CN116726180B

CN116726180B - Application of NAT10 inhibitor in polycythemia vera

Info

Publication number: CN116726180B
Application number: CN202310973239.6A
Authority: CN
Inventors: 王芳; 余佳; 王妍然
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of CAMS
Priority date: 2023-08-04
Filing date: 2023-08-04
Publication date: 2023-10-27
Anticipated expiration: 2043-08-04
Also published as: CN116726180A

Abstract

The invention discloses application of an NAT10 inhibitor in polycythemia vera, and the invention discovers that the agent for inhibiting the expression of ribosome interaction protein NAT10 can obviously inhibit the differentiation of hematopoietic stem/progenitor cells to erythrocytes for the first time.

Description

Application of NAT10 inhibitor in polycythemia vera

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a NAT10 inhibitor in polycythemia vera.

Background

Protein composition can determine the fate and function of a particular cell, so accurate regulation of gene expression is critical to the normal function of the cell. mRNA translation is a key step in the regulation of gene expression, but unlike what has been thought in the past to encode a single protein molecule per mRNA and decode the same gene in the same way, a number of studies have demonstrated that translational activity can be regulated, which can occur intercellular, intergenic, and even intragenic: translation of the same mRNA in different cells is not the same, so that translation differences among cells important for cell processes such as differentiation are generated; different mRNAs from different sources are translated differently in the same cell, so that translation differences among genes are generated, and even mRNAs from the same gene can be translated into different proteins in different time or space of the same cell, so that translation differences in genes are formed.

In addition to factors internal to the ribosome such as gene mutation of rRNA, rRNA modification, differences in expression abundance and composition of ribosomal proteins (Ribosomal proteins, RP) and post-translational modification, various ribosomal interacting proteins (Ribosomal associated protein, RAP) that interact with the ribosome are also important factors for regulating the translation activity of the ribosome. RAP has been shown to play an important regulatory role in the processes of participating in the translation of ribosomal subcellular localization mRNA, the ribosome quality control process, the initiation and extension of translation, etc., and is a dynamic molecular mediator linking ribosomes with different biological processes, but at present, no detailed study has been made as to whether RAP participates in the translational regulatory network of erythroid differentiation and in what mechanism to play a regulatory role. In view of this, the invention mainly uses RAP as a research object, and discovers a new rule of translation regulation of erythroid differentiation from the brand new angle of RAP-mediated translation regulation by excavating the function and action mechanism of RAP in erythroid differentiation process, and discovers potential therapeutic targets of diseases related to erythroid abnormal proliferation.

At present, no related research or report on the application of NAT10 inhibitors in inhibiting the differentiation of hematopoietic stem/progenitor cells into erythrocytes is seen, and no related research or report on the application of NAT10 inhibitors in polycythemia vera is seen.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide the application of the NAT10 inhibitor in polycythemia vera.

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

in a first aspect, the invention provides the use of an agent which inhibits NAT10 expression in the manufacture of a medicament for the treatment and/or prophylaxis of polycythemia vera.

Further, the agent that inhibits NAT10 expression includes:

(1) shRNA directed against NAT10 gene;

(2) siRNA against NAT10 gene;

(3) An agent that inhibits NAT10 gene transcription activity;

(4) An agent that inhibits NAT10 mRNA transcription levels;

(5) An agent that promotes degradation of NAT10 mRNA;

(6) An agent that inhibits translation of NAT10 mRNA;

(7) An agent that specifically recognizes a targeting nucleic acid of the NAT10 gene and cleaves to reduce the expression level of NAT 10;

(8) An agent for partially or completely knocking out the NAT10 gene.

Further, the agent for inhibiting NAT10 expression is shRNA aiming at NAT10 gene.

Further, the sequence of the shRNA is shown as SEQ ID NO 1-2 or SEQ ID NO 3-4.

In some embodiments, the agents of the invention that inhibit NAT10 expression include, but are not limited to: agents that inhibit or reduce the expression level of a gene encoding a NAT10 protein, reduce the activity of a NAT10 protein, e.g., agents that inhibit or reduce the expression level of a NAT10 gene, include, but are not limited to: an agent that inhibits the transcriptional activity of the NAT10 gene, an agent that inhibits the transcript level of the NAT10 mRNA, an agent that promotes degradation of the NAT10 mRNA, siRNA against the NAT10 gene, shRNA against the NAT10 gene, an agent that inhibits translation of the NAT10 mRNA, an agent that specifically recognizes and cleaves a targeting nucleic acid of the NAT10 gene to reduce the expression level thereof, dsRNA against the NAT10 gene, microrna against the NAT10 gene, antisense nucleic acid against the NAT10 gene. In other embodiments, inhibition or reduction of NAT10 gene expression can be achieved by knocking out the NAT10 whole gene by administration of a targeting vector. In other embodiments, the agent that reduces NAT10 protein activity may be, for example, a specific antibody that targets NAT10 or a small molecule compound that has the ability to inhibit NAT10 protein activity. In a specific embodiment of the invention, the agent for inhibiting NAT10 expression is shRNA with a coding sequence shown as SEQ ID NO. 1-2 or as SEQ ID NO. 3-4.

In some embodiments, the activity of the NAT10 protein may also be reduced by introducing mutations into it. In other embodiments, mutations are introduced into the functional domain of the NAT10 protein that result in a reduction or loss of their corresponding activity. Mutations may be insertions, deletions or substitutions of 1 or several or even more (e.g. more than 10, more than 20, more than 30) amino acids. The functional domain of the encoded NAT10 protein can be made to have mutations that result in the attenuation or loss of its associated biological activity by administration of agents that act on the NAT10 gene. Such agents may alter the sequence of the NAT10 gene, resulting in corresponding mutations in the encoded NAT10 protein, thereby having reduced activity or loss of activity. For example, the wild-type NAT10 gene can be replaced with a mutated NAT10 gene by homologous recombination techniques, resulting in the expression of a poorly or inactive NAT10 protein.

In the present invention, NAT10 means N-Acetyltransferase 10 (N-Acetyltransferase 10, NAT 10), and NAT10 has a Gene ID 55226 in NCBI (https:// www.ncbi.nlm.nih.gov /). NAT10 can participate in the acetylation modification of proteins such as histone, tubulin, p53/TP53, MDM2 and the like under in vitro conditions, promotes proteasome degradation through the acetylation modification to regulate and control the copying of centrosomes during mitosis, is also the only acetyl transferase for catalyzing acetyl cytosine modification (N4-acetylcysteine, ac 4C) on RNA in a human body, has been found to catalyze 18S rRNA and ac4C modification on tRNA, and recent researches have also found that NAT10 can catalyze ac4C modification of CDS region on mRNA in HeLa cell lines so as to enhance mRNA stability and promote translation.

In the present invention, polycythemia vera (Polycythernia Vera, PV) is a clonal myeloproliferative disease originating from hematopoietic stem cells, characterized by abnormal proliferation of erythrocytes due to clonal proliferation of pluripotent hematopoietic stem/progenitor cells, which are clinically manifested by polycythemia, hypertension, splenomegaly, thrombosis, bleeding tendency, etc., due to increased numbers of erythrocytes. Laboratory tests have shown increased numbers of leukocytes and platelets in addition to increased numbers of erythrocytes. The determination of diagnosis is based on the measurement of the capacity of the isotope red blood cells. Polycythemia vera is a disease that is insidious, and progresses slowly, and may be transformed in advanced stages, including liver and spleen medullary metaplasia and portal hypertension. In a specific embodiment of the present invention, it was found for the first time that inhibition of the expression of the ribosomal interaction protein NAT10 significantly inhibits the differentiation of hematopoietic stem/progenitor cells into erythrocytes, i.e. that NAT10 inhibitors can be used in the treatment of polycythemia vera, and that NAT10 inhibitors can inhibit the abnormal proliferation of erythrocytes caused by the clonal proliferation of pluripotent hematopoietic stem/progenitor cells.

In the present invention, the shRNA refers to short hairpin RNAs, which include two short inverted repeats. shRNA cloned into shRNA expression vectors comprises two short inverted repeats, separated by a stem-loop (loop) sequence in the middle, constituting a hairpin structure, controlled by the pol iii promoter. Then, 5-6T's are attached as transcription terminators for RNA polymerase III. shRNA can be stably integrated into the genome of the cell, allowing for long-term gene knockout.

In the present invention, the siRNA refers to small interfering ribonucleic acids, i.e. double stranded nucleic acids of relatively short length or optionally longer precursors thereof. In some embodiments, the length of the siRNA useful in the present invention is preferably about 20 to 50 bp. However, there is no particular limitation on the length of the usable siRNA in the present invention. For example, the siRNA may initially be present in the cell in a precursor form that is substantially different from the final or processed form of the siRNA that exhibits and exerts gene silencing activity upon or after delivery to the target cell. For example, a precursor form of an siRNA can include a precursor sequence element that is processed, degraded, altered, cleaved, etc., upon or after delivery to produce an siRNA that has mediated gene silencing activity within a cell.

In the present invention, the dsRNA refers to double-stranded ribonucleic acid, and an RNA molecule formed by renaturation of two complementary strands can be cleaved by Dicer enzyme to form siRNA. dsRNA inhibits gene expression by RNA interference (RNAi), and dsRNA does not need to have 100% homology with the target gene sequence, so long as it can inhibit target gene expression.

In the present invention, the micrornas refer to micrornas (mirnas), which are non-coding RNAs about 22 nt long, widely present in various organisms ranging from viruses to humans. Mature mirnas are mainly responsible for negative regulation of post-transcriptional levels of genes, involved in many life processes such as cell proliferation, apoptosis, immunity, neuroendocrine, and stem cell differentiation by causing degradation of their target mRNA or disruption of the translation process.

In the present invention, the antisense nucleic acid means a nucleic acid containing a sequence complementary to a sequence encoding NAT 10. Antisense nucleic acids can be composed of DNA, RNA, or both. The antisense nucleic acid can contain non-complementary bases so long as it is capable of specifically hybridizing under stringent conditions. When an antisense nucleic acid is introduced into a cell, it binds to a target polynucleotide and inhibits transcription, RNA processing, or stability. In addition to antisense polynucleotides, antisense nucleic acids also include polynucleotide mimics that contain a modified backbone, and 3 'and 5' end portions. Such antisense nucleic acids can be appropriately designed based on the sequence information of NAT10 and prepared using methods well known to those skilled in the art.

In a second aspect, the invention provides the use of an agent that inhibits NAT10 expression for non-diagnostic and non-therapeutic purposes in vitro for inhibiting differentiation of hematopoietic stem/progenitor cells into erythrocytes.

Further, the agent for inhibiting NAT10 expression is the agent for inhibiting NAT10 expression described in the first aspect of the present invention.

In a third aspect the present invention provides a pharmaceutical composition for the treatment and/or prophylaxis of polycythemia vera.

Further, the pharmaceutical composition comprises an agent that inhibits NAT10 expression;

preferably, the agent for inhibiting NAT10 expression is an agent for inhibiting NAT10 expression according to the first aspect of the present invention;

more preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant;

most preferably, the pharmaceutical composition further comprises a second active ingredient capable of being used for the treatment and/or prevention of polycythemia vera;

most preferably, the second active ingredient comprises: hydroxyurea, IFN-alpha, lu Suoti Nib, pipobromineAlkyl, busulfan, and, ³² P, pontine, aspirin, lu Suoti, anagrelide.

Further, such pharmaceutically acceptable carriers and/or excipients are described in detail in Remington's Pharmaceutical Sciences (19 th ed., 1995) and are useful to aid stability of the formulation or to aid in enhancing its activity or bioavailability as desired, including but not limited to: buffers, e.g., neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, for example, glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, e.g., glycine; an antioxidant; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); a preservative; and any other pharmaceutically or physiologically acceptable carrier, diluent or excipient that can be used in pharmaceutical compositions as reported in any of the prior art.

In some embodiments, the pharmaceutical compositions of the present invention are suitably administered in a variety of doses depending on the method of formulation, the mode of administration, the age, weight, sex, disease state, diet, time of administration, route of administration, rate of excretion and sensitivity of response of the patient, and in general, the prescription and the dosage of the drug for which the prescription is effective for the desired treatment or prophylaxis can be readily determined by the skilled practitioner.

In some embodiments, the pharmaceutical compositions of the present invention may be formulated as a variety of clinical pharmaceutical dosage forms as needed as a medicament for the treatment and/or prevention of polycythemia vera, including but not limited to: parenteral dosage forms or oral formulations, including injection, aerosol, suppository or subcutaneous dosage forms; the oral preparation comprises tablets, capsules, pills, granules, micro-caplets, suspensions, dripping pills and oral liquid preparations, and in the specific embodiment of the invention, the pharmaceutical dosage form is preferably a parenteral administration dosage form.

In some embodiments, the route of administration of the pharmaceutical composition of the present invention is not limited as long as it can exert a desired therapeutic or prophylactic effect, and includes, but is not limited to: topical, transdermal, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intrathecal, intramuscular, intratracheal, subcutaneous, inhalation, pleural, mucosal, dermal, gastrointestinal, intra-articular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic. In some cases, the administration may be systemic, in some cases, local.

In some embodiments, the administration dosage of the pharmaceutical composition of the present invention is not limited as long as a desired therapeutic effect or prophylactic effect can be obtained, and can be appropriately determined according to the symptom, sex, age, etc. of the subject. The administration dose of the pharmaceutical composition of the present invention can be determined in detail using, for example, the therapeutic effect or the prophylactic effect on a disease as an index.

In the present invention, the treatment and/or prevention refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease (e.g., polycythemia vera). Desirable therapeutic effects include, but are not limited to: preventing occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

In the present invention, the effective amount refers to a therapeutically effective amount, and as used herein an effective amount refers to an amount of a pharmaceutical composition sufficient to treat a specified disorder, condition, or disease (e.g., one or more of ameliorating, reducing, attenuating, and/or delaying symptoms thereof). In some embodiments, the effective amount is an amount sufficient to delay the progression of a disease (e.g., polycythemia vera). In some embodiments, the effective amount is an amount sufficient to prevent or delay disease recurrence. An effective dose can be administered in one or more administrations. Pharmaceutically effective amounts depend on a variety of factors including, but not limited to: characteristic factors (e.g., height, weight, sex, age, and history of administration) of the subject (subject), severity of the disease, etc.

In a fourth aspect, the invention provides an agent for inhibiting the differentiation of hematopoietic stem/progenitor cells into erythrocytes for non-diagnostic and non-therapeutic purposes in vitro.

Further, the agent comprises an agent that inhibits NAT10 expression;

preferably, the agent for inhibiting NAT10 expression is an agent for inhibiting NAT10 expression described in the first aspect of the present invention.

In a fifth aspect, the invention provides the use of NAT10 as a target in the selection of a candidate for the treatment and/or prophylaxis of polycythemia vera.

Further, screening of candidate drugs is performed using the screening method described in the sixth aspect below.

A sixth aspect of the invention provides a method of any one of:

(1) A method of screening for a candidate drug for the treatment and/or prevention of polycythemia vera, the method comprising the steps of:

(1) treating a system expressing or containing NAT10 with a test substance;

(2) detecting expression of NAT10 in the system;

(3) selecting a test substance capable of inhibiting NAT10 expression as a candidate drug;

further, the system is selected from: a cellular system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

Further, the test substances described in step (1) include, but are not limited to: interfering molecules, nucleic acid inhibitors, small molecule compounds, etc. designed for NAT 10.

Further, the method for detecting NAT10 expression in step (2) includes, but is not limited to: reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, ribonuclease Protection Assay (RPA), northern blotting, and DNA chip.

Further, reagents for detecting NAT10 expression include, but are not limited to: primers, probes or antisense nucleotides. One skilled in the art can design primers, probes, or antisense nucleotides capable of specifically binding to NAT10 based on the sequence information of NAT 10.

Further, the candidate drug selected in step (3) is a test substance capable of inhibiting the expression level of NAT10 in the presence of the candidate drug compared to the expression level of NAT10 detected in the absence of the candidate drug.

(2) A method for inhibiting the differentiation of hematopoietic stem/progenitor cells into erythrocytes for non-diagnostic and non-therapeutic purposes in vitro, comprising the steps of: adding an effective amount of an agent that inhibits NAT10 expression to a hematopoietic stem/progenitor cell culture system;

The invention also provides the use of an agent for inhibiting NAT10 expression according to the first aspect of the invention in combination with other agents for the treatment and/or prophylaxis of polycythemia vera.

The present invention also provides a method for treating and/or preventing polycythemia vera, the method comprising the steps of: administering to a subject in need thereof an effective amount of an agent that inhibits NAT10 expression as described in the first aspect of the invention and/or a pharmaceutical composition as described in the third aspect of the invention.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention provides a new application of the NAT10 inhibitor in treating polycythemia vera for the first time. The invention discovers for the first time that inhibiting the expression of the ribosome interaction protein NAT10 can obviously inhibit the differentiation of hematopoietic stem/progenitor cells into erythrocytes, namely, the NAT10 inhibitor can be used for treating polycythemia vera, and in addition, the NAT10 can be used as a target point for developing a medicament for treating polycythemia vera. The invention provides a theoretical basis and a new treatment idea for treating polycythemia vera, and has wide clinical application prospect.

Drawings

FIG. 1 shows subcellular localization of ribosome-associated protein NAT10, wherein, panel A: intersection of the interaction proteomes of NAT10 with the collection of human ribosomal proteins; b, drawing: immunofluorescence identifies NAT10 subcellular localization; c, drawing: western Blot identification NAT10 subcellular localization after nuclear mass separation;

fig. 2 shows the expression of NAT10 in erythroid differentiation, wherein, figure a: expression of NAT10 with erythroid differentiation in public transcriptome data and public mass spectrometry data, panel B: western Blot detects the expression condition of NAT10 in the process of differentiating HSPCs into erythroid, and C diagram: standardized gray statistics of Western Blot detection results;

FIG. 3 is a graph showing that inhibition of NAT10 expression affects erythroid differentiation at the RNA level, wherein, graph A: the upper graph is NAT10-shRNA knockdown efficiency verification, and the lower graph is the standardized gray value statistical result of the upper graph; b, drawing: inhibiting the effect of NAT10 expression on red-related genes (ns represents no significant difference, P <0.05, P < 0.01, P < 0.005);

FIG. 4 is a graph showing the effect of inhibiting NAT10 expression on erythroid differentiation function proteins, wherein, graph A: knocking down NAT10 to prevent erythroid progenitor cells from differentiating, wherein megakaryon-erythroid progenitor cells are mainly arranged in an R1 gate in a flow result, burst colony forming erythrocytes are mainly arranged in an R2 gate, erythroid precursor cells are mainly arranged in an R3 gate, premature erythrocytes and reticulocytes are mainly arranged in an R4 gate, and late erythrocytes and reticulocytes are mainly arranged in an R5 gate; b, drawing: knocking down NAT10 suppresses HbF expression.

Detailed Description

The invention is further illustrated below in conjunction with specific examples, which are provided solely to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

EXAMPLE 1 high expression of the ribosomal interaction protein NAT10 at erythroid progenitor stage

1. Experimental method

The invention selects ribosome interaction protein NAT10 as a research object and selects CD34 ⁺ Hematopoietic stem/progenitor cells (Hematopoietic stem and progenitor cells, HSPCs) were extensively studied using cells induced to differentiate into erythroid cells in vitro for the fifth day as test materials. The purpose of this example is to further determine whether NAT10 acts as RAP for erythroid differentiation to regulate translation by the action of its RNA acetyltransferase and thus affect erythroid differentiation. The specific experimental method is as follows:

(1) Analysis of intersection of interaction proteomes of NAT10 with human ribosomal protein collections

In the intercellular spaces after the ultraviolet irradiation cleaning, 1×10 was collected ⁷ Cells were gently blotted with a pre-chilled lysis buffer (2.5% tris hydrochloride, 3% sodium chloride, 1.5% magnesium chloride, 10% triton X-100, 8% glycerol, 0.1% dithiothreitol, 1% protease inhibitor, 73.9% ultrapure water), lysed for 30 minutes at 4 ℃, centrifuged at 2000 rpm on a centrifuge at 4 ℃ for 15 minutes, transferred to ice, the supernatant cell lysate was transferred to a fresh centrifuge tube, and divided equally into two portions, each lysate was added with 6 μg of anti-NAT 10 antibody, isotype control anti-igg antibody, 60 μl protein a agarose beads, and incubated overnight on a 4 ℃ incubator. The antigen-antibody-magnetic bead complex is adsorbed by a magnetic rack for the next use, after the supernatant is removed, the complex is washed 3 times by a washing buffer (2.5 percent of tris hydrochloride, 6 percent of sodium chloride, 1.5 percent of magnesium chloride, 10 percent of triton X-100, 0.1 percent of dithiothreitol, 1 percent of protease inhibitor and 78.9 percent of ultrapure water), the complex is eluted by a polyacrylamide gel electrophoresis loading buffer, and after the denaturation treatment in a metal bath at 95 ℃ for 5 minutes, the magnetic beads are adsorbed and removed by the magnetic rack. And (3) separating a target strip from the eluted sample by polyacrylamide gel electrophoresis, immersing and dyeing the gel block by using coomassie brilliant blue staining solution after electrophoresis is finished, removing the staining solution, adding a decolorizing solution until the background is blue-removed, and cutting the target strip to perform protein mass spectrum detection after the clear and brilliant strip is presented. For the detection results, the proteins enriched in the NAT10 antibody group were compared with the isotype control antibody groupInformation, and analyzing the set of protein information by intersection with the reported ribosomal proteins.

(2) Immunofluorescence identification NAT10 subcellular localization

Collecting 5×10 cells after cleaning with ultraviolet radiation ⁵ The individual cells were centrifuged, and after resuspension of the cell pellet with 1 mL of 4% paraformaldehyde fixative, incubated at room temperature for 10 minutes. Transferring the incubated cells into a centrifuge for centrifugation, centrifugally washing for 3 times by using a precooled phosphate buffer solution, removing the supernatant as much as possible each time, re-suspending the cell sediment by using 200 mu L of phosphate buffer solution, adding the cell sediment into a slice throwing device, fixing the cell suspension on a glass slide by using the slice throwing device, rapidly airing, dripping a cell permeable liquid, penetrating for 10 minutes at room temperature, discarding the permeable liquid, rinsing for 3 times by using the phosphate buffer solution, discarding the liquid on the glass slide, dripping a sealing liquid, incubating for 30 minutes at room temperature, and carrying out the following steps: anti-NAT 10 antibody is prepared at 1000 dilution, and after the end of blocking, preset diluted antibody is added, and the mixture is incubated for 1 hour at room temperature. Removing liquid on the slide, rinsing 3 times with phosphate buffer containing tween-20, removing liquid on the slide, adding 1:2000 dilutions of the secondary antibody were incubated in a wet box at room temperature for 1 hour in the dark. Removing liquid on the glass slide, rinsing 3 times with phosphate buffer solution containing Tween-20 under light-shielding condition, removing liquid on the glass slide, sucking residual liquid with mirror cleaning paper, dripping anti-fluorescence attenuation tablet, capping glass slide, and sealing with nail polish. Immunofluorescence imaging was performed using a Leica DM6B forward fluorescence microscope.

(3) Western blot identification NAT10 subcellular localization

In the intercellular spaces after the ultraviolet irradiation cleaning, 1×10 was collected ⁷ After centrifugation of individual cells to obtain a cell pellet, 500. Mu.L of a separation buffer (2% hydroxyethyl piperazine acetic sulfuric acid solution, 2% potassium chloride solution, 0.2% magnesium chloride, 0.1% ethylenediamine tetraacetic acid solution, 0.1% ethylene glycol tetraacetic acid solution, 1% dithiothreitol, 1% protease inhibitor, 93.6% ultrapure water) was added thereto and incubated on ice for 15 minutes. After the cell suspension was sufficiently homogenized and dissolved by a 27-gauge needle using a1 mL syringe, it was left on ice for 20 minutes. The sample was centrifuged at 3000 rpm for 5 minutes. Extraction ofSupernatant was passed to a new centrifuge tube containing mainly cytoplasmic histones. After the remaining nuclear pellet was washed with 1 mL isolation buffer and blown off with a pipette tip, 500. Mu.L of the sample was separated as a nuclear histones sample, and the remaining 500. Mu.L of the sample was homogenized 10 times through a 25 gauge needle. And centrifuged at 3000 rpm for 10 minutes. After discarding the supernatant, the precipitate was resuspended in a buffered tris salt solution containing 0.1% sodium dodecyl sulfate to give a chromatin protein sample. Total protein in the sample was quantified using the biquinolinecarboxylic acid method. And after reagent configuration and sample addition incubation are carried out according to the operation of the kit, detecting the absorbance value of the sample at 562 and nm on the enzyme-labeled instrument, and calculating the concentration of the total protein in the sample to be detected through the standard curve and the absorbance value of the sample to be detected. After the protein concentration in the sample to be detected is homogenized, the sample is denatured in a metal bath at 95 ℃ for 5 minutes, agarose gel electrophoresis is carried out, proteins with different molecular weights in the sample are separated, protein strips on gel blocks are transferred onto a PVDF film by a wet transfer method, and the PVDF film is blocked with 5% skimmed milk for 1 hour at room temperature. After flushing PVDF membrane with buffer solution of tris (hydroxymethyl) aminomethane containing Tween, cutting membrane strips where NAT10 protein and nuclear reference protein SNRP70 are located, adding dilution primary anti-NAT 10 solution and dilution primary anti-reference protein solution, and incubating overnight in a shaker at 4 ℃. The next day, after discarding the primary antibody solution, rinsing the membrane strip three times by using a trimethylol aminomethane buffer salt solution containing tween, diluting the secondary antibody by using 5% skimmed milk, adding the secondary antibody into the membrane strip, incubating for 2 hours at room temperature, discarding, rinsing the membrane strip three times by using a trimethylol aminomethane buffer salt solution containing tween, dripping a developing solution, and developing in a dark place.

This example further analyzed the expression of NAT10 by common transcriptome and mass spectrometry data of hematopoietic lineage differentiation, and detected CD34 isolated from cord blood by Western Blot ⁺ The whole protein expression level of NAT10 in the process of inducing differentiation of HSPCs to erythroid is as follows:

(1) Public data source and data analysis

The public transcriptome data is derived from transcriptome data sets numbered GSE60101, GSE165863, GSE74246, GSE53938, respectively, in a high-throughput gene expression database (GEO); the mass spectrum data is derived from the proteome data set of PRIDE database number PXD017442, the obtained transcript reads are compared, and the gene expression value heatmap of different differentiation stages is drawn through R language.

(2) Western Blot detection of protein expression level of NAT10 at different stages of erythroid differentiation

Collecting umbilical cord blood, pouring into clean test tube containing hydroxyethyl starch solution, naturally settling red blood cells, sucking the separated supernatant, gently superposing on lymphocyte separation medium, obtaining "white membrane layer" containing mononuclear cells by density gradient centrifugation, and using CD34 ⁺ Magnetic beads to isolate and enrich for CD34 in mononuclear cells ⁺ Cells were cultured in erythroid differentiation medium (1% myo-glucose, 1% folic acid, 0.5% ferrous nitrate, 0.5% ferrous sulfate, 0.1% thioglycerol, 2% glutamine, 16% calf serum, 1% bisantibiotic, 77.9% IMDM medium), and interleukin-3, cortisol, erythropoietin and stem cell growth factor were supplemented to the medium from day 0 to day 7 of induced differentiation. On days 8 to 13 of induced differentiation, erythropoietin and stem cell growth factors are supplemented to the medium. Erythropoietin is supplemented to the medium from day 14 to day 16 of induced differentiation. Collecting 5×10 cells from cells after ultraviolet irradiation cleaning on day 3, day 5, day 6, day 9, day 12 and day 15 of induced differentiation ⁵ And (3) after obtaining cell sediment by centrifugation, adding a proper amount of cell lysate containing protease inhibitors on ice, uniformly mixing by blowing a gun head, then cracking for 30 minutes, and obtaining the cell lysate by centrifugation. Total protein in the sample was quantified using the biquinolinecarboxylic acid method. And after reagent configuration and sample addition incubation are carried out according to the operation of the kit, detecting the absorbance value of the sample at 562 and nm on the enzyme-labeled instrument, and calculating the concentration of the total protein in the sample to be detected through the standard curve and the absorbance value of the sample to be detected. Homogenizing the protein concentration in the sample to be detected, denaturing in a metal bath at 95 ℃ for 5 minutes, performing agarose gel electrophoresis, and separating proteins with different molecular weights in the sampleThe protein bands on the gel block were transferred to PVDF membrane using wet transfer, and blocked with 5% skim milk at room temperature for 1 hour. After flushing PVDF membrane with buffer solution of tris (hydroxymethyl) aminomethane containing Tween, cutting membrane strips where NAT10 protein and internal reference protein are located, adding anti-NAT 10 diluted primary antibody solution and anti-internal reference protein diluted primary antibody solution, and incubating overnight in a shaking table at 4 ℃. The next day, after discarding the primary antibody solution, rinsing the membrane strip three times by using a trimethylol aminomethane buffer salt solution containing tween, diluting the secondary antibody by using 5% skimmed milk, adding the secondary antibody into the membrane strip, incubating for 2 hours at room temperature, discarding, rinsing the membrane strip three times by using a trimethylol aminomethane buffer salt solution containing tween, dripping a developing solution, and developing in a dark place.

2. Experimental results

The interacting proteomes of NAT10 showed that the RP identified in its interacting proteins accounted for 70% (56/80) of all human known RPs, confirming its tight interactions with ribosomes (see FIG. 1A). In the embodiment, the subcellular localization of NAT10 is further detected through an immunofluorescence experiment, and Western Blot experiments after nuclear mass separation prove that NAT10 is mainly localized in nuclear cytoplasm (see fig. 1B-C), and the localization mode suggests that NAT10 can participate in post-transcriptional modification of mRNA.

Analysis of the public transcriptome and mass spectrometry data for each lineage of hematopoietic differentiation showed that NAT10 was specifically and highly expressed mainly in megakaryo-erythroid progenitor stage (see figure 2A). This example further detects CD34 isolated from cord blood by Western Blot ⁺ The overall protein expression level of NAT10 during the induction differentiation of HSPCs into erythroid demonstrated that NAT10 was mainly highly expressed at erythroid progenitor stage (see fig. 2B). NAT10 is presumed to play a specific regulatory role mainly in early erythroid differentiation.

Example 2 inhibition of NAT10 expression inhibits differentiation of hematopoietic Stem/progenitor cells into erythrocytes

1. Experimental method

To further investigate the specific function of NAT10 in erythroid differentiation, this example used lentivirus expressing NAT10shRNA to infect K562 cells, where the protein expression level inhibition effect of shRNA 10-2 and shRNA 10-4 on NAT10 was most pronounced (see fig. 3A), thus confirming that these two groups of lentiviral plasmid vectors could be used for subsequent functional experiments, where the sequence information of shRNA 10-2 and shRNA 10-4 were as follows:

sequence information of shNAT 10-2:

sense strand: 5'-CAGAGCCAGGTCAGCACCACTGCTGAGAA-3' (SEQ ID NO: 1);

antisense strand: 5'-TTCTCAGCAGTGGTGCTGACCTGGCTCTG-3' (SEQ ID NO: 2).

sequence information of shNAT 10-4:

sense strand: 5'-ATGAAGGCAGGTTTCCTTGTCTGGAGGAA-3' (SEQ ID NO: 3);

antisense strand: 5'-TTCCTCCAGACAAGGAAACCTGCCTTCAT-3' (SEQ ID NO: 4).

In this example, the influence on the erythroid phenotype after inhibition of NAT10 expression was first examined, and the erythroid key transcription factors (GATA 1, TAL1, KLF 1) and hemoglobin gene (HBA, HBG, HBB) were used as main indicators of the erythroid phenotype, and the specific experimental method was as follows:

(1) K562 cell culture

Immersing the part of the freezing tube higher than the O-shaped ring into a water bath kettle at 37 ℃, rapidly thawing the cells by shaking, diluting the cell freezing solution with a preheated K562 basal medium IMDM, centrifuging, discarding the supernatant, re-suspending the cell sediment with a K562 complete medium (10% calf serum, 1% double antibiotics and 89% IMDM), adding the cell suspension into a culture vessel containing the complete medium, uniformly mixing in a crisscross manner, placing into a 37 ℃ incubator saturated with 5% carbon dioxide concentration and humidity, observing the growth speed and morphology of the cells under a microscope every day, and for good-growth cells, generally carrying out one-time cell passage (passage ratio is 1:3) for 2 days and replacing fresh complete medium. For the adherent cells 293T, after resuscitating culture, the cells with good general state are required to be subjected to passage treatment through pancreatin digestion, and the cells are subjected to passage once in 2-3 days, wherein the passage ratio is 1:3 to 1:5,293T cells used DMEM as basal medium.

(2) Cell transfection

Selecting good growth state 16-20 hr before transfection293T cells were digested according to 1:3-1:4, so that the confluency of the cells reaches 70-90% during transfection. 2 hours prior to transfection, the medium in the petri dishes was replaced with freshly prepared pre-warmed complete medium (10% calf serum, 1% double antibiotics, 1% sodium pyruvate, 1% glutamine, 87% DMEM). Mu.g of the objective plasmid DNA (shCtrl, shNAT10-2 and shNAT10-4 corresponding plasmid DNA) was mixed with 4.5. Mu.g of psPAX2 and 1.5. Mu.g of pMD2.G plasmid DNA, and 300. Mu.L of serum replacement OPTI-MEM was added to prepare a plasmid mixture. 12. Mu.L of plasmid DNA transfection-promoting reagent was added and allowed to stand at room temperature for 15 minutes to allow the transfection-promoting reagent to react well with the plasmid DNA to form a stable complex. The mixture is added into 293T cell culture medium drop by drop, mixed gently, placed into a 37 ℃ incubator with 5% carbon dioxide concentration and saturated humidity for culturing, and after 4-6 hours, the liquid in the culture dish is all discarded, and the culture dish is replaced by a fresh complete culture medium. After culturing for 48-72 hr, collecting cell culture supernatant via 0.45 μm filter into high speed centrifuge tube, centrifuging at 4deg.C for 3 hr at 20000 rpm to concentrate virus to form precipitate, re-suspending the virus precipitate with IMDM culture medium, adding into K562 cell culture medium to be infected, and controlling cell density at 4X10 ⁵ About one per mL, adding polybrene to promote virus infection, culturing continuously after changing fresh complete culture medium for 12-16 hours after co-culturing virus and K562 cells, adding puromycin to screen cells with high virus infection positive rate after 48-72 hours of co-culturing, and collecting cell sediment for RNA extraction and RT-qPCR experiment.

(3) RT-qPCR (reverse transcription-quantitative polymerase chain reaction) detection of key gene expression condition

Cell pellet infected with lentivirus was obtained every 1×10 ⁷ Adding 1 mL of RNA extraction reagent TRIZOL into individual cells, obtaining a total RNA sample in the cells by referring to the method described above, verifying the quality of the extracted total RNA sample by an ultraviolet spectrophotometer, performing reverse transcription to obtain cDNA, taking GAPDH as an internal reference gene, detecting the expression quantity of a research object NAT10 relative to the internal reference gene by using a Roche real-time fluorescence quantitative PCR detection system to verify the knockdown efficiency, and simultaneously detecting key genes GATA1, TAL1, KLF1 and HBA, HBB, HBG relative to the internal reference geneThe expression level of the gene is used to describe the effect of knocking down NAT10 on the erythroid differentiation key gene.

In this example, the influence of NAT10 on erythroid differentiation cell populations in different stages is further knocked down by flow analysis, and according to the existing study, the expression of CD71 increases and then decreases with erythroid differentiation, while the expression of CD235a increases continuously, and the erythroid differentiation process can be further subdivided by integrating the expression change conditions of both: CD71 and CD235a double negative (CD 71) ^- /CD235 ^- ) The cell population was mainly megakaryo-erythroid precursor cell stage cells, with weak CD71 positive and CD235a negative (CD 71 ^low /CD235 ^- ) The cell population was mainly one that differentiated to the stage of burst colony formation into erythroid progenitors, with high expression of CD71 and CD235a positive (CD 71 ^high /CD235 ⁺ ) Is mainly more mature erythroid precursor cells, CD71 is moderately expressed and CD235a positive (CD 71 ^median /CD235 ⁺ ) Is early and mid erythroid cells differentiated into early and mid erythroid lines, CD71 is low or negative and CD235a positive (CD 71) ^low/- /CD235a ⁺ ) Is late juvenile red blood cells and reticulocytes entering the terminal differentiation stage. The specific experimental method is as follows:

flow analysis of the Effect of knock-down NAT10 on erythroid differentiation of cell populations at different stages

With reference to the above procedure, 293T cells were transfected with a plasmid vector expressing shCtrl (control group), shNAT10-2 and shNAT10-4 to obtain corresponding virus concentrates and infected with CD34 ⁺ Collecting cells infected by HSPCs on the 0 th day of erythroid induced differentiation, respectively collecting fresh cell precipitates infected by lentivirus on the 6 th day, the 9th day and the 12 th day of erythroid induced differentiation, re-suspending and washing once by using phosphate buffer, discarding supernatant, respectively adding 100 mu L of phosphate buffer, 2 mu L of CD71-APC antibody and 1 mu L of CD235a-PE antibody, incubating for 30 minutes at the dark place at 4 ℃, adding phosphate buffer to dilute antibody mixture, centrifuging to remove supernatant containing the antibody, re-suspending each cell precipitate by using 500 mu L of phosphate buffer, filtering by using a 0.45 mu m filter, and quantifying the expression conditions of CD71 and CD235a in a sample by using an Attune NxT flow cytometerAnd (5) detecting. According to the conditions of CD71 and CD235 expressed on the cell surface, the cells can be divided into 5 groups with unequal erythrocyte maturity, and the cell population change conditions of different stages of erythroid differentiation after knocking down NAT10 can be statistically depicted by comparing the grouping conditions of shNAT10-2 group and shNAT10-4 group compared with shCtrl group.

2. Experimental results

The results showed that when NAT10 expression was inhibited at early stage of erythroid differentiation, the transcript expression levels of erythroid key transcription factors GATA1, TAL1, KLF1 were significantly reduced, and HBB, HBG gene transcript expression encoding hemoglobin was also significantly inhibited (see fig. 3B). The results show that inhibiting NAT10 expression can obviously inhibit the expression of erythroid differentiation key genes, and the molecular phenotype is expressed as inhibiting erythroid differentiation.

After monitoring the expression of cell surface markers CD71 and CD235a at each time point of erythroid differentiation, it is found that inhibiting NAT10 expression can significantly affect normal differentiation of erythroid precursor cells into more mature premature erythroid and middle premature erythroid cells (see fig. 4A), and simultaneously significantly inhibit expression of Fetal hemoglobin (HbF) (see fig. 4B), and in combination with the result of high expression level of Fetal hemoglobin in progenitor cell stage, NAT10 mainly plays an important regulatory role in HSPC-erythroid progenitor cell differentiation stage, and inhibiting NAT10 expression can significantly inhibit differentiation of hematopoietic stem/progenitor cells into erythrocytes, namely NAT10 inhibitor can be used in treating polycythemia vera.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. Use of an agent that inhibits NAT10 expression in the manufacture of a medicament for the treatment and/or prevention of polycythemia vera, wherein the agent that inhibits NAT10 expression is shRNA directed against the NAT10 gene;

the sequence of the shRNA is shown as SEQ ID NO 1-2 or SEQ ID NO 3-4.

Use of NAT10 as target in the selection of a candidate drug for the treatment and/or prevention of polycythemia vera, wherein the candidate drug inhibits expression of NAT 10.

3. A method of screening for a candidate drug for the treatment and/or prevention of polycythemia vera, the method comprising the steps of:

(1) treating a system expressing or containing NAT10 with a test substance;

(2) detecting expression of NAT10 in the system;

(3) test substances capable of inhibiting NAT10 expression are selected as drug candidates.