CN111939264A - Application of c-Fos gene and c-Fos inhibitor in relapse of drug-resistant diffuse large B cell lymphoma - Google Patents

Abstract

The C-Fos inhibitor is used for inhibiting the expression of the C-Fos in the DLBCL cell, and further, the Difluorobenzocumin CDF micromolecule inhibitor and the LAQ824 are used in a combined and synergistic mode to kill the DLBCL cell, synergistically induce the apoptosis of the DLBCL cell, synergistically inhibit the proliferation and the growth of the DLBCL tumor cell in vivo, improve the effect of the tumor inhibition rate and further solve the problem of relapse drug resistance of diffuse large B cell lymphoma. The c-Fos is used as a potential drug-resistant core driving gene, the expression level of the c-Fos is highly related to the drug IC50, and the c-Fos can be used as an index for evaluating the drug sensitivity of the LAQ 824.

Description

Application of c-Fos gene and c-Fos inhibitor in relapse of drug-resistant diffuse large B cell lymphoma

Technical Field

The invention relates to the field of molecular therapy, in particular to application of a c-Fos gene and a c-Fos inhibitor in a drug for treating relapsing drug-resistant diffuse large B cell lymphoma.

Background

Diffuse large B-cell lymphoma (DLBCL) is a group of highly invasive and heterogeneous diseases that all differ in genetic alterations, clinical features, morphological manifestations, treatment response, and prognosis. Although the application of CAR-T and novel targeted drugs has shown some efficacy in the treatment of patients with relapsed or refractory (R/R) DLBCL in recent years, there is currently no optimal rescue treatment for R/R DLBCL patients. Tumor cell heterogeneity and clonal evolution are the core driving forces for the development, progression and malignant transformation of blood tumors, and are the root causes of their refractory, recurrent and drug-resistant properties. How to analyze the heterogeneity of DLBCL, excavate drug-resistant core driving genes, explore new treatment strategies and design novel treatment schemes to improve the clinical efficacy of patients of the type is a main difficult problem and a barrier to be overcome.

Disclosure of Invention

The subject group of the invention continuously researches the analysis of the heterogeneity of DLBCL, the excavation of drug-resistant core driving genes, the exploration of new treatment strategies and the design of novel treatment schemes for many years. Research of a subject group finds that:

the protooncogene c-Fos, one of the members of the Fos gene family, is located on human chromosome 14 and consists of 3 introns and 4 exons and encodes a phosphoprotein product consisting of 380 amino acid residues. The c-Fos belongs to early direct response genes, when cells are stimulated by external stimulus, the c-Fos genes are activated to be transcribed, then enter cytoplasm and are translated into protein, and after returning to nucleus, the c-Fos can form a heterodimer complex (AP-1) with c-Jun protein in a leucine zipper form and participate in cell proliferation, apoptosis and DNA damage repair. The c-Fos protein is used as an important component of AP-1 transcription protein and is involved in the generation and development of tumors. Wodrich et al initially analyzed and detected 173 untreated non-small cell lung cancer tissues, and found that c-Fos protein is abnormally highly expressed in lung cancer, and promotes the proliferation of tumor cells; later researches show that c-Fos plays a key role in regulating and controlling cell growth and differentiation, and overexpression of the c-Fos is proved to be related to occurrence, development and prognosis of various malignant tumors such as human ovarian cancer, oral squamous cell carcinoma, liver cancer and the like; liver abnormally expressing c-Fos can have necrotic foci, immune cell infiltration and morphological change of liver cells; yoshida et al found that formaldehyde can promote carcinogenesis by inducing phosphorylation of serine at position 10 (H3S 10) of histone H3 in the c-Fos promoter region of protooncogene to up-regulate c-Fos expression.

At present, no report is found about the action mechanism research of c-Fos in DLBCL development and drug resistance. To this end, the subject groups studied c-Fos by single cell transcriptome sequencing.

The topic group was found by single cell transcriptome sequencing: LAQ824 can cause the up-regulation of c-Fos expression in DLBCL cells under a certain concentration (0.1 mu M), and the drug treatment cannot further induce the apoptosis of tumor cells; c-Fos can be used as an index for evaluating drug sensitivity of LAQ824, and the expression level of the c-Fos is related to prognosis of a DLBCL patient; difluorobenzocumin (CDF) can effectively inhibit the expression of c-Fos in DLBCL cells, and the small molecule inhibitor and LAQ824 can be used in a combined manner to kill the DLBCL cells synergistically, so that the tumor inhibition rate is remarkably improved.

The single cell transcriptome sequencing technology is one new technology for amplifying and sequencing transcriptome at single cell level. The principle is to amplify the separated micro transcriptome RNA of a single cell and then carry out high-throughput sequencing. The emergence and maturation of single cell transcriptome sequencing (scRNA-seq) technology provides unprecedented resolution for analyzing human disease pathophysiology, can deepen understanding of disease mechanisms, and can guide new clinical application of currently available monoclonal antibody or small molecule inhibitor drugs according to changes of gene expression or pathways.

Therefore, the invention provides the application of the c-Fos inhibitor in the relapse of the drug-resistant diffuse large B cell lymphoma.

Further, the c-Fos inhibitor and LAQ824 are used in a combined mode to kill DLBCL cells synergistically, and the tumor inhibition rate is improved.

Further, LAQ824 synergistically with c-Fos inhibitors induced apoptosis of DLBCL cells.

Further, application of Difluorobenzocumin as a c-Fos inhibitor in relapse of drug-resistant diffuse large B cell lymphoma.

Further, the application of the Difluorobenzocumin small-molecule inhibitor in the treatment of large B-cell lymphoma is achieved by inhibiting the expression of c-Fos in DLBCL cells and relapse drug resistance.

Furthermore, the Difluorobenzocumin small molecule inhibitor and LAQ824 are used in a combined mode to kill DLBCL cells synergistically, and the tumor inhibition rate is improved.

Further, LAQ824 synergistically induces DLBCL apoptosis with diffulobe zocurcun small molecule inhibitors.

Further, LAQ824 and diffulobe zocurcin small molecule inhibitors synergistically inhibit DLBCL tumor cell proliferation and growth in vivo.

Further, the invention also provides the use of LAQ824 for up-regulating c-Fos expression in DLBCL cells.

Further, the concentration of LAQ824 was 0.1uM and above, and as the concentration of LAQ824 increased, c-Fos expression was globally up-regulated.

The invention also provides application of the c-Fos gene in evaluating the drug-resistant relapse of the diffuse large B-cell lymphoma DLBCL.

Further, the application of the reagent for detecting the expression quantity of the c-Fos in evaluating the drug-resistant relapse of the diffuse large B-cell lymphoma DLBCL is provided.

Preferably, the nucleotide sequence of the c-Fos gene is SEQ ID NO. 1;

preferably, the reagent for detecting the expression quantity of the c-Fos comprises SYBR Green and PCR amplification primers shown in SEQ ID NO. 2 and SEQ ID NO. 3;

c-Fos upstream primer sequence SEQ ID NO: 2: 5'-TTCAACGCAGACTACGAGGC-3', respectively;

c-Fos downstream primer sequence SEQ ID NO: 3: 5'-GAAGTTGGCACTGGAGACG-3' are provided.

Has the advantages that: the c-Fos is used as a potential index of DLBCL drug resistance relapse, and the c-Fos expression quantity is detected by a reagent for detecting the c-Fos expression quantity, so that the effect of detecting and evaluating the drug resistance relapse of Diffuse Large B Cell Lymphoma (DLBCL) is achieved. The C-Fos inhibitor is used for inhibiting the expression of the C-Fos in the DLBCL cell, and further, the Difluorobenzocumin CDF micromolecule inhibitor and the LAQ824 are used in a combined and synergistic mode to kill the DLBCL cell, synergistically induce the apoptosis of the DLBCL cell, synergistically inhibit the proliferation and the growth of the DLBCL tumor cell in vivo, improve the effect of the tumor inhibition rate and further solve the problem of relapse drug resistance of diffuse large B cell lymphoma.

The c-Fos is used as a potential drug-resistant core driving gene, the expression level of the c-Fos is highly related to the drug IC50, and the c-Fos can be used as an index for evaluating the drug sensitivity of the LAQ 824.

The invention verifies that the c-Fos inhibitor inhibits the expression of c-Fos in DLBCL cells, further kills the DLBCL cells by combining the Difluorobenzocumin CDF micromolecule inhibitor and LAQ824 for cooperation, synergistically induces the apoptosis of the DLBCL cells, synergistically inhibits the proliferation and the growth of DLBCL tumor cells in vivo, improves the effect of tumor inhibition rate, and further solves the problem of relapse drug resistance of diffuse large B cell lymphoma, and adopts the following test method:

CC8 detection

And adding 100u of DLBCL cells treated under different conditions into a 96-well plate, adding 10ul of CCK8 into each well of 3 multiple wells in each group under the condition of keeping out of the light, incubating for 2-4 hours, and reading the absorbance value at the wavelength of 450 nm.

Streaming

Taking 5-10 ten thousand of resuspended cells, centrifuging, removing supernatant, adding Annexin V-FITC binding solution to lightly resuspend the cells, adding 5 mu l of Annexin V-FITC and 10 mu l of propidium iodide staining solution, lightly mixing, incubating for 10-20 minutes at room temperature (20-25 ℃) in the dark, and performing flow cytometry detection on the machine.

And (3) collecting the cells after the drug treatment, cracking the cells by RIPA cell lysate, centrifuging the cells, collecting protein, and quantifying the protein by using a BCA method. Equal amounts of protein from each group were then electrophoretically separated, transferred to PVDF membrane, blocked with 5% skim milk for one hour, incubated overnight for the primary antibody, and incubated for one hour at room temperature for the secondary antibody. Protein expression was detected on ECL machines.

RNA extraction, RNA quality detection, cDNA synthesis and Real Time PCR reaction.

Construction of DLBCL mouse model

In this project, the subject group established a DLBCL mouse model by transplanting a DLBCL cell line into an NOD SCID mouse. Selecting male mice of 6-8 weeks old, and inoculating U2932 cells, 1 multiplied by 107 cells/mouse, subcutaneously through forelimb armpits. After the U2932 cells were resuspended in PBS to an appropriate density, the volume ratio of the adjusted cells to matrigel was 1: 1 for inoculation after mixing. After the tumor is visible, the tumor diameter is regularly measured to calculate the tumor volume, and the tumor size calculation formula is as follows: tumor volume (mm 3) =0.5 × (tumor major diameter × tumor minor diameter 2). When the tumor volume is 100-300 mm3, the mice are randomly divided into 3 groups, 6-10 mice in each group are set as a negative control group, LAQ824 (75 mg/kg), LAQ824+ CDF (10 mg/kg). LAQ824 is administered by tail vein injection, once every 5 days, and is treated for 4 times. CDF is administered by intraperitoneal injection once a day for 28 days. And (3) carrying out intravenous injection of 18F-FLT on the 3 rd day after administration, carrying out whole-body static scanning of small animal PET (polyethylene terephthalate) after 1 h, delineating an interested area including a tumor by using PMOD (polymethylene oxide) and obtaining the radioactive uptake value of each tissue. And detecting the tumor and the body weight 2 times a week at intervals of 2-3 days every time for 28 days continuously, stripping the tumor after the 28 th day of scanning, taking a picture, weighing the tumor weight, and determining the antitumor effect of the test medicament.

Evaluation of antitumor effect by tracing technology

The 18F-FLT PET is a sensitive imaging method showing high-grade lymphoma, has higher sensitivity for detecting the lymphoma, and can be used for effectively detecting the proliferation condition of the lymphoma in vivo. The 18F-FLT is used as a positron tracer agent for reflecting cell proliferation, is applied to PET imaging, can noninvasively and quantitatively observe the proliferation condition of tumors in an organism at a molecular level, improves the specificity of tumor diagnosis, and also provides a method for detecting tumor treatment response. In the research, a subject group carries out different condition treatment on the constructed DLBCL model mouse, 18F-FLT is intravenously injected before administration and 3 days after administration respectively, the whole body static scanning of the small animal PET is carried out after 1 h, the region of interest including tumors is delineated by PMOD, the radioactive uptake value of each tissue is obtained, and the anti-tumor curative effect of the medicine is evaluated.

Single cell transcriptome sequencing

In the present study, the subject group performed single cell transcriptome sequencing using the currently mature GEXSCOPETM microfluidic platform to prepare single cell suspensions with a concentration of 1 × 105 cells/ml PBS. And (3) loading the single cell suspension on a microfluid device, and constructing an scRNA-seq library by using a GEXSCOPETM single cell RNA library construction kit (the number of loaded cells is controlled to be 10000-20000, the average value of cell activity is more than 90%, and 5000-6000 cells are expected to be successfully captured). And after the quality control of the library is qualified, performing 150 bp double-end sequencing on the library by utilizing Illumina HiSeq X, and performing letter generation analysis on off-line data.

Bioinformatics analysis

The method comprises the steps of exploring the molecular expression characteristics of a single cell unit based on transcriptome data, carrying out cell type identification and cluster analysis by using a Seruat software, identifying cell subsets by adopting methods such as dimension reduction, unsupervised clustering and the like, exploring the proportion and change conditions of different cell subsets and discovering key target subsets. Table was used to introduce the expression matrix into R, followed by cell clustering analysis using findsclusterics, setting the parameter resolution 0.6. Identifying Differentially Expressed Genes (DEG) among different samples or subgroups by using FindMarkers, comparing the differentially expressed genes with known cell type markers, analyzing the change condition of the proportion of each cell subgroup under different conditions, screening specific molecular markers of the cell groups through differential expression gene analysis and gene expression correlation analysis, and performing related cell characteristic analysis by using a cell cycle reference gene library; obtaining a single cell clone evolution track through pseudo-time sequence analysis by using monocle2 software; GO functional enrichment analysis was performed on the genome using clusterProfiler software to find biological functions or signaling pathways that are significantly associated with specifically expressed genes.

Drawings

FIG. 1 is a diagram of the up-regulation of C-Fos expression of DLBCL cells caused by high concentration of LAQ824 and the heterogeneity of DLBCL cells under the action of scRNA-seq analysis of LAQ 824;

FIG. 2 is a graph of the expression profile of c-Fos in DLBCL under different conditions;

FIG. 3 is a graph showing the results of an experiment in which LAQ824 was used in combination with a c-Fos inhibitor to kill DLBCL cells;

FIG. 4 is a graph of the results of an experiment in which LAQ824 and CDF synergistically inhibit DLBCL tumor growth in vivo;

fig. 5 shows the calculated IC50 of LAQ824 detected in 11 DLBCL cell lines, respectively.

Detailed Description

Example 1: the application of c-Fos inhibitor in relapse of drug-resistant diffuse large B cell lymphoma is as follows:

experimental example 1 Single cell transcriptome sequencing (scRNA-seq) analysis of DLBCL cell heterogeneity and drug resistance mechanisms

FIG. 1 shows the heterogeneity and high concentration of DLBCL cells under the action of LAQ824 in scRNA-seq analysis

LAQ824 causes a profile of upregulation of c-Fos expression in DLBCL cells in which:

(A) t-SNE analysis, and typing according to different sample sources;

(B) t-SNE analysis, and typing according to different cell subtypes;

(C, E) t-SNE and violin plots showing the expression profile of C-Fos in different treatment groups and different cell subsets;

(D) the expression distribution of the relevant differential genes in different subpopulations;

(F) scRNA-seq analysis of differentially expressed genes.

Topic group respectively carries out scRNA-seq on DLBCL cell line U2932 (non-GCB type) processed by LAQ824 with different concentrations (0/0.01/0.1 mu M), firstly carries out overall analysis on a sequencing result, and respectively clusters according to different sample sources (figure 1A) and different cell subsets (figure 1B) by adopting methods such as t-SNE (t distribution field embedding algorithm) dimension reduction and reduction, unsupervised clustering and the like, wherein the result shows that the heterogeneity of residual tumor cells is enhanced under the condition of 0.1 mu M concentration; the subject group further analyzed the expression level and distribution of C-Fos at the single cell level, as shown in fig. 1C, with C-Fos exhibiting significantly high expression in the 0.1uM LAQ 824-treated group, and being highly enriched especially in sub-groups 3 and 5 (fig. 1D, E); clustering heatmaps (fig. 1F) show differentially expressed genes in different samples and corresponding cell subsets, and it can be seen that c-Fos was significantly upregulated under 0.1uM LAQ824 treatment conditions; the above results suggest that the up-regulation of the c-Fos gene under higher drug stress or is associated with increased heterogeneity of DLBCL cells and LAQ824 resistance. Therefore, the subject group further studied the molecular mechanism associated with drug resistance, centered on the c-Fos gene.

Test example 2: western Blot for detecting c-Fos protein expression condition in DLBCL cells

FIG. 2 shows the expression profile of c-Fos in DLBCL under different conditions.

Fig. 2(a, B) shows c-Fos protein and gene expression in DLBCL at different drug concentrations ([ P ] < 0.05, [ P ] < 0.01, [ P ] < 0.001, [ P ] < 0.0001).

Collecting cells treated by the medicine LAQ824 (0 uM, 0.01uM, 0.1 uM), collecting protein, quantifying, performing electrophoresis, performing membrane transfer, sealing, incubating primary antibody and secondary antibody, and performing on-machine detection on ECL. Western Blot verified the expression levels of c-Fos in different DLBCL cell lines treated with different drug concentrations in vitro, and demonstrated that c-Fos expression was up-regulated as the concentration of LAQ824 was increased (FIG. 2 (A)).

Test example 3: qRT-PCR detection of C-Fos protein expression in DLBCL cells

As shown in FIG. 2 (B), cells treated under different conditions were collected, and RNA extraction, RNA quality detection, cDNA synthesis, and Real Time PCR reaction were performed. The expression levels of c-Fos were verified by qRT-PCR for different DLBCL cell lines under treatment with different drug concentrations in vitro, and the results demonstrated that c-Fos expression was upregulated in whole with increasing LAQ824 concentration (fig. 2 (B)).

Test example 4: tumor cell proliferation inhibition assay

FIG. 3 is a graph showing the results of an assay for killing DLBCL cells using LAQ824 in combination with a c-Fos inhibitor, wherein:

(A) CCK8 detects the expression level of c-Fos of 4 DLBCL cell lines under different treatment conditions;

(B) flow-detecting the influence of LAQ824 and LAQ824 combined with CDF on DLBCL apoptosis; (. P < 0.05,. P < 0.01,. P < 0.001,. P < 0.0001).

After DLBCL cells in the logarithmic growth phase were incubated with LAQ824 (0 uM, 0.01uM, 0.1 uM) and CDF (0.01 uM) for 24h, 48h, and 72h, proliferation of DLBCL cells under different treatment conditions was detected by CCK8, and as a result, it was found that the survival rate of cells in the group of LAQ824 and c-Fos inhibitor was significantly decreased in different DLBCL cell lines, and tumor cells were further effectively killed (fig. 3 (a)).

Test example 5: LAQ824 induces apoptosis of DLBCL cells

As shown in FIG. 3, DLBCL cells in logarithmic growth phase were incubated with LAQ824 (0 uM, 0.01uM, 0.1 uM) and CDF (0.01 uM) for 48h, and then apoptosis was detected by flow cytometry.

Flow analysis found that LAQ824 and c-Fos inhibitor synergistically induced DLBCL apoptosis, as exemplified by cell line U2932 (fig. 3 (B)).

Test example 6: LAQ824 and CDF synergistically inhibit DLBCL tumor growth in vivo

FIG. 4 is a graph of the results of an experiment in which LAQ824 and CDF synergistically inhibit DLBCL tumor growth in vivo. Wherein:

(A) DLBCL tumors of day 28 excised LAQ824 treatment group, LAQ824+ CDF combination group, and control group;

(B) small animal PET whole body static scanning is carried out after the LAQ824 treatment group, the LAQ824+ CDF combination group and the control group are subjected to intravenous injection of 18F-FLT.

Subject groups established DLBCL mouse models by transplanting DLBCL cell lines into NOD SCID mice. Mice were randomly divided into 3 groups, which were negative control groups, LAQ824 (75 mg/kg), LAQ824+ CDF (10 mg/kg). And (3) carrying out intravenous injection of 18F-FLT on the 3 rd day after administration, carrying out whole-body static scanning of small animal PET (polyethylene terephthalate) after 1 h, delineating an interested area including a tumor by using PMOD (polymethylene oxide) and obtaining the radioactive uptake value of each tissue. Tumors and body weights were measured 2 times a week for 28 consecutive days and tumors were removed after the 28 th day scan, photographed and weighed to determine the antitumor efficacy of the test agent. The 18F-FLT PET can effectively detect the proliferation condition of the lymphoma in vivo. LAQ824 and CDF were found to synergistically inhibit DLBCL tumor cell proliferation and growth in vivo by 18F-FLT PET and tumor volume measurements (fig. 4A, B).

Example 2 the invention proposes the use of the c-Fos gene in the assessment of drug-resistant relapse in diffuse large B-cell lymphoma (DLBCL).

The nucleotide sequence of the c-Fos gene is SEQ ID NO. 1.

The reagent for detecting the expression quantity of the c-Fos comprises SYBR Green and PCR amplification primers shown in SEQ ID NO. 2 and SEQ ID NO. 3.

Upstream primer sequence for c-Fos: 5'-TTCAACGCAGACTACGAGGC-3' (SEQ ID NO: 2);

downstream primer sequence for c-Fos: 5'-TGAAGTTGGCACTGGAGACG-3' (SEQ ID NO: 3).

The experimental examples are as follows:

test example 1: CCK8 detection:

proliferation of 11 DLBCL cells treated with LAQ824 (0 uM, 0.01uM, 0.1 uM) was examined by CCK 8. Half maximal inhibitory concentrations (IC50) were calculated by plotting dose-response curves using GraphPad Prism 7 data processing software.

Test example 2: qRT-PCR detection

1. RNA extraction

11 DLBCL cells were collected and tested. Total RNA was extracted from the TIANAmp Virus RNA Kit (TIANGEN, Cat. # DP 315-R), and the detailed procedures are described in the specification.

2. Reverse transcription PCR

20 muL reverse transcription reaction system:

the reverse transcription procedure was: 25 ℃ for 10 min; c, 50 ℃ for 15 min; 85 ℃ and 5 min.

3. Real-time fluorescent quantitative PCR (qRT-PCR)

10 mu L qRT-PCR reaction system:

reaction procedure: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 10 s, annealing/extension at 60 ℃ for 30 s.

The combined CCK8 detection and qRT-PCR detection result shows that: IC50 of LAQ824 was calculated by separate assays in 11 DLBCL cell lines, and we found that c-Fos expression was highly positively correlated with IC50 of LAQ824 in DLBCL cells by correlation analysis as shown in fig. 5 (R2 =0.939,. xp < 0.0001). Combining the results of the previous studies, LAQ824 caused the up-regulation of c-Fos expression in DLBCL cells at higher concentrations, at which time drug treatment failed to further induce tumor cell apoptosis, and c-Fos served as a potential drug-resistant core driver, whose expression level was highly correlated with drug IC50, and was used as an index for evaluating the drug sensitivity of LAQ 824.

Sequence listing

<110> Jiangsu province national hospital (the first subsidiary hospital of Nanjing medical university)

Application of <120> c-Fos gene and c-Fos inhibitor in relapse of drug-resistant diffuse large B cell lymphoma

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<170> SIPOSequenceListing 1.0

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<212> DNA/RNA

<213> human (Homo sapiens)

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gcga 2104

Claims (14)

1. Use of a c-Fos inhibitor in relapsed drug resistant diffuse large B-cell lymphoma.
2. 2. Use according to claim 1, characterized in that: the c-Fos inhibitor and LAQ824 are used in a combined mode to kill DLBCL cells synergistically, and the tumor inhibition rate is improved.
3. 3. Use according to claim 1, characterized in that: LAQ824 synergistically with c-Fos inhibitors induced apoptosis of DLBCL cells.
4. 4. Use according to claim 1, characterized in that: application of Difluorobenzocumin as a c-Fos inhibitor in relapse of drug-resistant diffuse large B cell lymphoma.
5. 5. Use according to claim 4, characterized in that: application of Difluorobenzocumin small-molecule inhibitor in large B-cell lymphoma is achieved by inhibiting c-Fos expression in DLBCL cells and relapse drug resistance.
6. 6. Use according to claim 4, characterized in that: the Difluorobenzocumin small molecule inhibitor and LAQ824 are used in a combined mode to kill DLBCL cells synergistically, and the tumor inhibition rate is improved.
7. 7. Use according to claim 4, characterized in that: LAQ824 and Difluorobenzocumin small molecule inhibitor synergistically induce DLBCL apoptosis.
8. 8. Use according to claim 4, characterized in that: LAQ824 and Difluorobenzocumin small molecule inhibitor synergistically inhibit proliferation and growth of DLBCL tumor cells in vivo.
9. Use of LAQ824 for up-regulating c-Fos expression in DLBCL cells.
10. 10. Use according to claim 7, characterized in that: the concentration of LAQ824 was 0.1uM and above, and as the concentration of LAQ824 increased, c-Fos expression was globally up-regulated.
11. Use of the c-Fos gene in the assessment of drug resistant relapse of diffuse large B-cell lymphoma DLBCL.
12. 12. The application of the reagent for detecting the expression quantity of c-Fos in evaluating the drug-resistant relapse of diffuse large B-cell lymphoma DLBCL.
13. 13. Use according to claim 11, characterized in that: the nucleotide sequence of the c-Fos gene is SEQ ID NO. 1.
14. 14. Use according to claim 12, characterized in that: the reagent for detecting the expression quantity of the c-Fos comprises SYBR Green and PCR amplification primers shown in SEQ ID NO. 2 and SEQ ID NO. 3;

    c-Fos upstream primer sequence SEQ ID NO: 2: 5'-TTCAACGCAGACTACGAGGC-3', respectively;

    c-Fos downstream primer sequence SEQ ID NO: 3: 5'-GAAGTTGGCACTGGAGACG-3' are provided.

Images