Computer device and computer readable storage medium for identifying or assisting in identifying transformation of mushroom-like granulomegaly cells

Abstract

The invention discloses a computer device and a computer readable storage medium for identifying or assisting in identifying transformation of mushroom-like granulomegaly cells. The invention provides a computer device, which comprises a memory, a processor and a computer program stored on the memory, and the processor executes the following steps: S1) data receiving: receiving the percentage of FOXM1 positive cells in skin lesion tissues of a mushroom granuloma (MF) subject; and S2) data output: outputting result information from a computer based on the percentage of the FOXM1 positive cells, wherein the result information indicates whether the mushroom-like granuloma subject is or is candidate to be the transformation of the mushroom-like granuloma cells. Experiments prove that compared with a traditional pathological technology, immunohistochemical staining of FOXM1 can more accurately position large cells and quantify the proportion of the large cells, and subjective bias can be reduced. Therefore, the immunohistochemical staining of the FOXM1 can be used as one of auxiliary technologies for diagnosing the transformation of the mushroom-like granulomegaly cells.

Description

Technical Field

[0001] The 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 large cells. Background Art

[0002] Primary cutaneous T-cell lymphoma (CTCL) is a tumor disease that originates in the skin and is characterized by clonal proliferation of T lymphocytes and resistance to apoptosis. The incidence rate is about 6.4 / 1 million, and there is a trend of gradual increase. Mycosis fungoides (MF) is the most common subtype of CTCL, accounting for about 60% of CTCL. The disease is more common in men, and the risk of disease increases with age.

[0003] Clinically, the disease manifests itself as multiple reddish-brown patches and plaques in the early stage, and tumors or even erythroderma may appear in the later stage. In severe cases, lymph nodes may be involved and even the blood and visceral systems may be invaded. The overall disease progression of MF patients is slow, but some MF patients may progress rapidly. In these patients, the tumor cells transform from small cells to large cells in terms of tissue morphology, which are 4 times larger than normal small lymphocytes, and the proportion of large cells exceeds 25% or forms small nodules, which is called MF large cell transformation (LCT), and can be diagnosed as MF-LCT. Those who do not meet the above criteria are MF non-large cell transformation, recorded as MF-NLCT. The course of MF-LCT patients is often invasive, with widespread and rapidly growing nodules and masses throughout the body, and are more likely to invade lymph nodes, peripheral blood, bone marrow and internal organs, and the survival rate and median survival time of patients are significantly reduced. Although LCT can occur in various stages from early to late MF, the vast majority of LCT occurs in the advanced stage of MF (about 25% in IIB and about 50% in IV). According to statistics, the earlier the LCT occurs, the worse the prognosis of patients tends to be.

[0004] The causes of LCT in MF and its specific molecular mechanisms are still unclear, and the diagnosis of LCT often requires at least two senior dermatopathologists to jointly evaluate the percentage of large lymphocytes in at least four microscopic areas. Its morphological identification and counting statistics are difficult, and the overall process is time-consuming; therefore, how to accurately and quickly identify MF-LCT in pathology is a major problem that needs to be solved in clinical work. In addition, patients in the advanced stage or with large cell transformation (LCT) have an aggressive course of disease and are resistant to conventional treatment and chemotherapy. How to control the progression of MF has always been another major problem in the treatment of MF.

[0005] FOXM1, whose full name is Forkhead box protein M1, is a transcription factor that is essential for embryonic development. Previous studies have shown that FOXM1 is expressed in a variety of solid tumors, such as lung cancer, gastric cancer, glioma, breast cancer, etc. It accelerates tumor progression by promoting tumor cell growth, reducing apoptosis, and promoting epithelial-mesenchymal transition of malignant cells. Currently, no studies have proven that it is associated 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 lesions of subjects treated with 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 is 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 evaluate 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 according to the percentage of FOXM1 positive cells, and 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 undergoes 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 according to 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 FOXM1-positive cell percentage data 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 occurs in mycosis fungoides, the method comprising the following steps: receiving FOXM1-positive cell percentage data 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 large cell transformation occurs in the subject with mycosis fungoides.

[0023] In the above, the above-mentioned 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 to obtain the area percentage of FOXM1-positive cells; the details are as follows:

[0025] After immunohistochemical staining (the color developer is DAB) of the skin lesion tissue of the subject with mycosis fungoides, the sections were scanned by NanoZoomer microslice scanner (Hamamatsu Photonics, Japan), and then observed by NDP.view2 (U12388-01) software, and then three areas were randomly selected under a 20-fold field of view for image output to obtain three images; each image was quantitatively calculated by Image J software, and the process was as follows: DAB (brown) was defined as positive, and the positive threshold was set to 140, and then 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. Each section had three images and were calculated separately, and then the average of the area percentages of FOXM1-positive cells in the three images was taken, recorded as the area percentage of FOXM1-positive cells, and used as the percentage of FOXM1-positive cells.

[0026] The above-mentioned output of result information from the computer according to the FOXM1-positive cell percentage data is based on the comparison of the FOXM1-positive cell percentage of the skin lesion tissue of the mycosis fungoides subject with a threshold value. If it 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 it 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 does not have or is a candidate for large cell transformation; or the possibility of large cell transformation in MF patients whose FOXM1-positive cell percentage in skin lesion tissue is greater than 1% is greater than or the candidate is greater than that in MF patients whose FOXM1-positive cell percentage in 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 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 described in the fourth or fifth aspect;

[0030] 1) A system for identifying or assisting in identifying 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 of 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 activity of mycosis fungoides cells; increasing the apoptosis level of mycosis fungoides cells; or inhibiting the colony formation of mycosis fungoides cells, or down-regulating 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) in pathology often indicates faster disease progression and worse prognosis. In this study, RNA sequencing revealed that FOXM1 (a cell cycle-related transcription factor) was specifically elevated in MF-LCT, and the results were verified at the RNA and protein levels by increasing the sample size. Immunohistochemical staining with FOXM1 antibody showed that the percentage of FOXM1-positive cells was greater than 1%, which had a high diagnostic value for MF large cell transformation (MF-LCT), and the sample size was expanded for preliminary verification. Compared with traditional pathological techniques, immunohistochemical staining of FOXM1 can more accurately locate large cells and quantify the proportion of large cells, which can reduce subjective bias. Therefore, it is proposed that immunohistochemical staining of FOXM1 can be used as one of the auxiliary techniques for diagnosing MF-LCT. In addition, the use of FOXM1-specific small molecule inhibitors can reduce the survival and increase apoptosis of tumor cells in vitro. In vitro experiments have confirmed that FOXM1 is of great significance to cell survival, apoptosis resistance, and cell 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 2Volcano plot of gene expression differences in MF patients with large cell mutation / non-large cell mutation (a) and the difference in RNA expression levels of FOXM1 in tissues of MF patients with large cell mutation / 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 Comparison of FOXM1 and TOX immunofluorescence in MF and BID (MF: mycosis fungoides; BID: benign inflammatory skin disease).

[0041] Figure 5 The image comparison of FOXM1 immunohistochemical staining and Image J processing of MF-LCT and MF-NLCT (FOXM1 immunohistochemical staining is DAB system, brown is positive; Image J processed pictures, red area is 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 the protein level of FOXM1 (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] Fig. 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] Fig.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] Fig.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] Fig.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] Fig.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] Fig.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] Fig.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 complies with the principles of the World Medical Association Declaration of Helsinki and has been approved by the Clinical Ethics Committee of Peking University First Hospital (Ethics Review Number: 2021 Scientific Research 151), and all patients have signed informed consent. Among them, the clinical diagnosis, pathological diagnosis, and disease staging of mycosis fungoides are based on the revised standards recommended by the International Society of Cutaneous Lymphoma (ISCL) and the European Organization for Research and Treatment of Cancer (EORTC) in 2007. The diagnosis of specimens of all patients with mycosis fungoides included in the case group must be confirmed by two experienced dermatopathologists (as the gold standard).

[0057] A total of 65 patients with mycosis fungoides were included in the training group of this study. All tumor specimens were used for protein level validation experiments (MF-LCT: 29 cases; MF-NLCT: 36 cases) and also as training sets; 15 MF-LCT and 31 MF-NLCT tumor specimens were used for RNA level validation; 31 patients with mycosis fungoides (MF-LCT: 14 cases; MF-NLCT: 17 cases) in the validation set were recruited from the Research Center of Peking University First Hospital (18 cases), the Department of Dermatology of Sun Yat-sen Memorial Hospital of Sun Yat-sen University in Guangzhou (5 cases), and the Department of Dermatology of Qilu Hospital of Shandong University (8 cases). At the same time, this study also included skin tissues of 20 patients with BID (inflammatory skin disease) (including 8 cases of lichen planus, 8 cases of eczema, and 4 cases of connective tissue disease) as control validation. At the same time, this study also included peripheral blood mononuclear cells (PBMCs) from 2 healthy subjects as protein controls for protein immunoblotting detection.

[0058] Table 1 shows the inclusion criteria

[0059]

[0060] 2. Origin of CTCL cell lines

[0061] The MF cell lines of human cutaneous T-cell lymphoma used in this experiment include Myla and PB2B. Myla and PB2B cell lines are recorded in the following literature: BCL11B-Mediated Epigenetic Repression Is a Crucial Target for Histone Deacetylase Inhibitors in Cutaneous T-Cell Lymphoma; Wenjing Fu et al., 2017 Journal of Investigative Dermatology.10.1016 / j.jid.2017.02.980. Myla and PB2B cell lines were cultured with 90% RPMI-1640 basal medium + 10% fetal bovine serum + 1% penicillin (100U / ml)-streptomycin (0.1mg / ml) double antibody. The culture conditions used for all CTCL cell lines were 37°C constant temperature, 100% relative humidity, 5% CO 2 All cells grew in suspension in the culture medium, and the medium was changed and subcultured according to the growth conditions. The cells used in the experiment 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 is the antibody

[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 slicer to slice the skin lesion tissue into paraffin tissue slices and patches.

[0088] 2) Place all slices in a 65°C thermostat for 1-2 hours, first place them in xylene I for 15 minutes, take them out and drain them roughly, then place them in xylene II for 15 minutes.

[0089] 3) Treat the slices with anhydrous ethanol, 95% ethanol and 75% ethanol for 5 minutes respectively, and then wash them with deionized water for 5 minutes. A total of 3 washes should be performed.

[0090] 4) Add the prepared 2L sodium citrate / EDTA solution (antigen repair solution) into the pressure cooker in advance and heat it on the induction cooker. After the solution is completely boiled, put the slices into the pot and repair them under 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) Use 3% H 2 O 2 After incubation for 10-15 minutes in a dark environment, 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 with antibody diluent or 1× PBS solution) of corresponding concentration to the tissue and incubate overnight in a 4°C refrigerator.

[0094] 8) After overnight incubation, take out 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 DAB of standard concentration to develop color at room temperature. During the development process, use a microscope to observe whether the target tissue appears brown color. 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 slices with 75% ethanol, 95% ethanol and anhydrous ethanol for 1 min respectively.

[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 slices in a 65°C thermostat for 1-2 hours, first place them in xylene I for 15 minutes, take them out and drain them roughly, then place them in xylene II for 15 minutes.

[0103] 3) Treat the slices with anhydrous ethanol, 95% ethanol and 75% ethanol for 5 minutes respectively, and then wash them with deionized water for 5 minutes. A total of 3 washes should be performed.

[0104] 4) Add the prepared 2L sodium citrate / EDTA solution (antigen repair solution) into the pressure cooker in advance and heat it on the induction cooker. After the solution is completely boiled, put the slices into the pot and repair them under 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 the goat serum was dried, an appropriate amount of primary antibody (anti-FOXM1; diluted with antibody diluent or 1× PBS solution) of corresponding concentration was added to the tissue and incubated overnight in a 4°C refrigerator.

[0107] 7) After overnight incubation, take out 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 an appropriate amount of primary antibody of corresponding concentration ( Goat Ant i-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 it away from light for 10 minutes after sealing.

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

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

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

[0113] 2) Ficoll solution should be aspirated using a disposable syringe and 3 mL of Ficoll solution should be added to each centrifuge tube.

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

[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 of the PBMC layer to each centrifuge tube to resuspend the cells.

[0117] 6) Centrifuge at 80×g for 10 min. After centrifugation, discard the supernatant and retain the lower layer of liquid.

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

[0119] 4.4 RNA extraction and cDNA reverse transcription

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

[0121] cDNA reverse transcription strictly follows PrimeScript TM The RT Master Mix kit instructions were followed 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 using 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 pre-mix of the corresponding gene to each well of the 8-well reaction tube or 96-well plate used, and then add 5 μL of cDNA of the corresponding sample to each well again, and make sure to mix well.

[0129] 4) After closing the lid of the 8-well plate 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 was obtained using the Vii TM 7software provided by the ViiA 7Applied Biosystems Prism system to obtain the Ct values ​​of the sample target gene and the internal reference gene GAPDH, and the corresponding ΔCt was calculated using EXCEL, and the formula 10000 / Power(2,ΔCt) was used to calculate the relative expression value of the target gene in each sample. Two replicate wells were set for each sample, and the average was taken as the final result.

[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 measured by the BCA method (Pierce TM BCA Protein Assay Kit).

[0138] The extracted proteins were subjected to Western blotting detection:

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

[0140] 2) Take 6-8 μL of protein marker and use 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 hole on the left and right sides.

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

[0143] 5) Wet transfer: After taking out the gel, cut it according to the position of the band and transfer it to 1× electrotransfer buffer for later use. Prepare a PVDF membrane activated by methanol and cut it to a suitable size. In the environment of 1× electrotransfer buffer, place the transfer splint (black) - thick sponge gasket - filter paper - sample gel - PVDF membrane - filter paper - sponge gasket - transfer splint (red) in this order. During this period, a glass rod should be used to remove all bubbles. After completion, clamp the splint and place the entire splint in the electrotransfer tank filled with 1× electrotransfer buffer for transfer. The parameters are 400mA and 30min.

[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 skim milk powder of the required concentration and add 5 mL to each cell to completely soak the PVDF membrane. Shake at 60-70 rpm and seal for 1 hour.

[0147] 9) Prepare the primary antibody at the required concentration using the prepared skim milk powder solution, add 5 mL to each cell, 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, with a shaking speed of 90-110 rpm, 5 minutes each time, and a total of 5 washes.

[0149] 11) Prepare the secondary antibody of the required concentration using the prepared skim milk powder solution, add 5 mL to each cell, incubate at room temperature for 1 hour, shake at 60-70 rpm, and then wash with TBST solution for 5 min × 3 times.

[0150] 12) Prepare chemiluminescent solution according to the instructions of EasySee Western Blot Kit (TransGen Biotech): mix equal volumes of solution A and solution B, and the volume of solution C is (A+B) / 1000. Evenly cover the surface of the PVDF membrane with the prepared luminescent solution, incubate at room temperature in the dark for half a minute, and perform imaging analysis using the SYNGENE gel imaging analysis system.

[0151] 4.7 Lentivirus construction and transfection experiments

[0152] 1) Construction of lentivirus for knocking down FOXM1 gene

[0153] GeneCare Gene Company synthesized FOXM1-sh1, a lentivirus that knocked 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 RNA interference sequence sh1 with a titer of 1E+9Tu / mL; used to knock down the FOXM1 gene;

[0156] Lentivirus LV-FOXM1-RNAi (88730-1) expresses 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 the control lentivirus, and the control insertion sequence is: TTCTCCGAACGTGTCACGT; the titer is 1.0×10 9 Tu / mL.

[0158] 2) RNAi target design

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

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

[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, Fig.15 ) as a lentiviral cloning vector, clone the recombinant vector obtained by inserting the above RNA interference sequence sh1 or RNA interference sequence sh2 between the AgeI and EcoRI sites of GV493 and the virus packaging helper plasmid (Helper1.0 and Helper1.2 in the construction of RNAi lentiviral vector) into 293 cells to obtain the lentivirus. The element sequence of the lentiviral cloning vector GV493 is hU6-MCS-CBh-gcGFP-IRES-puromycin, and the cloning sites are AgeI and EcoRI. cGFP is a fluorescent marker used to indicate positively transduced cells for easy 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 cell solution contains 5×10 5 The number of live cells per transfection was 2×10 5 indivual).

[0170] b. Add 20 μL of HitransG A virus infection reagent (Genentech, Cat. No. REVG004), virus solution (MOI value is 100), 400 μL containing 2×10 5 The culture medium of each cell 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 cells / mL using complete RPMI 1640 medium. 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 a target drug was added separately.

[0175] 2) Before each sampling, the cells must be mixed and the cell viability test was performed at 0h, 24h, 48h, and 72h after cell plating / drug addition. Each time, 100μL of the completely mixed cell suspension was added to the 96-well plate, and 20μL MTS solution was added to each well and mixed. Incubate in a cell culture incubator at 37℃ for 2 hours. Two replicate wells were made for each cell line at the same time.

[0176] 3) After 2 hours, the 96-well plate was taken out 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 by EXCEL, and the relative cell viability was calculated by the formula: original OD / OD 0 Calculate the relative cell viability value, 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 with complete RPMI 1640 medium. 6 / 4mL or 1×10 5 pcs / 3mL, cultured in culture flasks or 6-well plates. The test is usually 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 for resuspending (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 at the same time, and incubate at room temperature and away from light for 15 minutes.

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

[0183] 5) Perform flow cytometry apoptosis detection, analyze the obtained data using FlowJo 10.4 software, and calculate the apoptosis rate.

[0184] 4.10 Cell colony formation assay

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

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

[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 / mL. Mix the diluted cell solution and pipette 0.2mL of the cell suspension into 3mL of the complete methylcellulose medium. Mix the medium and cells thoroughly, vortex vigorously for ≥4 seconds and let stand for at least 5 minutes to allow the bubbles in the medium to drain to the top.

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

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

[0190] 6) Take photos using the 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 6After 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 tube for fixation (2 h to overnight).

[0194] 3) Prepare staining solution at a ratio of RNaseA:PI=1:9, wash with 2 mL of pre-cooled PBS, add 300 μL of staining solution to each flow tube, filter with a 40 μm filter, and incubate at room temperature away from light for 30-60 min.

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

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

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

[0198] The sections after immunohistochemical staining were scanned by NanoZoomer microslide scanner (Hamamatsu Photonics, Japan), and then observed by NDP.view2 (U12388-01) software. Three areas were randomly selected under a 20x field of view for image output to obtain three images. Each image was then quantitatively calculated by Image J software. The process was as follows: DAB (brown) was defined as positive, and the positive threshold was set to 140. 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. Each section had three images that were calculated separately, and then the average of the area percentages of FOXM1-positive cells in the three images was taken and recorded as the area percentage of FOXM1-positive cells, which was used as the percentage of FOXM1-positive cells.

[0199] 5.2 Statistical methods:

[0200] All raw data of 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 drawing. The quantitative data of normally distributed data were expressed as mean ± standard deviation (Mean ± SD), and the quantitative data of non-normally distributed data were expressed as median and quartile (25% quantile, median, 75% quantile). The comparison of mean differences between the two groups was performed using Student's t-test or nonparametric Mann-Whitney U-test; Kaplan-Meier was used for survival analysis. p < 0.05 was considered statistically significant.

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

[0202] 1. Transcriptome sequencing revealed that FOXM1 was 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 2 groups according to the presence or absence of large cell transformation: 26 MF-LCT and 23 MF-NLCT.

[0204] Tumor tissue RNA was extracted from 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 Figure 2, transcriptome sequencing volcano plot analysis found that the RNA expression of FOXM1 gene was significantly higher in MF-LCT than in MF-NLCT ( Figure 2 a). The average FPKM of FOXM1 in MF-LCT was 18.30, and the average FPKM 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 level 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 lesions 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 level of FOXM1 in each sample.

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

[0210] 2. Immunohistochemical staining

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

[0212] The results are as follows Figure 4 As shown in the figure, in MF tissues, FOXM1 is mainly expressed in tumor cells that are positive for TOX staining. However, in BID, FOXM1 is basically not expressed (TOX is also almost not expressed). Moreover, FOXM1 is mainly expressed in tumor cells that have increased in size.

[0213] At the clinical specimen level, the above experiments, by expanding the sample size, confirmed 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 to identify MF-LCT.

[0215] Example 2: Application of FOXM1 in assisting 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 pathological technique commonly used 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 in the training group. The sections after immunohistochemical staining were scanned by NanoZoomer microscopy scanner (Hamamatsu Photonics, Japan), and then observed by NDP.view2 (U12388-01) software. Three areas were randomly selected under 20 times field of view for image output to obtain 3 images. Then, each image was quantitatively calculated by Image J software. The process was as follows: DAB (brown) was defined as positive, and the positive threshold was set as 140. Then, the percentage of FOXM1-positive cell area in the total area of ​​the image was calculated to obtain the area percentage of FOXM1-positive cells. Each section had three images for calculation, and then the average of the area percentage of FOXM1-positive cells in the three images was taken, recorded as the area percentage of FOXM1-positive cells, and used 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 by 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 showed that the optimal cutoff value for distinguishing MF-LCT from MF-NLCT was 1.056%, and the sensitivity and specificity of identifying MF-LCT could reach 86.21% and 97.22%, respectively. However, considering the convenience of clinical application, the threshold was set at 1%, that is, 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, and its 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 was a MF-LCT patient;

[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 or more likely to be 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 MF-LCT and 17 MF-NLCT patients in the validation group. The percentage of FOXM1-positive cells 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 gender ratio (see Table 1 for specific comparison.

[0242] 2. Evaluation of identification model

[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 the 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 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, MF cell lines Myla and PB2B were used to knock down FOXM1 at the RNA level to observe its effect on the phenotype of CTCL cell lines.

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

[0253] The protein expression of FOXM1 in MF cell lines (Myla and PB2B) was detected by Western blotting. Protein derived from PBMC of healthy subjects (protein extracted from PBMC of healthy subjects) was used as control.

[0254] The results are as follows Figure 7 As shown, 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 inhibiting tumor cells

[0256] 1) shRNA knockdown of FOXM1

[0257] Myla and PB2B cell lines with high expression of FOXM1 were selected as research objects. The expression of FOXM1 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 the control group transfected with control lentivirus sh0, sh1 and sh2 corresponded to the transfection with lentivirus FOXM1-sh1 and lentivirus FOXM1-sh2, respectively).

[0258] The lentivirus FOXM1-sh1, FOXM1-sh2 and lentivirus expressing sh0 entrusted to GeneCare Gene Co., Ltd. for knocking down the FOXM1 gene were transfected into various Myla and PB2B cell lines according to the above method. The FOXM1 knockdown group was 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 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 with Myla;

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

[0266] On the 5th day after transfection, the 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 8As shown, sh0 is PB2B-sh0 or Myla-sh0, and sh1 and sh2 in 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 in both sh1 and sh2 groups were significantly reduced, among which sh2 had a higher knockdown efficiency.

[0268] 2) Detection of cell viability

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

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

[0271] 3) Detection of cell apoptosis level

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

[0273] The results are as follows Fig.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 an 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 Fig.11 As shown, the right picture is a colony map, the first row Myla and Myla-sh0 are the control group, and the second row Myla-sh1 and Myla-sh2 are the knockdown group; the left picture is a colony counting map; colony counting found that the number of large colonies formed after FOXM1 knockdown was significantly reduced compared with the control group, and the overall colony number was also significantly reduced. This shows 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 apoptosis 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 tumor cell colony formation.

[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 line 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 inoculated 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 Fig.12 As shown, the survival ability 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 in a PB2B cell line culture system (same as above) and cultured for 72 hours.

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

[0289] The results are as follows Fig.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 can be used as 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 ModFitLT for Macintel was used to analyze the cell cycle distribution.

[0294] The results are as follows Fig.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 blot detection of 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 6 Myla cells were seeded in a 3 mL volume in a six-well plate and cultured for 72 hours.

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

[0299] The results are as follows Fig.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 adding drugs 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, characterized in that: The processor executes the computer program to implement the following steps: S1) Data received: percentage of FOXM1-positive cells in skin lesions of subjects with mycosis fungoides; S2) Data output: outputting result information from a computer based on the percentage of FOXM1-positive cells, wherein the result information is whether the subject with mycosis fungoides is or is a candidate for 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 in claim 1 when executed by a processor.

3. A computer program product, comprising a computer program, characterized in that: The computer program implements the steps in claim 1 when executed by a processor.

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 according to the percentage of FOXM1 positive cells, and 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 identification 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 according to 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 identifying large cell transformation of mycosis fungoides, the method comprising the following steps: receiving FOXM1-positive cell percentage data 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.

7. A method for identifying or assisting in identifying whether mycosis fungoides has undergone large cell transformation, the method comprising the following steps: receiving FOXM1-positive cell percentage data 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, wherein the result information is whether the subject with mycosis fungoides has undergone large cell transformation.

8. Any of the following systems, comprising the device according to claim 4 or 5; 1) A system for identifying or assisting in identifying 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 of 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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