CN116392572A - Application of integrin protein ITGA5 as marker in preparation of medicines for treating acute myelogenous leukemia with high leukocyte - Google Patents
Application of integrin protein ITGA5 as marker in preparation of medicines for treating acute myelogenous leukemia with high leukocyte Download PDFInfo
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Abstract
The invention discloses application of an integrin protein ITGA5 serving as a marker in preparation of a medicament for treating acute myelogenous leukemia with high leucocyte, and belongs to the technical field of medicines. The invention discovers that the specific high-expression integrin ITGA5 of the patients with the high-leukocyte acute myelogenous leukemia (HAML) can effectively inhibit the migration, invasion and proliferation of HAML cells by inhibiting the expression or activity of the whole ITGA5. The invention provides application of ITGA5 as a marker in preparing a medicament for treating HAML, application of an inhibitor of ITGA5 in preparing the medicament for treating HAML and medicament for inhibiting migration, invasion and proliferation of HAML white blood cells, and application of a reagent for detecting expression level of ITGA5 in preparing a diagnostic HAML reagent or a kit.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of an integrin protein ITGA5 serving as a marker in preparation of a medicine for treating acute myelogenous leukemia with high leucocyte.
Background
Acute myeloid leukemia (acute myeloid leukemia, AML) is a clonal hematopoietic stem cell malignancy characterized by accumulation of immature progenitor cells, a hindered differentiation, leading to inhibition of hematopoietic function. White blood cell count in AML peripheral blood image exceeds 100×10 9 at/L, acute myelogenous leukemia (hyperleukocytic acute myeloid leukemia, HAML), which is known as hypercellular carcinoma, accounts for about 5% -20% of AML. HAML has rapid onset, rapid progress, relatively low remission rate, high early death rate, and death rate up to 40% in one week, and belongs to the high-risk type in acute leukemia. Leukemia cells proliferate in bone marrow in large quantity and migrate to the periphery, and hypoxia and embolism are main causes of early death due to stasis in tissues, but the mechanism of malignant proliferation infiltration is not clear.
Integrins are one of the most important mechanical sensitive receptors on cell membranes, mediate the mechanical action of cells and surrounding matrixes or adjacent cells, transduce mechanical stimulation signals into biochemical signals, activate a series of response reactions in the cells, finally influence the functions of cell differentiation, migration and the like, and are of great importance in the research of cell micromechanics. Integrins and related focal adhesion proteins are fundamental and important links in mediating adhesion between cells and extracellular matrix (ECM) and mechanically conducting cell migration. There is currently no uniform understanding of the mechanical biological mechanisms of integrin-mediated cell migration behavior.
Due to the high early mortality rate, some HAML patients do not receive conventional and drug treatment opportunities, and effective markers would be of great significance to diagnosis and treatment of HAML.
Disclosure of Invention
The invention aims to provide an application of an integrin protein ITGA5 serving as a marker in preparing a medicine for treating acute myelogenous leukemia with high leucocyte. The invention also aims at providing the application of the integrin protein ITGA5 in preparing products for diagnosing the acute myelogenous leukemia with high leucocyte.
The aim of the invention is achieved by the following technical scheme:
according to the invention, through collecting bone marrow cells of a HAML patient, a NHAML (non-hypercellular acute myelogenous leukemia) patient and a healthy control patient, single-cell transcriptome and proteomic sequencing are carried out, a HAML single-cell molecular map is drawn, and HAML specific populations and abnormal expression molecules are analyzed, so that the abnormal expression of integrin ITGA5 in the HAML leukemia cells is found. Further expanding the sample size, collecting bone marrow CD34+ cells and bone marrow supernatant of normal people, primary treatment HAML patients and NHAML patients, extracting nucleic acid, and verifying the level expression of integrin genes by PCR, thereby defining the specific high-expression integrin ITGA5 of the HAML patients. The invention also discovers that inhibiting the expression or activity of the integrin protein ITGA5 can effectively inhibit the migration, invasion, proliferation and the like of HAML white blood cells.
The application of integrin ITGA5 as a marker in preparing HAML medicine.
Use of an inhibitor of integrin ITGA5 in the manufacture of a medicament for the treatment of HAML.
Use of an inhibitor of integrin protein ITGA5 in the preparation of a medicament for inhibiting HAML leukocyte migration, invasion, proliferation.
The medicine takes ITGA5 as a target to inhibit ITGA5 gene expression or inhibit ITGA5 protein activity.
The ITGA5 inhibitor comprises: substances inhibiting the expression of ITGA5 gene and substances inhibiting the activity of ITGA5 protein. Further, the inhibitor of ITGA5 is ATN161; or the inhibitor of ITGA5 is an antibody of ITGA5 protein.
Use of a reagent for detecting the expression level of the integrin protein ITGA5 in the preparation of a diagnostic HAML reagent or kit. The reagent for detecting the expression level of the integrin protein ITGA5 comprises QPCR primers.
The invention has the advantages and beneficial effects that: the invention can effectively inhibit migration invasion of HAML white blood cells by inhibiting the expression of integrin protein ITGA5. ITGA5 can be used as a detection target of HAML, and also can be used as a target for treating HAML.
Drawings
FIG. 1 is a graph showing that ITGA5 inhibitor ATN161 inhibits leukocyte migration in HAML patients.
FIG. 2 is a graph showing that ITGA5 inhibitor ATN161 inhibits leukocyte invasion in HAML patients.
FIG. 3 shows QPCR detection of ITGA5 expression in HAML/NHAML/normal human leukocytes.
FIG. 4 is a graph showing the detection of ITGA5 expression in HAML/NHAML/normal human bone marrow supernatant by ELISA.
FIG. 5 is a graph showing that ITGA5 expression was elevated in leukocytes of different HAML patients by transcriptome sequencing, and that baseline was normal.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Acquisition of white blood cells from HAML patients used in the following examples: the bone marrow of the HAML patient is extracted and stored at low temperature and sent to a laboratory for separation and culture as soon as possible. The bone marrow fluid was gently added to a 15mL sterile centrifuge tube containing an equal volume of Percoll working fluid (density 1.073 g/L), taking care to be as gentle and smooth as possible. Subsequently, two distinct layers were observed in the centrifuge tube, the upper layer being bone marrow and the lower layer being Percoll solution. And placing the centrifuge tube into a centrifuge, setting the rotation speed to be 500g, centrifuging for 25min, and dividing the centrifuge tube into four layers from top to bottom. The middle buffy coat was gently aspirated by a pipette and repeatedly washed 2 times with PBS buffer containing 2% FBS. Centrifuging for 10min and 5min at 250g, and discarding supernatant to leave mononuclear cell precipitate, which is the white blood cell of HAML patient.
EXAMPLE 1 integrin ITGA5 inhibitor inhibits leukocyte migration and invasion in HAML patients
(1) Cell migration experiments:
magnetic bead sorting of HAML patient's leukocytes isolate cd34+ cells; into the Transwell upper chamber was added 0.1mL of serum-free cell suspension to give a final cell concentration of 7.5X10 4 Adding ITGA5 inhibitor ATN161 with a certain concentration into cells/well; 0.8mL of culture solution containing 10% serum is added into a Transwell lower chamber to serve as chemoattracetant, and cells are attracted to migrate and secrete matrix protease attack; the cell culture plate was placed at 37℃with 5% CO 2 Incubating the incubator for 24-48 hours; removing the culture solution, washing twice with PBS, and gently wiping the non-migrated cells with a cotton swab; 1mL of 100% methanol was added and the mixture was fixed at room temperature for 30 minutes, and the mixture was washed with PBS for 2 times; 1mL of 0.1% crystal violet dye solution is added for dyeing for 20 minutes at room temperature, and then PBS is used for cleaning for 2 times; the ITGA5 inhibitor ATN161 was found to significantly reduce the number of cells migrating in the lower chamber and significantly inhibit HAML cell migration by transferring the Transwell onto a slide glass and counting the number of cells migrating under a microscope with random 6-9 field views (fig. 1).
(2) Cell invasion assay:
preparing 0.5 Xof C buffer solution, 1 Xof A glue and a Transwell device applicable to 24 pore plates in advance; diluting the gel A by using a buffer solution C at 37 ℃ for 15-20 times, adding 0.2mL of gel A dilution into the Transwell upper chamber according to the bottom area of the Transwell upper chamber, and then placing the gel A dilution into a refrigerator at 4 ℃ for incubation for 2-3 hours; into the Transwell upper chamber was added 0.1mL of serum-free cell suspension to give a final cell concentration of 7.5X10 4 A cell/well, and adding a certain amount of ITGA5 inhibitor ATN161; 0.8mL of culture solution containing 10% serum is added into a Transwell lower chamber to serve as chemoattracetant, and cells are attracted to migrate and secrete matrix protease attack; the cell culture plate was placed at 37℃with 5% CO 2 Incubating the incubator for 24-48 hours; removing culture solution, washing twice with PBS, and lightly wiping with cotton swabRemoving non-migrated cells; 1mL of 100% methanol was added and the mixture was fixed at room temperature for 30 minutes, and the mixture was washed with PBS for 2 times; 1mL of 0.1% crystal violet dye solution is added for dyeing for 20 minutes at room temperature, and then PBS is used for cleaning for 2 times; the number of migrated cells was counted by transferring the Transwell onto a slide and observing the cells under a microscope in random 6-9 fields, and it was found that ITGA5 inhibitor ATN161 significantly reduced the number of cells invaded in the lower chamber and significantly inhibited HAML cell invasion (fig. 2).
Example 2QPCR assay for ITGA5 expression in HAML patient leukocytes:
rna extraction and concentration detection: collection of 1X 10 7 Adding 1mL of TRizol into white blood cells of HAML patient (or NHAML patient or normal person), repeatedly blowing and mixing, reversing for 10 times, and standing at room temperature for 5min after the liquid is clarified; adding 0.2mL of chloroform, mixing the mixture upside down, standing the mixture at room temperature for 5min, and centrifuging the mixture at the temperature of 12000g for 15min; about 400. Mu.L of the upper aqueous phase was placed in a 1.5mL Ep tube of another RNAfree; adding isopropanol with the same volume, reversing and uniformly mixing, and standing at room temperature for 10min; centrifuging 12000g at 4deg.C for 10min, discarding supernatant, adding pre-cooled 75% ethanol (diluted with DEPC water) 1mL, centrifuging 7500g at 4deg.C for 5min; discarding the supernatant, air drying for 5-10min, dissolving in 20 μl DEPC water, and preserving at-80deg.C; the concentration of the RNA obtained was measured by a nucleic acid concentration measuring instrument, and the A260/280 value was preferably about 2.0.
cDNA Synthesis
(1)
Storing at 42deg.C for 2min (or at room temperature for 5 min) and 4deg.C.
(2) < TB Green qPCR method >
And (3) performing PCR: cooling at 37 deg.C for 15min-85 deg.C for 5 sec-4 deg.C, and preserving the synthesized product at-20 deg.C
RT-PCR reaction: take 20. Mu.L of the reaction system as an example.
Wherein, the primer sequence for detecting ITGA5 is as follows:
ITGA5-F：TGGAAGGTCAGCAGCTCCTATAT，
ITGA5-R：CTGCAGACTTTGGCTCTCTTGTT。
reaction conditions (three-step process): pre-denaturation at 95℃for 10min; denaturation at 95℃for 15s, annealing at 55-64℃for 30s, extension at 72℃for 32s,39 cycles.
The QPCR results are shown in fig. 3, with ITGA5 expressed higher in HAML patient cells than AML (NHAML) and normal.
EXAMPLE 3ELISA detection of ITGA5 expression in bone marrow supernatants of HAML patients, NHAML patients and normal humans
(1) Reagents, samples and standards were prepared.
(2) 100. Mu.L of standard or sample was added to each well and incubated at 37℃for 2 hours. The liquid in each well was sucked off without washing.
(3) mu.L of biotin antibody (1-fold) was added to each well. Incubate at 37℃for 1 hour. The liquid in each well was aspirated and washed 3 times.
(4) mu.L of HRP-avidin (1X) was added to each well. The culture was carried out at 37℃for 1 hour,
(5) The liquid in each well was aspirated and washed 5 times.
(6) mu.L of TMB substrate was added to each well. Incubate at 37℃for 15-30 minutes. Light is avoided.
(7) mu.L of stop solution was added to each well. Read at 450nm in 5 minutes.
The results are shown in FIG. 4, where ITGA5 is highly expressed in bone marrow supernatants of HAML patients, above that of AML (NHAML) and normal.
Example 4 transcriptome sequencing
(1) Sample preparation: after the HAML patient leucocyte, NHAML patient and normal human leucocyte are prepared, the experimental cells are uniformly mixed by using an enzyme-free gun head and transferred into an enzyme-free 1.5mL centrifuge tube, the low-temperature high-speed centrifuge is set to 13000rpm for 10s, and after centrifugation, the supernatant is completely sucked and discarded, and only cell sediment is left.
(2) Total RNA extraction: the kit is used for extracting RNA, and an enzyme-free gun head and an EP tube are used in the whole process. (1) The pellet was gently flicked, 350. Mu.L of lysate was aspirated using an enzyme-free gun head, and added to the cell pellet. Quickly blowing and evenly mixing, and oscillating for 30s by using a vortex device to fully crack the materials; (2) adding 350 mu L of 70% ethanol, blowing again, mixing, transferring the mixture into an adsorption column of a kit, setting a low-temperature high-speed centrifuge to 13000rpm for 30s, rapidly centrifuging, and directly pouring out the waste liquid at the lower layer; (3) 700. Mu.L deproteinized solution was added to the column, and the column was left at room temperature for 30 seconds without immediate centrifugation. After timing is finished, centrifuging for 30s by using a low-temperature high-speed centrifugal machine 13000rpm, and pouring the lower-layer waste liquid into a waste liquid barrel; (4) adding 500 mu L of rinsing liquid into the adsorption column for rinsing, pouring the lower layer waste liquid into a waste liquid barrel before centrifugation under the same conditions, repeatedly washing for 1 time, centrifuging to remove the waste liquid, and centrifuging for 2min under the condition of 13000 rpm; (5) transferring the upper adsorption column into a new enzyme-free EP tube, adding proper amount of DEPC water, generally 30-50 μl, into the adsorption column according to the amount of cell precipitation, and avoiding low extraction concentration due to too much DEPC water. And (3) placing the sample at room temperature, timing for 1min, and centrifuging at 13000rpm for 1min by a low-temperature high-speed centrifuge to obtain the extracted RNA. Subpackaging the extracted RNA, and storing in a refrigerator at-80deg.C to avoid repeated freezing and thawing.
(3) Transcriptome sequencing: the extracted RNA was submitted to commercial sequencing companies.
(4) Analysis of differential genes: the readcount data was normalized with DESeq2 to obtain differential gene results. Screening criteria were fold difference greater than 2, corrected P values less than 0.05, resulting in significantly differentially expressed genes (Differentially expressed gene, DEG). And drawing a differential gene volcanic image and a differential gene clustering heat image by using R language.
(5) Differential gene enrichment analysis: gene Ontology (GO) analysis and kyoto encyclopedia of genes and genome (Kyoto Encyclopedia of Genes and Genomes, KEGG) analysis were performed on differential genes using a DAVID database. The results are shown in FIG. 5, where ITGA5 expression is elevated in different HAML patients.
Claims (9)
1. The application of integrin ITGA5 as a marker in preparing HAML medicine.
2. Use of an inhibitor of integrin ITGA5 in the manufacture of a medicament for the treatment of HAML.
3. Use of an inhibitor of integrin protein ITGA5 in the preparation of a medicament for inhibiting HAML leukocyte migration, invasion, proliferation.
4. A use according to any one of claims 1-3, characterized in that: the medicine takes ITGA5 as a target to inhibit ITGA5 gene expression or inhibit ITGA5 protein activity.
5. A use according to claim 2 or 3, characterized in that: the ITGA5 inhibitor comprises: substances inhibiting the expression of ITGA5 gene and substances inhibiting the activity of ITGA5 protein.
6. The use according to claim 5, characterized in that: the inhibitor of ITGA5 is ATN161.
7. The use according to claim 5, characterized in that: the inhibitor of ITGA5 is an antibody of ITGA5 protein.
8. Use of a reagent for detecting the expression level of the integrin protein ITGA5 in the preparation of a diagnostic HAML reagent or kit.
9. The use according to claim 8, characterized in that: the reagent for detecting the expression level of the integrin protein ITGA5 comprises QPCR primers.
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