccugguu gauaa 25

<210> 2

<211> 25

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcggaggaau auagccucua ugaag 25

Claims (10)

1. A diagnostic product comprising a reagent for detecting OTULIN;

preferably, the reagent comprises a reagent for detecting the OTULIN expression level in the sample by digital imaging technology, protein immunization technology, dye technology, nucleic acid sequencing technology, nucleic acid hybridization technology, chromatographic technology and mass spectrometry technology;

preferably, the reagent comprises:

a probe that specifically recognizes OTULIN mRNA; or

Primers for specifically amplifying OTULIN mRNA; or

An antibody that specifically binds to an OTULIN protein;

preferably, the product comprises a chip, a kit;

preferably, the kit comprises a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry kit, an immunohistochemical detection kit, an ELISA kit and an electrochemiluminescence detection kit.

2. A pharmaceutical composition comprising an enhancer of OTULIN functional expression;

preferably, the promoter is an agent comprising a nucleic acid encoding a functional OTULIN protein, an activator of an OTULIN protein, an agent comprising an OTULIN protein;

preferably, the promoter is an expression vector containing an OTULIN nucleic acid.

3. A pharmaceutical composition comprising an inhibitor of the functional expression of OTULIN;

preferably, the inhibitor reduces OTULIN mRNA or egg levels, or reduces OTULIN activity;

preferably, the inhibitor comprises a nucleic acid inhibitor, a protein inhibitor, a proteolytic enzyme, a protein binding molecule;

preferably, the inhibitor is a nucleic acid inhibitor;

preferably, the nucleic acid inhibitor is selected from shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid or a construct thereof;

preferably, the nucleic acid inhibitor is selected from the group consisting of small interfering RNA or a construct thereof;

preferably, the sequence of the small interfering RNA is as shown in SEQ ID NO: 1 or 2;

preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

4. A method of screening for a candidate agent for treating a cilia-related disorder, comprising the steps of:

treating a system expressing or containing an OTULIN gene or protein encoded thereby with a substance to be screened; and detecting the expression or activity of the OTULIN gene or protein encoded thereby in said system;

wherein, if the candidate substance can inhibit the expression or activity of OTULIN, the candidate substance is a candidate drug for treating cilia-related diseases;

preferably, the cilia-related disease is a primary cilia formation disorder disease.

5. A method of inhibiting cilia formation, comprising administering an effective amount of an accelerant for OTULIN;

preferably, the promoter is an agent comprising a nucleic acid encoding a functional OTULIN protein, an activator of an OTULIN protein, an agent comprising an OTULIN protein;

preferably, the promoter is an expression vector comprising an OTULIN nucleic acid;

preferably, the cilia are primary cilia.

6. A method of promoting cilia formation, comprising administering an effective amount of an inhibitor of OTULIN;

preferably, the inhibitor reduces OTULIN mRNA or egg levels, or reduces OTULIN activity;

preferably, the inhibitor comprises a nucleic acid inhibitor, a protein inhibitor, a proteolytic enzyme, a protein binding molecule;

preferably, the inhibitor is a nucleic acid inhibitor;

preferably, the nucleic acid inhibitor is selected from shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid or a construct thereof;

preferably, the nucleic acid inhibitor is selected from the group consisting of small interfering RNA or a construct thereof;

preferably, the sequence of the small interfering RNA is as shown in SEQ ID NO: 1 or 2;

preferably, the cilia are primary cilia.

7. Use according to any one of the following:

1) use of a diagnostic product according to claim 1 in the manufacture of a means for diagnosing a cilia-related disease;

2) use of the pharmaceutical composition of claim 2 for the preparation of a model for constructing a cilia-related disease;

3) use of the pharmaceutical composition of claim 2 in the manufacture of a product for inhibiting cilia formation;

4) use of the pharmaceutical composition of claim 3 in the manufacture of a medicament for treating a cilia-related disease;

5) use of a pharmaceutical composition according to claim 3 in the manufacture of a product for promoting cilia formation;

6) use of the method of claim 4 for screening a candidate agent for the treatment of a cilia-related disease;

7) use of the method of claim 5 for constructing a cilia-associated disease model.

8. The use according to claim 7, wherein said cilia-related disease comprises polycystic kidney disease, nephronophhitis disease, Joubert syndrome, Meckel-Gruber syndrome, Bardet Biedl syndrome.

9. The use according to claim 7, wherein the cilia in 3) or 5) are primary cilia.

10. The use of claim 7, wherein the model in 2) or 7) is a cellular or animal model;

preferably, the cell model is an RPE-1 cell model.

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