Computer device and computer readable storage medium for identifying or assisting in identifying large cell transformation of mycosis fungoides

Abstract

The present invention discloses a computer device and computer-readable storage medium for identifying or assisting in the identification of large cell transformation of mycosis fungoides. The present invention provides a computer device comprising a memory, a processor, and a computer program stored in the memory, wherein the processor performs the following steps: S1) data reception: receiving the percentage of FOXM1-positive cells in skin lesions of a subject with mycosis fungoides (MF); S2) data output: outputting result information from the computer based on the percentage of FOXM1-positive cells, wherein the result information indicates whether the subject with mycosis fungoides has, or is a candidate for, large cell transformation of mycosis fungoides. Experiments in the present invention demonstrate that immunohistochemical staining for FOXM1 can more accurately locate large cells and quantify the proportion of large cells compared to traditional pathological techniques, thereby reducing subjective bias. Therefore, it is proposed that immunohistochemical staining for FOXM1 can be used as an auxiliary technique for diagnosing large cell transformation of mycosis fungoides.

Description

Technical Field

[0001] The present invention belongs to the field of bioinformatics and relates to a computer device and a computer-readable storage medium for identifying or assisting in identifying the transformation of mycosis fungoides into large cells. Background Art

[0002] Primary cutaneous T-cell lymphoma (CTCL) is a tumor that arises in the skin and is characterized by the clonal proliferation of T lymphocytes that are resistant to apoptosis. Its incidence is approximately 6.4 per million and is increasing. Mycosis fungoides (MF) is the most common subtype of CTCL, accounting for approximately 60% of CTCL cases. It is more common in men, and the risk increases with age.

[0003] Clinically, the disease manifests in its early stages as multiple reddish-brown patches and plaques, while later stages may develop tumors or even erythroderma. Severe cases can involve lymph nodes and even invade the blood and internal organs. MF generally progresses slowly, but some patients experience rapid progression. In these patients, tumor cells histologically transform from small to large cells, becoming four times larger than normal small lymphocytes, with a proportion of large cells exceeding 25%, or forming small nodules. This is called large cell transformation (LCT) and is diagnosed as MF-LCT. Patients who do not meet these criteria are classified as MF-non-large cell transformation, designated MF-NLCT. MF-LCT often has an aggressive course, with widespread, rapidly growing nodules and masses throughout the body. They are more likely to invade the lymph nodes, peripheral blood, bone marrow, and internal organs, resulting in significantly reduced survival rates and median survival times. Although LCT can occur at any stage of MF, from early to late, the vast majority occur in advanced MF (approximately 25% in stage IIB and 50% in stage IV). According to statistics, the earlier the LCT occurs, the worse the prognosis of patients tends to be.

[0004] The causes and specific molecular mechanisms of LCT in MF remain unclear. Diagnosis of LCT often requires the joint assessment of the percentage of large lymphocytes in the total lymphocyte population by at least two experienced dermatopathologists in at least four microscopic areas. Morphological identification and counting of LCT are challenging and time-consuming. Therefore, accurate and rapid pathological identification of MF-LCT remains a major challenge in clinical practice. Furthermore, patients with advanced MF or large cell transformation (LCT) have an aggressive disease course and are resistant to conventional treatment and chemotherapy. Controlling MF progression remains another major challenge in MF treatment.

[0005] FOXM1, or Forkhead box protein M1, is a transcription factor crucial for embryonic development. Previous studies have shown that FOXM1 expression is elevated in various solid tumors, including lung cancer, gastric cancer, glioma, and breast cancer. It accelerates tumor progression by promoting tumor cell growth, reducing apoptosis, and promoting epithelial-mesenchymal transition in malignant cells. However, no studies have yet demonstrated a correlation with MF or its subtypes.

[0006] Therefore, finding evaluation methods for MF-LCT or developing therapeutic targets for MF-LCT has become a research hotspot. Summary of the Invention

[0007] The object of the present invention is to provide a computer device for identifying or assisting in identifying large cell transformation of mycosis fungoides.

[0008] In a first aspect, the present invention provides a computer device comprising a memory, a processor, and a computer program stored in the memory, wherein the processor executes the computer program to implement the following steps:

[0009] S1) Data received: Percentage of FOXM1-positive cells in skin lesional tissues of subjects receiving mycosis fungoides (MF);

[0010] S2) Data output: Outputting result information from a computer based on the percentage of FOXM1-positive cells, wherein the result information indicates whether the subject with mycosis fungoides is or is a candidate for mycosis fungoides-large cell transformation (MF-LCT), or whether the subject with mycosis fungoides has large cell transformation (LCT).

[0011] The computer device is used to assist in identifying or distinguishing large cell transformation of mycosis fungoides, or to assist in identifying or distinguishing whether large cell transformation occurs in MF, or to assess the occurrence of large cell transformation in MF.

[0012] The above evaluation of the occurrence of large cell transformation in MF is the probability of occurrence.

[0013] In a second aspect, the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein the computer program implements the steps in the first aspect when executed by a processor.

[0014] In a third aspect, the present invention provides a computer program product, comprising a computer program, which implements the steps in the first aspect when executed by a processor.

[0015] In a fourth aspect, the present invention provides an apparatus for assisting in identifying or distinguishing large cell transformation of mycosis fungoides, comprising a data receiving module and a data output module;

[0016] The data receiving module is used to receive the percentage of FOXM1 positive cells in the skin lesion tissue of the subject with mycosis fungoides;

[0017] The data output module is used to output result information based on the percentage of FOXM1-positive cells, wherein the result information is whether the mycosis fungoides subject is or is a candidate for mycosis fungoides large cell transformation.

[0018] In a fifth aspect, the present invention provides a device for assisting in identifying or distinguishing whether MF has undergone macrocellular transformation, comprising a data receiving module and a data output module;

[0019] The data receiving module is used to receive the percentage of FOXM1 positive cells in the skin lesion tissue of the subject with mycosis fungoides;

[0020] The data output module is used to output result information based on the percentage of FOXM1-positive cells, and the result information is whether the mycosis fungoides subject has undergone large cell transformation.

[0021] In a sixth aspect, the present invention provides a method for identifying or assisting in identifying large cell transformation of mycosis fungoides, the method comprising the following steps: receiving data on the percentage of FOXM1-positive cells in skin lesion tissue of a subject with mycosis fungoides; and then outputting result information from a computer based on the FOXM1-positive cell percentage data, the result information being whether the subject with mycosis fungoides is or is a candidate for large cell transformation of mycosis fungoides.

[0022] In a seventh aspect, the present invention provides a method for identifying or assisting in identifying whether large cell transformation has occurred in mycosis fungoides, the method comprising the following steps: receiving data on the percentage of FOXM1-positive cells in the skin lesion tissue of a subject with mycosis fungoides, and then outputting result information from a computer based on the FOXM1-positive cell percentage data, wherein the result information is whether large cell transformation has occurred in the subject with mycosis fungoides.

[0023] In the above, the FOXM1-positive cells are detected by immunohistochemical staining, wherein the detection reagent includes an antibody that specifically binds to the FOXM1 protein and a color developer. In an embodiment of the present invention, the color developer is DAB.

[0024] The above percentage of FOXM1-positive cells is obtained by immunohistochemical staining and the percentage of FOXM1-positive cells is as follows:

[0025] Sections of skin lesion tissue from the mycosis fungoides subjects were immunohistochemically stained (with DAB as the developer) and then scanned using a NanoZoomer microslide scanner (Hamamatsu Photonics, Japan). The sections were then observed using NDP.view2 (U12388-01) software. Three areas were randomly selected at a 20x field of view for image output, yielding three images. Each image was quantitatively calculated using Image J software using the following procedure: DAB (brown) was defined as positive, the positive threshold was set at 140, and the percentage of the area of FOXM1-positive cells to the total area of the image was calculated to obtain the area percentage of FOXM1-positive cells. Three images were calculated for each section, and the average of the area percentages of FOXM1-positive cells in the three images was taken as the FOXM1-positive cell area percentage, which was then used as the FOXM1-positive cell percentage.

[0026] The above-mentioned output of result information from the computer based on the FOXM1-positive cell percentage data is based on the comparison of the FOXM1-positive cell percentage in the skin lesion tissue of the mycosis fungoides subject with a threshold value. If the percentage is greater than the threshold value, the mycosis fungoides subject to be tested is or is a candidate for large cell transformation of mycosis fungoides, or the mycosis fungoides subject to be tested has or is a candidate for large cell transformation; if the percentage is less than or equal to the threshold value, the mycosis fungoides subject to be tested is not or is not a candidate for large cell transformation of mycosis fungoides, or the mycosis fungoides subject to be tested has not or is a candidate for not having large cell transformation; or the mycosis fungoides subject to be tested has not or is a candidate for not having large cell transformation; or, the likelihood of a MF patient whose FOXM1-positive cell percentage in the skin lesion tissue is greater than 1% for large cell transformation is greater than or is a greater candidate for large cell transformation than a MF patient whose FOXM1-positive cell percentage in the skin lesion tissue is less than or equal to 1%.

[0027] The threshold is obtained based on the percentage of FOXM1-positive cells obtained after immunohistochemical staining of a training set consisting of 29 cases of MF-LCT and 36 cases of MF-NLCT. In the embodiment of the present invention, the threshold is specifically 1%.

[0028] The use of the above-mentioned reagents for detecting FOXM1 protein or FOXM1-positive cells in the preparation of a kit or product for assisting in identifying or distinguishing large cell transformation of mycosis fungoides, or assisting in identifying or distinguishing whether large cell transformation occurs in mycosis fungoides, is also within the scope of protection of the present invention.

[0029] In an eighth aspect, the present invention provides any of the following systems, comprising the device according to the fourth or fifth aspect;

[0030] 1) A system for identifying or assisting in the identification of large cell transformation of mycosis fungoides;

[0031] 2) A system for identifying or assisting in identifying whether mycosis fungoides has undergone large cell transformation;

[0032] 3) A system for evaluating large cell transformation in mycosis fungoides.

[0033] In a ninth aspect, the present invention provides a substance that inhibits the expression, content or activity of FOXM1 protein, or a substance that inhibits the expression of FOXM1 mRNA, or a substance that inhibits the expression of the gene encoding FOXM1, and their use in the preparation of a product for treating mycosis fungoides.

[0034] The above-mentioned treatment of mycosis fungoides is embodied in any of the following: inhibiting the viability of mycosis fungoides cells; increasing the apoptosis level of mycosis fungoides cells; or inhibiting the colony formation of mycosis fungoides cells, or downregulating the S and G2 phases of mycosis fungoides tumor cells.

[0035] The substance is shRNA that interferes with and inhibits the expression of FOXM1 mRNA, or a vector or virus expressing the same, or an inhibitor targeting FOXM1. In the embodiment of the present invention, the substance is shRNA, and its nucleotide sequence is sequence 1 or 2.

[0036] Mycosis fungoides (MF) is the most common subtype of primary cutaneous T-cell lymphoma. The presence of large cell transformation (LCT) pathologically often indicates more rapid disease progression and a worse prognosis. This study, using RNA sequencing, revealed that FOXM1 (a cell cycle-related transcription factor) expression is specifically elevated in MF-LCT, and this was validated at both the RNA and protein levels by expanding the sample size. Immunohistochemical staining with a FOXM1 antibody demonstrated that a percentage of FOXM1-positive cells greater than 1% has a high diagnostic value for MF-LCT, and this was preliminarily validated by expanding the sample size. Compared to traditional pathological techniques, FOXM1 immunohistochemical staining allows for more precise localization and quantification of large cells, reducing subjective bias. Therefore, FOXM1 immunohistochemical staining is proposed as an adjunctive technique for the diagnosis of MF-LCT. Furthermore, the use of a FOXM1-specific small molecule inhibitor reduced tumor cell survival and increased apoptosis in vitro. In vitro studies have confirmed the importance of FOXM1 in cell survival, apoptosis resistance, and tumorigenesis. Therefore, FOXM1 may serve as one of the future therapeutic targets for MF. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A computer flow chart of a method for identifying or assisting in the identification of large cell transformation of mycosis fungoides.

[0038] Figure 2Figure 3 Volcano plot of gene expression differences in MF patients with large cell / non-large cell mutation (a) and RNA expression level differences of FOXM1 in tissues of MF patients with large cell / non-large cell mutation (b) (NLCT: non-large cell mutation; LCT: large cell mutation, FPKM: RNA expression level; |log2(fold change)|≥0.58, adjusted p value<0.05).

[0039] Figure 3 Comparison of RNA expression levels of MF-LCT and MF-NLCT (****P<0.0001).

[0040] Figure 4 Comparative immunofluorescence images of FOXM1 and TOX in MF and BID (MF: mycosis fungoides; BID: benign inflammatory skin disease).

[0041] Figure 5 Comparison of FOXM1 immunohistochemical staining and Image J processing of MF-LCT and MF-NLCT (FOXM1 immunohistochemical staining uses the DAB system, brown indicates positive; in Image J-processed images, the red area indicates the counted area).

[0042] Figure 6 ROC curves of the MF training set (a) and validation set (b) (AUC: area under the curve).

[0043] Figure 7 Western blotting was used to detect FOXM1 protein levels (PB2B and Myla are MF cell lines, and PBMC1 and PBMC2 correspond to PBMCs from two healthy volunteers, respectively).

[0044] Figure 8 Protein expression levels after lentivirus-mediated shRNA knockdown of FOXM1 in Myla (a) and PB2B (b) cell lines.

[0045] Figure 9 The relative cell viability of the FOXM1 knockdown group and the control group in Myla (a) and the relative cell viability of the FOXM1 knockdown group and the control group in PB2B (b) (**P<0.005).

[0046] Figure 10 The apoptosis levels of cells in the FOXM1 knockdown group and the control group in Myla (a) and the apoptosis levels of cells in the FOXM1 knockdown group and the control group in PB2B (b) (**P<0.005).

[0047] Figure 11Comparison of the number of colonies formed by FOXM1 knockdown group cells and control group cells in semi-solid culture medium (a. Scale bar = 10000 μm) and the appearance of colonies under the microscope with or without FOXM1 knockdown (b) (*P < 0.05).

[0048] Figure 12 Relative cell viability of PB2B tumor cells treated with different concentrations of FDI-6 (a) (5 μM, 10 μM, 20 μM), RCM1 (b) (10 μM, 50 μM, 100 μM) and DMSO, as well as untreated tumor cells (*P < 0.05).

[0049] Figure 13 The apoptosis levels in Myla cell line after treatment with 10 μM FDI-6 and 50 μM RCM1 for 72 hours (a) and the apoptosis levels in PB2B cell line after treatment with 10 μM FDI-6 and 50 μM RCM1 for 72 hours (b) (****P<0.0001).

[0050] Figure 14 The percentage change of cell cycle (a) and the expression level of FOXM1 protein (b) in Myla cell line after treatment with 50 μM FDI-6 and 100 μM RCM1 for 72 hours (***P<0.001).

[0051] Figure 15 It is a lentiviral vector GV493. DETAILED DESCRIPTION

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0053] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0054] Some of the materials in the following examples are as follows:

[0055] 1. Source of clinical specimens

[0056] This experimental study fully adhered to the principles of the World Medical Association Declaration of Helsinki and was approved by the Clinical Ethics Committee of Peking University First Hospital (Ethics Review Number: 2021 Scientific Research 151). All patients provided signed informed consent. The clinical diagnosis, pathological diagnosis, and disease staging of mycosis fungoides were based on the revised criteria recommended by the International Society for Cutaneous Lymphoma (ISCL) and the European Organization for Research and Treatment of Cancer (EORTC) in 2007. The confirmed diagnosis of mycosis fungoides specimens from all patients included in the case group was confirmed by two experienced dermatopathologists (the gold standard).

[0057] A total of 65 patients with mycosis fungoides were included in the training set. All tumor specimens from these patients were used for protein validation experiments (MF-LCT: 29; MF-NLCT: 36) and also served as the training set. Tumor specimens from 15 MF-LCT and 31 MF-NLCT were used for RNA validation. The validation set included 31 patients with mycosis fungoides (MF-LCT: 14; MF-NLCT: 17), recruited from the Research Center of Peking University First Hospital (18), the Department of Dermatology of Sun Yat-sen Memorial Hospital, Guangzhou (5), and the Department of Dermatology of Qilu Hospital, Shandong University (8). Skin tissues from 20 patients with inflammatory skin diseases (BIDs) (including 8 cases of lichen planus, 8 cases of eczema, and 4 cases of connective tissue disease) were also included as control validation samples. Peripheral blood mononuclear cells (PBMCs) from two healthy subjects were also included as protein controls for western blotting.

[0058] Table 1 shows the inclusion criteria

[0059]

[0060] 2. Origin of CTCL cell lines

[0061] The human cutaneous T-cell lymphoma MF cell lines used in this experiment, including Myla and PB2B, are described in the following references: BCL11B-Mediated Epigenetic Repression Is a Crucial Target for Histone Deacetylase Inhibitors in Cutaneous T-Cell Lymphoma; Wenjing Fu et al., Journal of Investigative Dermatology, 2017, 10.1016 / j.jid.2017.02.980. Myla and PB2B cell lines were cultured in 90% RPMI-1640 basal medium supplemented with 10% fetal bovine serum and 1% penicillin (100 U / ml)-streptomycin (0.1 mg / ml). All CTCL cell lines were cultured at 37°C, 100% relative humidity, and 5% CO2. All cells were grown in suspension in the culture medium and passaged according to growth. Cells used in experiments were in the logarithmic growth phase.

[0062] 3. Test instruments and consumables

[0063] 3.1 Instruments used in this experiment

[0064] Table 2 shows the instruments used in this experiment.

[0065]

[0066]

[0067] 3.2 Consumables used in this experiment

[0068] Table 3 shows the consumables used in this experiment.

[0069]

[0070] 3.3 Reagents used in this experiment

[0071] Table 4 shows the reagents used in this experiment

[0072]

[0073]

[0074] 3.4 Kits used in this experiment

[0075] Table 5 shows the kits used in this experiment

[0076]

[0077]

[0078] 3.5 Antibodies

[0079] Table 6 shows antibodies

[0080]

[0081] 3.6 Preparation and storage of main reagents

[0082] Table 7 shows the preparation of the main reagents

[0083]

[0084]

[0085] 4. Experimental methods in the embodiments:

[0086] 4.1 Immunohistochemistry (IHC)

[0087] 1) After preparing the cationic anti-shedding sheet, use a microtome to slice the skin lesion tissue into paraffin tissue slices and apply the patch.

[0088] 2) Place all sections in a 65°C incubator for 1-2 hours. First, place them in xylene I for 15 minutes. After draining them, place them in xylene II for 15 minutes.

[0089] 3) Treat the sections with anhydrous ethanol, 95% ethanol, and 75% ethanol for 5 minutes each, followed by a 5-minute wash with deionized water. This should be done three times in total.

[0090] 4) Add 2L of sodium citrate / EDTA solution (antigen retrieval solution) to a pressure cooker and heat on an induction cooker. Once the solution is completely boiling, place the sections in the cooker and retrieval using high temperature and high pressure for 120 seconds.

[0091] 5) After cooling to room temperature, wash three times with the prepared PBST solution, each time for 5 minutes.

[0092] 6) Incubate with 3% H2O2 in the dark for 10-15 minutes, then wash three times with the prepared PBST solution, each time for 5 minutes.

[0093] 7) Add an appropriate amount of primary antibody (anti-FOXM1; diluted in antibody diluent or 1× PBS solution) to the tissue and incubate overnight in a 4°C refrigerator.

[0094] 8) After overnight incubation, remove the specimen and wash it three times with the prepared PBST solution, each time for 5 minutes.

[0095] 9) Add the corresponding secondary antibody ( Goat Anti-Rabbit IgG (H+L), diluted with antibody diluent or 1× PBS solution), let stand at room temperature for 30 minutes, and then wash with PBS solution three times, each time for 5 minutes.

[0096] 10) Use standard concentration of DAB to develop color at room temperature. During the development process, observe the target tissue under a microscope to see if brown color appears. If so, stop the development immediately.

[0097] 11) Immerse the entire specimen in 2% hydrochloric acid alcohol for 5 seconds, then immerse it in ammonia water for 30 seconds to turn blue, and then wash it with deionized water for 3 minutes. A total of 3 washes should be performed.

[0098] 12) Treat the sections with 75% ethanol, 95% ethanol, and anhydrous ethanol for 1 min each, in that order.

[0099] 13) Add an appropriate amount of neutral resin to the tissue surface, seal the slide, and observe under a microscope.

[0100] 4.2 Tissue immunofluorescence staining (IF)

[0101] 1) After preparing the cationic anti-shedding film, use a microtome to slice and mount the paraffin tissue.

[0102] 2) Place all sections in a 65°C incubator for 1-2 hours. First, place them in xylene I for 15 minutes. After draining them, place them in xylene II for 15 minutes.

[0103] 3) Treat the sections with anhydrous ethanol, 95% ethanol, and 75% ethanol for 5 minutes each, followed by a 5-minute wash with deionized water. This should be done three times in total.

[0104] 4) Add 2L of sodium citrate / EDTA solution (antigen retrieval solution) to a pressure cooker and heat on an induction cooker. Once the solution is completely boiling, place the sections in the cooker and retrieval using high temperature and high pressure for 120 seconds.

[0105] 5) Cover the tissue completely with blocking goat serum and incubate at room temperature for 30 minutes.

[0106] 6) After drying the goat serum, add an appropriate amount of primary antibody (anti-FOXM1; diluted in antibody diluent or 1× PBS solution) to the tissue and incubate overnight in a 4°C refrigerator.

[0107] 7) After overnight incubation, remove the specimen and wash it three times with the prepared PBST solution, each time for 5 minutes.

[0108] 8) Incubate with the corresponding secondary antibody diluted in PBS at room temperature for 2 hours in the dark, then wash with PBST solution in the dark for 3 times, 5 minutes each time. Add the appropriate amount of primary antibody of the corresponding concentration ( Goat Anti-Rabbit IgG (H+L), diluted with antibody diluent or 1× PBS solution) and incubated overnight in a 4°C refrigerator.

[0109] 9) Add an appropriate amount of DAPI-containing sealing glue to the tissue surface and place the slide in the dark for 10 minutes.

[0110] 10) Using a Leica laser confocal microscope, select the corresponding laser segment to observe the staining of the corresponding indicators in the target cells.

[0111] 4.3 Extraction of peripheral blood mononuclear cells (PBMCs)

[0112] 1) Take a blood sample with a volume of 1× PBS solution equal to the blood sample volume, transfer the mixed liquid to a new centrifuge tube, and finally fill the liquid to a total volume of 8 mL and mix thoroughly.

[0113] 2) Use a disposable syringe to draw out the Ficoll solution and add 3 mL of Ficoll solution to each centrifuge tube.

[0114] 3) Pipette 4 mL of the mixed solution and slowly add it to the centrifuge tube containing only 3 mL of Ficoll solution. During the addition, keep the Ficoll tube tilted at 45 degrees with your left hand and slowly push the blood into the centrifuge tube with a syringe to ensure the liquid is in a stratified state.

[0115] 4) Centrifuge at 400×g, 18-20°C for 40 min.

[0116] 5) The PBMC layer should be flocculent. Pipette 2-3 mL of the PBMC layer into a new centrifuge tube. Then, add 3 times the volume of cold 1× PBS solution as the PBMC layer to each centrifuge tube to resuspend the cells.

[0117] 6) Centrifuge at 80 × g for 10 min. Discard the supernatant and retain the lower layer.

[0118] 7) Add an appropriate volume of CELLBANKER to the system to resuspend the cells according to the cell quantity, and transfer the cell suspension into a cell freezing box.

[0119] 4.4 RNA extraction and cDNA reverse transcription

[0120] In this study, the EZNA total RNA kit II was used to extract RNA from skin lesions of patients with mycosis fungoides, and the RNeasy Mini Kit was used to extract total RNA from PBMCs and CTCL cell lines. The entire experiment was performed strictly according to the kit instructions.

[0121] cDNA reverse transcription strictly follows PrimeScript TM The RT Master Mix kit was used to perform the operation to ensure the accuracy and consistency of the results.

[0122] 4.5 Quantitative real-time reverse transcriptase-PCR

[0123] A) Steps

[0124] 1) Dilute the primer to 10 μM with sterile water and mix equal volumes of upstream primer and downstream primer.

[0125] 2) Prepare pre-mix:

[0126] Table 8 is the pre-MIX

[0127]

[0128] 3) Add 15 μL of the corresponding gene premix to each well of the 8-well reaction tube strip or 96-well plate used, and then add 5 μL of the corresponding sample cDNA to each well again, making sure to mix thoroughly.

[0129] 4) After closing the lid of the 8-well strip or attaching the membrane of the 96-well plate, centrifuge at 1000 rpm for 2 minutes.

[0130] 5) Data acquisition and processing:

[0131] Raw data were acquired using Vii™ 7 software (available with the Applied Biosystems Prism system). Ct values for the target gene and the internal reference gene GAPDH were obtained. Corresponding ΔCt values were calculated using Excel. The relative expression value of the target gene in each sample was calculated using the formula 10000 / Power(2,ΔCt). Two replicate wells were set for each sample, and the final result was the mean.

[0132] Primer sequences

[0133] The reaction primer sequences are as follows:

[0134] Table 9 shows the reaction primer sequences

[0135]

[0136] 4.6 Western Blot

[0137] Protein was extracted according to the instructions of the Keygen protein extraction kit (KGP250), and the protein concentration was determined by BCA method (Pierce TM BCA Protein Assay Kit).

[0138] The extracted proteins were detected by Western blotting:

[0139] 1) Calculate the sample loading volume based on the lowest protein concentration and prepare a mixture of 6× loading buffer and 1× loading buffer to a total volume of 30-40 μL. Next, place the small EP tube containing this mixture in a 100°C metal bath for 8 minutes and then return it to ice.

[0140] 2) Take 6-8 μL of protein marker and mix it with 1× buffer to make the same volume of marker as in step 1), and prepare electrophoresis solution at the same time.

[0141] 3) Gently pull out the comb that comes with the precast gel and insert it into the corresponding electrophoresis stand. Add electrophoresis solution to the electrophoresis tank and load the sample. Add the configured marker to the second well on the left and right sides.

[0142] 4) Run electrophoresis at 120 V for 60 min.

[0143] 5) Wet transfer: After removing the gel, cut the gel according to the band position and transfer it to 1× electrotransfer buffer for later use. Prepare a methanol-activated PVDF membrane and cut it to the appropriate size. In 1× electrotransfer buffer, arrange the transfer splint (black) - thick sponge gasket - filter paper - sample gel - PVDF membrane - filter paper - sponge gasket - transfer splint (red) in this order. Use a glass rod to remove all bubbles during this process. Once completed, clamp the splint and place the entire splint in an electrotransfer tank filled with 1× electrotransfer buffer for transfer at 400mA for 30 minutes.

[0144] 6) After electroporation, cut the PVDF membrane according to the location of the target protein.

[0145] 7) Wash the cut PVDF membrane with the prepared TBST solution using a shaker at a speed of 90-110 rpm for 5 minutes each time, for a total of 4 washes.

[0146] 8) Prepare the desired concentration of skim milk powder and add 5 mL to each well, ensuring the membrane is completely soaked. Shake at 60-70 rpm for 1 hour.

[0147] 9) Prepare the desired concentration of primary antibody using the prepared skim milk powder solution, add 5 mL to each well, and incubate overnight at 4°C for 12-18 hours.

[0148] 10) The next day, wash the PVDF membrane after the primary antibody reaction with the prepared TBST solution on a shaker at 90-110 rpm for 5 minutes each time, for a total of 5 washes.

[0149] 11) Prepare the desired concentration of secondary antibody using the prepared skim milk powder solution, add 5 mL to each well, incubate at room temperature for 1 hour on a shaker at 60-70 rpm, and then wash with TBST solution for 5 minutes x 3 times.

[0150] 12) Prepare chemiluminescent solution according to the EasySee Western Blot Kit (TransGen Biotech) instructions: Mix equal volumes of Solution A and Solution B, and use a volume of Solution C equal to (A + B) / 1000. Evenly coat the surface of the PVDF membrane with the prepared solution. Incubate at room temperature in the dark for half a minute, and analyze the gel using the SYNGENE Gel Image Analysis System.

[0151] 4.7 Lentivirus Construction and Transfection Experiment

[0152] 1) Construction of lentivirus to knock down FOXM1 gene

[0153] GeneCare Gene Company synthesized FOXM1-sh1, a lentivirus that knocks down the FOXM1 gene

[0154] (LV-FOXM1-RNAi(88729-1)) and lentivirus FOXM1-sh2 (LV-FOXM1-RNAi(88730-1)) as well as control lentivirus sh0.

[0155] Lentivirus LV-FOXM1-RNAi (88729-1) expresses the RNA interference sequence sh1 at a titer of 1E+9Tu / mL; used to knock down the FOXM1 gene;

[0156] Lentivirus LV-FOXM1-RNAi (88730-1) expresses the RNA interference sequence sh2 with a titer of 6E+8Tu / mL; used to knock down the FOXM1 gene;

[0157] The control lentivirus sh0 (con313) is a control lentivirus with a control insert sequence of TTCTCCGAACGTGTCACGT; the titer is 1.0×10 9 Tu / mL.

[0158] 2) RNAi target design

[0159] GeneCare designed and provided the RNA interference sequences targeting the target gene FOXM1 used in this experiment. The target sequences of the RNA interference sequence sh1, RNA interference sequence sh2, and synthetic oligo sequences are detailed in Table 10 below:

[0160] Table 10 shows the sequences of sh1, sh2 and synthetic oligos

[0161]

[0162]

[0163] 3) Lentiviral vector map

[0164] The above FOXM1-RNAi(88729-1)-a and FOXM1-RNAi(88729-1)-b were annealed to obtain FOXM1-RNAi(88729-1), which expressed the RNA interference sequence sh1;

[0165] The above FOXM1-RNAi(88730-1)-a and FOXM1-RNAi(88730-1)-b were annealed to obtain FOXM1-RNAi(88730-1), which was recorded as RNA interference sequence sh2;

[0166] The lentivirus used to knock down the FOXM1 gene in this study was GV493

[0167] (http: / / www.genechem.com.cn / service / index.php?ac=gene&at=vector_search&ke yword=GV493, Figure 15 ) as a lentiviral cloning vector. The recombinant vector, obtained by inserting the aforementioned RNA interference sequence sh1 or sh2 between the AgeI and EcoRI sites of GV493 cells, and the viral packaging helper plasmids (Helper1.0 and Helper1.2 used in RNAi lentiviral vector construction), were cloned and packaged in 293 cells to produce the resulting lentivirus. The GV493 lentiviral cloning vector contains the following sequence: hU6-MCS-CBh-gcGFP-IRES-puromycin, with cloning sites at AgeI and EcoRI. cGFP is a fluorescent marker used to indicate positively transduced cells, facilitating screening.

[0168] 4) Lentiviral transfection to knock down FOXM1 gene

[0169] a. Count the suspended cells to be transfected in the logarithmic growth phase and adjust the cell density with complete RPMI 1640 medium so that each 1000 μL of cell solution contains 5×10 5 viable cells (the number of cells transfected each time was 2×10 5 indivual).

[0170] b. Add 20 μL of HitransG A virus infection reagent (Jikai Company, Catalog No. REVG004) to a 24-well plate, virus solution (according to the MOI value of 100), 400 μL containing 2×10 5 The culture medium of 10 cells was added and the total volume was made up to 500 μL with complete RPMI1640 medium.

[0171] c. After culturing for 24 hours in a normal culture environment, centrifuge and completely discard the supernatant, and replace the culture conditions with complete RPMI 1640 medium.

[0172] d. Flow cytometry was used to measure the proportion of fluorescence-positive cells 72 hours after transfection and calculate the transfection efficiency.

[0173] 4.8 Cell viability assay

[0174] 1) Count the suspended cells in the logarithmic growth phase and adjust the cell density to 6×10 4 / 3mL or 1×10 5 Then, 3 mL of the corresponding cell solution was added to each well of the 6-well plate. When conducting drug experiments, a control drug (DMSO) or target drug was added separately.

[0175] 2) Mix cells thoroughly before each sampling. Perform cell viability assays at 0, 24, 48, and 72 hours after plating / drug addition. Pipette 100 μL of thoroughly mixed cell suspension into a 96-well plate. Add 20 μL of MTS solution to each well and mix thoroughly. Incubate in a 37°C cell culture incubator for 2 hours. Perform two replicate wells for each cell line.

[0176] 3) After 2 hours, the 96-well plate was removed and placed in a microplate reader to measure the absorption peak OD of each well at 490 nm.

[0177] 4) The relevant data obtained by the microplate reader were standardized using EXCEL, and the relative cell viability value was calculated using the formula relative cell viability = original OD / OD0, OD 0h Refers to the OD value of the cells in this group measured at 0 hours.

[0178] 4.9 Flow cytometry apoptosis detection

[0179] 1) Count the suspended cells in the logarithmic growth phase and adjust the cell density to 1×10 6 / 4mL or 1×10 5 100 μg / 3 mL, cultured in culture flasks or 6-well plates. Detection is generally performed 72 hours after lentiviral transfection or on the 2nd to 3rd day of cell culture.

[0180] 2) At the scheduled time, add about 0.5 mL of the cell solution of the sample to be tested to the flow tube, and add 2 mL of pre-cooled PBS for washing. After centrifugation, discard the supernatant. Repeat the above steps once, add 50 μL of 1× Binding Buffer to each tube and resuspend (cell concentration is about 1×10 5 / mL).

[0181] 3) Add 2.5 μL of APC Annexin V to the sample tube to be tested and the APC Annexin V single-positive tube respectively, mix the cells and APC Annexin V dye, and incubate at room temperature in the dark for 15 minutes.

[0182] 4) After incubation, add 200 μL of 1× Binding Buffer to make up to 250 μL.

[0183] 5) Flow cytometry apoptosis detection was performed, and the obtained data were analyzed using FlowJo 10.4 software to calculate the apoptosis rate.

[0184] 4.10 Cell colony formation assay

[0185] 1) Prepare one sterile 100 mm Petri dish and three sterile 35 mm Petri dishes.

[0186] 2) Place two 35mm dishes with lids and one 35mm dish without lid in a 100mm Petri dish with 2-3 ml of sterile water.

[0187] 3) Take an appropriate amount of cell sample, centrifuge and resuspend in IMDM medium containing 2% FBS, count and adjust the cell density to 3×10 3 Mix the diluted cell suspension thoroughly and add 0.2 mL of the cell suspension to 3 mL of aliquoted complete methylcellulose medium. Thoroughly mix the medium and cells. Vortex vigorously for ≥ 4 seconds and let stand for at least 5 minutes to allow any bubbles in the medium to dissipate to the top.

[0188] 4) Place three 35 mm culture dishes into a 100 mm culture dish. Use a 5 mL syringe with a 16-gauge blunt needle to extract the mixed cell solution. Inject 1.2 mL of the semi-solid culture medium containing the cells into each of the two 35 mm culture dishes. Slowly shake the dishes to evenly distribute the semi-solid culture medium throughout the dish.

[0189] 5) Add 2-3 ml of sterile water for injection to the third 35 mm dish in the 100 mm dish, remove the dish cover, and culture in a cell culture incubator for 10-14 days. Observe cell colonies under a microscope and count them according to colony size.

[0190] 6) Take photos using a Cytation 5 cell imaging microplate reader.

[0191] 4.11 Cell cycle detection

[0192] 1) Count the suspended cells in the logarithmic growth phase and take about 2 mL (at least 10 6 After adding the cell solution of the sample to be tested (cells), the tube was washed once with 2 mL of pre-cooled PBS.

[0193] 2) After centrifugation, the supernatant was completely discarded and 500 μL of 70% cold ethanol was added to each flow cytometry tube for fixation (2 h to overnight).

[0194] 3) Prepare staining solution at a ratio of RNase A:PI = 1:9. Wash with 2 mL of pre-chilled PBS. Add 300 μL of staining solution to each flow cytometry tube, filter with a 40 μm filter, and incubate at room temperature in the dark for 30-60 minutes.

[0195] 4) Cell cycle detection was performed on the computer, and the data were analyzed using ModFit LT for Macintel.

[0196] 5. Drawing and statistical methods in the embodiments:

[0197] 5.1 The immunohistochemical scoring process is as follows:

[0198] Immunohistochemically stained sections were scanned using a NanoZoomer microslide scanner (Hamamatsu Photonics, Japan) and observed using NDP.view2 (U12388-01) software. Three areas were randomly selected at a 20x field of view for image output, resulting in three images. Each image was then quantitatively calculated using Image J software using the following procedure: DAB (brown) was defined as positive, the positive threshold was set at 140, and the percentage of the area of FOXM1-positive cells in the total area of the image was calculated to obtain the area percentage of FOXM1-positive cells. Three images were taken for each section and the area percentage of FOXM1-positive cells in the three images was averaged and recorded as the area percentage of FOXM1-positive cells, which was then used as the percentage of FOXM1-positive cells.

[0199] 5.2 Statistical methods

[0200] All raw data from tests and observations must be recorded. GraphPad Prism 8.0 (GraphPad Software, La Jolla, California, USA) and SPSS Statistics 26.0 (SPSS Inc., Chicago, IL, USA) were used for data processing, statistical analysis, and graphing. Normally distributed data were expressed as mean ± standard deviation (mean ± SD), while non-normally distributed data were expressed as median and quartiles (25th percentile, median, 75th percentile). Differences in means between groups were compared using the Student's t-test or the nonparametric Mann-Whitney U-test. Survival analysis was performed using the Kaplan-Meier test. A p < 0.05 was considered statistically significant.

[0201] Example 1: Discovery of FOXM1 Identification of MF-LCT

[0202] 1. Transcriptome sequencing revealed that FOXM1 is elevated in MF-LCT

[0203] To explore the abnormally expressed molecules and / or signal transduction pathways in MF-LCT, 49 MF patients were divided into two groups according to the presence or absence of large cell transformation: 26 MF-LCT and 23 MF-NLCT.

[0204] RNA was extracted from tumor tissues of 26 cases of MF-LCT (denoted as LCT in the figure) and 23 cases of MF-NLCT (denoted as NLCT in the figure), and then traditional transcriptome sequencing was performed.

[0205] The results are as follows Figure 2 As shown in the volcano plot analysis of transcriptome sequencing, the RNA expression level of FOXM1 gene was significantly increased in MF-LCT compared with MF-NLCT ( Figure 2 a). The average FOXM1 RNA expression level (FPKM) in MF-LCT was 18.30 and in MF-NLCT was 8.384, with significant difference between the two groups (P<0.0001) ( Figure 2 b).

[0206] 2. Verification of FOXM1 expression levels in MF-LCT using clinical specimens

[0207] 1. Real-time fluorescence quantitative PCR detection

[0208] To further expand the sample size, we extracted RNA from lesional skin of 15 cases of MF-LCT and 31 cases of MF-NLCT, and then reverse transcribed it to obtain cDNA. The aforementioned real-time fluorescence quantitative PCR was used to detect the RNA expression of FOXM1 in each sample.

[0209] The results are as follows Figure 3 As shown in the results, the average RNA expression level of FOXM1 in the MF-LCT group was 57.55, while that in the MF-NLCT group was 31.15, indicating significant differences between the two groups.

[0210] 2. Immunohistochemical staining

[0211] To further clarify the expression localization of FOXM1, double immunofluorescence staining of FOXM1 and TOX was performed in lesional tissues of MF patients, using benign inflammatory skin disease (BID) lesions as controls. TOX is known to be a nuclear protein that is highly expressed specifically in MF tumor cells.

[0212] The results are as follows Figure 4 As shown, in MF tissue, FOXM1 is primarily expressed in TOX-positive tumor cells. However, in BID, FOXM1 is essentially absent (TOX is also barely expressed). Furthermore, FOXM1 is primarily expressed in enlarged tumor cells.

[0213] At the clinical specimen level, the above experiments, by expanding the sample size, reconfirmed at the RNA level that FOXM1 expression in MF-LCT was higher than that in MF-NLCT; the protein level also confirmed that FOXM1 was mainly expressed in MF tumor cells, and mainly in larger tumor cells.

[0214] The above results indicate that FOXM1 (Genbank number: NM_202002.3, 2019.06.02) may serve as an auxiliary identification of MF-LCT.

[0215] Example 2: Application of FOXM1 in Assisted Identification of MF-LCT

[0216] Figure 1 A computer flow chart of a method for identifying or assisting in the identification of large cell transformation of mycosis fungoides.

[0217] 1. FOXM1 as a marker for assisting in the identification of MF-LCT

[0218] Immunohistochemistry is a commonly used pathological technique in clinical practice. The present invention uses immunohistochemistry to detect FOXM1 protein expression and quantify FOXM1 protein expression to find the optimal cutoff value for assisting in the identification of MF-LCT.

[0219] 1. Immunohistochemical staining

[0220] Immunohistochemical staining was performed on 29 MF-LCT and 36 MF-NLCT lesion tissues from the training group. The sections were scanned using a NanoZoomer microslide scanner (Hamamatsu Photonics, Japan) and observed using NDP.view2 (U12388-01) software. Three areas were randomly selected at a 20x field of view for image output, resulting in three images. Quantification of each image was performed using Image J software as follows: DAB (brown) was defined as positive, and the positive threshold was set at 140. The percentage of FOXM1-positive cell area in the total image area was calculated to obtain the percentage of FOXM1-positive cells. Three images were collected for each section, and the average of the three images was recorded as the percentage of FOXM1-positive cells.

[0221] like Figure 5 As shown, the left side is the original image of FOXM1 immunohistochemical staining of MF-LCT and MF-NLCT (brown is positive), and the right side is the corresponding image processed by Image J software. The red area is the positive cell area identified by the software.

[0222] The results of FOXM1 immunohistochemical staining quantitatively calculated using Image J software are shown in Table 11.

[0223] Table 11 shows the percentage of FOXM1 positive cells in the MF training set

[0224]

[0225] 2. Draw the ROC curve

[0226] The percentage of FOXM1-positive cells in the above 29 patients with MF-LCT and 36 patients with MF-NLCT was plotted as a ROC curve.

[0227] The results are as follows Figure 6 As shown in (a), it can be seen that with MF-NLCT as the control group, the area under the ROC curve for identifying MF-LCT was 0.96 (P < 0.001).

[0228] Furthermore, the Youden Index determined that the optimal cutoff for distinguishing MF-LCT from MF-NLCT was 1.056%, with a sensitivity of 86.21% and a specificity of 97.22% for identifying MF-LCT. However, considering the convenience of clinical application, a cutoff of 1% was set, meaning that when the percentage of FOXM1-positive cells was greater than 1%, the diagnosis of MF-LCT was more likely.

[0229] Therefore, FOXM1 protein can be used as a marker to assist in the identification of MF-LCT. The specific applications are as follows:

[0230] Immunohistochemistry was used to detect the expression of FOXM1 in the skin lesions of MF patients, and the percentage of FOXM1-positive cells in each patient was used to assist in identifying whether the patient had MF-LCT.

[0231] If the percentage of FOXM1-positive cells in the skin lesions of the MF patient to be tested is greater than 1%, the MF patient to be tested is a candidate for MF-LCT;

[0232] If the percentage of FOXM1-positive cells in the skin lesions of the MF patient to be tested is less than or equal to 1%, the MF patient to be tested is not a candidate for MF-LCT;

[0233] Alternatively, the likelihood of LCT in MF patients whose percentage of FOXM1-positive cells in lesional tissue is greater than 1% is greater than that in MF patients whose percentage of FOXM1-positive cells in lesional tissue is less than or equal to 1%.

[0234] 2. Identification and Validation of FOXM1 in MF-LCT

[0235] To verify the application value of FOXM1-positive cell percentage greater than 1% in clinical identification of MF-LCT, 31 MF clinical skin lesion specimens (including MF-LCT: 14 cases and MF-NLCT: 17 cases) were recruited from three medical centers as a validation set.

[0236] 1. Immunohistochemical staining

[0237] Immunohistochemical staining was performed on the skin lesions of 14 patients with MF-LCT and 17 patients with MF-NLCT in the validation group. The percentage of FOXM1-positive cell area was calculated using Image J software to obtain the percentage of FOXM1-positive cells.

[0238] The results are shown in Table 12:

[0239] Table 12 shows the percentage of FOXM1 positive cells in the MF validation set

[0240]

[0241] There is no significant difference between the validation set and the training set in basic demographic characteristics such as age and sex ratio (see Table 1 for a specific comparison.

[0242] 2. Evaluation of identification models

[0243] An overview of the FOXM1 percentage in the validation group and the current gold standard for identification is shown in Table 13 In the validation group, it was confirmed that the percentage of FOXM1-positive cells >1% in the MF training group had a sensitivity of 92.9%, a specificity of 76.5%, a positive predictive value of 92.9%, a negative predictive value of 92.9%, and a Youden index of 0.694 in identifying MF-LCT.

[0244] The above results indicate that a percentage of FOXM1-positive cells greater than 1% obtained by immunohistochemical staining has a high identification value in the identification of MF-LCT and is worthy of further clinical promotion.

[0245] Table 13 is an overview of the percentage of FOXM1-positive cells and the gold standard judgment in the validation group

[0246]

[0247] The percentage of FOXM1-positive cells in the above 14 MF-LCT and 17 MF-NLCT patients was evaluated by fitting the ROC curve with the gold standard.

[0248] The results are as follows Figure 6 As shown in b, the area under the ROC curve was 0.98, P < 0.001, indicating that the percentage of FOXM1-positive cells > 1% has a high accuracy in assisting the identification of MF-LCT.

[0249] Example 3: Application of FOXM1 as a therapeutic target for MF

[0250] 1. Application of FOXM1 knockdown in the treatment of MF

[0251] To explore the effect of FOXM1 on the biological phenotype of MF tumor cells, the MF cell lines Myla and PB2B were used to observe the effect of FOXM1 knockdown on the phenotype of CTCL cell lines by RNA level.

[0252] 1. Expression of FOXM1 in MF cell lines (Myla and PB2B)

[0253] Western blotting was used to detect the protein expression of FOXM1 in MF cell lines (Myla and PB2B). Proteins derived from PBMCs of healthy subjects (proteins extracted from PBMCs of healthy subjects) were used as controls.

[0254] The results are as follows Figure 7 As shown in the Figure 3, it can be seen that the expression of FOXM1 protein in MF tumor cell lines is significantly higher than that in PBMCs of healthy subjects.

[0255] 2. Application of Inhibiting FOXM1 Expression in Suppressing Tumor Cells

[0256] 1) shRNA knockdown of FOXM1

[0257] Myla and PB2B cell lines that highly express FOXM1 were selected as research objects. Their FOXM1 expression was inhibited by lentivirus-mediated shRNA, and the degree of knockdown at the protein level was verified (Myla and PB2B were control groups without virus transfection, sh0 was a control group transfected only with the control lentivirus sh0, and sh1 and sh2 corresponded to the control groups transfected with the lentivirus FOXM1-sh1 and lentivirus FOXM1-sh2, respectively).

[0258] The lentivirus FOXM1-sh1, FOXM1-sh2 and lentivirus expressing sh0, which were commissioned by GeneCare Gene to knock down the FOXM1 gene, were transfected into various Myla and PB2B cell lines according to the above method. The FOXM1 knockdown groups were specifically divided into the following groups:

[0259] Myla-sh1: transfect Myla with lentiviral FOXM1-sh1;

[0260] Myla-sh2: transfect Myla with lentiviral FOXM1-sh2;

[0261] PB2B-sh1: transfect PB2B with lentiviral FOXM1-sh1;

[0262] PB2B-sh2: transfect PB2B with lentiviral FOXM1-sh2;

[0263] The Myla-sh0 group, PB2B-sh0 group, Myla cells not transfected with the virus (denoted as Myla in the figure), and PB2B cell lines (denoted as PB2B-con or PB2B in the figure) were used as control groups.

[0264] Myla-sh0 group: Lentivirus sh0 was transfected into Myla;

[0265] PB2B-sh0 group: PB2B was transfected with lentivirus sh0.

[0266] On the 5th day after transfection, proteins of each transfected cell were extracted, and the expression level of FOXM1 was detected by Western blotting.

[0267] The results are as follows Figure 8 As shown, sh0 is PB2B-sh0 or Myla-sh0, and sh1 and sh2 of each cell correspond to Myla-sh1, Myla-sh2, PB2B-sh1 and PB2B-sh2, respectively; a is the protein expression level of FOXM1 after lentivirus-mediated shRNA knockdown in Myla cells; b is the protein expression level of FOXM1 after lentivirus-mediated shRNA knockdown in PB2B cells; it can be seen that compared with sh0, the FOXM1 protein expression levels of both sh1 and sh2 groups were significantly reduced, among which sh2 had a higher knockdown efficiency.

[0268] 2) Cell viability detection

[0269] The relative cell viability of the cells in each FOXM1 knockdown group and the control group in 1) above was detected at 0h, 24h, 48h and 72h after transfection.

[0270] The results are as follows Figure 9 As shown, it can be seen that compared with the control Myla and PB2B, the growth trend of cells decreased to varying degrees after knocking down FOXM1, and there was a significant difference in the relative cell viability between the FOXM1 knockdown group cells and the control group cells at 72 h.

[0271] 3) Detection of cell apoptosis levels

[0272] Annexin V APC and flow cytometry were used to detect the apoptosis levels of cells in each FOXM1 knockdown group and control group.

[0273] The results are as follows Figure 10 As shown, it can be seen that compared with the control Myla and PB2B, the proportion of apoptotic cells in the FOXM1 knockdown group was significantly increased. This part of the results reversely suggests that FOXM1 has the effect of reducing apoptosis of tumor cells.

[0274] 4) In vitro cell colony formation assay

[0275] The Myla FOXM1 knockdown groups and the control group were subjected to in vitro cell colony formation assay.

[0276] The results are as follows Figure 11 As shown, the right picture is a colony image, the first row of Myla and Myla-sh0 are the control group, and the second row of Myla-sh1 and Myla-sh2 are the knockdown group; the left picture is a colony count. Colony counting revealed that the number of large colonies formed after FOXM1 knockdown was significantly reduced compared to the control group, and the overall colony number was also significantly reduced, indicating that FOXM1 has a promoting effect on the colony formation of tumor cells themselves.

[0277] The above-mentioned in vitro cell experiments confirmed that after FOXM1 knockdown, the cell growth ability decreased, the number of apoptotic cells increased, and the colony formation decreased.

[0278] Therefore, inhibiting FOXM1 protein expression can inhibit the growth of mycosis fungoides tumor cells, promote apoptosis, and inhibit the formation of tumor cell colonies.

[0279] 2. Application of FOXM1 inhibitors in the treatment of MF

[0280] FDI-6 (Sigma-Aldrich, SML1392) and RCM1 (Tocris, 339163-65-4) were selected as specific small molecule inhibitors for the FOXM1 target.

[0281] Two small molecule inhibitors, FDI-6 and RCM1, were selected and added to the MF cell lines cultured in vitro to observe whether they could affect tumor cell growth by inhibiting FOXM1.

[0282] 1. Relative cell viability assay

[0283] 5 μM, 10 μM, 20 μM FDI-6 and 10 μM, 50 μM, 100 μM RCM1 were added to the PB2B cell line culture system (4×10 5 PB2B cell line (cells / mL) was seeded in a 3 mL volume in a six-well plate and cultured.

[0284] The relative cell viability of the treatment groups and the control group was measured using the MTS method at 0h, 24h, 48h and 72h after addition.

[0285] The results are as follows Figure 12 As shown, the viability of tumor cells PB2B decreased with increasing drug concentrations when treated with FDI-6 (a) and RCM-1 (b) in vitro.

[0286] 2. Apoptotic Cell Detection

[0287] 10 μM FDI-6 and 50 μM RCM1 were added to the Myla cell line culture system (1.2×10 6 Myla cells were seeded in a 3 mL volume in a six-well plate) or cultured in the PB2B cell line culture system (same as above) for 72 hours.

[0288] Apoptotic cells were stained with Annexin V-APC, and the apoptosis level was detected by flow cytometry.

[0289] The results are as follows Figure 13 As shown, it can be seen that the proportion of apoptotic cells increased in the FDI-6 (a) and RCM1 (b) treatment groups.

[0290] These results indicate that FOXM1-specific small molecule inhibitors FDI-6 and RCM1 can inhibit the survival of MF tumor cells by increasing cell apoptosis, suggesting that FOXM1 may be a potential therapeutic target for MF.

[0291] 3. Cell cycle detection

[0292] 50 μM FDI-6 and 100 μM RCM1 were added to the Myla cell line culture system (1.0×10 6 Myla cells were seeded in a 3 mL volume in a six-well plate and cultured for 72 hours.

[0293] The staining solution was prepared using RNaseA and PI, and the cell cycle was detected by flow cytometry, and then the cell cycle distribution was analyzed using ModFitLT for Macintel.

[0294] The results are as follows Figure 14 As shown in a, it can be seen that in the FDI-6 (a) and RCM1 (b) treatment groups, the proportion of cells in the G0 / G1 phase increased, and the proportion of cells in the S+G2 / S phase decreased.

[0295] The above results indicate that FOXM1-specific small molecule inhibitors FDI-6 and RCM1 can inhibit tumor cell growth by downregulating the S and G2 phases of tumor cells, suggesting that FOXM1 can be used as a potential therapeutic target for MF.

[0296] 4. Western blotting to detect FOXM1 expression after drug treatment of Myla

[0297] 50 μM FDI-6 and 100 μM RCM1 were added to the Myla cell line culture system (1.0×10 6Myla cells were seeded in a 3 mL volume in a six-well plate and cultured for 72 hours.

[0298] The expression of FOXM1 was detected by protein extraction, BCA method for protein concentration determination, Western blotting, anti-FOXM1 and anti-GAPDH antibody incubation, secondary antibody incubation, and color development.

[0299] The results are as follows Figure 14 b shows that after Myla was treated with FDI-6, RCM1, etc., the expression of FOXM1 was significantly reduced compared with the control group without drug addition and the control group with DMSO.

[0300] The above results indicate that FOXM1-specific small molecule inhibitors FDI-6 and RCM1 can both cause a decrease in FOXM1 protein levels.

Claims

1. A computer device comprising a memory, a processor, and a computer program stored in the memory, wherein: The processor executes the computer program to implement the following steps: S1) Data received: Percentage of FOXM1-positive cells in skin lesional tissues of subjects receiving mycosis fungoides; S2) Data output: Outputting result information from a computer based on the percentage of FOXM1-positive cells, wherein the result information indicates whether the subject with mycosis fungoides has or is a candidate for having large cell transformation of mycosis fungoides, or outputting whether the subject with mycosis fungoides has large cell transformation.

2. A computer-readable storage medium having a computer program stored thereon, characterized in that: The computer program implements the steps of claim 1 when executed by a processor.

3. A computer program product comprising a computer program, characterized in that: When the computer program is executed by a processor, the steps of claim 1 are implemented.

4. A device for assisting in the identification or differentiation of large cell transformation of mycosis fungoides, comprising a data receiving module and a data output module; The data receiving module is used to receive the percentage of FOXM1 positive cells in the skin lesion tissue of the subject with mycosis fungoides; The data output module is used to output result information based on the percentage of FOXM1-positive cells, wherein the result information is whether the mycosis fungoides subject is or is a candidate for mycosis fungoides large cell transformation.

5. A device for assisting in the identification or determination of whether a patient with mycosis fungoides has undergone large cell transformation, comprising a data receiving module and a data output module; The data receiving module is used to receive the percentage of FOXM1 positive cells in the skin lesion tissue of the subject with mycosis fungoides; The data output module is used to output result information based on the percentage of FOXM1-positive cells, and the result information is whether the mycosis fungoides subject has undergone large cell transformation.

6. A method for identifying or assisting in the identification of large cell transformation of mycosis fungoides, the method comprising the following steps: receiving data on the percentage of FOXM1-positive cells in skin lesions of a subject with mycosis fungoides; and outputting result information from a computer based on the FOXM1-positive cell percentage data, the result information indicating whether the subject with mycosis fungoides has or is a candidate for having large cell transformation of mycosis fungoides.

7. A method for identifying or assisting in identifying whether large cell transformation has occurred in mycosis fungoides, the method comprising the following steps: receiving data on the percentage of FOXM1-positive cells in skin lesions of a subject with mycosis fungoides, and outputting result information from a computer based on the FOXM1-positive cell percentage data, the result information indicating whether large cell transformation has occurred in the subject with mycosis fungoides.

8. Any of the following systems, comprising the apparatus according to claim 4 or 5; 1) A system for identifying or assisting in the identification of large cell transformation of mycosis fungoides; 2) A system for identifying or assisting in identifying whether mycosis fungoides has undergone large cell transformation; 3) A system for evaluating large cell transformation in mycosis fungoides.

9. Use of a substance that inhibits the expression, content, or activity of FOXM1 protein, or a substance that inhibits the expression of FOXM1 mRNA, or a substance that inhibits the expression of the gene encoding FOXM1, in the preparation of a product for treating mycosis fungoides.

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