CN114404616A - ITGA4 gene inhibitor and application thereof in preparation of drug for treating refractory or recurrent acute myeloid leukemia - Google Patents
ITGA4 gene inhibitor and application thereof in preparation of drug for treating refractory or recurrent acute myeloid leukemia Download PDFInfo
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- CN114404616A CN114404616A CN202111573516.1A CN202111573516A CN114404616A CN 114404616 A CN114404616 A CN 114404616A CN 202111573516 A CN202111573516 A CN 202111573516A CN 114404616 A CN114404616 A CN 114404616A
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Abstract
The invention provides an ITGA4 gene inhibitor and application thereof in preparing a drug for treating refractory or relapsed acute myeloid leukemia, belonging to the technical field of biological medicines. The invention discovers that ITGA4 is a potential therapeutic target for refractory or recurrent leukemia, so that an inhibitor comprising shRNA and/or natalizumab with the nucleic acid sequence of SEQ ID NO. 1 is selected. And the inhibitor is applied to the preparation of the drug for treating refractory or relapsing acute myelogenous leukemia. The invention definitely reduces the expression or activity of ITGA4, and the proliferation of leukemia cells is inhibited; the survival of the leukemia mice is obviously prolonged; reducing the homing of leukemia cells.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an ITGA4 gene inhibitor and application thereof in preparation of a drug for treating refractory or relapsed acute myeloid leukemia.
Background
Acute Myeloid Leukemia (AML) is a hematological tumor in which myeloid leukocytes abnormally proliferate, and is one of the most common hematological malignancies in adults, and the mortality rate accounts for the first ten digits of various tumors. About 10-40% of young AML patients and 40-60% of elderly patients fail to achieve complete remission from induced chemotherapy, and after intensive chemotherapy, the disease-free survival rate in 10 years for AML patients under the age of 60 is 16%, and the disease-free survival rate in 10 years for patients over the age of 60 is only 2%. Approximately 50% of patients relapse after induction of chemotherapy. Patients with relapsed or refractory AML have a poor prognosis with an overall 3-year survival (OS) of no more than 10%.
In recent years, with the development of medical treatment, hematopoietic stem cell transplantation, targeted drugs, and cellular immunotherapy have improved the prognosis of some patients. Patients who are refractory or relapsed still lack effective treatment. The effect of the reinforcement chemotherapy for the patients who are difficult to treat or relapse is poor, and the Complete Remission (CR) rate of the rescue chemotherapy is lower than 50 percent. The existing treatment target coverage rate of the patients is low, and the probability of targeted treatment is lower than 40%. The prognosis of the refractory patients after hematopoietic stem cell transplantation is improved to a limited extent, and the survival rate of the refractory patients 5 years after transplantation is lower than 20%. Whereas AML patients who relapse after transplantation survive worse, with a 5-year survival rate of no more than 10%. In addition, CAR-T treatment of AML has the problems of lack of effective targets, poor treatment effect, off-target, relapse and the like. Therefore, finding therapeutic targets for refractory or relapsed AML is an urgent need to improve the survival of such patients.
Leukemia cells have a strong ability to home and migrate, they can quickly home in the bone marrow microenvironment, disturb the microenvironment structure and function of normal hematopoietic stem cells, establish an abnormal bone marrow microenvironment (leukemia microenvironment) and "hijack" normal hematopoietic stem cells to "trap" them in the leukemia microenvironment. The establishment of leukemia microenvironment further promotes the massive proliferation of leukemia stem cells and accelerates the development of leukemia. The dimer VLA-4 consisting of integrin α 4(ITGA4 gene) and integrin β 1 is highly expressed in leukemia stem cells and leukemia cells, which mediates adhesion of leukemia cells to extracellular matrix or mesenchymal cells. VLA-4 is highly expressed in leukemia and its Leukemic Stem Cells (LSCs), and in AML, chronic lymphocytic leukemia and acute B lymphocytic leukemia (ALL), high expression of VLA4 is strongly associated with poor prognosis.
Since VLA-4-VCAM1 mediates the migration of immune cells in the inflammatory response, VLA-4 is a major target for the treatment of autoimmune diseases. The blocking antibody Natalizumab (NZM) for VLA-4 was FDA approved in 2004 for the treatment of multiple sclerosis and crohn's disease, however, was withdrawn from the market due to the rare "progressive multifocal leukoencephalopathy" resulting from side effects of the drug. In 2006 it was re-marketed under the urgent need for disease treatment. The small molecule inhibitor of VLA-4, BIO5192, was reported to inhibit VLA-4 mediated homing of hematopoietic stem cells, mobilizing hematopoietic stem cells into peripheral blood. Since VLA-4 is highly expressed in leukemia cells and is closely associated with prognosis, multiple studies have shown that decreasing the expression of VLA-4 or inhibiting the binding of VLA-4 to VCAM1 can significantly increase the sensitivity of chemotherapy-resistant ALL cells, CLL cells and AML cells to chemotherapeutic drugs.
However, the role of VLA-4 in refractory or relapsed leukemia has not been reported, and it is unclear whether VLA-4 could be a target for treatment of refractory or relapsed AML.
Disclosure of Invention
In order to solve the technical problems, the invention provides an ITGA4 gene inhibitor and application thereof in preparing a medicament for treating refractory or relapsed acute myeloid leukemia.
An ITGA4 gene inhibitor, which comprises shRNA and/or natalizard monoclonal antibody with the nucleic acid sequence of SEQ ID NO. 1.
A recombinant vector comprising a sequence capable of transcribing the shRNA, said sequence being embedded in a vector.
In one embodiment of the invention, the sequence SEQ ID NO 1 is GCTCCGTGTTATCAAGATTATCTCGAGATAATCTTGATAACACGGAGCTTTTTG.
In one embodiment of the invention, the vector is a lentiviral vector.
In one embodiment of the invention, the lentiviral vector is selected from the group consisting of pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-tagCFP, pLKO.1-puro-CMV-tagYFP, pLKO.1-puro-CMV-tagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-tagFP, pLKO-puro-1 xLacO, pLKO-PURO-IPTG-3xLacO, pLKLP 34, pLKO-uP-2-dNa-GCIJV, pLKO-GCIJV-3976, pLKO-GLYnLKO-GCH, pLKO-GCH-LKO, pLKO-JV-3, pLKOGpLKO-JV-3, pLKO-JV-3, pLKOTpLKOTpLKO-3, pLKOTpLKOTpLKOTpLKO-JV 3, pLJV-JV, pLKOTpLJV 3, pLJV-JV, pLJV-JW-JV, pLJV-JV-3, pLJV-JV-3, pLJV-JV-3, pLJV-JV-3, pLJV-JV-3, pLJV-JV, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.
A lentivirus obtained by cell packaging of the recombinant vector.
A pharmaceutical composition comprising the ITGA4 gene inhibitor, the recombinant vector, or the lentivirus.
In one embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In one embodiment of the invention, the carrier is selected from one or more of disintegrants, diluents, lubricants, binders, wetting agents, flavouring agents, suspending agents, surfactants and preservatives.
A kit comprising the ITGA4 gene inhibitor, the recombinant vector, the lentivirus, or the pharmaceutical composition.
The invention also provides application of the ITGA4 gene inhibitor, the recombinant vector, the lentivirus, the pharmaceutical composition or the kit in preparation of drugs for treating refractory or relapsed acute myelogenous leukemia.
The invention provides a potential target point for treating refractory or recurrent acute myeloid leukemia and application thereof in inhibiting proliferation and migration adhesion of refractory or recurrent leukemia cells.
Specifically, gene knockdown of expression of VLA-4 α 4 subunit (ITGA4) or inhibition of binding to VCAM-1 with NZM was used to inhibit clonal colony formation of AML cell line MV4-11, to inhibit proliferation of AML cells, and to block the AML cell cycle to G1.
Specifically, knockdown of expression of ITGA4 or NZM treatment was used to inhibit migration and adhesion ability of AML cells.
Specifically, the knockdown of the expression of ITGA4 is used for prolonging the survival of AML mice constructed by MV4-11 cells, inhibiting the disease reconstruction of AML cells and inhibiting the infiltration of AML cells in the liver and spleen.
Specifically, knockdown of ITGA4 expression was used to inhibit the homing of AML cells to bone marrow and spleen hematopoietic tissues.
In particular, NZM is used to inhibit clonal colony formation, migration and adhesion of leukemia cells and leukemia stem cells in refractory and relapsed patients.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the invention provides a potential target point for treating refractory or recurrent acute myeloid leukemia and application thereof in inhibiting proliferation and migration adhesion of refractory or recurrent leukemia cells. In the aspect of AML cell strain MV4-11, leukemia cell animal models and leukemia cells of patients difficult to treat or relapse AML, the ITGA4 is determined to be a potential treatment target point of difficult-to-treat or relapse leukemia by interfering the expression of ITGA4 gene with shRNA or inhibiting the activity of ITGA4 with NZM through proliferation, migration, adhesion and homing experiments.
Interfering the expression of ITGA4 or inhibiting the activity of NZM, obviously inhibiting the proliferation of AML cells, weakening the clonogenic capacity of AML cells, reducing the homing of AML cells, obviously prolonging the survival of AML mice and delaying the progression of leukemia. Especially in refractory or relapsing AML cells lacking therapeutic means, inhibition of ITGA4 activity also significantly reduced the proliferative and migratory adhesion capacity of AML cells in refractory or relapsing patients. ITGA4 is a potential therapeutic target for refractory or relapsed leukemia.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a graph of the number of clonal colonies of MV4-11 cells that have been knocked down for ITGA4 expression or for natalizumab treatment according to the invention. Knockdown of ITGA4 expression or natalizumab-treated group clone colonies were significantly reduced in number compared to the no-knockdown group or IgG-treated control group, where # indicates P ≧ 0.05, P <0.05, and P < 0.001.
FIG. 2 is a dynamic proliferation curve of MV4-11 cells cultured for 7 days with the knockdown of ITGA4 expression or the treatment of natalizumab with MV4-11 cells. Knockdown of ITGA4 expression or natalizumab-treated groups cells grew slowly and proliferation was inhibited compared to the non-knock-out group or IgG-treated control group, where indicates P < 0.05.
FIG. 3 shows that the ratio of cells in G1 phase in the invention with knockdown of ITGA4 expression or natalizumab treatment group is significantly increased compared with the control group without knockdown or IgG treatment by knocking down the expression of ITGA4 or treating MV4-11 cells with natalizumab, wherein # indicates that P is greater than or equal to 0.05 and # indicates that P is less than 0.05.
FIG. 4 shows that the ratio of cells with knockdown of ITGA4 expression or migration of the natal mab-treated group was reduced compared to the non-knocked-down group or IgG-treated control group by knocking down ITGA4 expression or natal mab-treated MV4-11 cells according to the present invention. # denotes P.gtoreq.0.05, P <0.05, and P < 0.001.
FIG. 5 shows that the ratio of cells with knockdown of ITGA4 expression or adhesion of the natal mab-treated group was reduced compared to the non-knocked-down group or IgG-treated control group by knocking down ITGA4 expression or natal mab-treated MV4-11 cells according to the present invention. # denotes P.gtoreq.0.05, # denotes P <0.05, # denotes P < 0.01.
FIG. 6 shows that the MV4-11 cells with the knockdown of ITGA4 expression of the invention were transplanted into immunodeficient mice, and the survival time of mice in the knockdown of ITGA4 expression group was prolonged compared with that of the control in the non-knocked-out group.
FIG. 7 shows that MV4-11 cells with the knockdown of ITGA4 expression of the invention were transplanted into immunodeficient mice, and that leukemia cell reconstitution in bone marrow, liver and spleen 15 days after the transplantation of mice in the knockdown of ITGA4 expression group was reduced compared with that in the non-knockout group. Denotes P <0.05, denotes P < 0.001.
FIG. 8 shows that MV4-11 cells with the knockdown of ITGA4 expression of the invention were transplanted into immunodeficient mice, and that leukemia cell infiltration in bone marrow, liver and spleen was reduced 15 days after the transplantation of mice with the knockdown of ITGA4 expression group, compared with the non-knockout group.
FIG. 9 shows that MV4-11 cells with the knockdown of ITGA4 expression of the invention were transplanted into immunodeficient mice, and that the percentage of leukemia cells homing in the bone marrow and spleen 16 hours after the transplantation was reduced in mice with the knockdown of ITGA4 expression group compared to the non-knockout control. Denotes P <0.01, denotes P < 0.001.
FIG. 10 shows that natalizumab-treated or relapsed AML cells of the invention significantly reduced the number of clonal colonies compared to IgG-treated controls. Denotes P <0.01, denotes P < 0.001.
FIG. 11 shows that natalizumab-treated or relapsed AML cells of the invention have a reduced proportion of cells that migrated compared to IgG-treated controls. Denotes P <0.01, denotes P < 0.001.
FIG. 12 shows that natalizumab-treated or relapsed AML cells of the invention have a reduced proportion of cells adhered compared to IgG-treated controls. Denotes P <0.01, denotes P < 0.001.
FIG. 13 shows that natalizumab of the present invention treated leukemic stem cells with refractory or relapsed AML, significantly reduced numbers of clonal colonies in the natalizumab-treated group compared to the IgG-treated control group. P <0.05, P <0.01, P < 0.001.
FIG. 14 shows that natalizumab-treated groups of leukemic stem cells treated with natalizumab of the invention have a reduced proportion of cells migrating compared to IgG-treated control groups. # denotes P.gtoreq.0.05, # denotes P <0.05, # denotes P <0.01, and # denotes P < 0.001.
FIG. 15 shows that natalizumab-treated or relapsed AML leukemia stem cells of the invention have a reduced proportion of cells adhered compared to IgG-treated controls. Denotes P <0.05, denotes P < 0.001.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Examples
Firstly, the method comprises the following steps: materials and methods
(I) cell line
Human AML cell line MV4-11 was cultured in RPMI1640 complete medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2Culturing in saturated humidity cell culture box (0.4-1) x 106in/mL culture, passage every 2 days, logarithmic growth phase cells were used for the experiment.
(II) clinical specimens
Bone marrow mononuclear cell specimens of AML patients treated at the first hospital affiliated suzhou university from month 1 in 2018 to month 7 in 2020 were collected and complete clinical data of AML patients were collected. The study was approved by the ethical committee of the first hospital affiliated with the university of suzhou, and all patients signed informed consent.
Primary cells and leukemia stem cells from AML patients were treated with StemBan containing 1% AA, 20ng/mL recombinant human TPO, 20ng/mL recombinant human SCF and 20ng/mL recombinant human FLT3-LigandTMSFEM is as 5X 106Culture at a density of/mL.
(III) Experimental animals
Healthy SPF (specific pathogen free) NOD/SCID female mice, 6-8 weeks old, 20-22 g in weight, were purchased from Shanghai Slek laboratory animal center. The mouse feed and padding are subjected to irradiation sterilization, sterilized acidified water is drunk, and neomycin sulfate is added into the transplanted drinking water. Mice were housed in the SPF-grade environment at the experimental animal center of suzhou university. All animal experiments were approved by the ethics committee of the university of suzhou.
(IV) Main reagent and consumable
NZM (natalizumab ): the Absolute antibody, Inc. of UK
IgG antibody: merck Millipore, USA
Fetal Bovine Serum (FBS): israel Biological Industries, Inc
RPMI (Roswell park Memori Institute, Roseviv park Community) -1640 medium: thermo Hyclone in USA
IMDM (Iscove's Modified Dulbecco's Medium, Modified Dulbecco's Medium of Iscove): thermo Hyclone in USA
Penicillin: shanghai bioengineering Ltd
Streptomycin: shanghai bioengineering Ltd
StemSpanTMSFEM (Serum-free medium for culture and expansion of hematopoietic cells): StemCell Technologies, Inc. USA
Recombinant Human SCF (Stem cell factor): PeproTech, USA
Recombinant HumanFLT3-ligand (Fms-like tyrosine kinase 3 ligand): PeproTech, USA
Recombinant Human TPO (thrombopoetin): PeproTech, USA
MethoCultTMH4100: STEMCELL Technologies, Canada
MethoCultTMH4435: STEMCELLTechnologies, Canada
fibrigen: sigma Co of USA
Transwell: corning Inc. of USA
Giemsa rui staining solution: solarbio, China
Second, Experimental methods
1. Drug-treated cells
MV4-11 cells or primary leukemia cells were added to natalizumab at a concentration of 10. mu.g/mL, and control group was added with IgG at the same concentration as control, and in vitro experiments were performed after 1-3 days of treatment.
2. Cell proliferation
Cells were seeded in 96-well plates with 3 to 5 wells per group, 20000 cells/well, 100 μ L/well. Culturing at 37 deg.C in 5% CO2 incubator, counting cells every day, continuously detecting proliferation level of cells for 7 days, detecting at the same time every day, and changing cell culture solution for each well from the third day.
3. Cell cycle
Cells were cultured at 5X 105/mL and the cell cycle was examined when growth was in log phase. Cells were centrifuged at 500g for 5min, the supernatant discarded, washed once with PBS, 200. mu.L of Cytofix/CytopermBuffer was fixed to rupture the membrane, frozen for 30min, stopped with 1mL of 1 XBD Perm/WashBuffer, centrifuged at 500g for 5min, the supernatant discarded, 200. mu.L of PBS containing 1% BSA and 2. mu.L of Ki67-APC antibody were added, frozen for 30min, stopped with 3mL of PBS containing 1% BSA, centrifuged at 500g for 5min, the supernatant discarded, 200. mu.L of PBS containing 1% BSA and 2. mu.L of DAPI were added, 20min at room temperature, stopped with 3mL of PBS containing 1% BSA, centrifuged at 500g for 5min, the supernatant discarded, 500. mu.L of PBS containing 1% BSA was added, the cell cycle was measured by flow cytometry, and the rate of the supernatant was lower than 100/s.
4. Clone formation experiments
Preparing a MethoCultH4100 semisolid culture medium, adding 10mL of FBS and 1mL of double antibody into 40mL of stock solution, adding IMDM culture medium to supplement 100mL, violently shaking, standing until bubbles disappear, and subpackaging into 3mL tubes. The cells were counted, 600 were resuspended in 300 μ l complete medium, slowly added dropwise to H4100 semisolid medium, shaken vigorously, left to stand until the air bubbles disappeared, and the mixture was transferred to 3 35mm petri dishes, 200 cells/dish, using a syringe. The 3 dishes were placed in 15cm cell culture dishes and 3 additional water trays were placed to maintain medium humidity. After culturing at 37 ℃ in a 5% carbon dioxide incubator for 7 to 10 days, the number of colony colonies grown was counted under a microscope. Primary cells were cultured in H4435 semisolid medium at 250. mu.L/well in 24-well plates, typically 2000CD34+ populations/well. The surrounding wells were hydrated to maintain medium humidity. After 10 to 14 days at 37 ℃ in a 5% carbon dioxide incubator, the number of colony colonies grown was counted under a microscope.
5. Cell migration assay
Transwell plates, 6.5mm diameter, 8 μm pore size, were prepared and the Transwell chamber was partitioned with 20g/mL of bone Fibrin (FN) (PBS) incubated at 4 ℃And (5) breeding over night. The following day was washed 3 times with IMDM medium containing 0.5% BSA. 3X 105The AML cells were resuspended in 100 μ L of IMDM medium containing 0.5% BSA and added to the upper chamber. The lower chamber was supplemented with 600. mu.L of IMDM medium containing 0.5% BSA. Each group is provided with 3 multiple holes. Separately prepare 3X 105The AML cells were resuspended in 600. mu.L of IMDM containing 0.5% BSA and used as input control without the chamber. Transwellplants were incubated at 37 ℃ with 5% CO2Culturing for 12 h. The cells AML and input control cells migrated to the lower chamber were collected in a flow tube, and 100. mu.L of GFP-bearing MV4-11 NSC cells (1X 10 cells) were added thereto5The percentage of GFP-cells in the flow assay was calculated as 100% based on the input control, and the migration rate of AML cells was calculated.
6. Adhesion test
The wells were pre-plated with 10. mu.g/mL FN in 96-well plates, 70. mu.L/well, overnight at 4 ℃ and washed 3 times with PBS, blocked with 1% BSA at 37 ℃ for 1h and washed 3 times with PBS. IMDM containing 0.5% BSA resuspended cells, adjusted to a concentration of 5X 105Each of the cells was inoculated in 50000 cells/well in a 96-well plate, 3 wells per group, and after 1 hour at 37 ℃, the non-adherent cells were gently washed away with PBS. Adding 100 mu L of methanol into each hole, fixing for 15min, adding 50 mu L of the Giemsa rapae dye solution A into each hole, dyeing for 30s, adding 50 mu L of the Giemsa rapae dye solution B into each hole, dyeing for 5min, washing off the dye solution by using a culture medium, randomly taking 10 visual fields in each hole under an inverted microscope, photographing, counting the number of adhered cells, and counting the result.
7. Mouse transplantation
6-8 weeks NOD/SCID mice were purchased and kept in a stable state in SPF-grade animal housing for 2 weeks. NOD/SCID mice to be transplanted are irradiated to 2.0Gy with a half lethal dose in an X-ray irradiator, and anti-CD122 antibody is injected intraperitoneally at a dose of 10 ug/g of mice after irradiation to eliminate innate immunity (NS122 immunodeficient mice, sources of construction methods: Duan CW, Shi J, Chen J, Wang B, Yu YH, Qin X, et al, Leukemia prolating cells rebuilt an animal amino acid in response to cancer Cell2014,25(6):778 + 793). Tail vein transplantation of sorted GFP within 24 hours+Leukemia cells. The cells were resuspended in PBS containing 1% BSA at 100. mu.L per mouse, 1 in105Cell amount, homing assay 150. mu.L, 2X 10 cells per mouse6The amount of cells was resuspended. Cells were injected into mice along the tail vein using a 1mL syringe or insulin syringe and the mice were earmarked. The mice were returned to the SPF environment and fed two weeks of sterile acidified water containing 1.1mg/mL neomycin sulfate. The survival status of the mice was observed daily and the body weight of the mice was recorded. When the weight of the mouse is obviously reduced, the mouse is attacked when the states of arch back, hair explosion, slow movement and the like appear.
8. Obtaining bone marrow cells from mice
The mice were sacrificed by cervical dislocation, sprayed with 75% alcohol, and fixed on the operating table. Skin and fascia are cut open, femur, tibia and ilium are cut off, muscle is removed, bone tissue is placed in a mortar filled with pre-cooled IMDM containing 1% BSA, and bone is ground to release bone marrow cells. The suspension was filtered through a 70 μm sieve to obtain a bone marrow cell suspension, which was placed on ice. Centrifuging the cell suspension at 600g for 5min, removing the supernatant, adding 5mL of erythrocyte lysate ACK, standing at room temperature for 10min, and adding 10mL of PBS to terminate lysis. Centrifuge at 600g for 5min, discard the supernatant, resuspend with IMDM containing 1% BSA, and place on ice until needed.
9. Obtaining of mouse liver and spleen cells
Killing mice by cervical dislocation and spraying 75% alcohol, cutting skin and peritoneum, cutting liver and spleen related ligaments, taking down liver and spleen, respectively placing into precooled IMDM containing 1% BSA, gently grinding with frosted glass slide, filtering the suspension with 70 μm filter screen to obtain liver and spleen cell suspension, and placing on ice. Centrifuging the cell suspension at 600g for 5min, discarding the supernatant, adding 5mL of erythrocyte lysate ACK, standing at room temperature for 10min, and adding 10mL of PBS to terminate lysis. Centrifuge at 600g for 5min, discard the supernatant, resuspend with IMDM containing 1% BSA and 2mM EDTA, and place on ice until use.
10. Flow assay mouse reconstruction
Taking 50-100 mu L of mouse bone marrow, liver and spleen cells, resuspending in 200 mu L of PBS containing 1% BSA, adding CD45-APC antibody according to a ratio of 1:100, incubating for 30min at 4 ℃ in a dark place, adding 3ml PBS for termination, centrifuging for 600g5min, discarding supernatant, and resuspending in 200 mu L of PBSLast Novocyte flow cytometer for detecting CD45+GFP+A population of cells.
11. Plasmid amplification
The plasmid was synthesized by Kingchi corporation, and the small interfering RNA of ITGA4 (sh-ITGA4) and the non-interfering oligonucleotide sequence (NSC) were inserted into plko.1-EGFP lentiviral vectors, respectively.
And (3) plasmid transformation: LB medium containing 100. mu.g/. mu.L ampicillin and solid plates containing 100. mu.g/. mu.L ampicillin were prepared. The competent ice was thawed, and 10. mu.L of competent plasmid was added to 1. mu.L (100 ng/. mu.L). Standing the mixture on ice for 30min, thermally shocking the mixture for 90s at 42 ℃, and placing the mixture on ice for 2-3 min. 500. mu.L of LB medium containing 100. mu.g/. mu.L of ampicillin was added, 250. mu.L of the mixture was applied to an LB solid plate containing ampicillin, and the plate was uniformly coated with a sterilized coating rod or a ball. After the bacterial solution is absorbed, the plate is inverted and cultured at 37 ℃ for 12 to 16 hours.
After the strain at the long part of the culture plate, a monoclonal colony is picked, cultured in 3mL LB culture medium containing 100 mug/mL ampicillin, shaken in a shaker at 37 ℃ and 250rpm for 6-8 h, transferred into 200mL LB culture medium containing 100 mug/mL ampicillin, and shaken in a shaker at 37 ℃ and 250rpm for 12-16 h.
Plasmid extraction: centrifuging the bacterial liquid in a high-speed centrifuge at 8000g for 10min at 4 deg.C, discarding the supernatant, washing with PBS, centrifuging, and discarding the supernatant. Using MN plasmid extraction kit, resuspending the bacterial pellet with 8mL of resuspension solution RES-EF containing RNase, adding 8mL of alkaline lysis solution LYS gently, and slowly inverting for 5 to 8 times, wherein the time can not exceed 5 min. Add 8mL of acid stop buffer NEU and gently invert until the indicated color disappeared. Standing on ice for 5 min. Soaking the filter paper in the endotoxin filter column with 15mL of salt equilibrium liquid EQU, shaking the cleavage product to be homogeneous, pouring the cleavage product into the center of the filter paper, and washing the filter paper with 5mLFIL-EF when the filter column does not drip any more. When the dripping was completed, the filter paper was discarded, and 35mL of ENDO-EF was added to the endotoxin filter column. After the addition, 15ml of LASH-EF was added. After the completion of the dropping, 5mL of ELU-EF preheated to 65 ℃ was added, and the filtrate was collected by a clean centrifuge tube. 3.5mL of isopropanol was added, mixed by inversion, and centrifuged at 15000g for 40min at 4 ℃ at high speed. The supernatant was discarded, 2mL of 70% EtOH was added, and the mixture was centrifuged at 15000g for 10min4 ℃. The supernatant was aspirated (200. mu. L H)2O-EF resuspending the pellet, Nanodrop for DNA concentrationAnd (4) degree. The plasmid can be stored at-20 ℃.
12. Lentivirus packaging, concentration, titer determination, infection of cells
293T cells were prepared and transfected 24 hours after passage when the cells grew to a confluence of about 60% to 80%. For cells in 1-dish 10cm cell culture dish, plasmid-calcium chloride mixture was prepared as follows:
after mixing, 2 XHBSS 500. mu.L was gently dropped on the liquid surface, and the mixture was left to stand for 30 min. Dropping 1mL of mixed solution onto 293T liquid surface, shaking the cross-shaped culture dish up and down and left and right for 5-10 times, adding 5% CO at 37 deg.C2An incubator. After 6 hours, 8mL of medium was aspirated from 293T and fresh complete medium pre-warmed to 37 ℃ was added. After 48 hours, GFP fluorescence was observed under a fluorescence microscope, and the supernatant of the produced virus was collected and supplemented with fresh complete medium. The culture was continued for 72 hours and the virus-containing medium was collected. The collected virus solution was filtered through a 0.45 μm filter head.
Centrifuging the virus supernatant with ultracentrifuge at 25000rpm for 1h 30min at 4 deg.C, discarding the supernatant, resuspending the virus pellet with 1% BSA-containing PBS at a ratio of 1:500, and storing the concentrated virus at minus 80 deg.C without repeated freeze thawing.
293T cells were passaged to 6-well plates and 24 hours later virus titer was determined. 1.3. mu.L of the concentrated virus was taken and diluted to 1000-fold gradient 10 in complete medium4Multiple, 105Multiple sum of 106Multiple 4 groups. One of 293T cells in a 6-well plate was counted, 1mL of the medium was discarded for each of the remaining wells, and 1mL of the diluted virus solution was added to the cells and placed in an incubator. After 48 hours, the GFP positivity of each well cell was measured by flow cytometry. Groups with percentages ranging from 1% to 10% were selected to calculate viral titers: viral titer-GFP positive rate × dilution fold × number of cells before infection.
Leukemia cells were arranged at 5X 105concentration/mL was resuspended in complete medium and virus concentration was calculated as MOI 50The amount of fluid required, i.e. number of cells x MOI/viral titer. Adding virus concentrated solution, blowing, beating, mixing, and adding 5% CO at 37 deg.C2Culturing in an incubator. After 24 hours, the medium was replaced with fresh complete medium and the culture was continued. Cells were sorted after 72 hours complete expression of GFP protein.
13. Flow cytometric sorting
14. Statistical analysis:
comparison of control and experimental groups was performed using independent sample t test, survival of transplanted mice was compared using Kaplan-Meier analysis, log-rank test. All experiments were repeated three more times and the results are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS software, Graphpad prism software was plotted, P <0.05 is statistically different and is indicated by x, P <0.01 is indicated by x, and P <0.001 is indicated by x.
Third, experimental results
1. Decreasing the expression or activity of ITGA4 inhibits the expansion of AML cells.
In AML cell line MV4-11, shRNA was used to interfere with the expression of ITGA4 or to block the activity of ITGA4 with NZM. The number of clones of MV4-11 cells was significantly reduced after knockdown or inhibition of ITGA 4. (FIG. 1) MV4-11 cells after the above treatment were cultured in vitro for 7 days, and dynamic cell number monitoring showed significant inhibition of cell proliferation. (FIG. 2) treated MV4-11 cells were fixed for membrane rupture, labeled DAPI and ki67, and flow analysis showed an increase in the proportion of cells in the G1 phase. (FIG. 3)
2. Decreasing the expression or activity of ITGA4 inhibits migration and adhesion ability of AML cells.
Following knockdown or inhibition of ITGA4, the number of MV4-11 cells migrated to the lower chamber of the transwell plate was significantly reduced, (fig. 4) the number of MV4-11 cells adhering to the fibrinectin coated plate was significantly reduced (see fig. 5).
3. Knockdown of expression of ITGA4 prolonged survival of AML mice constructed from MV4-11 cells.
ITGA4 shRNA and NSC control virus were transfected into MV4-11 cells and GFP was flow-sorted+The population of (1). Injection through tail vein of 1.0 × 105cells/NS 122 mice irradiated to 2.0Gy only to half lethal dose, mice survival time was monitored. Survival time was significantly prolonged in mice of knockdown ITGA4 group compared to NSC group. (FIG. 6) on day 15 after transplantation, mice were sacrificed, bone marrow hematopoietic cells, spleen cells and liver cells of the mice were isolated, human CD45 was labeled, and the ratio of CD45+ GFP + leukemia was flow-tested. Compared with the NSC group, the mice with the knockdown ITGA4 group have obviously reduced proportion of leukemia cells in bone marrow cells and obviously reduced proportion of leukemia cells in liver and spleen. (FIG. 7) the bone marrow, liver and spleen tissue sections of mice transplanted with NC and sh-ITGA4 cells for 15 days were subjected to HE staining, and as a result, the bone marrow, liver and spleen tissue structures of the mice in the NSC group were destroyed, and a large amount of leukemia cells were infiltrated, and the leukemia infiltrates of the bone marrow, liver and spleen tissues of the mice in the knocked-down ITGA4 group were significantly reduced (FIG. 8).
4. Knockdown of ITGA4 expression inhibited homing of AML cells to bone marrow and spleen hematopoietic tissues.
Tail vein injection of MV4-11 cells knocked-down in ITGA4 or NC into semi-lethal dose irradiated immunodeficient mice, 2.0X 10 per transplanted mouse6Cells, mice sacrificed 16 hours after transplantation, and the proportion of leukemia cells in the bone marrow and spleen of the mice were flow-tested. The proportion of MV4-11 cells that knockdown ITGA4 home to both bone marrow and spleen was significantly reduced compared to the NSC control group. (FIG. 9)
NZM inhibits clonal colony formation, migration and adhesion of leukemia cells and leukemia stem cells in refractory and relapsed patients.
Bone marrow cells of patients with refractory or relapsed AML were collected, and the specimens with numbers #61, #58 and #59 were refractory AML, and the specimen with number # 11 was relapsed AML. Leukemia cells were isolated and treated with IgG or NZM for 48 hours before colony formation, migration and adhesion. After NZM treatment, clonal colony formation of AML cells was significantly reduced in refractory or relapsed patients (fig. 10), the proportion of migrating cells was reduced (fig. 11), and the number of adherent cells was significantly reduced (fig. 12) compared to the IgG group.
Flow sorting of CD34+ leukemia stem cell populations from refractory or relapsed patients. The specimens with numbers #60, #62, #56 and #63 were of refractory AML and the specimen with number # 69 was of relapsed AML. The sorted leukemic stem cells were treated with IgG or NZM for 48 hours and subjected to colony formation experiments, migration and adhesion experiments. Similar to leukemia cells, NZM treatment also inhibited clonal colony formation (fig. 13), migration (fig. 14), and adhesion (fig. 15) of AML leukemia stem cells in refractory or relapsed patients.
In conclusion, the present invention evaluated ITGA4 as a target for treatment of refractory or relapsed AML and its use. The invention definitely reduces the expression or activity of ITGA4, and the proliferation of leukemia cells is inhibited; the survival of the leukemia mice is obviously prolonged; reducing the homing of leukemia cells. The blocking antibody NZM of ITGA4 significantly inhibited the clonogenic, migratory and adherent formation of leukemia cells and leukemia stem cells in refractory or relapsed AML patients.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
SEQUENCE LISTING
<110> Suzhou university
<120> ITGA4 gene inhibitor and application thereof in preparation of drug for treating refractory or relapsed acute myeloid leukemia
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 54
<212> DNA
<213> (Artificial Synthesis)
<400> 1
gctccgtgtt atcaagatta tctcgagata atcttgataa cacggagctt tttg 54
Claims (10)
1. An ITGA4 gene inhibitor, which is characterized by comprising shRNA with a nucleic acid sequence of SEQ ID NO. 1 and/or a natalub monoclonal antibody.
2. A recombinant vector comprising a sequence capable of transcribing the shRNA of claim 1 embedded in a vector.
3. The recombinant vector according to claim 2, wherein the vector is a lentiviral vector.
4. The recombinant vector according to claim 3, wherein the lentiviral vector is selected from the group consisting of pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagFP, pLKO.1-puro-TagFP 635, pLKO.1-puro-UbCoGFP, pDNAO.1-puro-UbC-TagFP 635, pLKO-puro-IPTG-1xLacO, pLKO-IPTG-3-pDNAxLatO, pDNA34-pDNAxlaPG, pDNAz-3976-LJV-3975, pLKO-3975/29-LerlaGW/32, pLKO-LerluWt-3, pLKO-3-pDNAz-3, pDNAz-3-LernyVal, pLKO-3-LELKO, pLKO-LKO, pLKO-3, pLKO-JV, pLKO, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.
5. A lentivirus, which is obtained by cell packaging of the recombinant vector of claim 2.
6. A pharmaceutical composition comprising the ITGA4 gene inhibitor of claim 1, the recombinant vector of claim 2, or the lentivirus of claim 5.
7. The pharmaceutical composition of claim 6, further comprising a pharmaceutically acceptable carrier.
8. The pharmaceutical composition of claim 7, wherein the carrier is selected from one or more of disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants, and preservatives.
9. A kit comprising the ITGA4 gene inhibitor of claim 1, the recombinant vector of claim 2, the lentivirus of claim 5 or the pharmaceutical composition of claim 6.
10. Use of the ITGA4 gene inhibitor according to claim 1, the recombinant vector according to claim 2, the lentivirus according to claim 5, the pharmaceutical composition according to claim 6 or the kit according to claim 9 for the preparation of a medicament for the treatment of refractory or relapsed acute myeloid leukemia.
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| US20100266587A1 (en) * | 2009-04-17 | 2010-10-21 | Biogen Idec Ma Inc. | Compositions and Methods to Treat Acute Myelogenous Leukemia |
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| WO2003097097A1 (en) * | 2002-05-15 | 2003-11-27 | Yoshiro Niitsu | Method of treating and/or preventing acute leukemia using medicinal composition for therapeutic use containing vla4 antagonist, and method of diagnosing prognosis of acute leukemia using vla4 as indication |
| US20100278837A1 (en) * | 2009-03-09 | 2010-11-04 | The Regents Of The University Of California | Compositions And Methods For Reducing Cancer And Inflammation |
| US20100266587A1 (en) * | 2009-04-17 | 2010-10-21 | Biogen Idec Ma Inc. | Compositions and Methods to Treat Acute Myelogenous Leukemia |
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