CN112089842A - Target point c-FOS related to leukemia treatment and application thereof - Google Patents

Target point c-FOS related to leukemia treatment and application thereof Download PDF

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CN112089842A
CN112089842A CN202010945739.5A CN202010945739A CN112089842A CN 112089842 A CN112089842 A CN 112089842A CN 202010945739 A CN202010945739 A CN 202010945739A CN 112089842 A CN112089842 A CN 112089842A
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bach2
fos
leukemia
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nalm
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张寒
冯敏
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Institute of Medical Biology of CAMS and PUMC
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Abstract

The invention discloses a target point c-FOS related to leukemia diagnosis and treatment and application thereof. Experiments prove that the inhibition of the expression of the c-FOS gene or the inhibition of the activity of the c-FOS protein can improve the sensitivity of leukemia cells to chemotherapeutic drugs or reduce the drug resistance of the leukemia cells to the chemotherapeutic drugs. The invention also discloses that the c-FOS gene is regulated and controlled by the transcription inhibition of a tumor suppressor BACH2 in leukemia cells; the tumor suppressor BACH2 is involved in transcriptional repression regulation by combining the c-FOS gene promoter and two MARE sequences in the 5' -UTR. The c-FOS is expected to become a potential target point for anti-leukemia therapy, and has very wide application prospect in the field of medical research.

Description

Target point c-FOS related to leukemia treatment and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a target point c-FOS related to leukemia diagnosis and treatment and application thereof.
Background
Leukemia is a malignant clonal disease of hematopoietic stem cells. Due to differentiation disorder, uncontrolled proliferation, and impaired apoptosis, malignant cells proliferate in large amounts in bone marrow and other hematopoietic tissues, causing changes in the microenvironment of the bone marrow, and thus causing severe clinical symptoms and complications. Among them, acute leukemia is one of the most common malignant tumors and major causes of death in childhood. The morbidity of the leukemia of children in China is 4-6/10 ten thousands, and the morbidity of the leukemia of children is on a rising trend year by year. Among them, Acute Lymphoblastic Leukemia (ALL) in children is the most common type, accounting for about 75% or more of leukemia in children. With the application of combined chemotherapy and supportive therapy, the cure rate of children ALL reaches more than 80% in China. Nevertheless, leukemia resistance and relapse have led to the bottleneck phase of childhood ALL treatment with no significant improvement in overall cure rates. The drug resistance and the recurrence rate of the leukemia of the children are as high as about 15-20 percent, which become the most key factors influencing the recovery of children with leukemia, and bring irreparable serious injury and inharmonious factors to the children, families and society.
In childhood leukemia chemotherapy regimens, cytarabine (Ara-C) is a pyrimidine antimetabolite that acts on the proliferative phase of cells S to interfere with cell proliferation by inhibiting cellular DNA synthesis. Cytarabine is mainly used for acute leukemia, has curative effects on acute myelocytic leukemia, acute lymphocytic leukemia and acute monocytic leukemia, and belongs to a core drug for inducing chemotherapy in the treatment scheme of the diseases. Nevertheless, long-term and large-dose application of cytarabine is easy to cause drug resistance reaction of children patients; in addition, the high-risk subtype of children with leukemia is very easy to have drug resistance reaction and disease recurrence due to high tumor malignancy, and the curative effect and disease outcome of the chemotherapy scheme for leukemia are seriously influenced.
Activator protein-1 (AP-1) is a heterodimer composed of c-JUN and c-FOS, and plays a role of a transcription activator in human cells. Among them, c-JUN shows high expression in malignant tumor, and participates in the generation and development of tumor by regulating cell malignant transformation, apoptosis, angiogenesis and DNA methylation. Compared with c-JUN, c-FOS has similar tumor promotion effect, and plays a key regulation and control role in bone cell proliferation, differentiation and bone marrow microenvironment reconstruction. Studies have shown that c-fos knock-out in mice can induce bone remodeling defects leading to severe osteopetrosis. Currently in the country, c-FOS protein inhibitors include nordihydroguaiaretic acid (NDGA) and curcumin (curcumin). Wherein, nordihydroguaiaretic acid is a natural substance and is widely present in various resin-containing plants; in addition, chemical synthesis of this substance has also been reported. Nordihydroguaiaretic acid is an antioxidant, has various biological functions and therapeutic effects, and particularly has obvious effects on proliferation inhibition and induced differentiation of tumors. Curcumin is a compound separated from turmeric, has wide pharmacological activities of anti-inflammation, antioxidation, blood fat reduction, anti-tumor, anti-infection and the like in medicine, and has low toxicity and small adverse reaction. Scientific research shows that the nordihydroguaiaretic acid and the curcumin are both inhibitors targeting c-FOS and can effectively inhibit the activation function of the c-FOS.
BTB and CNC homology protein 2(BTB and CNC homology 2, BACH2) are B-cell specific transcription factors, encoded by BACH2 gene, that play a key role in the process of B-cell directed development. At the lymphoid progenitor stage, BACH2 inhibits myeloid developmental programs, driving progenitor cell differentiation into B cells. In the pre-B cell stage, BACH2 competed with BCL6 for the regulatory gene promoter region, and B cells were negatively selected. During the subsequent B cell maturation phase, BACH2 further cooperates with BCL6 to prevent B cell differentiation into plasma cells and to regulate human somatic hypermutation and class switch recombination. Recent studies have shown that BACH2 is still involved in T cell-mediated immune responses and maintenance of the resting state of T cells. It can be seen that BACH2 plays an important regulatory role in lymphocyte development and differentiation.
BACH2 is a class of proteins with basic region leucine zipper (bZIP) domains. It forms heterodimer with small Maf protein in vivo, and inhibits the transcription of downstream target gene by identifying Maf recognition element (MARE, 5 '-TGCTGA [ G/C ] TCAGCA-3') on DNA sequence, thus playing a key role in regulation. During B cell development and differentiation, BACH2 is regulated by a number of upstream transcription factors, such as PAX5, E2A, and the like. In addition, BACH2 effects modulation of B cell development and differentiation by modulating downstream target genes such as PRDM1, HMOX1, and the like. These upstream regulatory factors and downstream target genes together contribute to the B cell-plasma cell gene regulatory network of BACH2, enabling BACH2 to regulate B cell development, maturation and differentiation. Therefore, the deep understanding of the gene regulatory network of BACH2 is helpful to further understand the occurrence mechanism and treatment of related diseases, such as leukemia.
Disclosure of Invention
The first purpose of the invention is to provide a new application of a substance for inhibiting the activity of c-FOS protein or a substance for reducing the content of c-FOS protein or a substance for silencing or knocking out or mutating c-FOS gene or a substance for inhibiting the expression of c-FOS gene.
The invention provides an application of a substance for inhibiting the activity of c-FOS protein or a substance for reducing the content of the c-FOS protein or a substance for silencing or knocking out or mutating c-FOS gene or a substance for inhibiting the expression of the c-FOS gene in any one of the following 1) or 2):
1) preparing a product for improving the sensitivity of leukemia cells to chemotherapeutic drugs;
2) preparing a product for reducing the drug resistance of leukemia cells to chemotherapeutic drugs.
The second purpose of the invention is to provide a product, the active ingredients of which are substances for inhibiting the activity of the c-FOS protein or substances for reducing the content of the c-FOS protein or substances for silencing or knocking out or mutating the c-FOS gene or substances for inhibiting the expression of the c-FOS gene;
the product has the function of improving the sensitivity of the leukemia cells to the chemotherapeutic drugs or reducing the drug resistance of the leukemia cells to the chemotherapeutic drugs.
In any of the above uses or products, the increasing the sensitivity of the leukemia cells to the chemotherapeutic agent is synergistically increasing the sensitivity of the leukemia cells to cytarabine.
The drug resistance of the leukemia cells to the chemotherapeutic drugs is reduced by the drug resistance reaction of cytarabine resistant cells or the drug resistance reaction of the leukemia cells to cytarabine in a drug resistance co-culture medium.
In any of the above applications or products, the substance for inhibiting the activity of c-FOS protein or the substance for reducing the content of c-FOS protein may be any substance known to those skilled in the art that can inhibit the synthesis of c-FOS protein or promote the degradation of c-FOS protein or inhibit the function of c-FOS protein, such as protein (e.g., c-FOS antibody), polypeptide or small molecule compound (e.g., c-FOS inhibitor), etc.
The substance for silencing or knocking out or mutating the c-FOS gene or the substance for inhibiting the expression of the c-FOS gene can be any substance which can reduce the expression quantity of the c-FOS gene or cause deletion mutation or insertion mutation or base substitution of the c-FOS gene, such as nucleic acid molecules (such as miRNA, siRNA, dsRNA, shRNA and the like) or CRISPR/Cas9 system and the like, which are well known to those skilled in the art.
In the embodiment of the invention, the substance for inhibiting the activity of the c-FOS protein is an inhibitor targeting c-FOS, in particular nordihydroguaiaretic acid or curcumin.
In any of the above uses or products, the c-FOS gene has at least one of the following a) and b) properties:
a) is regulated by the transcriptional inhibition of a tumor suppressor BACH2 in leukemia cells;
b) binds to tumor suppressor BACH2 in leukemia cells.
Further, the sequence of the c-FOS gene which binds to the tumor suppressor BACH2 in leukemia cells is located in the c-FOS gene promoter and 5 'untranslated region (5' -UTR).
Furthermore, the sequence of the c-FOS gene which is combined with the tumor suppressor BACH2 in leukemia cells is a DNA molecule shown in a sequence 5 and/or a DNA molecule shown in a sequence 6.
In any of the above uses or products, the chemotherapeutic agent is cytarabine.
In any of the above applications or products, the leukemia cell can be a human B lymphocyte leukemia cell or a human B lymphocyte leukemia cytarabine resistant cell.
The human B lymphocyte leukemia cell can be a human B lymphocyte leukemia cell derived from a cell line or a B lymphocyte leukemia bone marrow cell derived from a patient.
The human B lymphocyte leukemia cytarabine drug-resistant cells are human B lymphocyte leukemia cells for knocking down BACH 2.
In any of the above applications or products, the amino acid sequence of the tumor suppressor BACH2 is sequence 1 in the sequence table, and the nucleotide sequence of the BACH2 gene is sequence 2 in the sequence table.
The amino acid sequence of the c-FOS protein is a sequence 3 in a sequence table, and the nucleotide sequence of the c-FOS gene is a sequence 4 in the sequence table.
The application of the c-FOS gene as a target point in the development or design of products for treating or assisting in treating leukemia also belongs to the protection scope of the invention.
The application of the c-FOS gene as a downstream target gene of a tumor suppressor BACH2 in participating in BACH2 gene network regulation also belongs to the protection scope of the invention.
The present invention first examined the combined efficacy of small doses of cytarabine (20nM) and two c-FOS inhibitors (nordihydroguaiaretic acid or curcumin). The results show that: leukemia cells were cultured in RPMI-1640 medium (normal medium) containing 10% FBS, and the human B-lymphocytic leukemia cell line Nalm-6/BACH2 was treated with 20nM cytarabine (Ara-C)ConCytarabine drug-resistant strain cell line Nalm-6/BACH2 of human B lymphocyte leukemiaKD-2 and two cases of children with acute B lymphocyte leukemia (Pt1 and Pt2) with leukemia cell survival rates of 47.03%, 77.29%, 53.15% and 46.65%, respectively; with proto-oncoprotein c-FOS as a target spot, Nalm-6/BACH2 is treated by combining 5 mu M nordihydroguaiaretic acid and 20nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells are 7.88%, 18.71%, 12.74% and 8.79%, respectively; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 20nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells were 37.70%, 35.77%, 43.12% and 38.33%, respectively. Leukemia cells were cultured in drug-resistant coculture media, and Nalm-6/BACH2 was treated with 20nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rate of leukemia cells is 90.72%, 97.70% and 92.98% respectivelyAnd 91.60%; with proto-oncoprotein c-FOS as a target spot, Nalm-6/BACH2 is treated by combining 5 mu M nordihydroguaiaretic acid and 20nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells being 31.30%, 34.69%, 35.68% and 33.26%, respectively; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 20nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells were 63.38%, 61.60%, 67.54% and 63.77%, respectively.
The present invention further examined the combined efficacy of large doses of cytarabine (200nM) with two c-FOS inhibitors (nordihydroguaiaretic acid or curcumin). The results are as follows: leukemia cells were cultured in normal medium and Nalm-6/BACH2 was treated with 200nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells being 21.06%, 50.90%, 27.13% and 22.85%, respectively; with proto-oncoprotein c-FOS as a target spot, Nalm-6/BACH2 is treated by combining 5 mu M nordihydroguaiaretic acid and 200nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells are 5.34%, 9.63%, 9.22% and 6.79%, respectively; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 200nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells were 17.47%, 19.20%, 22.82% and 19.05%, respectively. Similarly, leukemia cells were cultured in drug-resistant coculture media with 200nM cytarabine Nalm-6/BACH2Con、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells are 65.95%, 79.99%, 71.93% and 65.16%, respectively; with proto-oncoprotein c-FOS as a target spot, Nalm-6/BACH2 is treated by combining 5 mu M nordihydroguaiaretic acid and 200nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells being 32.24%, 34.94%, 34.04% and 35.68%, respectively; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 200nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells were 60.30%, 65.87%, 69.05% and 62.77%, respectively.
The invention also adopts Compuyn software to calculate drug Combination Index (CI) to analyze the combination effect of the cytosine arabinoside combined with the nordihydroguaiaretic acid or the curcumin. Experiments prove that leukemia cells are cultured in a common culture medium, and Nalm-6/BACH2 is treated by combining 20nM cytarabine and 200nM cytarabine with 5 mu M nordihydroguaiaretic acid respectivelyConThe CI values of the cells are 0.36 and 0.23 respectively, and are both less than 1; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 20nM and 200nM cytarabine, respectivelyConThe CI values of the cells were 0.90 and 0.67, respectively, both less than 1. Similarly, leukemia cells were cultured in normal medium with 5. mu.M nordihydroguaiaretic acid-treated Nalm-6/BACH2 in combination with 20nM cytarabine and 200nM cytarabine, respectivelyKD-2 cells had CI values of 0.12 and 0.07, respectively, both less than 1; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 20nM and 200nM cytarabine, respectivelyKDCI values for-2 cells were 0.06 and 0.12, respectively, both less than 1. In addition, leukemia cells were cultured in drug-resistant coculture media with 5 μ M nordihydroguaiaretic acid-treated Nalm-6/BACH2 in combination with 20nM and 200nM cytarabine, respectivelyConThe CI values of the cells are 0.02 and 0.12 respectively, and are both less than 1; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 20nM and 200nM cytarabine, respectivelyConThe CI values of the cells were 0.26 and 0.61, respectively, both less than 1. Similarly, leukemia cells were cultured in drug-resistant coculture media with 5 μ M nordihydroguaiaretic acid-treated Nalm-6/BACH2 in combination with 20nM and 200nM cytarabine, respectivelyKD-2 cells had CI values of 0.01 and 0.08, respectively, both less than 1; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 20nM and 200nM cytarabine, respectivelyKDCI values for-2 cells were 0.03 and 0.36, respectively, both less than 1.
The invention finally researches the regulation mechanism of the tumor suppressor BACH2 on proto-oncoprotein c-FOS. In the first step, a real-time fluorescent quantitative PCR (qRT-PCR) method is adopted to detect a stably-transformed recombinant leukemia cell line Nalm-6/BACH2KDmRNA level of c-FOS gene in-2, results show that recombinant leukemia cell line Nalm-6/BACH2 is stably transferredKDThe mRNA level of the c-FOS gene in-2 was significantly higher than that of the control group, and a negative correlation was shown between the two. Secondly, in the human kidney epithelial cell line 293T cell, the BACH2 recombinant expression plasmid pcDNA3.1(+) -BACH2 is respectively pre-mixed with the plasmid carrying different MAREsThe c-FOS gene luciferase recombinant plasmids (pGL3-MARE1 and pGL3-MARE2) of the site-measuring sites and the renilla luciferase expression vector plasmid pRL-SV40 were co-transfected into 293T cells, and the results show that the transcriptional activity of pGL3-MARE2 carrying 2 different MARE predicted sites is reduced by about 25 times compared with the control group, and the obvious transcriptional inhibition effect is presented. Thirdly, the cell line Nalm-6 of the human B lymphocyte leukemia is adopted to carry out CUT&Tag high-throughput sequencing and analysis results show that compared with a control group, a significant BACH2 protein enrichment peak exists in a c-FOS gene promoter and a 5 'untranslated region (5' -UTR). The fourth step adopts CUT&PCR verification is carried out on the Tag library, and the result shows that the BACH2 protein is combined with the c-FOS gene promoter and two MARE sequences on the 5' -UTR respectively.
The experimental result shows that the drug resistance of the cytarabine resistant strain cell and the leukemia cell in the drug resistant co-culture medium can be effectively reduced by treating the leukemia cell with the proto-oncoprotein c-FOS inhibitor (nordihydroguaiaretic acid or curcumin), and a new way for overcoming the drug resistant reaction is provided for the leukemia treatment; on the other hand, the tumor suppressor BACH2 is shown to be capable of inhibiting the expression of the c-FOS gene in a transcription mode, the tumor suppressor BACH2 participates in transcription inhibition regulation and control by combining a c-FOS gene promoter and two MARE sequences in 5' -UTR, a new BACH2 downstream target gene is provided for leukemia research, and the downstream target gene is expected to become a potential target point for leukemia resistance treatment, so that the application prospect in the field of medical research is quite wide.
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FIG. 1 shows the Western Blot result of tumor suppressor BACH2 in RNA interference stable recombinant leukemia cells. The first row is a detection tumor suppressor BACH2 with BACH2 monoclonal antibody as a primary antibody, and the second row is a detection internal reference GAPDH with GAPDH monoclonal antibody as a primary antibody. Wherein, BACH2KD-1 represents the stable recombinant leukemia cell Nalm-6/BACH2KD-1；BACH2KD-2 represents the stable recombinant leukemia cell Nalm-6/BACH2KD-2；BACH2ConRepresenting stably transformed recombinant leukemia cell Nalm-6/BACH2Con。
FIG. 2 shows the detection of RNA interference stable-transformation recombinant leukocyte by flow cytometryThe cellular ratio of 7-AAD (-)/Annexin V (+) to 7-AAD (+)/Annexin V (+) in the leukemic cells. Wherein NS is normal saline, Ara-C is chemotherapy drug cytarabine. Wherein, BACH2KD-2 represents the stable recombinant leukemia cell Nalm-6/BACH2KD-2；BACH2ConRepresenting stably transformed recombinant leukemia cell Nalm-6/BACH2Con。
FIG. 3 is a statistical chart of the survival rate of stably transformed recombinant leukemia cells treated by different concentrations of drugs and the half inhibitory concentration of the drugs. Wherein Ara-C is Cytarabine and Red IC50Value BACH2KD-2 semi-inhibitory concentration of drug in cells, black IC50Value BACH2ConThe semi-inhibitory concentration of the drug in the cell. Wherein, BACH2KD-2 represents the stable recombinant leukemia cell Nalm-6/BACH2KD-2；BACH2ConRepresenting stably transformed recombinant leukemia cell Nalm-6/BACH2Con。
FIG. 4 is a statistical chart of the survival rate of leukemia cells treated with different concentrations of drugs and the half inhibitory concentration of the drugs. Wherein Ara-C is Cytarabine, BACH2ConRepresenting stably transformed recombinant leukemia cell Nalm-6/BACH2Con、BACH2KD-2 represents the stable recombinant leukemia cell Nalm-6/BACH2KD-2, black IC50The value is the drug half-inhibitory concentration, red IC, of leukemia cells in the common culture medium50The values are the drug half inhibitory concentration of leukemia cells in co-culture medium.
FIG. 5 is a statistical chart of the survival rate of leukemia cells treated with drugs in a common culture medium. Wherein, Control is normal saline (Control group), Ara-C is chemotherapy drug cytarabine, NDGA is proto-oncoprotein C-FOS inhibitor nordihydroguaiaretic acid, and curcumin is proto-oncoprotein C-FOS inhibitor curcumin. Wherein, the four groups on the left adopt 20nM cytarabine; the right four groups of data were performed with 200nM cytarabine. The results of the experimental data were averaged ± standard deviation, wherein the cytarabine combined nordihydroguaiaretic acid and cytarabine combined curcumin groups were compared with the cytarabine treated group, respectively. The common medium represents RPMI-1640 medium containing 10% FBS.
FIG. 6 is a statistical graph of the survival rate of leukemia cells treated with drugs in co-culture medium. Wherein, Control is normal saline (Control group), Ara-C is chemotherapy drug cytarabine, NDGA is proto-oncoprotein C-FOS inhibitor nordihydroguaiaretic acid, and curcumin is proto-oncoprotein C-FOS inhibitor curcumin. Wherein, the four groups on the left adopt 20nM cytarabine; the right four groups of data were performed with 200nM cytarabine. The results of the experimental data were averaged ± standard deviation, wherein the cytarabine combined nordihydroguaiaretic acid and cytarabine combined curcumin groups were compared with the cytarabine treated group, respectively. The co-culture medium represents a culture medium obtained by co-culturing a human bone marrow stromal cell line HS-5 and a human B lymphocyte leukemia cell line Nalm-6.
FIG. 7 is a statistical chart of drug combination index in a common medium. Wherein, A + N represents the combination of cytarabine and nordihydroguaiaretic acid, A + C represents the combination of cytarabine and curcumin, Fa represents the inhibition rate, and CI represents the combination index. The common medium represents RPMI-1640 medium containing 10% FBS.
FIG. 8 is a statistical plot of drug combination index in co-culture medium. Wherein, A + N represents the combination of cytarabine and nordihydroguaiaretic acid, A + C represents the combination of cytarabine and curcumin, Fa represents the inhibition rate, and CI represents the combination index. The co-culture medium represents a culture medium obtained by co-culturing a human bone marrow stromal cell line HS-5 and a human B lymphocyte leukemia cell line Nalm-6.
FIG. 9 shows that the mRNA level of c-FOS gene stably knocks down the recombinant leukemia cell line Nalm-6/BACH2 of the tumor suppressor BACH2KD-real-time fluorescent quantitative PCR (qRT-PCR) assay results in 2. Wherein, BACH2KD-2 represents the stable recombinant leukemia cell Nalm-6/BACH2Con；BACH2ConRepresenting stably transformed recombinant leukemia cell Nalm-6/BACH2Con。
FIG. 10 shows the statistical results of dual-luciferase reporter gene assays. Wherein, the c-FOS fragment (position-1050-0) carrying 1 MARE prediction site (5'-CTGAGACAGGA-3' at positions-212 to-202) is inserted into a pGL3-basic vector and named as pGL3-MARE 1; inserting a c-FOS fragment (positions-1050-412) carrying 2 MARE prediction sites (5'-CTGAGACAGGA-3' of positions-212 to-202; 5'-AAGACTGAGCCG-3' of positions 32-43) into a pGL3-basic vector, and naming the fragment as pGL3-MARE 2; the empty luciferase reporter plasmid pGL3-basic served as blank control. The BACH2 recombinant expression plasmid pcDNA3.1(+) -BACH2 is constructed by subcloning the open reading frame sequence coding for the human BACH2 protein from a commercially available Lenti-ORF-BACH2 plasmid into an expression vector pcDNA3.1(+), which is named pcDNA3.1-BACH 2; the no-load expression vector plasmid pcDNA3.1(+) was designated pcDNA3.1 as a negative control.
FIG. 11 shows the result of CUT & Tag high-throughput sequencing using the cell line Nalm-6 of human B-lymphocytic leukemia. Using the human GRCh38 genome as a reference, the human c-FOS gene region was analyzed for BACH2 protein enrichment peaks using the visual analysis software IGV v2.8.3. The results show that compared with the IgG control group, the BACH2 protein shows two obvious enrichment peaks in the promoter and 5' -UTR regions of the c-FOS gene respectively, and the regions corresponding to the peaks are matched with two predicted MARE sequences (MARE1 and MARE 2).
FIG. 12 shows the results of PCR verification using the CUT & Tag library. The top panel shows a simplified diagram of the positions of the different DNA fragments on the c-FOS gene; the lower panel shows the results of PCR using the CUT & Tag library for each DNA fragment and the relative expression ratio. The results showed that both the c-FOS-b fragment containing the predicted site of MARE1 and the c-FOS-c fragment containing the predicted site of MARE2 bound to the BACH2 protein; compared with the IgG control group, the binding multiple of the c-FOS-b fragment and the BACH2 protein is about 12.16; compared with the IgG control group, the binding ratio of the c-FOS-c fragment to the BACH2 protein was about 4.98.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The experiments in the following examples were set up in triplicate and the results averaged ± standard deviation. The statistical analysis used the t-test. Denotes a p value of less than 0.05; denotes a p value of less than 0.01; indicates that the p value is less than 0.001.
The expression vector plasmid pcDNA3.1(+) in the following examples is a product of Invitrogen, USA; the BACH2-ORF plasmid is a product of GE Dharmacon, USA; luciferase expression vector plasmid pGL3-basic, Renilla luciferase expression vector plasmid pRL-SV40 and dual luciferase reporter gene detection kit are products of Promega corporation, USA.
The human B-lymphocytic leukemia cell line Nalm-6 in the examples below is a product of the German Collection of microorganisms and cell cultures (DSMZ). The human kidney epithelial cell line 293T is a product of Kunming animal research institute, Chinese academy of sciences. Human bone marrow stromal cell line HS-5 is a product of the institute of Biotechnology, Beijing Beinan, and Association. The cell lines are identified and confirmed by cell STR identification.
Lenti-BACH2-shRNA-1, Lenti-BACH2-shRNA-2 and Lenti-NS-shRNA, lentivirus-mediated shRNA plasmids in the following examples, are all products of GE Dharmacon, USA. Wherein, the Clone numbers (Clone ID) of Lenti-BACH2-shRNA-1 and Lenti-BACH2-shRNA-2 are V3LHS _363286 and V3LHS _409004 respectively. Mature antisense strand sequence of V3LHS _ 363286: AAATTCTGAATACAGTCCA are provided. Mature antisense strand sequence of V3LHS _ 409004: ACTTCGGAACAGTATTGCT are provided.
The lentiviral packaging plasmids pMD2.G and psPAX2 in the examples below are both products of Invitrogen, USA.
The Acute Lymphoblastic Leukemia (ALL) chemotherapeutic drug cytarabine (Ara-C) in the following examples was cytarabine for injection produced by Saedsa, production lot 7ND 5121. Nordihydroguaiaretic acid (NDGA) is a product from Sigma-Aldrich, under the trade designation 74540-1G. Curcumin (curcumin) is a product of Sigma-Aldrich, cat # 8203540010.
The RPMI-1640 medium in the examples described below was a product of Thermo Fisher Scientific, cat # C11875500 BT.
The DMEM medium in the examples described below is a product of Biological Industries, Inc. under the trade designation 06-1055-57-1 ACS.
The amino acid sequence of the tumor suppressor BACH2 in the following examples is sequence 1 in the sequence table, and the coding gene sequence is sequence 2 in the sequence table.
The amino acid sequence of proto-oncoprotein c-FOS in the following embodiment is sequence 3 in the sequence table, and the coding gene sequence is sequence 4 in the sequence table.
Half Inhibitory Concentrations (IC) in the following examples50) Refers to the drug concentration at which 50% of tumor cell growth is inhibited.
The drug combination index CI in the examples below refers to the quantitative determination of the extent of drug interaction at a certain end-point of effect measurement when combined with chemotherapy. If CI is less than 1, indicating that there is a synergistic effect between the drugs; if CI equals 1, indicating that there is an additive effect between the drugs; if CI is greater than 1, it indicates that there is antagonism between the drugs.
Example 1 preparation of test cells and culture Medium
First, collection and processing of clinical samples
1. Collection of clinical specimens
Two primary bone marrow specimens were collected from two leukemic children hospitalized in the Beijing Children hospital during the 11 month period of 2018.
2. Diagnosis and typing of infant patients in leukemia initial diagnosis
Two cases of leukemia patients were confirmed to be common B-ALL (c-B-ALL) by clinical, morphological, cytochemical staining and immunological methods. The diagnosis and classification standard is according to national standard (diagnosis and treatment suggestion of acute leukemia in children, China J.pediatrics 1999,137(5): 305-307). And (3) detecting 29 genes formed by chromosome structure distortion in the bone marrow samples of the primary diagnosis of the two ALL patients by adopting a multiple nested reverse transcription PCR method, wherein the detection result is negative. The details of the infant patients are shown in table 1.
TABLE 1
Numbering Sex First diagnosis age (year of age) Immunotyping Chromosomal translocation Fusion gene
1 Woman 2.6 c-B-ALL － －
2 Woman 3.3 c-B-ALL － －
3. Collection of bone marrow specimens
Under sterile conditions, 2ml of bone marrow of ALL patient infants is extracted (from different sites, such as sternum or ilium), and anticoagulant Edetate (EDTA) is added. Collecting bone marrow specimens: EDTA anticoagulation, bone marrow amount greater than 2ml, no blood clot and specimen storage time less than 24 hours.
4. Extraction of mononuclear cells
Extracting mononuclear cells from collected bone marrow, which comprises the following specific operation steps:
A. and (3) cracking red blood cells: taking 2ml of bone marrow specimen, putting the bone marrow specimen into an extraction tube, adding 10ml of erythrocyte lysate (20 mg of EDTA sodium salt, 4.2g of ammonium chloride and 0.5g of potassium bicarbonate, adding distilled water to a constant volume of 500ml) into the extraction tube, fully mixing with the bone marrow, standing at room temperature for 3 minutes, and centrifuging at the normal temperature of 1000rpm for 3 minutes. After the centrifugation, the supernatant liquid was discarded.
B. Observing mononuclear cells extracted from the bottom layer of the extraction tube, if more erythrocytes are mixed therein, repeating the step A1-2 times to remove the excess erythrocytes.
C. And (3) washing a lysate: placing 10ml of normal saline into an extraction tube, blowing and beating bottom layer cells by using a suction tube to fully mix the bottom layer cells with the normal saline, centrifuging at the normal temperature of 1000rpm for 3 minutes, and discarding the upper layer liquid after the centrifugation is finished.
D. Adding small amount of physiological saline into the extraction tube, blowing with a pipette to mix with the bottom layer cells, transferring into 1.5mL EP storage tube, centrifuging at 4000rpm at room temperature for 5 s, discarding the upper layer physiological saline, and extracting mononuclear cells with the number of 107Several orders of magnitude.
E. And storing the extracted mononuclear cell specimen in a refrigerator at the temperature of minus 80 ℃ for later use.
Second, construction of RNA interference stable transfection cell line of tumor suppressor BACH2
1. Packaging and preparation of RNA interference lentivirus of tumor suppressor BACH2
1) Culture of cells for transfection
24 hours before transfection, DMEM medium containing 10% Fetal Bovine Serum (FBS) was used in 100mm dishes at 37 ℃ and 5% CO2The human kidney epithelial cell line 293T is transfected when 60% -70% is cultured under the condition.
2) Packaging and preparation of lentiviruses
Transfecting lentivirus shRNA plasmids Lenti-BACH2-shRNA-1 and Lenti-BACH2-shRNA-2 of a targeting tumor suppressor BACH2 and shRNA plasmids Lenti-NS-shRNA-2 of a non-silencing control into the cells cultured in the step 1) respectively to obtain virus solutions for expressing Lenti-BACH2-shRNA-1, Lenti-BACH2-shRNA-1 and Lenti-NS-shRNA.
The specific method of transfection is as follows:
two sterile 1.5ml EP tubes were taken, and one of the tubes was diluted 12. mu.g of Lenti-BACH2-shRNA-1 or Lenti by adding 300. mu.l of serum-free DMEM medium-BACH2-shRNA-2 or Lenti-NS-shRNA plasmid, 6 μ g of packaging plasmid-pmd 2.g and 6 μ g of packaging plasmid-bipax 2; another EP tube was filled with 300. mu.l serum-free DMEM medium and 72. mu.l Lipofectamine2000 reagent. The two tubes were mixed well and allowed to stand at room temperature for 5 minutes. Adding 600 μ l of the mixture dropwise into a 100mm cell culture dish, shaking the dish gently, mixing well, transferring to 37 deg.C, and 5% CO2And continuing culturing in the cell culture box. The lentivirus-containing supernatant was collected 48 hours after transfection and filtered into 50ml sterile centrifuge tubes using a 0.45 μm filter. After centrifugation at 25000rpm for 90 minutes using a 4 ℃ ultracentrifuge, the supernatant was carefully removed, and the viral particles at the bottom of the tube were retained. 1ml of pre-cooled 1 XPBS buffer was added to the bottom of the tube and the tube was refrigerated overnight at 4 ℃. The next day, the lysed virus solution was frozen in a-80 ℃ freezer for subsequent use by infected cells.
2. Screening and establishment of RNA interference stable transfection cell line of tumor suppressor BACH2
Respectively infecting the virus liquid obtained in the step 1 with a human B lymphocyte leukemia cell line Nalm-6 to establish a stable transfer recombinant leukemia cell line Nalm-6/BACH2KD-1、Nalm-6/BACH2KD-2, and the control cell line Nalm-6/BACH2Con. The method comprises the following specific steps:
1) culture of cells for infection
Using RPMI-1640 medium containing 10% FBS in T-25 gas-permeable cell culture flask, 37 deg.C and 5% CO2Culturing B lymphocyte leukemia cell line Nalm-6 under the condition.
2) Lentivirus infection
Counting the cell density of Nalm-6 cultured in the step 1), and taking the cell with 2 x 106The culture medium of each cell was centrifuged at 1000rpm for 5 minutes to remove the medium. The cells were resuspended in 100mm culture dishes using 10ml RPMI-1640 medium containing 10% FBS. Add polybrene (polybrene) 8. mu.g/ml to the cell suspension, let stand at 37 ℃ with 5% CO2Culturing in a cell culture box. After 1 hour, the cells were removed, 300. mu.l of the virus solution was added, centrifuged at 2500rpm for 1 hour at room temperature, transferred to a T-25 vented cell culture flask, and placed at 37 ℃ in 5% CO2The cultivation in the cell incubator was continued for 24 hours.
3) Screening and establishment of stable transfection (stable transformation) recombinant leukemia cell line
Removing the cell line cultured in step 2), removing the culture medium, adding 10ml of fresh RPMI-1640 containing 10% FBS, resuspending the cells, further standing at 37 deg.C and 5% CO2Culturing in a cell culture box. After 48 hours, the cells in the T-25 flask were transferred to a T-75 gas cell culture flask and continued to be cultured by supplementing 10ml of fresh RPMI-1640 medium containing 10% FBS. After 24 hours, the medium was removed, 20ml of fresh medium was replaced to resuspend the cells, and 2. mu.g/ml puromycin (puromycin) was added to screen the cell lines, and the medium was replaced every 2 to 3 days for 10 to 14 days.
3. Western Blot detection of expression of tumor suppressor BACH2 in stably transfected recombinant leukemia cell line
Taking the stable transfer recombinant leukemia cell line Nalm-6/BACH2 established in the step 2KD-1、Nalm-6/BACH2KD-2 and Nalm-6/BACH2ConTotal proteins were extracted separately, and Western blot detection was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference. The primary antibody for detecting tumor suppressor BACH2 was BACH2 (molecular weight 130KDa) monoclonal antibody (available from Cell Signaling Technology, USA), and the primary antibody for detecting internal reference GAPDH was GAPDH monoclonal antibody (available from Cell Signaling Technology, USA), and the results are shown in FIG. 1.
The results show that: stable transfer recombinant leukemia cell line Nalm-6/BACH2 established by Lenti-BACH2-shRNA-1 plasmidKD-1 and stable transfer recombinant leukemia cell line Nalm-6/BACH2 established by adopting Lenti-BACH2-shRNA-2 plasmidKDThe expression level of BACH2 protein in-2 is obviously lower than that of a stable leukemia cell line Nalm-6/BACH2 established by a control Lenti-NS-shRNA lentivirusCon。
The specific method for extracting total protein is as follows:
centrifuging 90g for 5 minutes to collect stably expressed leukemia cells, washing twice with precooled 1 XPBS buffer, adding 150-300. mu.l RIPA buffer, cracking on ice for 30min, centrifuging at 12000rpm for 30min at 4 ℃; sucking the supernatant, and performing protein quantification by using a Bradford method; 20 μ g of each total protein was subjected to Western blot detection.
The specific method for detecting the Western blot is as follows:
1) electrophoresis and membrane conversion: and (3) carrying out SDS-PAGE electrophoresis on the protein sample to be detected, firstly carrying out electrophoresis for 30 minutes under the voltage of 80V, and after the front edge of the dye enters the separation gel, increasing the voltage to 120V and continuing the electrophoresis for about 1-1.5 hours until the bromophenol blue reaches the bottom of the separation gel. After the electrophoresis was completed, the separated protein sample was transferred to a nitrocellulose membrane (NC membrane) by an electrotransfer method at 400mA for 2 hours.
2) Sealing the membrane: the NC membrane was washed with PBS-T (2 ml of Tween-20 was added to 1 XPBS buffer and the volume was adjusted to 1L) for 10-15 minutes. The NC membrane was placed in a PBS-T solution containing 5% BSA and blocked at room temperature for 1 hour.
3) Immunohybrid a, primary antibody incubation: BACH2 (molecular weight 130KDa) monoclonal antibody (or GAPDH monoclonal antibody) was diluted 1:1000 in PBS-T solution, incubated overnight with NC membrane on a destaining shaker at 4 ℃ in a cold room, and the NC membrane was washed with 5ml of PBS-T solution for 10 minutes, repeated 3 times.
B. And (3) secondary antibody incubation: goat anti-rabbit IgG antibody conjugated with horseradish peroxidase was diluted 1:5000 in PBS-T solution, incubated with NC membrane on a destaining shaker at room temperature for about 45 minutes, and the NC membrane was washed with 5ml of PBS-T solution for 10 minutes, repeated 3 times.
4) ECL reagent color development: NC membrane color reaction was performed using ECL protein hybridization detection kit (BioRad, usa) with reference to the protocol.
Preparation of the three, Co-Medium
1. Culture of the cells used
DMEM medium containing 10% FBS in 100mm dishes at 37 ℃ and 5% CO2Culturing a human bone marrow stromal cell line HS-5 under the condition; in a T-25 gas-permeable cell culture flask, 37 ℃ and 5% CO in RPMI-1640 medium (normal medium) containing 10% FBS2The cell line Nalm-6 of human B lymphocyte leukemia is cultured under the condition.
2. Preparation of the Co-Medium
Adding 2X 10 cells after the HS-5 cells cultured in the step 1 form a monolayer of adherent cells6The Nalm-6 suspension cells cultured in the step 1,a co-culture system was obtained, in which case the medium in the co-culture system was a mixed medium obtained by mixing a DMEM medium containing 10% FBS and an RPMI-1640 medium containing 10% FBS at a volume ratio of 1: 1. The CO-culture system was placed at 37 ℃ in 5% CO2Culturing in a cell culture box, collecting the culture medium after 48 hours of co-culture, centrifuging at room temperature of 10000rpm to remove suspended cells and cell debris to obtain the co-culture medium for subsequent use.
Example 2 detection of drug resistance in test cells and Medium
First, detection of apoptosis level of chemotherapy drug treated stable recombinant leukemia cells
Taking the stably transformed recombinant leukemia cell Nalm-6/BACH2 established in the second step of the example 1KD-2 with Nalm-6/BACH2ConThe test cells were cultured in 3mL of RPMI-1640 medium containing 10% FBS at a cell density of 5X 10 in 6-well plates5And (3) adding 20nM cytarabine (Ara-C) and Normal Saline (NS) which are clinically common chemotherapy drugs for Acute Lymphoblastic Leukemia (ALL) into each hole, respectively, harvesting the cells after 48 hours, and carrying out apoptosis detection on the cells by adopting a flow cytometer. The apoptosis detection kit is a 7-AAD/Annexin V apoptosis kit of BD Biosciences in the United states. The results are shown in FIG. 2.
The results show that: after Ara-C treatment, recombinant leukemia cells Nalm-6/BACH2 are stably transformedKD-2 and Nalm-6/BACH2ConThe early apoptosis ratio of (1) is 25.63% and 35.24%, respectively; the proportion of late apoptosis is 12.19 percent and 25.44 percent respectively; the overall apoptosis ratio was 37.82% and 60.68%, respectively. After NS treatment, recombinant leukemia cells Nalm-6/BACH2 are stably transformedKD-2 and Nalm-6/BACH2ConThe early apoptosis ratio of (1.85%) and (2.17%) respectively; the proportion of late apoptosis was 1.45% and 3.44%, respectively, and the proportion of total apoptosis was 3.3% and 5.61%, respectively.
Second, detection of leukemia cell survival rate by chemotherapy drug treatment
Taking the stably transformed recombinant leukemia cell Nalm-6/BACH2 established in the second step of the example 1KD-2 with Nalm-6/BACH2ConThe test cells were cultured in 1mL of the culture mediumCell density of 5X 10 in 24-well plates of RPMI-1640 medium containing 10% FBS4Ara-C (0, 2.5nM, 5nM, 10nM, 25nM, 50nM, 100nM, 200nM, 500nM and 1000nM) was added to each well at different drug concentrations, and the cells were harvested after 48 hours, tested for cell viability and calculated as the drug half Inhibitory Concentration (IC) using the CellTiter-Blue cell viability assay kit from Promega, USA50). The results are shown in FIG. 3.
The results show that: after being treated by Ara-C with different concentrations, the recombinant leukemia cell Nalm-6/BACH2 is stably transformedKD-2 cells have a significantly reduced sensitivity to Ara-C; stable transfer recombinant leukemia cell Nalm-6/BACH2KD-2 and Nalm-6/BACH2ConIC for Ara-C50191.44nM and 22.68nM, respectively. The expression of the interference tumor suppressor BACH2 is proved to be capable of inhibiting B-ALL cells from apoptosis, reducing the sensitivity of leukemia cells to chemotherapeutic drugs and increasing the drug resistance of leukemia cells to chemotherapeutic drugs. The stable transfer recombinant leukemia cell line Nalm-6/BACH2 constructed by the inventionKD-2 has obvious drug resistance of cytarabine and is used for subsequent evaluation of the curative effect of the combined chemotherapy drug.
Third, the survival rate of leukemia cells in the chemotherapy drug treated common culture medium and the co-culture medium is compared
Taking the stable transfer recombinant leukemia cell line Nalm-6/BACH2 established in the second step of the example 1KD-2 with Nalm-6/BACH2ConAs test cells, the cells were cultured in 1mL of 24-well plates containing a common medium (RPMI-1640 medium containing 10% FBS) and the co-medium prepared in step three of example 1 at a cell density of 5X 104Per well, cytarabine Ara-C was added at different drug concentrations (0, 2.5nM, 5nM, 10nM, 25nM, 50nM, 100nM, 200nM, 500nM and 1000nM), and after 48 hours the cells were harvested, the viability of the cells was examined and the half Inhibitory Concentration (IC) of the drug was calculated using CellTiter-Blue cell viability assay kit from Promega, USA50). The results are shown in FIG. 4.
The results show that: after Ara-C treatment in a common culture medium, the recombinant leukemia cell line Nalm-6/BACH2 is stably transferredKD-2 and Nalm-6/BACH2ConIC of50191.44nM and 22.68nM, respectively; after Ara-C treatment in the co-culture medium, the recombinant leukemia cell line Nalm-6/BACH2 was stably transferredKD-2 and Nalm-6/BACH2ConIC of501237.42nM and 688.99nM, respectively. The result shows that the co-culture medium has obvious drug resistance of the cytarabine and is named as a drug-resistant co-culture medium for subsequent evaluation of the curative effect of the combined chemotherapy drugs.
Example 3 detection of leukemia cell survival and drug combination index in combination with chemotherapeutic drug treatment
Leukemia cell survival rate detection by combination of chemotherapy drug treatment
1. Taking mononuclear cell samples Pt1 and Pt2 of two bone marrow patients prepared in the first step of example 1 and a stable transfer recombinant leukemia cell line Nalm-6/BACH2 established in the second stepKD-2 and Nalm-6/BACH2ConAs test cells, the cells were cultured in 1mL of 24-well plates containing a common medium at a cell density of 5X 104Each test cell is divided into the following treatment groups according to the different added drugs:
treatment group 1: 20nM cytarabine Ara-C.
Treatment group 2: 20nM cytarabine Ara-C + 5. mu.M nordihydroguaiaretic acid (NDGA).
Treatment group 3: 20nM cytarabine Ara-C +5 μ M curcumin (curcumin).
Treatment group 4: 200nM cytarabine Ara-C.
Treatment group 5: 200nM cytarabine Ara-C + 5. mu.M nordihydroguaiaretic acid (NDGA).
Treatment group 6: 200nM cytarabine Ara-C +5 μ M curcumin (curcumin).
Meanwhile, the test cells without drug treatment were used as a Control (Control).
After 48 hours of treatment, the cells were harvested and the cell viability was measured using the CellTiter-Blue cell viability assay kit from Promega, USA. The results are shown in FIG. 5.
2. Taking mononuclear cell samples Pt1 and Pt2 of two bone marrow patients prepared in the first step of example 1 and a stable transfer recombinant leukemia cell line Nalm-6/BACH2 established in the second stepKD-2 and Nalm-6/BACH2ConTest cells were cultured in 1mL of 24-well plates containing a drug-resistant co-culture medium at a cell density of 5X 104Each test cell is divided into the following treatment groups according to the different added drugs:
treatment group 1: 20nM cytarabine Ara-C.
Treatment group 2: 20nM cytarabine Ara-C + 5. mu.M nordihydroguaiaretic acid (NDGA).
Treatment group 3: 20nM cytarabine Ara-C +5 μ M curcumin (curcumin).
Treatment group 4: 200nM cytarabine Ara-C.
Treatment group 5: 200nM cytarabine Ara-C + 5. mu.M nordihydroguaiaretic acid (NDGA).
Treatment group 6: 200nM cytarabine Ara-C +5 μ M curcumin (curcumin).
Meanwhile, the test cells without drug treatment were used as a Control (Control).
After 48 hours of treatment, the cells were harvested and the cell viability was measured using the CellTiter-Blue cell viability assay kit from Promega, USA. The results are shown in FIG. 5.
The results show that: Nalm-6/BACH2 was treated with 20nM cytarabine in normal mediumCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells being 47.03%, 77.29%, 53.15% and 46.65%, respectively; Nalm-6/BACH2 was treated with 5. mu.M nordihydroguaiaretic acid in combination with 20nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells are 7.88%, 18.71%, 12.74% and 8.79%, respectively; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 20nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells were 37.70%, 35.77%, 43.12% and 38.33%, respectively.
Treatment of Nalm-6/BACH2 with 20nM cytarabine in drug-resistant coculture MediumCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells being 90.72%, 97.70%, 92.98% and 91.60%, respectively; uses proto-oncoprotein c-FOS as target spot and adopts 5 mu M nordihydroguaiareticTreatment of Nalm-6/BACH2 with combinations of Wood acid and 20nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells being 31.30%, 34.69%, 35.68% and 33.26%, respectively; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 20nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells were 63.38%, 61.60%, 67.54% and 63.77%, respectively.
Nalm-6/BACH2 was treated with 200nM cytarabine in normal mediumCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells being 21.06%, 50.90%, 27.13% and 22.85%, respectively; with proto-oncoprotein c-FOS as a target spot, Nalm-6/BACH2 is treated by combining 5 mu M nordihydroguaiaretic acid and 200nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells are 5.34%, 9.63%, 9.22% and 6.79%, respectively; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 200nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells were 17.47%, 19.20%, 22.82% and 19.05%, respectively.
Treatment of Nalm-6/BACH2 with 200nM cytarabine in drug-resistant coculture MediumCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells are 65.95%, 79.99%, 71.93% and 65.16%, respectively; with proto-oncoprotein c-FOS as a target spot, Nalm-6/BACH2 is treated by combining 5 mu M nordihydroguaiaretic acid and 200nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells being 32.24%, 34.94%, 34.04% and 35.68%, respectively; treatment of Nalm-6/BACH2 with 5. mu.M curcumin in combination with 200nM cytarabineCon、Nalm-6/BACH2KD-2, Pt1 and Pt2, the survival rates of leukemia cells were 60.30%, 65.87%, 69.05% and 62.77%, respectively.
Detection of drug combination index
Taking the stably transformed recombinant leukemia cell Nalm-6/BACH2 established in the second step of the example 1ConWith Nalm-6/BACH2KD-2 as test cells, separately cultured in a common medium,the cells were treated by adding cytarabine Ara-C (0, 2.5nM, 5nM, 10nM, 25nM, 50nM, 100nM, 200nM, 500nM and 1000nM) at different concentrations, respectively; Nalm-6/BACH2 is got in stepConWith Nalm-6/BACH2KD-2 as test cells, respectively cultured in a common medium, and the cells were treated with different concentrations of nordihydroguaiaretic acid NDGA (0, 5nM, 50nM, 500nM, 5000nM and 25000nM), respectively; taking Nalm-6/BACH2ConWith Nalm-6/BACH2KD-2 as test cells, respectively culturing in common culture medium, respectively adding curcumin (0, 5nM, 50nM, 500nM, 5000nM and 25000nM) with different concentrations to treat cells; finally, Nalm-6/BACH2 is takenConWith Nalm-6/BACH2KD-2 as test cells, respectively culturing in common culture medium, respectively adding different drug combinations (20nM cytarabine Ara-C and 5 μ M NDGA, 200nM cytarabine Ara-C and 5 μ M NDGA, 20nM cytarabine Ara-C and 5 μ M curcumin, 200nM cytarabine Ara-C and 5 μ M curcumin) to treat cells in combination; after 48 hours, the treated cells were harvested, and the cell viability was measured by using a CellTiter-Blue cell viability measuring kit of Promega, USA.
Taking the stably transformed recombinant leukemia cell Nalm-6/BACH2 established in the second step of the example 1ConWith Nalm-6/BACH2KD-2 as test cells, cultured in drug-resistant co-culture media, respectively, and treated with cytarabine Ara-C (0, 2.5nM, 5nM, 10nM, 25nM, 50nM, 100nM, 200nM, 500nM and 1000nM) at different concentrations, respectively; Nalm-6/BACH2 is got in stepConWith Nalm-6/BACH2KD-2 as test cells, cultured in drug-resistant co-culture medium, respectively, and treated with nordihydroguaiaretic acid NDGA (0, 5nM, 50nM, 500nM, 5000nM and 25000nM) at different concentrations, respectively; taking Nalm-6/BACH2ConWith Nalm-6/BACH2KD-2 as test cells, respectively culturing in drug-resistant co-culture medium, respectively adding curcumin (0, 5nM, 50nM, 500nM, 5000nM and 25000nM) with different concentrations to treat cells; finally, Nalm-6/BACH2 is takenConWith Nalm-6/BACH2KD-2 as test cells, separately cultured in a drug-resistant co-culture medium, separately addedTreating the cells with different drug combinations (20nM cytarabine Ara-C and 5. mu.M NDGA, 200nM cytarabine Ara-C and 5. mu.M NDGA, 20nM cytarabine Ara-C and 5. mu.M curcumin, 200nM cytarabine Ara-C and 5. mu.M curcumin); after 48 hours, the treated cells were harvested, and the cell viability was measured by using a CellTiter-Blue cell viability measuring kit of Promega, USA. Inputting the data into Compuyn software to calculate the drug combination index CI, wherein the calculation formula is as follows:
Figure BDA0002675221310000121
wherein n represents the number of combinations; (D)x)iA dose representing x% inhibition of drug alone; (D)irepresents the dose at which the i drug inhibits x% in the combination therapy. A CI of less than 1 indicates a synergistic effect between the drugs; if CI equals 1 indicates that there is an overlap between drugs; a CI greater than 1 indicates an antagonistic effect between the drugs.
The results are shown in FIGS. 7 and 8.
The results show that: leukemia cells were cultured in normal medium with 5 μ M NDGA treatment of Nalm-6/BACH2 with 20nM and 200nM cytarabine, respectively, in combination with 5 μ M NDGA treatment of nordihydroguaiaretic acidConThe CI values of the cells are 0.36 and 0.23 respectively, and are both less than 1; Nalm-6/BACH2 treated with 20nM and 200nM cytarabine, respectively, in combination with 5 μ M curcuminConThe CI values of the cells were 0.90 and 0.67, respectively, both less than 1. Similarly, leukemia cells were cultured in normal medium with 5 μ M NDGA treatment of Nalm-6/BACH2 with 20nM cytarabine in combination with 200nM cytarabine, respectivelyKD-2 cells had CI values of 0.12 and 0.07, respectively, both less than 1; Nalm-6/BACH2 treated with 20nM and 200nM cytarabine, respectively, in combination with 5 μ M curcuminKDCI values for-2 cells were 0.06 and 0.12, respectively, both less than 1.
In addition, leukemia cells were cultured in drug-resistant coculture media with 5 μ M NDGA-treated Nalm-6/BACH2 in combination with 20nM and 200nM cytarabine, respectivelyConCI of cellThe values are 0.02 and 0.12, respectively, both less than 1; Nalm-6/BACH2 treated with 20nM and 200nM cytarabine, respectively, in combination with 5 μ M curcuminConThe CI values of the cells were 0.26 and 0.61, respectively, both less than 1. Similarly, leukemia cells were cultured in drug-resistant coculture media with 5 μ M NDGA treatment of Nalm-6/BACH2 with 20nM and 200nM cytarabine, respectivelyKD-2 cells had CI values of 0.01 and 0.08, respectively, both less than 1; Nalm-6/BACH2 treated with 20nM and 200nM cytarabine, respectively, in combination with 5 μ M curcuminKDCI values for-2 cells were 0.03 and 0.36, respectively, both less than 1.
The results show that the combination of cytarabine and the proto-oncoprotein c-FOS inhibitor can synergistically increase the sensitivity of the leukemia cells to the chemotherapeutic drugs in the leukemia cells and effectively reduce the drug resistance of the leukemia cells to the chemotherapeutic drugs in the cytarabine drug-resistant strain cells and the drug-resistant co-culture medium.
Example 4 qRT-PCR detection of expression level of c-FOS Gene in Steady-transgenic recombinant leukemia cell line
Taking the stable transfer recombinant leukemia cell line Nalm-6/BACH2 established in the second step of the example 1KD-2 and Nalm-6/BACH2ConTotal RNAs were extracted separately, and qRT-PCR was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal control. The forward primer for detecting the c-FOS gene is 5'-AGAATCCGAAGGGAAAGGAA-3', and the reverse primer is 5'-CTTCTCCTTCAGCAGGTTGG-3'; the forward primer for detecting GAPDH gene is 5'-GACAGTCAGCCGCATCTTCT-3', and the reverse primer is 5'-GCGCCCAATACGACCAAATC-3'.
The above-mentioned total RNA extraction was carried out using Direct-zol RNA MiniPrep Plus kit from Zymo Research, USA. The specific operation method comprises the following steps:
1) centrifuging at 1000rpm for 5min to collect leukemia cells, adding 200 μ l Trizol reagent, shaking thoroughly, mixing, and lysing at room temperature for 5 min;
2) adding 200 mul of 100% ethanol solution, fully shaking and uniformly mixing;
3) sleeving a filtrate Tube (Zymo-Spin IIICG Column) into a Collection Tube (Collection Tube), transferring the mixed solution into the filtrate Tube, and centrifuging at 12000rpm for 1min at room temperature;
4) discarding the collecting pipe containing the filtrate, and sleeving the filtrate pipe into another clean collecting pipe;
5) adding 400 μ l of RNA pre-wash solution (Direct-zol RNA PreWash) into the filtrate tube, and centrifuging at 12000rpm for 1min at room temperature; discarding the filtrate, and repeatedly cleaning once;
6) add 700. mu.l of RNA Wash Buffer (RNA Wash Buffer) to the filtrate tube and centrifuge at 12000rpm for 2min at room temperature;
7) transferring the filtrate tube to a 1.5ml EP tube without RNase, adding 50. mu.l of purified water without DNA/RNase to the center of the filtrate tube, and centrifuging at 12000rpm for 1min at room temperature;
8) 100ng of total RNA was taken for qRT-PCR detection.
The specific steps of qRT-PCR detection are as follows:
1) qPT-PCR detection Using SYBR One-Step fluorescent quantitative PCR kit (One Step SYBR PrimeScript PLUS RT-PCR kit) from Takara, Japan.
2) The reaction solutions were prepared on ice according to the instructions, and the specific amounts and final concentrations used are shown in Table 2.
TABLE 2
Reagent Amount used (ul) Final concentration
2×One Step SYBR RT-PCR Buffer 4 10 1×
TaKaRa Ex Taq HS Mix 1.2
PrimeScript PLUS RTase Mix 0.4
Forward primer (10. mu.M) 0.8 0.4μM
Reverse primer (10. mu.M) 0.8 0.4μM
ROX Reference Dye 0.4
Total RNA (50 ng/. mu.l) 2
Pure water containing no RNase 4.4
Total amount of 20
3) A PCR reaction program was set on a Real-Time fluorescent quantitative PCR instrument (Applied Biosystems 7500Fast Real-Time PCR System), and the specific reaction conditions are shown in Table 3.
TABLE 3
Figure BDA0002675221310000141
4) By using 2-ΔΔCtThe method calculates the relative expression of c-FOS gene, normalized by GAPDH gene. The results are shown as Nalm-6/BACH2 relative to control cellsConThe ratio of c-FOS mRNA levels in (A). The results are shown in FIG. 9.
The results show that: stable transfer recombinant leukemia cell line Nalm-6/BACH2KDThe expression level of the c-FOS gene in the-2 is obviously higher than that of a control recombinant leukemia cell line Nalm-6/BACH2ConA clear negative correlation was shown between BACH2 and the c-FOS gene.
Example 5 detection of Dual luciferase reporter genes
Construction of luciferase report recombinant plasmid and BACH2 recombinant expression plasmid
1. Construction of recombinant plasmids pGL3-MARE1 and pGL3-MARE2
1) Construction of pGL3-MARE1 recombinant plasmid
The c-FOS promoter fragment (position-1050-0) carrying 1 MARE prediction site (5'-CTGAGACAGGA-3' at positions-212 to-202) was inserted into pGL3-basic vector to obtain recombinant plasmid pGL3-basic-FOS (recombinant plasmid pGL3-basic-FOS is a product of GeneCopoeia, Guangzhou). Wherein, the upstream primer of the c-FOS promoter introduces a restriction enzyme MluI sequence (underlined partial sequence), namely: 5' -CGACGCGTAAGCACACCCCGCCTTGTCA-3'; the downstream primer introduces the restriction enzyme HindIII sequence (underlined sequence), namely 5' -CCAAGCTTGGAGTACGAGGCGCCGC-3'. The recombinant plasmid pGL3-basic-FOS is used as a template, a c-FOS promoter fragment (position-1050-0) containing MluI and HindIII enzyme cutting sites is amplified by adopting a PCR technology, and the PCR fragment is inserted into a luciferase reporter gene pGL3-basic vector by respectively adopting restriction enzymes MluI and HindIII.
2) Construction of pGL3-MARE2 recombinant plasmid
The c-FOS promoter carrying 2 MARE prediction sites (5'-CTGAGACAGGA-3' (SEQ ID NO: 5) at positions-212 to-202; 5'-AAGACTGAGCCG-3' (SEQ ID NO: 6) at positions 32 to 43) and the 5 ' -UTR fragment (SEQ ID NO: 1050 to 412) were inserted into a pGL3-basic vector to obtain a pGL3-MARE2 recombinant plasmid (pGL3-MARE2 recombinant plasmid is a product of GeneCopoeia, Guangzhou).
2. Construction of recombinant expression plasmid pcDNA3.1(+) -BACH2
The open reading frame sequence of BACH2 protein in a Lenti-ORF-BACH2 plasmid is subcloned into an expression vector pcDNA3.1(+) by respectively adopting restriction enzymes BamHI and XhoI by taking an Open Reading Frame (ORF) expression plasmid Lenti-ORF-BACH2(Lenti-ORF-BACH2 is GE Dharmacon, USA, and the cloning number is PLOHS-100066339) of human BACH2 protein as a template, so as to obtain a recombinant expression plasmid pcDNA3.1(+) -BACH 2.
The primer synthesis and the recombinant plasmid sequencing are both completed by the Oncorks biology company.
Detection of dual-luciferase reporter gene
1. Culture of cells for transfection
24 hours before transfection, 96-well cell culture plates were incubated with DMEM medium containing 10% Fetal Bovine Serum (FBS) at 37 ℃ and 5% CO2The human kidney epithelial cell line 293T is transfected when 60% -70% is cultured under the condition.
2. Transient transfection of recombinant plasmids
And (3) co-transfecting 293T cells with the luciferase reporter recombinant plasmids pGL3-MARE1 and pGL3-MARE2 obtained in the step one and the recombinant expression plasmid pcDNA3.1(+) -BACH2 and the renilla luciferase expression vector plasmid pRL-SV40 respectively. The empty luciferase reporter plasmid pGL3-basic and the empty expression vector plasmid pcDNA3.1(+) were used as a blank control and a negative control, respectively.
The specific method of transfection is as follows: taking two sterile 1.5ml EP tubes, adding 100 μ l serum-free DMEM medium into one of the EP tubes to dilute 100ng recombinant expression plasmid pcDNA3.1(+) -BACH2 or pcDNA3.1(+), 100ng luciferase report recombinant plasmid pGL3-MARE1 or pGL3-MARE2, 20ng renilla luciferase expression vector plasmid pRL-SV 40; another EP tube was filled with 100. mu.l serum-free DMEM medium and 0.66. mu.l Lipofectamine 2000. Mixing the two tubes, standing at room temperatureFor 5 minutes. 200. mu.l of the mixture was added dropwise to 2 parallel wells of a 96-well cell culture plate, 100. mu.l per well. Gently shake the plate and mix well, then transfer to 37 ℃, 5% CO2Culturing in a cell culture box.
3. Detection of Dual luciferase reporter genes
The cells were harvested 48 hours after transfection, and the activities of Firefly Luciferase (FL) and Renilla Luciferase (RL) were measured on the samples using a dual-luciferase assay kit from Promega, usa, and the relative luciferase activity (FL/RL) was calculated as shown in fig. 10.
The specific operation method for determining the luciferase activity by adopting the dual-luciferase reporter gene detection kit comprises the following steps:
1) carefully remove the supernatant from the cell wells;
2) adding 100 mul/hole 1 XPBS buffer solution to rinse the cells, and removing the rinse solution;
3) adding 20 μ l/well 1 × Lysis solution (PLB), placing on a shaking bed at room temperature, and shaking gently for 15 min;
4) adding 100 μ l/well fluorogenic substrate reagent (LARII), measuring light output value for 10 s with instrument to obtain FL activity, and recording measured value;
5) adding 100 μ l/well Stop Reagent (Stop & Glo Reagent), measuring the light output value for 10 seconds by an instrument to obtain RL activity, and recording the measured value;
6) correcting the transfection efficiency error of each well cell by the internal control RL activity, namely dividing the measured FL activity value by the internal control RL activity value to obtain the relative activity (FL/RL) after correcting the transfection efficiency; and after the relative activity of the luciferase is calculated, further calculating the activity change multiple through the relative activity of the luciferase of the control group, namely dividing the relative activity of the luciferase of the experimental group by the relative activity of the luciferase of the control group to obtain the activity multiple. The results are shown in FIG. 10.
The results show that: compared with the no-load expression vector plasmid pcDNA3.1(+) (control group), the transcription activity of the co-transfected BACH2 recombinant expression plasmid pcDNA3.1(+) -BACH2 and pGL3-MARE2 is reduced by about 25 times, and obvious transcription inhibition effect is presented.
Example 6 CUT & Tag high throughput detection and PCR validation of human B-lymphoid leukemia cells
First, CUT & Tag high-flux detection of human B lymphocyte leukemia cell
The establishment of the CUT & Tag library and high throughput sequencing were performed using the human B-lymphocytic leukemia Cell line Nalm-6, to which a monoclonal antibody to the tumor suppressor BACH2 (primary antibody, available from Cell Signaling Technology, USA) was added, for the detection of DNA fragments interacting with BACH2 protein.
The establishment of the CUT & Tag library and the high throughput sequencing were performed by Beijing Baimaike Biotechnology Ltd. After the sequencing was completed, the sequencing raw data were imported into visual analysis software IGV v2.8.3, and the human c-FOS gene region was locked with reference to the human GRCh38 genome, and the enrichment peak of BACH2 protein was observed, as shown in FIG. 11.
The results showed that the BACH2 protein showed two distinct enrichment peaks in the promoter and 5' -UTR regions of the c-FOS gene, respectively, compared to the IgG control group, and the regions corresponding to the peaks (indicated by arrows) matched with the two predicted MARE sequences (MARE1 and MARE 2).
Second, PCR detection verification of CUT & Tag library of human B lymphocyte leukemia cell
And (2) respectively designing 5 DNA fragments with different sizes of 140-180 bp and amplification primers thereof on a near-end promoter, a 5' -UTR and a near-end translation region (position-1050-1000) of the human c-FOS gene by taking the CUT & Tag library established in the step one as a template. Each fragment was amplified separately by PCR, with the primer sequences shown in Table 4.
TABLE 4
Figure BDA0002675221310000161
The specific method for PCR detection is as follows:
1) PCR was carried out using PrimeSTAR Max DNA Polymerase kit (Takara, Japan).
2) The reaction solutions were prepared according to the instructions, and the specific amounts and final concentrations thereof were as shown in Table 5.
TABLE 5
Reagent Amount used (ul) Final concentration
2×PrimeSTAR Max Premix 10 1×
Forward primer (1. mu.M) 1 0.5μM
Reverse primer (1. mu.M) 1 0.5μM
Stencil (100 ng/. mu.l) 1
Sterilized distilled water 7
Total amount of 20
3) The reaction program was set on the PCR machine, and the specific reaction conditions are shown in Table 6.
TABLE 6
Figure BDA0002675221310000171
4) Preparing agarose gel with the concentration of 3%, and carrying out electrophoresis on the PCR product obtained in the step 3. And after the electrophoresis is finished, recording the result by adopting an ultraviolet analyzer.
The results are shown in FIG. 12. The results show that: the c-FOS-b fragment containing the predicted site of MARE1 and the c-FOS-c fragment containing the predicted site of MARE2 both bound to the BACH2 protein; compared with the IgG control group, the binding multiple of the c-FOS-b fragment and the BACH2 protein is about 12.16; compared with the IgG control group, the binding ratio of the c-FOS-c fragment to the BACH2 protein was about 4.98.
The above examples 4-6 demonstrate that the tumor suppressor BACH2 can transcriptionally repress the expression of the c-FOS gene, and that BACH2 protein is involved in transcriptional repression regulation by combining the c-FOS gene promoter and two MARE sequences in the 5' -UTR. The invention provides a new BACH2 downstream target gene for leukemia research, and the downstream target gene is expected to become a potential target for anti-leukemia treatment, and has very wide application prospect in the field of medical research.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of medical science and biology of China academy of medical sciences
<120> target point c-FOS related to leukemia diagnosis and treatment and application thereof
<160> 6
<170> PatentIn version 3.5
<210> 1
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Met Ser Val Asp Glu Lys Pro Asp Ser Pro Met Tyr Val Tyr Glu Ser
1 5 10 15
Thr Val His Cys Thr Asn Ile Leu Leu Gly Leu Asn Asp Gln Arg Lys
20 25 30
Lys Asp Ile Leu Cys Asp Val Thr Leu Ile Val Glu Arg Lys Glu Phe
35 40 45
Arg Ala His Arg Ala Val Leu Ala Ala Cys Ser Glu Tyr Phe Trp Gln
50 55 60
Ala Leu Val Gly Gln Thr Lys Asn Asp Leu Val Val Ser Leu Pro Glu
65 70 75 80
Glu Val Thr Ala Arg Gly Phe Gly Pro Leu Leu Gln Phe Ala Tyr Thr
85 90 95
Ala Lys Leu Leu Leu Ser Arg Glu Asn Ile Arg Glu Val Ile Arg Cys
100 105 110
Ala Glu Phe Leu Arg Met His Asn Leu Glu Asp Ser Cys Phe Ser Phe
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Leu Gln Thr Gln Leu Leu Asn Ser Glu Asp Gly Leu Phe Val Cys Arg
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Lys Asp Ala Ala Cys Gln Arg Pro His Glu Asp Cys Glu Asn Ser Ala
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Gly Glu Glu Glu Asp Glu Glu Glu Glu Thr Met Asp Ser Glu Thr Ala
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Lys Met Ala Cys Pro Arg Asp Gln Met Leu Pro Glu Pro Ile Ser Phe
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Glu Ala Ala Ala Ile Pro Val Ala Glu Lys Glu Glu Ala Leu Leu Pro
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Glu Pro Asp Val Pro Thr Asp Thr Lys Glu Ser Ser Glu Lys Asp Ala
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Leu Thr Gln Tyr Pro Arg Tyr Lys Lys Tyr Gln Leu Ala Cys Thr Lys
225 230 235 240
Asn Val Tyr Asn Ala Ser Ser His Ser Thr Ser Gly Phe Ala Ser Thr
245 250 255
Phe Arg Glu Asp Asn Ser Ser Asn Ser Leu Lys Pro Gly Leu Ala Arg
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Gly Gln Ile Lys Ser Glu Pro Pro Ser Glu Glu Asn Glu Glu Glu Ser
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Ile Thr Leu Cys Leu Ser Gly Asp Glu Pro Asp Ala Lys Asp Arg Ala
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Gly Asp Val Glu Met Asp Arg Lys Gln Pro Ser Pro Ala Pro Thr Pro
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Thr Ala Pro Ala Gly Ala Ala Cys Leu Glu Arg Ser Arg Ser Val Ala
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Ser Pro Ser Cys Leu Arg Ser Leu Phe Ser Ile Thr Lys Ser Val Glu
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Leu Ser Gly Leu Pro Ser Thr Ser Gln Gln His Phe Ala Arg Ser Pro
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Ala Cys Pro Phe Asp Lys Gly Ile Thr Gln Gly Asp Leu Lys Thr Asp
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Tyr Thr Pro Phe Thr Gly Asn Tyr Gly Gln Pro His Val Gly Gln Lys
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Glu Val Ser Asn Phe Thr Met Gly Ser Pro Leu Arg Gly Pro Gly Leu
405 410 415
Glu Ala Leu Cys Lys Gln Glu Gly Glu Leu Asp Arg Arg Ser Val Ile
420 425 430
Phe Ser Ser Ser Ala Cys Asp Gln Val Ser Thr Ser Val His Ser Tyr
435 440 445
Ser Gly Val Ser Ser Leu Asp Lys Asp Leu Ser Glu Pro Val Pro Lys
450 455 460
Gly Leu Trp Val Gly Ala Gly Gln Ser Leu Pro Ser Ser Gln Ala Tyr
465 470 475 480
Ser His Gly Gly Leu Met Ala Asp His Leu Pro Gly Arg Met Arg Pro
485 490 495
Asn Thr Ser Cys Pro Val Pro Ile Lys Val Cys Pro Arg Ser Pro Pro
500 505 510
Leu Glu Thr Arg Thr Arg Thr Ser Ser Ser Cys Ser Ser Tyr Ser Tyr
515 520 525
Ala Glu Asp Gly Ser Gly Gly Ser Pro Cys Ser Leu Pro Leu Cys Glu
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Phe Ser Ser Ser Pro Cys Ser Gln Gly Ala Arg Phe Leu Ala Thr Glu
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His Gln Glu Pro Gly Leu Met Gly Asp Gly Met Tyr Asn Gln Val Arg
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Pro Gln Ile Lys Cys Glu Gln Ser Tyr Gly Thr Asn Ser Ser Asp Glu
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Ser Gly Ser Phe Ser Glu Ala Asp Ser Glu Ser Cys Pro Val Gln Asp
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Arg Gly Gln Glu Val Lys Leu Pro Phe Pro Val Asp Gln Ile Thr Asp
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Leu Pro Arg Asn Asp Phe Gln Met Met Ile Lys Met His Lys Leu Thr
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atgtctgtgg atgagaagcc tgactccccc atgtatgtgt atgagtccac agtccactgc 60
accaacatcc tcctgggcct caatgaccag cggaaaaagg atattctctg tgacgtgact 120
ttgatcgtgg agaggaagga gttccgggcc caccgggctg tgctggccgc atgcagtgaa 180
tatttttggc aggcgctggt tggacagaca aaaaatgatt tggtggtcag cttgcctgag 240
gaggtcacag ccaggggctt tgggccgctg ttacagtttg cctacactgc caagctgtta 300
ctcagcagag aaaacatccg cgaggtcatc cgctgtgctg agttcctgcg catgcacaac 360
ctggaggact cctgcttcag cttcctgcag acccagctcc tgaacagtga ggatggcctg 420
tttgtgtgcc ggaaggatgc tgcgtgccag cgcccacacg aggactgcga gaactctgca 480
ggagaggagg aggatgaaga ggaggagacg atggattcag agacggccaa gatggcttgc 540
cccagggacc agatgcttcc agagcccatc agctttgagg ccgccgccat ccccgtagca 600
gagaaggaag aagccctgct gcccgagcct gacgtgccca cagacaccaa ggagagctca 660
gaaaaggacg cgttaacgca gtaccccaga tacaagaaat accagcttgc atgtaccaag 720
aatgtctata atgcatcatc acacagtacc tcaggttttg caagcacatt ccgggaagat 780
aactctagca acagcctcaa gccggggctt gccagggggc agattaaaag tgagccgccc 840
agtgaagaga atgaggaaga gagcatcacg ctctgcctgt ctggagatga gcctgacgcc 900
aaggacagag cgggggatgt cgagatggac cggaaacagc ccagccctgc ccctaccccc 960
acggccccag ctggggccgc ctgcctggag agatccagga gcgtggcctc gccctcctgc 1020
ttaaggtctc tgttcagcat aacgaaaagt gtggagctgt ctggcctgcc cagtacatct 1080
cagcagcact ttgccaggag tccagcctgc ccttttgaca aggggatcac tcagggtgac 1140
cttaaaactg actacacccc tttcacaggg aattatggac agccccacgt gggccagaag 1200
gaggtgtcca acttcaccat ggggtcgccc ctcagggggc ctgggttgga ggctctctgt 1260
aaacaggagg gagagctgga ccggaggagc gtgatcttct cctccagcgc ttgtgaccaa 1320
gtgagcacct cggtgcattc ttattctggg gtgagcagtt tggacaaaga cctctctgag 1380
ccggtgccaa agggtctgtg ggtgggagcc ggccagtccc tccccagctc gcaggcctac 1440
tcccacggtg ggctgatggc cgaccacttg ccaggaagga tgcggcccaa caccagctgc 1500
ccggtaccaa tcaaagtctg ccctcgctca ccccccttgg agaccaggac caggacttcc 1560
agctcctgct cttcctattc ctacgcggag gacgggagcg ggggctcacc ctgcagcctc 1620
cctctctgtg agttctcctc ctcgccctgt tcccagggag ccagattcct tgccacagaa 1680
catcaggaac caggcctgat gggagatgga atgtacaacc aagtgcggcc ccaaattaaa 1740
tgtgagcagt cttatggaac caactccagt gacgaatccg gatcgttctc ggaagcagac 1800
agtgagtcgt gtcctgtgca ggacaggggc caggaggtaa aacttccttt tcctgtagat 1860
caaatcacag atcttccaag gaacgatttc cagatgatga ttaaaatgca caagctaacc 1920
tcagaacagt tagagtttat tcatgatgtc cgacggcgca gcaagaaccg catcgcggcc 1980
cagcgctgcc gcaaaaggaa actggactgt attcagaatt tagaatgtga aatccgcaaa 2040
ttggtgtgtg agaaagagaa actgttgtca gagaggaatc aactgaaagc atgcatgggg 2100
gaactgttgg acaacttctc ctgcctttcc caggaagttt gccgagacat ccagagcccc 2160
gagcagatcc aggccctgca tcggtattgc cctgtcctca gacccatgga cttgcccacg 2220
gcctccagta ttaaccctgc gcccttgggt gctgagcaga acattgcggc ctcccaatgc 2280
gcagtggggg aaaacgtgcc ctgctgcttg gagccaggcg cggctccccc cggacccccc 2340
tgggcaccca gcaacacctc cgagaattgt acctctggga ggagactaga aggcactgac 2400
ccgggaacct tctcagagag aggacctcct cttgaaccca ggagccaaac agtgaccgtg 2460
gacttctgcc aggaaatgac tgataagtgt acaactgacg aacagcccag gaaagattat 2520
acctag 2526
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<212> PRT
<213> Artificial Sequence
<400> 3
Met Met Phe Ser Gly Phe Asn Ala Asp Tyr Glu Ala Ser Ser Ser Arg
1 5 10 15
Cys Ser Ser Ala Ser Pro Ala Gly Asp Ser Leu Ser Tyr Tyr His Ser
20 25 30
Pro Ala Asp Ser Phe Ser Ser Met Gly Ser Pro Val Asn Ala Gln Asp
35 40 45
Phe Cys Thr Asp Leu Ala Val Ser Ser Ala Asn Phe Ile Pro Thr Val
50 55 60
Thr Ala Ile Ser Thr Ser Pro Asp Leu Gln Trp Leu Val Gln Pro Ala
65 70 75 80
Leu Val Ser Ser Val Ala Pro Ser Gln Thr Arg Ala Pro His Pro Phe
85 90 95
Gly Val Pro Ala Pro Ser Ala Gly Ala Tyr Ser Arg Ala Gly Val Val
100 105 110
Lys Thr Met Thr Gly Gly Arg Ala Gln Ser Ile Gly Arg Arg Gly Lys
115 120 125
Val Glu Gln Leu Ser Pro Glu Glu Glu Glu Lys Arg Arg Ile Arg Arg
130 135 140
Glu Arg Asn Lys Met Ala Ala Ala Lys Cys Arg Asn Arg Arg Arg Glu
145 150 155 160
Leu Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Leu Glu Asp Glu Lys
165 170 175
Ser Ala Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys Glu Lys
180 185 190
Leu Glu Phe Ile Leu Ala Ala His Arg Pro Ala Cys Lys Ile Pro Asp
195 200 205
Asp Leu Gly Phe Pro Glu Glu Met Ser Val Ala Ser Leu Asp Leu Thr
210 215 220
Gly Gly Leu Pro Glu Val Ala Thr Pro Glu Ser Glu Glu Ala Phe Thr
225 230 235 240
Leu Pro Leu Leu Asn Asp Pro Glu Pro Lys Pro Ser Val Glu Pro Val
245 250 255
Lys Ser Ile Ser Ser Met Glu Leu Lys Thr Glu Pro Phe Asp Asp Phe
260 265 270
Leu Phe Pro Ala Ser Ser Arg Pro Ser Gly Ser Glu Thr Ala Arg Ser
275 280 285
Val Pro Asp Met Asp Leu Ser Gly Ser Phe Tyr Ala Ala Asp Trp Glu
290 295 300
Pro Leu His Ser Gly Ser Leu Gly Met Gly Pro Met Ala Thr Glu Leu
305 310 315 320
Glu Pro Leu Cys Thr Pro Val Val Thr Cys Thr Pro Ser Cys Thr Ala
325 330 335
Tyr Thr Ser Ser Phe Val Phe Thr Tyr Pro Glu Ala Asp Ser Phe Pro
340 345 350
Ser Cys Ala Ala Ala His Arg Lys Gly Ser Ser Ser Asn Glu Pro Ser
355 360 365
Ser Asp Ser Leu Ser Ser Pro Thr Leu Leu Ala Leu
370 375 380
<210> 4
<211> 1143
<212> DNA
<213> Artificial Sequence
<400> 4
atgatgttct cgggcttcaa cgcagactac gaggcgtcat cctcccgctg cagcagcgcg 60
tccccggccg gggatagcct ctcttactac cactcacccg cagactcctt ctccagcatg 120
ggctcgcctg tcaacgcgca ggacttctgc acggacctgg ccgtctccag tgccaacttc 180
attcccacgg tcactgccat ctcgaccagt ccggacctgc agtggctggt gcagcccgcc 240
ctcgtctcct ccgtggcccc atcgcagacc agagcccctc accctttcgg agtccccgcc 300
ccctccgctg gggcttactc cagggctggc gttgtgaaga ccatgacagg aggccgagcg 360
cagagcattg gcaggagggg caaggtggaa cagttatctc cagaagaaga agagaaaagg 420
agaatccgaa gggaaaggaa taagatggct gcagccaaat gccgcaaccg gaggagggag 480
ctgactgata cactccaagc ggagacagac caactagaag atgagaagtc tgctttgcag 540
accgagattg ccaacctgct gaaggagaag gaaaaactag agttcatcct ggcagctcac 600
cgacctgcct gcaagatccc tgatgacctg ggcttcccag aagagatgtc tgtggcttcc 660
cttgatctga ctgggggcct gccagaggtt gccaccccgg agtctgagga ggccttcacc 720
ctgcctctcc tcaatgaccc tgagcccaag ccctcagtgg aacctgtcaa gagcatcagc 780
agcatggagc tgaagaccga gccctttgat gacttcctgt tcccagcatc atccaggccc 840
agtggctctg agacagcccg ctccgtgcca gacatggacc tatctgggtc cttctatgca 900
gcagactggg agcctctgca cagtggctcc ctggggatgg ggcccatggc cacagagctg 960
gagcccctgt gcactccggt ggtcacctgt actcccagct gcactgctta cacgtcttcc 1020
ttcgtcttca cctaccccga ggctgactcc ttccccagct gtgcagctgc ccaccgcaag 1080
ggcagcagca gcaatgagcc ttcctctgac tcgctcagct cacccacgct gctggccctg 1140
tga 1143
<210> 5
<211> 11
<212> DNA
<213> Artificial Sequence
<400> 5
ctgagacagg a 11
<210> 6
<211> 12
<212> DNA
<213> Artificial Sequence
<400> 6
aagactgagc cg 12

Claims (10)

1. The application of a substance for inhibiting the activity of c-FOS protein or a substance for reducing the content of the c-FOS protein or a substance for silencing or knocking out or mutating c-FOS gene or a substance for inhibiting the expression of the c-FOS gene in any one of the following 1) or 2):
1) preparing a product for improving the sensitivity of leukemia cells to chemotherapeutic drugs;
2) preparing a product for reducing the drug resistance of leukemia cells to chemotherapeutic drugs.
2. Use according to claim 1, characterized in that: the chemotherapeutic drug is cytarabine;
or the substance inhibiting the activity of the c-FOS protein is a c-FOS inhibitor;
or, the c-FOS inhibitor is nordihydroguaiaretic acid or curcumin.
3. Use according to claim 1 or 2, characterized in that: the c-FOS gene has at least one of the following characteristics a) and b):
a) is regulated by the transcriptional inhibition of a tumor suppressor BACH2 in leukemia cells;
b) binds to tumor suppressor BACH2 in leukemia cells.
4. Use according to claim 3, characterized in that: in the b), the sequence of the c-FOS gene which is combined with the tumor suppressor BACH2 in leukemia cells is a DNA molecule shown in a sequence 5 and/or a DNA molecule shown in a sequence 6.
5. A product contains active ingredients of substances for inhibiting c-FOS protein activity or substances for reducing c-FOS protein content or substances for silencing or knocking out or mutating c-FOS gene or substances for inhibiting c-FOS gene expression;
the product has the function of improving the sensitivity of the leukemia cells to the chemotherapeutic drugs or reducing the drug resistance of the leukemia cells to the chemotherapeutic drugs.
6. The product of claim 5, wherein: the chemotherapeutic drug is cytarabine;
or the substance inhibiting the activity of the c-FOS protein is a c-FOS inhibitor;
or, the c-FOS inhibitor is nordihydroguaiaretic acid or curcumin.
7. The product according to claim 5 or 6, characterized in that: the c-FOS gene has at least one of the following characteristics a) and b):
a) is regulated by the transcriptional inhibition of a tumor suppressor BACH2 in leukemia cells;
b) binds to tumor suppressor BACH2 in leukemia cells.
8. The product of claim 7, wherein: in the b), the sequence of the c-FOS gene which is combined with the tumor suppressor BACH2 in leukemia cells is a DNA molecule shown in a sequence 5 and/or a DNA molecule shown in a sequence 6.
9. Use according to any one of claims 1 to 4 or a product according to any one of claims 5 to 8, wherein: the leukemia cell is human B lymphocyte leukemia cell or human B lymphocyte leukemia cytarabine drug-resistant cell.
The application of the c-FOS gene as a target point in developing or designing a product for treating or assisting in treating leukemia;
or, the c-FOS gene is used as a downstream target gene of a tumor suppressor BACH2 in participating in the regulation of a BACH2 gene network.
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