A BRD4 exon deletion model to study the functions of different BRD4 isoforms and tumor development

Abstract

The present invention provides a BRD4 exon deletion model for studying the functions of different BRD4 subtypes and tumor development, and relates to the technical field of biological model construction. The model is used to study the occurrence and development of tumors, and the deleted exon is exon 14 of human BRD4 or exon 15 of mouse BRD4. By establishing a stable cell line model in which different exons of human BRD4 and different exons corresponding to mouse BRD4 are deleted for studying the occurrence and development of tumors, the effects of different exon regions on cell phenotypes such as tumor proliferation, migration, adhesion, and differentiation stemness are studied for a long time. At the same time, we can also conveniently use it for long-term drug screening of some cell lines. The animal model is also suitable for long-term metastasis research of tumors in vivo.

Description

Technical Field

[0001] The present invention relates to the technical field of biological model construction, and in particular to a BRD4 exon deletion model for studying the functions of different BRD4 subtypes and tumor development. Background Art

[0002] The terminal stage of tumor refers to the latest stage of tumor development, when tumors often spread and metastasize to tissues and organs in the body. Cancer is the main cause of death, and metastatic cancer is the most advanced form of the disease, which is usually fatal. Therefore, understanding the process of tumor metastasis and the key regulatory factors involved, as well as ultimately finding strategies to block metastasis, is an important way to overcome cancer. Even for cancer cells, the metastasis process is an extremely complex and highly inefficient process, with only a few cells in many cancer cells successfully colonizing at a distant site. The metastasis process includes a series of major steps, mainly five processes: local invasion, tumor cell intravasation, survival in the circulation, tumor cell extravasation, and micrometastasis formation. Cancer cells detaching from the tumor mass and penetrating the surrounding stroma is the first step in metastasis. Adhesion molecules and mechanical extrusion are believed to play a key role in detaching from the tumor mass, but how spontaneous cell detachment occurs and its molecular regulation are less studied.

[0003] Bromodomain-containing protein 4 (BRD4) is a promising cancer therapeutic target currently in clinical trials. In clinical studies, BRD4 inhibitors inhibit the function of BRD4 to achieve the effect of inhibiting tumor development. However, clinical experimental results show that there are huge individual differences. This is because BRD4 has two subtypes, BRD4L and BRD4S. At present, BRD4 inhibitors inhibit the function of BRD4L (BRD4 long, BRD4 long subtype and BRD4short, BRD4 short subtype) at the same time, but studies have found that there are great differences in the functions of BRD4L and BRD4S, and sometimes they are opposite. Therefore, in-depth research on the functional differences between BRD4L and BRD4S is the key to improving the therapeutic effect of BRD4 inhibitors and conducting personalized treatment. BRD4S is a short subunit, which has less carboxyl terminal sequence than the amino acid sequence of BRD4L, and the rest of the sequence is the same. Therefore, by knocking out the amino acid part unique to BRD4L, the amount of BRD4S and BRD4L in the cell can be changed, so as to study the functional differences between BRD4S and BRD4L and their effects on tumor development. In general tumor research, since the functions of BRD4L and BRD4S are not distinguished, it is subjectively believed that the role of BRD4 in cancer is mainly attributed to the long isoform (BRD4-L). Through isoform-specific knockout and endogenous protein detection and transgenic expression technology, it was found that the short isoform of BRD4 (BRD4-S) has a carcinogenic effect, while BRD4-L has a tumor suppressor effect in the proliferation and migration of breast cancer cells and the formation and metastasis of breast tumors. BET family proteins containing bromodomains (BD domains) and ET domains are acetyl-lysine readers that mainly bind to acetylated chromatin and transcription factors. Chromosomal translocation can cause BRD4 to mistakenly recruit histone-modifying enzymes to the promoter region, thereby causing many tumors. BRD4 helps maintain the chromatin structure of daughter cells during mammalian cell mitosis. In addition, BRD4 also controls the expression of some important oncogenes, such as c-myc, bcl-2, and cyclinD1. Therefore, BRD4 is considered to be a potential oncogenic protein [5]. The structural difference between BRD4-L and BRD4-S is that the former has an additional C-terminal low complexity domain, namely the LCD domain. However, it is still unclear what effect the LCD domain has on tumor migration and spread.

[0004] The C-terminal low complexity domain (LCD domain) of BRD4 is the main structural component that distinguishes BRD4 protein from other members of the BET protein family. However, previous studies have not clearly explained the function of BRD4-LCD. BRD4 inhibitors such as JQ1 and dBETi are drugs that act on the bromo (BD) domain, which is a common domain of the BET protein family. This also leads to clinical broad-spectrum BET inhibitors that inhibit the entire BET family of proteins. Therefore, inhibitors that act on the bromo domain have greater side effects. Summary of the invention

[0005] The purpose of the present invention is to solve the defect of the prior art that the functions of BRD4L and BRD4S cannot be distinguished, and the amount of BRD4L and BRD4S in cells is changed by knocking out the exons specific to BRD4L, studying the role of BRD4L and BRD4S in tumors, and understanding the intrinsic mechanism of functional differences.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A BRD4 exon deletion model for studying the functions of different BRD4 isoforms and tumor development, wherein the model is used to study the occurrence and development of tumors.

[0008] Preferably, the deleted exon is exon 14 of human BRD4 or exon 15 of mouse BRD4.

[0009] Preferably, the model includes a cell line with a deletion of exon 14 of human BRD4, a cell line with a deletion of exon 15 of mouse BRD4, and a mouse melanoma cell metastasis model.

[0010] Preferably, the cell line lacking exon 14 of BRD4 is obtained by precisely cutting out exon 14 in HEK293T cells using CRISPR-CAS9 technology.

[0011] Preferably, the cell line with exon 15 deletion of mouse BRD4 is mouse melanoma cell-B16.

[0012] Preferably, the mouse melanoma cell metastasis model is established by subcutaneous and intraperitoneal methods.

[0013] Preferably, the specific method for constructing the mouse melanoma cell metastasis model is as follows:

[0014] B6 mice are raised in an animal room to ensure the comfort and health of the animals, and the required B16 cell line is cultured at the same time, wherein the B16 cell line is a B16 cell line in which exon 15 of mouse BRD4 is deleted;

[0015] Select WT B6 mice of the same age and sex and inject the same amount of tumor cells. Before injection, disinfect the mouse's lower abdomen or subcutaneous tissue with alcohol cotton, then blow the cell suspension evenly with a gun tip, draw the suspension with a 1mL syringe, and inject it into the mouse's abdominal cavity or subcutaneous tissue.

[0016] In this application, by establishing a stable cell line model for studying the occurrence and development of tumors, different exons of human BRD4 and different exons corresponding to mouse BRD4 are deleted, and the effects of different exon regions on cell phenotypes such as tumor proliferation, migration, adhesion, and differentiation stemness are studied for a long time. At the same time, we can also conveniently use it for long-term drug screening of some cell lines. Animal models are also suitable for long-term metastasis studies of tumors in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Genotyping (A) and Western blot (B) were used to verify the HEK293T cell line in which exon 14 and exon 12-14 of human BRD4 were knocked out in one embodiment of the present invention;

[0018] Figure 2 The morphology of HEK293T cells deficient in SgVector and BRD4 E14 and E12-14 under a light microscope in one embodiment of the present invention;

[0019] Figure 3 A scratch assay and an adhesion assay comparing SgVector and BRD4 E14 and E12-14 deficient HEK293T cells in one embodiment of the present invention;

[0020] Figure 4 To find differentially expressed genes between SgVector and BRD4 E14 by RNA-Seg sequencing in one embodiment of the present invention;

[0021] Figure 5 RT-PCR detection of S100A10 transcription expression level (A); Western bolting (B) and immunofluorescence detection of S100A10 translation expression level (C), immunofluorescence S100A10 (Green);

[0022] Figure 6 In one embodiment of the present invention, S100A10 and ANXA2 plasmids with FLAG tags were transfected into HEK293T cells to achieve exogenous overexpression of genes (A), and the differences in cell morphology between the control group and the exogenous overexpression of S100A10 and ANXA2 groups were compared under a light microscope (B);

[0023] Figure 7The scratch test (A) and adhesion test (B) in one embodiment of the present invention compare the differences in migration and adhesion ability between the control group and the S100A10 overexpression and ANXA2 overexpression cell groups;

[0024] Figure 8 In one embodiment of the present invention, a cell line in which exon 15 of human BRD4 is knocked out is verified by genotyping in B16 cells;

[0025] Fig. 9 RT-PCR detection of S100A10 transcription expression level in one embodiment of the present invention (A); Western boltting detection of S100A10 translation expression level (B);

[0026] Fig.10 In one embodiment of the present invention, a scratch assay (A) and an adhesion assay (B) compare the phenotypes of SgVector and BRD4 E15-deficient B16 cells;

[0027] Fig.11 In one embodiment of the present invention, 4 pairs of C57BL / 6 mice of the same sex and age were intraperitoneally injected with an equal amount of 1x106 SgVector and BRD4 E15-deficient B16 cells, and the abdominal cavity of the mice was dissected 14 days later (A) and a picture of the size of the abdominal tumor (B);

[0028] Fig.12 Histochemical staining (A) compares S100A10 and S100A10 (Green) in tumors with SgVector and BRD4 E15 deficiency in one embodiment of the present invention; Immunohistochemistry (B) compares the expression of S100A10 in tumors with SgVector and BRD4 E15 deficiency;

[0029] Fig.13 In one embodiment of the present invention, HEK293T cells were transfected with Sg-MYC plasmid, and a stable cell line was constructed by Puro drug screening. Western boltting verified the reduction of MYC, and the protein expression of S100A10 was detected by Western boltting (A). Similarly, Western boltting was used to detect the protein expression of MYC, S100A10, and ANXA2 in B16 (B);

[0030] Fig.14 In one embodiment of the present invention, the relationship between BRD4 and MYC was evaluated by co-immunoprecipitation using anti-BRD4 antibodies in control and BRD4-E14-deficient cells. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments.

[0032] A BRD4 exon deletion model for studying the functions of different BRD4 subtypes and tumor development, wherein the model is used for studying the functions of different BRD4 subtypes and the occurrence and development of tumors.

[0033] The deleted exon is exon 14 of human BRD4 or exon 15 of mouse BRD4.

[0034] The models include a cell line with a deletion of exon 14 of human BRD4, a cell line with a deletion of exon 15 of mouse BRD4, and a mouse melanoma cell metastasis model.

[0035] Among them, the cell line with exon 14 deletion of human BRD4 was obtained by precisely cutting out exon 14 in HEK 293T cells using CRISPR-CAS9 technology.

[0036] The cell line in which exon 15 of mouse BRD4 is deleted is mouse melanoma cell-B16.

[0037] The mouse melanoma cell metastasis model is established by subcutaneous and intraperitoneal methods.

[0038] In one embodiment, B6 mice are raised in an animal room to ensure the comfort and health of the animals, and the desired B16 cell line is cultured at the same time, wherein the B16 cell line is a B16 cell line in which exon 15 of mouse BRD4 is deleted;

[0039] Select WT B6 mice of the same age and sex and inject the same amount of tumor cells. Before injection, disinfect the mouse's lower abdomen or subcutaneous tissue with alcohol cotton, then blow the cell suspension evenly with a gun tip, draw the suspension with a 1mL syringe, and inject it into the mouse's abdominal cavity or subcutaneous tissue.

[0040] The present application is described below in conjunction with specific embodiments:

[0041] Example 1: Construction and verification of a cell line with deletion of exon 14 of human BRD4

[0042] In one embodiment, HEK 293T cells are divided into three groups.

[0043] Two of the groups used CRISPR-CAS9 technology to precisely cut out exon 14 and exons 12-14 in HEK 293T cells;

[0044] The three groups of cells were transfected separately and stable cell lines were established.

[0045] Then, Western boltting and genotyping were used to verify the three groups of cell lines. The results are as follows Figure 1 shown.

[0046] Then, the phenotypic differences between the KO cell lines (i.e., the cell lines in which exon 14 (E14) was cleaved and the cell lines in which exons 12-14 (E12-14) were cleaved) and the control group (SgVector) were compared.

[0047] The phenotypic differences include comparisons of cell shape, cell migration speed, and adhesion ability;

[0048] Specifically, use a light microscope to take pictures of SgVector and BRD4 E14 and E12-14 deficient HEK293T cells for comparison. Figure 2 It can be seen that the absence of exon 14 of human BRD4 can cause cells to undergo spindle-like changes.

[0049] The scratch test and cell adhesion test were used to compare the migration speed and adhesion ability of cells. Figure 3 As shown, the cell migration speed was significantly accelerated and the adhesion ability was significantly decreased.

[0050] In one embodiment, RNA-seq analysis of differentially expressed genes was performed on SgVector and SgE14 cells, and the results were as follows: Figure 4 As shown, the expression of adhesion-related genes S100A10 and ANXA2 was increased in SgE14 cells.

[0051] The protein level of S100A10 was detected by Western boltting and immunofluorescence experiments, and the transcription level of S100A10 was detected by RT-PCR. Figure 5 As shown, S100A10 was elevated at both the protein level and the RNA level, and this result was consistent with the RNA-Seq sequencing results.

[0052] In order to verify that the increased expression of S100A10 in the cell line with human BRD4 exon 14 deletion leads to changes in cell adhesion ability, in one embodiment, HEK 293T cells were transfected with plasmids overexpressing S100A10 and ANXA2, and the cell adhesion reduction model was verified. At 48 hours after transfection, the cells underwent a long spindle-shaped change ( Figure 6 ), while overexpression of S100A10 and ANXA2 in HEK293T cells can lead to a decrease in cell adhesion and an increase in migration ability ( Figure 7). The experimental results strongly support the model provided by the present application, that is, when exon 14 of BRD4 is deleted, the expression of S100A10 and ANXA2 proteins in cells will increase, which will then lead to a weakening of the cell's adhesion ability and an enhancement of its migration ability.

[0053] Example 2: Cell line with deletion of exon 15 of mouse BRD4

[0054] In one embodiment, a stable cell line defective in exon 15 (E15) was constructed in mouse melanoma cells-B16 cells, and verified by Westernboltting and Genotyping. The results are as follows Figure 8 As shown;

[0055] Then, RT-PCR was used to detect the transcriptional expression level of S100A10, and Western blotting was used to detect the translational expression level of S100A10. The results showed that E15 deficiency led to increased expression of S100A10.

[0056] Adhesion and scratch tests were then performed to compare the adhesion and migration abilities of E15-deficient cells. Fig.10 As shown, the adhesion ability of E15-deficient cells was weakened, while the migration ability was enhanced.

[0057] Example 3: Construction of mouse model

[0058] In this example, in order to study the role of E15 in tumor development in vivo, the E15-deficient cells obtained in Example 2 were injected into the peritoneal cavity of mice. Specifically,

[0059] The model was established by subcutaneous and intraperitoneal injection. B6 mice were raised in the animal room to ensure the comfort and health of the animals, and the required B16 cell lines were cultured at the same time. WT B6 mice of the same age and gender were selected and injected with equal amounts of tumor cells. Before injection, the lower abdomen or subcutaneous part of the mouse was disinfected with alcohol cotton, and then the cell suspension was blown evenly with a gun tip, and the suspension was sucked with a 1mL syringe and injected into the mouse peritoneal cavity or subcutaneous part. The mice were killed two weeks later, and the size and metastasis of the tumors in the two groups of mice were compared.

[0060] See also Fig.11 It can be seen that the number and size of tumor colonies in E15-deficient cells are greater than those in the control group, which indicates that E15 has a stronger ability to spread tumors locally than the control group.

[0061] In addition, immunofluorescence and immunohistochemistry experiments were used for validation, see Fig.12, the level of S100A10 protein increased in E15-deficient tumors. The research results of this application show that the E14 (E15 in mice) coding region of BRD4 is crucial to its tumor suppressor function in vivo and in vitro. So far, this application has relatively completely constructed a human BRD4 exon 14 and mouse BRD4 exon 15 deletion model for studying tumor occurrence and development.

[0062] Embodiment 4:

[0063] To further explain the potential mechanism of the above model, the present application detected the potential transcription factors of BRD4-E14. The present application compared the up-regulated and down-regulated genes in BRD4-E14-deficient cells.

[0064] A large number of genes affected, both up-regulated and down-regulated, are targets of MYC, and MYC expression levels were also found to be reduced in BRD4-E14-deficient cells.

[0065] Based on this, the present application further explores the specific regulatory relationship between BRD4-MYC-S100A10. Specifically, in one embodiment, MYC was knocked out in HEK293T cells, and an increase in S100A10 protein expression was found. The same result can be repeated in B16 cells ( Fig.13 ). Therefore, in the model of the present application, it was found that MYC can negatively regulate the expression of S100A10. In previous studies, it has been known that BRD4, as an upstream gene of MYC, can effectively bind to it. The upregulation of S100A10 expression caused by the loss of BRD4 E14 guided us to consider whether the loss of E14 caused the loss of interaction between BRD4 and MYC, which in turn caused MYC to lose its inhibition of S100A10. The IP results of E14 and the control group confirmed the above conjecture of the present application ( Fig.14 ).

[0066] So far, the present application has a deeper understanding of the regulatory relationship between BRD4-MYC-S100A10. BRD4 cooperates with MYC to inhibit the expression of S100A10. This regulatory relationship limits the ability of tumor cells to spread. Therefore, when facing the targeted treatment of tumors in clinical practice, do not destroy the BRD4 E14 region to avoid local spread of tumors.

[0067] Some of the experiments in this application are as follows:

[0068] Cell culture

[0069] HEK293T and B16 cells were cultured in DMEM containing 10% serum and 1% penicillin-streptomycin mixture, placed in an incubator and set at 37°C and 5% carbon dioxide concentration. Cells were rinsed with PBS during the liquid replacement process, and the cell processing process was completed on a clean bench. Sterile operation was observed throughout the process, and cell morphology and cleanliness were observed regularly.

[0070] Cell adhesion assay

[0071] Digest the cells with trypsin, count the same amount of cells with abalone counting plate, add to 12-well plate, and shake gently to make the cells evenly distributed. Take a photo of the cells at this time and record it as the photo of 0h. At this time, the cells are completely non-adherent. After 2h, gently blow and wash twice with PBS preheated to 37°, take a photo of the cells at this time and record it as the photo of 2h. Count the number of cells again after trypsin digestion. Compare the similarities and differences in the proportion of adherent cells of different cells.

[0072] Cell floating experiment

[0073] Inoculate an equal number of cells in a 12-well plate, and after 24 hours, the cells are completely attached to the wall and well stretched. Wash twice with PBS, then add 1 ml of 37° preheated PBS to the 12-well plate, select typical fields of view at 0, 2, 4, 8, and 12 hours to take pictures, and compare the proportion of floating cells at the same time point for different cells.

[0074] Cell scratch assay

[0075] After the cells are digested, they are inoculated in a 6-well plate to ensure that the cells cover the six-well plate. After 24 hours, the cells are completely attached to the wall and stretch well. Use the tip of the gun to make a scratch perpendicular to the cells in the 6-well plate, keeping the width of the scratch consistent. Discard the culture medium and rinse twice with PBS to wash away the cell debris produced by the scratch. Add culture medium and select a fixed position to take pictures of the scratch size at 0, 12, 24, and 48 hours.

[0076] RT-PCR

[0077] RNA was extracted using the Trizol method, and the sample was reverse transcribed using a reverse transcription kit to obtain cDNA. Finally, an excess of SYBR fluorescent dye was added to the PCR reaction system, and the RNA expression level of the target gene was detected on the machine.

[0078] Westernboltting

[0079] Collect the cells, add lysis buffer RIPA and protease inhibitor PMSF. Sonicate on ice 1-3 times, 10s each time. Then place it on a shaker for 30min, centrifuge at 12000Rpm for 15min, add 5X loading buffer after removing the precipitate, and denature the protein at 105° metal bath for 5min. Place the sample in the SDS-PAGE gel of the corresponding concentration for electrophoresis, activate the PVDF membrane with methanol, depolarize it in DDH2O, and transfer the protein to the PVDF membrane. Block with 5% skim milk at room temperature for 2h and incubate with primary antibody overnight. Recover the primary antibody the next day and rinse 3 times with 1xTBST. Incubate with secondary antibody of the corresponding species for 2h, rinse again 3 times with TBST, and develop with ultrasensitive ECL chemiluminescent detection reagent.

[0080] Genomic genotyping

[0081] Collect cells into centrifuge tubes, add DNA lysis solution (50μL 1xMouse Buffer + 1μLProteinase K), place in 55° water bath for 30min, 95° metal bath for 5min, centrifuge and keep the supernatant as sample DNA. Add PCR premix solution, primers, DDH2O and DNA sample to the eight-tube strip, mix well and place in PCR instrument. Prepare agarose gel and add PCR products. After the electrophoresis is completed, place the gel in the developer for development.

[0082] Immunofluorescence

[0083] Place a round glass slide in a 24-well plate and inoculate the cells. After the cells are completely attached and well stretched, rinse with PBS three times, fix with 4% paraformaldehyde for 15 minutes, and rinse again with PBS three times. Use 0.5% TritonX-100 solution for permeabilization for 5 minutes. Prepare 1% BSA blocking solution and block at room temperature for 1 hour. Discard the blocking solution, add the primary antibody, and incubate at 4° for 24 hours. Discard the primary antibody and rinse with PBS, add the secondary antibody of the corresponding species, incubate for 2 hours, discard the secondary antibody and rinse with PBS. Finally, counterstain the nucleus with Hoechst and incubate in the dark for 30 minutes, discard the Hoechst staining solution and rinse three times with PBS. Seal the slide with an anti-fade agent and place it under an inverted fluorescence microscope for photography.

[0084] Transfection and establishment of stable cell lines

[0085] Inoculate cells in a six-well plate until the confluence is 70%-80%, and start transfection when the cells are fully stretched. Take a sterilized 1.5ml EP tube, add Opti-MEM culture medium and 1.5μg plasmid, mix well and let stand for 5min. Add 3μl Lipo-2000 reagent, mix well and let stand for 20min to make a transfection solution. Then slowly add the transfection solution to the six-well plate, place it in a cell culture incubator, and change the solution every 6-8h. After 48h, the cell fluorescence can be observed under an inverted fluorescence microscope, and puromycin can be added to screen the successfully transfected cells. Increase the concentration of puromycin successively until all WT cells die, stop adding the drug and continue to culture the successfully transfected cells. When the cells grow well, collect cell samples and verify the successful establishment of the stable cell line through WB, Genotyping and sequencing.

[0086] Co-IP

[0087] Collect cells, add IP lysis buffer, lyse on ice for 30 minutes, centrifuge and keep the supernatant. Take 200μL supernatant, add 5X loading buffer and place in a 105° metal bath for 5 minutes. This specimen is used as an input control. Add Protein A to the remaining supernatant, take the supernatant after centrifugation, add 1.5μg IgG and 1.5μg primary antibody respectively, and incubate overnight with 4° rotation. The next day, wash 3 times with IP lysis buffer, add 5X loading buffer and place in a 105° metal bath for 5 minutes. Use western bolt to detect the target protein.

[0088] In this application, by establishing a stable cell line model for different exons of human BRD4 and different exons corresponding to mouse BRD4 for studying the occurrence and development of tumors, the effects of different exon regions on cell phenotypes such as tumor proliferation, migration, adhesion, and differentiation stemness are studied for a long time. At the same time, we can also conveniently use it for long-term drug screening of some cell lines. Animal models are also suitable for long-term metastasis studies of tumors in vivo. The regulatory relationship of BRD4L-MYC-S100A10 was first proposed, and BRD4L plays a tumor-suppressing role through its unique exon 14. However, BRD4S, because it is shorter and does not contain relevant domains, plays a more tumor-promoting role. Therefore, the clinical use of BRD4 inhibitors to simultaneously inhibit the functions of BRD4L and BRD4S may not be satisfactory. Therefore, it is necessary to use the model we established to further study the functional differences between BRD4L and BRD4S, so as to design a more precise treatment for BRD4. Corresponding to the function and mechanism of BRD4L and BRD4S, can also help us understand the occurrence and development of tumors.

Claims

1. A BRD4 exon deletion model for studying the functions of different BRD4 isoforms and tumor development, characterized by: The model is used to study the occurrence and development of tumors. The deleted exon is exon 14 of human BRD4 or exon 15 of mouse BRD4. The model includes a cell line with a deletion of exon 14 of human BRD4 and a cell line with a deletion of exon 15 of mouse BRD4. The cell line with a deletion of exon 14 of BRD4 is obtained by precisely cutting out exon 14 in HEK 293T cells using CRISPR-CAS9 technology, and the cell line with a deletion of exon 15 of mouse BRD4 uses mouse melanoma cells-B16.

2. A method for constructing a BRD4 exon deletion model for studying the functions of different BRD4 isoforms and tumor development, characterized in that: The BRD4 exon deletion model for studying the functions of different BRD4 subtypes and tumor development is a mouse melanoma cell metastasis model. The mouse melanoma cell metastasis model is constructed by subcutaneous and intraperitoneal methods. The specific construction method of the mouse melanoma cell metastasis model is as follows: B6 mice are raised in an animal room to ensure the comfort and health of the animals, and the required B16 cell line is cultured at the same time, wherein the B16 cell line is a B16 cell line in which exon 15 of mouse BRD4 is deleted; WT B6 mice of the same age and sex were selected and injected with the same amount of tumor cells. Then inject the B16 cell line. Before the injection, disinfect the mouse's lower abdomen or subcutaneous tissue with alcohol cotton, then blow the cell suspension evenly with the gun tip, draw the suspension with a 1 mL syringe, and inject it into the mouse's abdominal cavity or subcutaneous tissue.

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