Use of ncbp1 as an auxiliary diagnostic and / or prognostic marker for diffuse large b-cell lymphoma

Abstract

The application provides application of NCBP1 as an auxiliary diagnosis and / or prognosis marker of diffuse large B-cell lymphoma (DLBCL), and belongs to the technical field of medicinal biology and molecular biology. The application first discovers that NCBP1 is highly expressed in DLBCL patients, the high expression of NCBP1 has a significant influence on the proliferation of DLBCL cells, and is related to poor prognosis; NCBP1 enhances the proliferation of DLBCL cells in a METTL3-dependent manner, NCBP1 enhances the m6A catalytic function of METTL3 by maintaining the stability of the oncogene METTL3 mRNA; the NCBP1 / METTL3 / m6A / c-MYC axis may be a mechanism for the development of DLBCL, which provides a new idea for molecular targeted therapy of DLBCL, enriches the understanding of the m6A modification system in DLBCL, and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmacognosy medicine and molecular biology, and particularly relates to application of NCBP1 as an auxiliary diagnosis and / or prognosis marker for diffuse large B-cell lymphoma. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of generally presenting the context of the application. The information disclosed in this Background section is not to be necessarily taken as an acknowledgement or any form of suggestion that this information forms part of the prior art already known to a person skilled in the art.

[0003] Diffuse large B-cell lymphoma (DLBCL) is a malignant tumor characterized by malignant proliferation of B lymphocytes, with an annual incidence of 7 per 100,000, accounting for about 40% of all non-Hodgkin's lymphoma. About 60% of DLBCL patients can be cured by rituximab combined with the classic chemotherapy regimen CHOP. However, to date, 30%-40% of patients relapse or have primary drug resistance, and such patients have poor prognosis. New treatment regimens are still needed, but so far, new generation CD20 antibody drugs such as atumumab, veltuzumab and obinutuzumab, and other drugs such as bortezomib, lenalidomide, ibrutinib, everolimus, etc. have not achieved better results than the traditional R-CHOP. Therefore, it is necessary to develop markers for early diagnosis or prognosis, and to find new treatment targets through pathogenesis and causes, which is an effective means to improve the survival of patients with diffuse large B-cell lymphoma.

[0004] The nuclear cap-binding complex binds to the 5' end "cap structure" of newly synthesized messenger RNA, and this binding affects the final fate of mRNA. The nuclear cap-binding complex family currently consists of nuclear cap-binding protein 1 (NCBP1), 2 (NCBP2) and C17orf85 (NCBP3). In eukaryotic cells, NCBP1 binds to mature mRNA precursors and is involved in various biological behaviors of mRNA maturation, such as precursor mRNA splicing, translation regulation, nonsense mRNA decay, and mRNA export from the nucleus. It has been reported that NCBP1 binds to ribonucleoprotein particles, regulates cell growth and proliferation, and makes it more adaptable to environmental changes; promotes the translation of oncogenes and plays an antiviral role in plants; and affects the sensitivity of solid tumors to chemotherapeutic drugs by affecting the expression of downstream genes. However, the role of NCBP1 in hematological tumors DLBCL has not been reported.

[0005] The m6A modification of RNA is the most extensive and most studied RNA modification in eukaryotic cells. The m6A methylation is a dynamic catalytic reduction process catalyzed by a variety of methyltransferase complexes. METTL3 is the most representative RNA m6A modification enzyme, which has been widely reported to play a major role in various biological processes. For example, METTL3 regulates autophagy by enhancing the m6A modification of ATG7, thereby promoting osteoarthritis; METTL3 affects BCL2 stability through YTHDF1-mediated m6A modification, thereby inhibiting apoptosis; METTL3 regulates lncRNA stability and promotes CUL4B nuclear transport. Although it is reported in other diseases, for DLBCL, only one report shows that METTL3 increases the occurrence of DLBCL by regulating the m6A modification of PEDF. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application provides the application of NCBP1 as an auxiliary diagnostic and / or prognostic marker for diffuse large B-cell lymphoma. The present application discloses that NCBP1 plays a promoting role in the occurrence of DLBCL, and proves that NCBP1 can increase the stability of the cancer-promoting molecule METTL3 mRNA, and verifies that NCBP1 affects the m6A modification degree and expression of the classic oncogenic molecule c-MYC by affecting the RNAm6A modification function of METTL3, thereby affecting the occurrence and development of diffuse large B-cell lymphoma. Therefore, by exploring the function of NCBP1 in DLBCL, the application of NCBP1 as an auxiliary diagnostic and / or prognostic marker for diffuse large B-cell lymphoma is provided.

[0007] Specifically, the present application relates to the following technical solutions:

[0008] In a first aspect of the present application, the application of NCBP1 as an auxiliary diagnostic and / or prognostic marker for diffuse large B-cell lymphoma is provided.

[0009] The application of the above first aspect includes but is not limited to any one of the following modes:

[0010] (1) By detecting the expression content of NCBP1 in the patient detection sample, whether the patient is a diffuse large B-cell lymphoma patient is diagnosed, and the speed of tumor progression of the patient is predicted;

[0011] (2) By detecting the expression content of NCBP1 in the diffuse large B-cell lymphoma patient detection sample, the prognosis of the patient is evaluated;

[0012] (3) The detection reagent of NCBP1 is used as the application of preparing an auxiliary diagnostic and / or prognostic detection reagent for diffuse large B-cell lymphoma.

[0013] In the application of the first aspect, the detection sample of the diagnostic marker is a clinical biological sample of a patient, including one or more of serum, plasma, whole blood, secretion, cotton swab, pus, body fluid, tissue, organ, paraffin section. In one of the verified ways of the application, the detection sample is a lesion tissue sample or a suspected lesion tissue sample of a patient.

[0014] According to the research results of the application, the expression content of NCBP1 in the lesion sample of a diffuse large B-cell lymphoma patient is higher than that in an inflammatory lymph node, and the increase in the expression content is positively correlated with the proliferation activity of diffuse large B-cell lymphoma cells and indicates poor prognosis. Therefore, in the application of each aspect, the increase in the content of NCBP1 in the detection sample means that the patient with diffuse large B-cell lymphoma has a faster tumor proliferation and a poor natural course prognosis.

[0015] In the second aspect of the application, a product for assisting in the diagnosis and / or prognosis of diffuse large B-cell lymphoma is provided, and the product comprises substances for detecting the expression amount of NCBP1 and METTL3.

[0016] The product of the second aspect further comprises substances suitable for other biomarkers for predicting diffuse large B-cell lymphoma, including genetic factor-related biomarkers, endocrine factor-related biomarkers, infection factor-related biomarkers (such as any serious infection that can cause bacteremia or viremia), immune function-related biomarkers (such as anti-phospholipid antibodies, anti-nuclear antibodies, anti-DNA antibodies, anti-sperm antibodies, anti-thyroid antibodies, increased number and activity of natural killer (NK) cells, abnormal macrophage function, abnormal dendritic cell function, complement system abnormalities, lack of blocking antibodies, abnormal T and B lymphocytes, abnormal Th1 / Th2 cytokines, etc.), pre-thrombotic state-related biomarkers (such as mutations in factor V and factor II (thrombin) genes, protein S deficiency), and other systemic diseases of the subject. By using other biomarkers in combination, the interference of other physiological and pathological states can be further excluded, thereby improving the sensitivity and specificity of the detection and meeting the requirements of precision medicine.

[0017] In the detection of the expression amount of NCBP1 and METTL3 in the second aspect, the detection methods include but are not limited to RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip, or gene sequencing for detecting the expression level of NCBP1 and METTL3.

[0018] The product of the second aspect includes but is not limited to primers, probes, chips, nucleic acid membrane strips, preparations, or kits for detecting the expression level of NCBP1 and METTL3 in the sample to be tested.

[0019] The detection sample is a clinical biological sample of a patient, including one or more of serum, plasma, whole blood, secretion, cotton swab, pus, body fluid, tissue, organ, paraffin section. The tissue section detection sample is a lesion tissue sample or a suspected lesion tissue sample of a patient.

[0020] In a third aspect, the present application provides a system for assisting in the diagnosis and / or prognosis of diffuse large B-cell lymphoma, characterized in that the system comprises:

[0021] i) an analysis unit comprising: determining the expression levels of NCBP1 and METTL3 in a test sample of a subject, and;

[0022] ii) an evaluation unit comprising: determining whether the subject has a poor prognosis according to the expression levels of NCBP1 and METTL3 determined in i).

[0023] In the analysis unit of the third aspect, other biomarkers suitable for the prognosis of diffuse large B-cell lymphoma are also included, including genetic factor-related biomarkers, endocrine factor-related biomarkers, infection factor-related biomarkers, immune function-related biomarkers, pre-thrombotic state-related biomarkers, and other systemic diseases of the subject.

[0024] The evaluation unit of the third aspect specifically includes the following evaluation process:

[0025] Compared with the reference, if the expression levels of NCBP1 and METTL3 in the test sample of the subject are up-regulated, the subject is or is a candidate for a poor prognosis; otherwise, the subject is not or is not a candidate for a poor prognosis.

[0026] In a fourth aspect, the present application provides the use of a substance that inhibits the expression level and / or reduces the activity of NCBP1 and METTL3 in the preparation of a product.

[0027] The function of the product is any one or more of the following:

[0028] (a1) inhibiting the proliferation of diffuse large B-cell lymphoma cells;

[0029] (a2) inhibiting the cell cycle progression of diffuse large B-cell lymphoma cells;

[0030] (a3) inhibiting the expression of METTL3;

[0031] (a4) inhibiting the m6A catalytic function of METTL3

[0032] (a5) inhibiting the expression of c-MYC;

[0033] (a6) preventing and / or treating diffuse large B-cell lymphoma.

[0034] In a fifth aspect of the present application, there is provided a product, the active ingredient of which comprises a substance that inhibits the expression level and / or reduces the activity of NCBP1 and METTL3;

[0035] The function of the product is any one or more of the following:

[0036] (a1) inhibiting the proliferation of diffuse large B-cell lymphoma cells;

[0037] (a2) inhibiting the cell cycle progression of diffuse large B-cell lymphoma cells;

[0038] (a3) inhibiting the expression of METTL3;

[0039] (a4) inhibiting the m6A catalytic function of METTL3

[0040] (a5) inhibiting the expression of c-MYC;

[0041] (a6) preventing and / or treating diffuse large B-cell lymphoma.

[0042] In a sixth aspect of the present application, there is provided the use of a substance that promotes the expression level and / or improves the activity of NCBP1 and METTL3 in the preparation of a product;

[0043] The function of the product is any one or more of the following:

[0044] (a1) promoting the proliferation of diffuse large B-cell lymphoma cells;

[0045] (a2) promoting the cell cycle progression of diffuse large B-cell lymphoma cells;

[0046] (a3) promoting the expression of METTL3;

[0047] (a4) promoting the m6A catalytic function of METTL3

[0048] (a5) promoting the expression of c-MYC;

[0049] (a6) pre-establishing an animal model of diffuse large B-cell lymphoma.

[0050] In a seventh aspect of the present application, there is provided a product, the active ingredient of which comprises a substance that promotes the expression level and / or improves the activity of NCBP1 and METTL3;

[0051] The function of the product is any one or more of the following:

[0052] (a1) promoting the proliferation of diffuse large B-cell lymphoma cells;

[0053] (a2) promoting cell cycle progression of diffuse large B-cell lymphoma cells;

[0054] (a3) promoting METTL3 expression;

[0055] (a4) promoting m6A catalytic function of METTL3

[0056] (a5) promoting expression of c-MYC;

[0057] (a6) pre-building a diffuse large B-cell lymphoma animal model.

[0058] The product in the above fourth to seventh aspects can be a drug.

[0059] The beneficial technical effects of one or more of the above technical solutions are:

[0060] The present application first found that NCBP1 is highly expressed in DLBCL patients and is related to poor prognosis, and also found that high expression of NCBP1 has a significant effect on the proliferation of DLBCL cells. Further studies have shown that NCBP1 enhances the proliferation of DLBCL cells in a METTL3-dependent manner, and NCBP1 enhances the RNAm6A catalytic function of METTL3 by maintaining the stability of the oncogene METTL3 mRNA. Mechanistically, the expression of the proto-oncogene c-MYC is regulated by METTL3 modification, and NCBP1 enhances the expression of METTL3, thereby affecting the proto-oncogene c-MYC to play a role in promoting tumors. The NCBP1 / METTL3 / m6A / c-MYC axis may be the mechanism of DLBCL development, providing a new idea for molecular targeted therapy of DLBCL, enriching the understanding of m6A modification system in DLBCL, and having important clinical application value.

[0061] In summary, NCBP1 as a potential new diagnostic and prognostic biomarker for DLBCL, and may become a therapeutic target, thus playing an important role in the diagnosis, prediction, and treatment of diffuse large B-cell lymphoma, and therefore has good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0062] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the illustrative embodiments of the present application and their descriptions serve to explain the present application and do not constitute an improper limitation on the present application.

[0063] Figure 1 is a graph showing overexpression of NCBP1 in DLBCL patients;

[0064] wherein A indicates that the GEPIA database indicates that there is a significant difference in the expression of NCBP1 in DLBCL tumors and normal tissues,

[0065] B is the expression of NCBP1 in 50 cases of DLBCL tissues and 14 cases of inflammatory lymph nodes in patients admitted to Qilu Hospital of Shandong University is also different,

[0066] C is the difference in overall survival of DLBCL patients grouped by high (red) or low (blue) NCBP1 expression, high NCBP1 expression is associated with shorter survival time;

[0067] Figure 2 is an in vitro experiment to verify the role of NCBP1 in promoting DLBCL cell proliferation;

[0068] A and B are confirmed by RT-qPCR and western blotting analysis that the efficiency of NCBP1 lentivirus transduction in SU-DHL-4 cells,

[0069] C is the cell proliferation activity of SU-DHL-4 cells after stable transfection measured by CCK8 method,

[0070] D is the flow cytometry EdU method to evaluate the proliferation difference of SU-DHL-4 cells after virus transfection,

[0071] E is the semi-solid culture method to analyze the influence of NCBP1 different expression on the proliferation potential of SU-DHL-4 cells;

[0072] Figure 3 is a graph showing the influence of NCBP1 on METTL3 expression in DLBCL cells;

[0073] A is a volcano plot showing that METTL3 expression is affected by NCBP1 in SU-DHL-4 cells,

[0074] B is RT-qPCR to confirm that METTL3 is down-regulated with siNCBP1,

[0075] C is that METTL3 expression is significantly correlated with NCBP1 (r=0.7) (data from GEPIA database),

[0076] D is the correlation between NCBP1 and METTL3 mRNA levels in DLBCL tumor specimens analyzed by RT-qPCR,

[0077] E is the half-life of METTL3 mRNA in lentivirus transfected SU-DHL4 cells is affected by NCBP1 expression (T1 / 2);

[0078] Figure 4 is a graph showing that NCBP1 affects the proliferation of DLBCL cells through METTL3;

[0079] Wherein, A is the expression difference of METTL3 in DLBCL tumor and normal tissue, from GEPIA database,

[0080] B is the expression of METTL3 in 50 DLBCL tissues and 14 inflammatory lymph node tissues collected by Qilu Hospital of Shandong University detected by RT-qPCR

[0081] C is the differential expression of METTL3 in DLBCL tissues and inflammatory lymph nodes shown by immunohistochemistry (IHC);

[0082] D is the correlation of METTL3 expression with overall survival in DLBCL patients, high (red) or low (blue) METTL3 expression difference is significant,

[0083] E, F are the efficiency of transfection of SU-DHL-4 cells with METTL3 lentivirus confirmed by RT-qPCR and western blotting,

[0084] G, H are the cell viability of transfected SU-DHL-4 cells at the specified time point determined by CCK8 method,

[0085] I is the proliferation of transfected SU-DHL-4 cells evaluated by flow cytometry with EdU;

[0086] Figure 5 is the diagram that METTL3 promotes the proliferation of DLBCL cells by increasing the expression of c-MYC;

[0087] Wherein, A is the GEPIA website data suggesting that c-MYC expression is positively correlated with METTL3,

[0088] B is the collected DLBCL specimens showing that c-MYC mRNA is correlated with METTL3 expression,

[0089] C is the immunohistochemical staining image of c-MYC expression in DLBCL tissues and inflammatory lymph nodes,

[0090] D, E are the effectiveness of c-MYC lentivirus transduction in SU-DHL-4 cells confirmed by RT-qPCR and western blotting,

[0091] F, G are the cell viability of transfected SU-DHL-4 cells at the specified time point determined by CCK8 method,

[0092] H, I are the proliferation of transfected SU-DHL-4 cells evaluated by EdU;

[0093] Figure 6is the figure of NCBP1 affecting its m6A modification function on c-MYC mRNA through METTL3;

[0094] A is the influence of NCBP1 on the degree of m6A methylation, (methylene blue (MB) staining as a loading control);

[0095] B is the website (http: / / rna.sysu.edu.cn / rmbase / ) providing c-MYC m6A modification sites,

[0096] C is the potential m6A high modification region of c-MYC mRNA,

[0097] D is the influence of NCBP1 expression on the abundance of m6A on c-MYC mRNA transcript in SU-DHL-4 cells detected by MeRIP-qPCR. DETAILED DESCRIPTION

[0098] The present application is further explained by the following examples, which do not limit the present application. It should be understood that these examples are used to illustrate the present application and not to limit the scope of the present application.

[0099] EXAMPLE

[0100] I. Experimental techniques:

[0101] 1. GEPIA database was used to evaluate the expression difference of NCBP1 and METTL3, and the expression of NCBP1 was verified by clinical patient specimens.

[0102] 2. Lentivirus was used to up-regulate and down-regulate the expression of NCBP1 in DLBCL cell line SU-DHL-4, CCK8 experiment was used to detect cell proliferation, EdU experiment was used to detect DNA replication rate, and semi-solid culture experiment was used to detect cell clonogenicity.

[0103] 3. RNA-seq was used to screen NCBP1 downstream molecules, and RT-qPCR experiment was used for verification.

[0104] 4. Data analysis was used to screen the key molecule METTL3 related to NCBP1, and the expression of METTL3 was verified by clinical specimens. Lentivirus was used to up-regulate and down-regulate the expression of METTL3 in SU-DHL-4 cells, and cell proliferation function was detected. At the same time, NCBP1 and METTL3 were transfected reversely, and the regulation of NCBP1 on cell proliferation ability was verified to depend on the expression level change of METTL3 through recovery experiment.

[0105] 5. Dot-blot was used to detect the level of m6A in cells, and the change related to the expression of NCBP1 was detected.

[0106] 6. m6A database analysis METTL3 regulates the expression of downstream molecules cMYC through m6A methylation modification of the action site, and verified by MeRIP experiment.

[0107] 7. RNA stability experiment detects that the up-regulation mechanism of METTL3 mRNA is related to the expression of NCBP1.

[0108] II. Specific materials and methods

[0109] 1. Cell lines and culture

[0110] SU-DHL-4 cells were purchased from American Type Culture Collection (ATCC) with STR identification certificate. RPMI 1640 medium (Gibco, USA); fetal bovine serum (FBS, Gibco, USA); penicillin / streptomycin mixture (100x, Shanghai Solabio); the culture conditions of SU-DHL-4 cells are 10% FBS, 100 U / mL streptomycin and penicillin in 1640 medium, and the cells are cultured in a cell incubator at 37°C with 5% CO2. After virus transfection, the SU-DHL-4 cells stably expressing NCBP1 or METTL3 were subjected to puromycin pressure treatment.

[0111] 2. Virus infection and stable screening

[0112] For suspension cells, collect the suspension cells in the logarithmic growth phase and in good condition, centrifuge, resuspend and adjust the cell density to 2x10 5 Lipofectamine 2000 transfection reagent (Invitrogen, USA), Opti-MEM serum-free medium (Gibco, USA). According to the virus titer, calculate the required virus amount for MOI=50, add it to the cell culture medium, shake gently, infect in the incubator for 48 hours, centrifuge and replace with fresh complete medium. After 3 days of cell transfection, add puromycin (Beijing Solabio) to the culture medium to a final concentration of 1 μg / mL for screening of stable expression cell lines.

[0113] Lentivirus was purchased from (Shanghai Jikai). From GenePharma, obtain siRNA oligonucleotides targeting NCBP1, METTL3 and c-MYC and the corresponding control siRNA. The sequences are shown in Table 1 below. After efficiency verification, the interference fragments were sent to the company for lentivirus packaging. Lipofectamine 2000 was used to help virus transfection of SU-DHL-4 cells for 24 hours, and puromycin was used for selection.

[0114] Table 1: Lentivirus sequences

[0115]

[0116] 3. RNA extraction and real-time quantitative reverse transcription polymerase chain reaction

[0117] Total RNA was extracted by TRIzol method. TRIzol (Invitrogen, USA), 5x Evo M-MLV RT Master Mix (AG, China), 2x SYBR Green Pro Taq HS Premix (AG, China), DEPC water (Solepia, Beijing). All primers in this part were synthesized by Shanghai Biosun Biotech Co., Ltd. and dissolved in DEPC water to prepare 100 mM stock solution. After reverse transcription of RNA to cDNA, appropriate primers (see Table 2 below) were selected, SYBR Green PCR Master Mix was added to prepare a 10 ul reaction system, and Applied Biosystem 7900HT System (ABI) was used for amplification to obtain readings.

[0118] Specific process:

[0119] 5x10 6 Cells were placed in a 1.5 mL RNase-free EP tube, and 1 mL TRIzol reagent was aspirated into the tube with an RNase-free gun tip, and the solution was dissolved thoroughly to remove solid substances. The solution was equilibrated to room temperature.

[0120] 200 μL chloroform was added to the EP tube, and the tube was tightly capped and shaken vigorously for about 15 seconds. The liquid in the tube was milky white and emulsified. The liquid was allowed to stand at room temperature for 5 minutes to preliminarily separate the layers. The centrifuge was pre-cooled to 4°C, and the EP tube was centrifuged at 12000 rpm for 15 minutes to allow the liquid in the tube to separate into distinct layers. The upper clear liquid was gently aspirated and transferred to a new RNase-free EP tube. The total volume of the transferred liquid was recorded, and the white membrane layer in the middle was not aspirated.

[0121] Isopropanol was added to the EP tube at a volume ratio of isopropanol: upper clear liquid = 1:1. The tube was tightly capped and gently inverted 10 times, then placed on ice for 5-10 minutes for precipitation. Then, the tube was centrifuged at 12000 rpm in a pre-cooled centrifuge for 10 minutes, and the RNA was retained at the bottom of the tube. The supernatant was removed.

[0122] 500 μL of 80% ethanol was added to dissolve the residual isopropanol in the RNA. The tube was centrifuged at 12000 rpm for 5 minutes, and the supernatant was aspirated. The tube cap was opened, and the liquid on the tube wall and the surface of the RNA was thoroughly air-dried until the RNA precipitate was translucent. An appropriate amount of DEPC water was added, and the RNA was dissolved by blowing.

[0123] After setting blank control with DEPC water, detect RNA concentration and purity one by one to ensure that RNA purity (A260 / A280) is between 1.8-2.0.

[0124] cDNA reverse transcription

[0125] The configuration method of 10 μL RNA reverse transcription system is: 500 ng RNA sample, 2 μL reverse transcriptase 5x Evo M-MLVRT Master Mix, and the volume is made up to 10 μL with DEPC water. The system is placed in a RNase-free EP tube, mixed and centrifuged, then reacted at 37℃ for 15 minutes to fully activate the enzyme activity, and then inactivated at 85℃ for 10 seconds to obtain cDNA, which is stored at -20℃.

[0126] RT-qPCR

[0127] qRT-PCR reaction system configuration:

[0128] The configuration method of 10 μL qRT-PCR reaction system is: 1 μL cDNA template, 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM), 5 μL SYBR Green Pro Taq HS Premix, and 3.2 μL deionized water.

[0129] Sample addition: After premixing upstream and downstream primers, SYBR Green Pro Taq HS Premix and deionized water, add 9 μL of the above premix and 1 μL of cDNA to each well of the 96-well plate for PCR sample addition. Note that each sample has two duplicate wells. After sample addition, cover the plate with sealing film and centrifuge at 2000 rpm for 3 minutes.

[0130] PCR amplification: The PCR amplification conditions are set as three parts of pre-denaturation, denaturation, annealing and extension, and melting curve. Among them, the pre-denaturation condition is 95℃ for 3 minutes, 1 cycle; the denaturation condition is 95℃ for 15 seconds, the annealing condition is 60℃ for 15 seconds, and the extension condition is 72℃ for 45 seconds, which is repeated for 40 cycles; the melting curve condition is 72℃ for 3 minutes, 1 cycle.

[0131] PCR data analysis: First, analyze the melting curve of the reaction to ensure that the melting curve is a specific single peak. Then calculate the Ct value of the amplification curve, and take GAPDH as the internal reference to calculate the relative expression of the target gene according to the following formula: -ΔCt )

[0132] 2 -ΔCT = 2 -(Ct目的-Ct内参)

[0133] Table 2 RT-qPCR primer sequences

[0134]

[0135] 4. Immunoblotting experiment

[0136] 4.1 Materials: Protein extraction reagent M-PER™ Mammalian Protein Extraction Reagent (Thermo Scientific, USA), protease and phosphatase inhibitors (Beyotime, Shanghai), protein concentration determination kit (Bi Yun Tian, Shanghai), SDS-PAGE protein loading buffer (Bi Yun Tian, Shanghai), PAGE gel rapid preparation kit (Ya Zeng, Shanghai), TBS powder, electrophoresis buffer powder, electrotransfer buffer powder (Sewell, Wuhan), Tween 20 (Solebao, Beijing), nitrocellulose (NC) membrane (Millipore, USA), ECL luminescent solution (Millipore, USA), protein electrophoresis marker (Thermo Scientific, USA), skimmed milk powder for blocking (Fei Jing, Fuzhou), antibody diluent (Sewell, Wuhan).

[0137] 4.2 Preparation of reagents:

[0138] 1x electrophoresis buffer: dissolve one bag of electrophoresis buffer powder (containing 3.03 g Tris-Base, 14.4 g glycine, 1 g SDS) in distilled water, stir with a stirring rod for 5 minutes to ensure complete dissolution, and finally dilute to 1 L with distilled water.

[0139] 1x electrotransfer buffer: dissolve one bag of electrotransfer buffer powder (containing 3.03 g Tris-Base, 14.4 g glycine) in distilled water, stir with a stirring rod for 5 minutes to ensure complete dissolution, and finally dilute to 0.8 L with distilled water. Before use, add 0.2 L of methanol, mix appropriately, and use on ice.

[0140] TBST solution: dissolve TBS powder (containing 2.42 g Tris-Base, 17.56 g NaCl) in distilled water, stir with a stirring rod for 5 minutes, and dilute to 2 L with distilled water. Add 2 mL of Tween 20, stir to dissolve, and use at room temperature.

[0141] 5% skimmed milk: accurately weigh 5 g of skimmed milk powder for blocking, add to a beaker containing 100 mL of TBST solution, and stir with a stirring rod until the insoluble material is fully dissolved. Use at room temperature.

[0142] Prepare cell lysis solution: cell lysis solution: protease inhibitor: phosphatase inhibitor at a ratio of 1000:4:4, and store on ice for later use.

[0143] Lyse cells: Obtain cell pellet, 1×10⁻⁶ 6 Add 40 μL of cell lysis buffer to each cell, mix thoroughly by pipetting, and incubate on ice for 20 minutes, vortexing or pipetting for 30 seconds every 5 minutes during lysis. After lysis, centrifuge at 15,000 rpm for 15 minutes in a pre-cooled (4°C) high-speed centrifuge. Discard the residue at the bottom of the tube and retain the clear supernatant in a clean 1.5 mL EP tube.

[0144] Protein concentration determination: Prepare BCA working solution in advance (solution A:solution B = 100:2) and equilibrate to room temperature. Add 0.5 mg / mL of protein standard to a 96-well plate and set the standard concentration gradient according to the instructions. Simultaneously, add 2 μL of protein sample to 18 μL of PBS solution. Then, quickly add 200 μL of BCA working solution to each sample using a micropipette and incubate at 37°C in the dark for 30 minutes. Measure the OD value of each reaction system at 570 nm using a microplate reader. Calculate the concentration-OD570 standard curve and fitting formula based on the OD570 values ​​corresponding to different concentrations of protein standard in the wells. Use this formula and the OD570 value of the sample to calculate the corresponding protein concentration.

[0145] Protein sample denaturation: Mix the protein supernatant with 5×SDS PAGE loading buffer at a ratio of 1:4 and thoroughly mix by pipetting. Heat in a 95°C metal bath for 5 minutes to denature the protein by depolymerization.

[0146] Western blotting:

[0147] PAGE Gel Preparation: Determine the PAGE gel concentration based on the molecular weight of the target protein. Mix the lower gel solution and lower gel buffer from the kit in equal proportions to prepare a 5 mL lower gel working solution. Add 60 μL of the coagulant from the kit, mix thoroughly by pipetting, and carefully pour the mixture along one side of the gel casting plate, avoiding air bubbles, ensuring the liquid level is approximately 1 cm from the top edge of the short glass plate. Then, gently add double-distilled water to press the gel. After approximately 15 minutes, the lower gel solidifies; discard the water. Take 0.4 mL of upper gel solution and an equal volume of upper gel buffer, add 15 μL of modified coagulant, mix well, and pour into the gel casting plate, inserting a comb to avoid air bubbles. After 15 minutes, the upper gel solidifies; remove the comb to complete the PAGE gel preparation.

[0148] Protein electrophoresis: According to the protein concentration, 20 μg of protein corresponds to the sample volume. PAGE gel is inserted into the electrophoresis tank, and the electrophoresis buffer is added. In the sample well of the PAGE gel, add the denatured protein sample in order from left to right, and add protein marker on both sides of the edge sample. After constant voltage electrophoresis at 80V for 30 minutes, switch the voltage to 120V, and continue constant voltage electrophoresis for about 1 hour, until the desired band position is clearly separated.

[0149] Electrotransfer: pry open the glass plate, remove the gel, cut the gel with a gel cutting plate, and place it on the black side of the electrotransfer clamp pad with a fiber pad and filter paper. Cover the cut NC membrane on the gel, remove the bubbles, cover the filter paper and fiber pad, remove the bubbles layer by layer, close the white plate, and prepare a "sandwich". Place it in the electrotransfer tank, add cold electrotransfer buffer, and electrotransfer at 220mA constant current. Keep the electrotransfer device running at low temperature.

[0150] Blocking: completely immerse the NC membrane successfully electrotransferred in the blocking milk balanced to room temperature in advance, and place it in a shaker to slowly shake the membrane in sufficient contact with the blocking solution and floating movement. Incubate at room temperature for more than 1 hour. Wash with TBST solution for 3 times, each for 5 minutes.

[0151] Antibody incubation: place the NC membrane in the antibody incubation bag, add the diluted primary antibody solution (primary antibodies include anti-GAPDH (Abways, AB0037), anti-NCBP1 (Abeam ab154532, Proteintech 10349-1-AP), anti-METTL3 (Abeam ab195352, BETHYL A301-568A), and anti-cMYC (Proteintech 10828-1-AP, Cell Signaling Technology #9402). Then use the enzyme-labeled secondary antibody (Servicebio GB23302)), squeeze out the air in the incubation bag, seal the bag, and place it flat in the 4°C refrigerator for overnight incubation. The next morning, wash the membrane with TBST solution. Then incubate in the diluted secondary antibody solution at room temperature for 1-2 hours, and repeatedly wash with TBST solution. Prepare the ECL working solution by mixing the A and B solutions in the ECL kit in equal proportions, drop it on the NC membrane, and evenly spread it. Use the FluroChem E chemiluminescence imager to image the bands. Use ImageJ (version 1.8.0) software to calculate the gray value and semi-quantitatively analyze.

[0152] 5. Clinical specimens

[0153] Fifty DLBCL patients and 14 inflammatory lymph node tissue samples who were treated in Qilu Hospital of Shandong University between 2013 and 2019 were selected. The specimens were obtained from puncture biopsy or lymph node resection. The inflammatory control samples were lymph nodes without any malignant lesions. All clinical specimen-related experiments in this study were approved by the Ethics Committee of Qilu Hospital of Shandong University. The diagnosis was confirmed by two experienced pathologists. Immunohistochemical detection of METTL3 and c-MYC. RNA was extracted from paraffin-embedded sections, and RT-qPCR was used to evaluate the relationship between survival and NCBP1 / METTL3 / c-MYC expression.

[0154] 6. RNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples

[0155] The pathological tissue was rapidly fixed in 4-10% formalin. The fixation time was limited to 14-24 hours (the longer the fixation time, the more likely it will cause DNA fragmentation, which is not conducive to downstream experiments). The fixed tissue was thoroughly dehydrated.

[0156] Operation flow:

[0157] Removing paraffin: melting in xylene and removing;

[0158] Lysis: incubation under denaturing conditions and proteinase K;

[0159] Heating 90°C: reversing cross-linking;

[0160] Binding to the column: DNA binds to the silica gel membrane column in a suitable solution environment;

[0161] Washing: washing residual contaminants;

[0162] Elution: purifying and concentrating RNA.

[0163] Subsequent reverse transcription and RT-qPCR methods are the same as before.

[0164] 7. Cell counting reagent-8 (CCK8) cell proliferation experiment

[0165] CCK8 cell proliferation and cytotoxicity assay kit (Shanghai BestBio). To assess tumor cell proliferation, equal amounts of SU-DHL-4 cells from different intervention groups were placed into 96-well plates and incubated in a 37 °C incubator. At least 3 replicates were set up for each group, and 10 μL of CCK-8 (BestBio, Shanghai, China) was added to each well in a 37 °C incubator for 3 hours. The OD value at 450 nm was detected using a microplate reader (Thermo Scientific). The OD value of each day was plotted as a curve, and the cell proliferation curve was calculated.

[0166] 8. EdU cell proliferation experiment

[0167] According to the instructions of the iClick TM EdU Andy Fluro 647 Flow Cytometry Assay Kit.

[0168] The cell suspension was collected, washed and resuspended with PBS. The cells were incubated with 10 μM EdU-AF647 staining solution at 37 °C for 3 hours. Centrifugation, collection of cells and EP tube, addition of 100 μL fixing solution for 15 minutes. Prepare 1% BSA PBS washing solution, wash once, then add 100 μL membrane breaking solution for 15 minutes, and wash the washing solution once. Prepare iClick reaction mixture, prepare fresh, take 100 μL and add to the cells, incubate at room temperature for 30 minutes. Wash with washing solution, resuspend with 300 μL PBS solution, analyze the stained cells using a BD FACSAria III flow cytometer (BD Biosciences, San Jose, CA, USA), and analyze the data using Kaluza software.

[0169] 9. Immunohistochemical staining

[0170] To perform immunohistochemical evaluation, according to the standard protocol of the histochemical kit (ZSGB-BIO), the antibodies were selected as follows: NCBP 1 (Abeam ab154532, Proteintech 10349-1-AP), anti-METTL3 (Abeam ab195352, BETHYL A301-568A) and cMYC (Proteintech 10828-1-AP, Cell Signaling Technology #9402).

[0171] The process is as follows:

[0172] Paraffin-embedded sections were baked in an oven at 65 degrees for 30 minutes.

[0173] De-waxing: After the paraffin sections were removed from the oven, they were immediately placed in the environmentally friendly de-waxing solution for 15 minutes, then placed in a new environmentally friendly de-waxing solution for 15 minutes. Next, they were placed in anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, then rinsed with double-distilled water for 3-4 minutes, and finally dipped in PBS solution once.

[0174] Antigen retrieval: The sections were placed in a microwave oven and heated to boiling in 0.01 M sodium citrate buffer or EDTA buffer (recommended according to the antibody instructions) for 5 minutes, then heated for 10 seconds and cooled for 50 seconds, repeated for a total of 15 minutes, so that the liquid temperature reached above 95°C, and attention was paid not to boil. The sections were naturally cooled to room temperature with the buffer.

[0175] After the sections were cooled, they were rinsed with PBS for 3 minutes, a total of 3 times.

[0176] 3% H2O2 was prepared and added dropwise to the tissues. The reaction was carried out in a 37°C incubator for 15 minutes, and the main role of this step was to eliminate endogenous peroxidase activity. The tissues were rinsed with distilled water and PBS.

[0177] Blocking: 5% goat serum working solution was added dropwise to the tissues, completely covering the tissue surface, to block non-specific antigens, and the tissues were incubated in a 37°C incubator for 30 minutes.

[0178] Primary antibody incubation: The serum on the sections was shaken off, 100 μL of diluted primary antibody (see above for specific antibodies) was added, and the sections were placed in a wet box and incubated at 4°C overnight. The next day, the sections were incubated at 37°C for 30 minutes. PBS was used to rinse the sections for 5 minutes, a total of 3 times.

[0179] Secondary antibody incubation: Biotinylated secondary antibody working solution was added to completely cover the tissues. The wet box was incubated at room temperature for 20 minutes. The liquid was poured off, and the tissues were soaked in PBS for 3 minutes each time, a total of 3 times. Streptavidin-horseradish peroxidase working solution was then used to cover the tissues, which were incubated at room temperature for 20 minutes and then rinsed with PBS until clean.

[0180] DAB staining: DAB staining working solution was added dropwise, and the tissues were observed under a microscope in the dark. When the positive cells were significantly stained brown-yellow and the background was clean, the staining was stopped, and the sections were placed in water.

[0181] Counterstaining: Hematoxylin staining solution was used to stain the sections for 1-2 minutes, and then the sections were rinsed with water. If the counterstaining was too deep, the sections could be decolorized with 1% hydrochloric acid alcohol for 2-5 seconds, and then with 1% ammonia water for 10-25 seconds.

[0182] Dehydration and mounting: The sections were passed through the following reagents in order: 80% ethanol (30 seconds), 90% ethanol (1-2 minutes), 95% ethanol (2 minutes), absolute ethanol (5 minutes), absolute ethanol (5 minutes), environmental de-waxing solution (5 minutes), environmental de-waxing solution (5 minutes). The sections were mounted with neutral resin.

[0183] The slides were observed and photographed under an optical microscope (Nikon, Ni-U).

[0184] 10. RNA-seq experiment and bioinformatics analysis

[0185] RNA extraction kit TransZol Up Plus RNA Kit (Trans, USA). RNA purification used RNase-Free DNase Set (QIAGEN, Germany), and the machine was completed by Huada Gene Company. According to the requirements of the sequencing sample, RNA extraction was performed on NC and shNCBP1 SU-DHL-4. The raw data was filtered by quality. Then the cleaned data was mapped to GRCh38 for further analysis. The R package DESeq2 (v 1.20.0) was used to obtain differentially expressed transcripts from the original read count, with a false discovery rate (FDR) cutoff of 0.05. Custom scripts were used in R to compare the exon counts of differentially up-regulated and down-regulated transcripts. The expression differences of NCBP1, METTL3, and cMYC in DLBCL were obtained from the GEPIA public database (http: / / gepia.cancer-pku.cn / index.html).

[0186] 11. Dot-blot experiment

[0187] Dot-blot experiment was used to detect the m6A modification level of cell RNA.

[0188] RNA was extracted using TRIzol, and the RNA concentration was accurately measured. Then 2 μL of RNA sample was dropped on the positive side of the positively charged nylon transfer membrane, and after drying, the nylon transfer membrane was subjected to two times of ultraviolet crosslinking. The nylon membrane was blocked with blocking solution for 1 hour. Then it was placed in TBST solution and washed 3 times. Then it was incubated with diluted m6A antibody (ab195352, 1:1000) at 4°C overnight. The next day, the nylon membrane was washed with TBST 3 times, 5 minutes each time. Then it was placed in the corresponding secondary antibody solution for 1 hour. TBST was washed 3 times, 8 minutes each time. Then the film was developed and photographed using ECL luminescent solution.

[0189] 12. Clonogenic assay

[0190] To detect the effect of NCBP1 expression on the colony formation ability of DLBCL cells, the logarithmic growth phase of SU-DHL-4 cells stably transfected were taken, resuspended using semi-solid medium, vortexed and mixed, and then inoculated in a 6-well plate at a density of 2x10 5 / mL, and a DMSO control group was set up, with attention paid to not generating bubbles. Water was added around the cell-inoculated well plate to maintain humidity, and the well plate was placed in a humidified constant-temperature incubator and left undisturbed for 7 days. After 7 days, the formation of colonies was observed under a microscope at 400x magnification, and the total number of colonies with more than 50 cells in a single colony was counted.

[0191] 13. m6A methylation RNA immunoprecipitation (MeRIP) experiment

[0192] Cell preparation

[0193] About 1x10 7 cells of lentivirus stably transfected SU-DHL-4 cells and corresponding control cells were collected, washed 3 times with PBS, and then centrifuged and dissolved in 2 mL of TRIzol reagent (Invitrogen).

[0194] RNA extraction

[0195] 200 μL of chloroform was added to each 1 mL of TRIzol, and the mixture was shaken vigorously for 15 seconds. After standing for 3 minutes, the mixture was centrifuged at 13000 g for 10 minutes in a 4°C pre-cooled centrifuge. The upper clear liquid was aspirated, and an equal amount of isopropanol was added. The mixture was precipitated at -20°C for 2 hours. The RNA precipitate was obtained by centrifuging the mixture at 13000 g for 10 minutes in a 4°C pre-cooled centrifuge, and the supernatant was discarded. 1 mL of 75% alcohol was immediately added, and the mixture was mixed well by inverting. The mixture was centrifuged at 13000 g for 5 minutes in a 4°C pre-cooled centrifuge, and the supernatant was discarded. The mixture was left to dry in a flow-through environment for 15 minutes, and 200 μL of RNase-free water was added to dissolve the RNA precipitate.

[0196] RNA fragmentation

[0197] Add 440 μL fragmentation reagent to the sample, react at 94°C for 2 min to cleave the RNA into fragments of about 300 nt, and then add EDTA to terminate the reaction. Add 2 μL glycogen, 60 μL sodium acetate, and 1.2 mL ethanol, and precipitate at -20°C for 2 h. Centrifuge at 13000g at 4°C for 30 min, discard the supernatant, add 1 mL of pre-cooled 75% ethanol, invert to mix, centrifuge at 13000g at 4°C for 5 min, discard the supernatant, repeat the centrifugation and aspiration of the supernatant, and dry in air for 10 min. Add 850 μL IP buffer 1 and 4 μL RNase inhibitor to dissolve the RNA sample. Take 50 μL of the RNA sample as the input, and divide the remaining sample into 400 μL / tube as the IP group and the IgG group.

[0198] Immunoprecipitation

[0199] In the IP group, add 20 μL of IP buffer 2 and 4 μg of m6A antibody (ab151230). In the IgG group, add the same amount of IP buffer 2 and IgG antibody, and incubate at 4°C for 4 h.

[0200] Protein A / G magnetic bead washing and blocking

[0201] Add 40 μL of Protein A / G magnetic beads and 500 μL of IP buffer 3 to the EP tube, and blow several times. Use a magnetic stand to adsorb the magnetic beads, and remove the buffer. Add BSA solution, and incubate at 4°C for 4 h to block the magnetic beads. Adsorb the magnetic beads on the magnetic stand, aspirate the supernatant, and wash once with IP buffer 3, then divide 200 μL of IP buffer 3 into two tubes.

[0202] Protein A / G and antibody binding

[0203] In the IP group and the IgG group, add the prepared Protein A / G magnetic beads, and incubate at 4°C for 1 h. Adsorb the magnetic beads on the magnetic stand, aspirate the supernatant, and wash three times with IP buffer 3, then add 200 μL of elution buffer and 2 μL of proteinase K, respectively, and digest at 55°C for 30 min. Adsorb the magnetic beads on the magnetic stand, and transfer the supernatant to a new RNase-free EP tube.

[0204] RNA extraction

[0205] Input sample was added with 150 μL eluent. Three tubes of sample were added with 200 μL RNA extraction liquid (phenol: chloroform: isopropyl alcohol = 25:24:1) and mixed well by rapid inversion for 15 seconds. The uppermost liquid was taken after running the pre-cooled high-speed centrifuge at 13000g for 10 minutes, 1 μL Glycogen, 20 μL sodium acetate and 400 μL ethanol were added and mixed well. The RNA was precipitated in the refrigerator at -20°C for about 2 hours, and then placed in the high-speed centrifuge at 4°C with the parameter set at 13000g for 30 minutes. The liquid was discarded and the precipitated part was left. 1 mL of pre-cooled 75% ethanol was added and mixed by inversion, and then placed in the high-speed centrifuge at 4°C with the parameter set at 16000g for 5 minutes. The liquid was discarded and the precipitated part was left, and the centrifugation was repeated once to completely remove the residual liquid. The tube wall was dried in a ventilated environment for about 15 minutes. 20 μL of RNA-dissolving water was used to dissolve the lower RNA of the tube, and it was stored on ice.

[0206] RNA quantitative detection

[0207] DNA quantification was performed by qRT-PCR using designed specific PCR primers (as shown in Table 3 below). The reaction system and program conditions refer to the RT-qPCR experimental method.

[0208] Table 3: c-MYC primer sequences

[0209]

[0210] 14. RNA stability experiment

[0211] The SU-DHL-4 cells in the logarithmic growth phase and in good condition were inoculated in a 6-well plate at a density of 1-2 x 10 5 / mL, 2 mL per well, and then 5 μg / mL of actinomycin D (Sigma) was added at 6 hours, 4 hours, 2 hours and 0 hours before the cells were harvested to block the synthesis of RNA. The cells were collected, the RNA was extracted by TRIzol method, and the relative expression amount of METTL3 mRNA was detected by RT-qPCR. The linear fitting was performed according to the time and the expression amount of METTL3 mRNA by using Graphpad Prism 8 software, and the fitting curve and half- decay time (t1 / 2) were calculated.

[0212] 15. Statistical method

[0213] For normally distributed experimental data, mean ± standard deviation was used to describe, and t-test was used to analyze the differences between groups. For non-normally distributed experimental data, median ± interquartile range was used to describe, and non-parametric test was used to analyze the differences between groups. Survival analysis used Kaplan-rank test method. All data were repeated three times or more. P<0.05 was considered statistically significant.

[0214] III. Experimental results

[0215] 1. NCBP1 is overexpressed in DLBCL patients, and is related to poor prognosis of patients

[0216] By querying in the TCGA database, NCBP1 high expression was related to low survival rate and short DFS time of DLBCL. In order to verify the relationship between NCBP1 expression and prognosis of DLBCL patients in the patients admitted, first observe the expression of NCBP1 in DLBCL in the GEPIA database, and find that the expression of NCBP1 in DLBCL tissue is higher than that in normal tissue (P<0.05) Figure 1 A).

[0217] Then, the result was verified by clinical samples, and the difference in expression of NCBP1 in the two groups was compared by extracting RNA from 50 cases of DLBCL tissue samples and 14 cases of inflammatory lymph node samples diagnosed and treated in Qilu Hospital of Shandong University. The results showed that the expression of NCBP1 in tumor tissue was significantly higher than that in inflammatory lymph nodes (P=0.0029) Figure 1 B).

[0218] In order to further verify the effect of high expression of NCBP1 on the prognosis of DLBCL patients, Kaplan-Meier analysis was used to compare the difference in overall survival rate between 50 cases of DLBCL patients with high expression and low / medium expression of NCBP1 (cut off=50%) collected in Qilu Hospital of Shandong University Figure 1 C), it can be found that the survival time of DLBCL patients with high expression of NCBP1 is significantly shorter than that of patients with low / medium expression (P<0.001).

[0219] In summary, it is proved that NCBP1 is overexpressed in DLBCL patients, and is related to poor prognosis of patients.

[0220] 2. In vitro experiment verifies that NCBP1 promotes the proliferation of DLBCL cells

[0221] To investigate whether NCBP1 affects the proliferation of DLBCL cells, the DLBCL cell line SU-DHL-4 was selected as the experimental cell line. SU-DHL-4 cells transfected with the virus and stably expressing NCBP1 (upregulated or downregulated) were cultured to the logarithmic growth phase. mRNA and protein were extracted, and RT-qPCR and Western blot were performed to analyze the NCBP1 regulatory efficiency. Figure 2 After successful transfection (A, B), the cells were seeded in 96-well plates, and the effect of NCBP1 on SU-DHL-4 cell viability was detected using the CCK-8 assay. Figure 2 C). The results showed that, compared with the control transfection, downregulation of NCBP1 significantly inhibited cell proliferation at 24, 48, 72 and 96 h (P<0.05). Conversely, transfection with lentivirus overexpressing NCBP1 significantly increased tumor cell proliferation (P<0.05).

[0222] Furthermore, flow cytometry analysis of EdU proliferation yielded similar results to the CCK-8 assay, indicating that overexpression / silencing of NCBP1 in DLBCL cells did indeed increase / decrease DLBCL cell proliferation. Figure 2 D).

[0223] Furthermore, in semi-solid culture experiments, interfering with NCBP1 expression inhibited the formation of SU-DHL4 cell communities, while NCBP1 overexpression resulted in larger cell communities. Figure 2 E).

[0224] In conclusion, NCBP1 significantly promoted the proliferation of DLBCL cells in vitro.

[0225] 3. NCBP1 affects the expression of METTL3 in DLBCL cells.

[0226] To investigate how NCBP1 affects DLBCL proliferation, RNA-seq was performed on SU-DHL-4 cells transfected with NCBP1 interference virus. The results showed that 223 genes were upregulated and 163 genes were downregulated in shNCBP1-transfected SU-DHL-4 cells. Among these downregulated genes, METTL3 was one of them, exhibiting significant differential expression in the differential gene volcano plot. Figure 3 A). Then, RT-qPCR was used to demonstrate that, compared with the NC group, the expression of METTL3 mRNA in shNCBP1-transfected SU-DHL-4 cells was significantly reduced ( Figure 3 B). These results suggest that METTL3 may be a downstream target of NCBP1 in DLBCL.

[0227] Furthermore, linear regression analysis using DLBCL tumor tissues from the GEPIA database revealed a significant positive correlation between METTL3 and NCBP1, exhibiting a moderate positive correlation (r = 0.7; P < 0.0001). Figure 3 C). Meanwhile, in the collected clinical DLBCL tissue samples, elevated NCBP1 mRNA expression was also found in patients with previously elevated METTL3 expression, and a positive correlation was found between the two. Figure 3 D).

[0228] Further validation revealed that NCBP1 specifically binds to the Cap structure of precursor RNA and regulates gene expression. RNA half-life experiments were conducted by incubating cells with actinomycin-D to block RNA synthesis, and then the time required for METTL3 mRNA levels to decrease to 50% of their initial levels was measured to calculate RNA stability. The results showed that NCBP1 improves the stability of METTL3 mRNA. Figure 3 E).

[0229] In conclusion, NCBP1 enhances the expression of METTL3 in DLBCL cells.

[0230] 4. NCBP1 affects the proliferation of DLBCL cells via METTL3.

[0231] Before investigating whether NCBP1 promotes DLBCL proliferation through METTL3, the effect of METTL3 on DLBCL proliferation was first studied. (Source: TCGA database) Figure 4 A) and our clinical samples ( Figure 4 Data from B) showed that METTL3 mRNA was highly expressed in DLBCL. Similarly, immunohistochemical staining ( Figure 4 C) This demonstrates high METTL3 protein expression in tumor tissues. Furthermore, Kaplan-Meier analysis compared the overall survival rates of 50 DLBCL patients treated at Qilu Hospital of Shandong University, showing a significant difference between DLBCL patients with high METTL3 expression and those with low / moderate METTL3 expression (cutoff = 50%). Figure 4 D), the survival of DLBLC patients with high METTL3 expression was significantly lower than that of patients with low / moderate METTL3 expression (P<0.001). DLBLC cells were transfected with lentiviruses that overexpress or underexpress METTL3, and the METTL3 regulatory efficiency was verified by RT-qPCR and Western blotting. Figure 4 E, F). CCK-8 assay results showed that interfering with METTL3 expression inhibited DLBCL cell proliferation, while increasing METTL3 expression significantly promoted tumor cell proliferation.Figure 4 G)。

[0232] All results prove the significant influence of METTL3 on DLBCL.

[0233] It is also important that the study found that interfering with the expression of METTL3 can partially reverse the effect of NCBP1 overexpression on promoting cell proliferation in DLBCL Figure 4 H) Similarly, flow cytometry EdU detection also shows that METTL3 can effectively reverse the enhancement of NCBP1 overexpression on DLBCL cell proliferation Figure 4 I).

[0234] In summary, the above results prove that the influence of NCBP1 on DLBCL proliferation is exerted through METTL3.

[0235] 5. METTL3 promotes the proliferation of DLBCL cells by increasing the expression of c-MYC

[0236] Through literature review, it is known that many literatures report that c-MYC is a modification target of METTL3 m6A and plays an important role in many tumor formation processes. In order to study whether METTL3 mediates DLBCL cell proliferation through c-MYC, the present application first studies the mRNA expression data from the GEPIA dataset and our clinical samples.

[0237] The results show that by linear regression analysis, the expression of c-MYC is significantly positively correlated with METTL3 in DLBCL Figure 5 A, B), and immunohistochemical staining shows that the expression level of c-MYC protein in DLBCL tissues is high Figure 5 C).

[0238] Then, by down-regulating the expression of c-MYC in DLBCL cell line SU-DHL4 cells, the down-regulation efficiency is verified Figure 5 D, E), CCK-8 Figure 5 F, G) and EdU verification Figure 5 H, I) infection of c-MYC can also reverse the promotion of DLBCL cell proliferation caused by METTL3 overexpression.

[0239] In summary, the research results show that in DLBCL, c-MYC is also an important target of METTL3 to exert the function of promoting tumor proliferation, and may also play an important role in the promotion of DLBCL cell proliferation mediated by NCBP1.

[0240] 6. NCBP1 affects its m6A modification function on c-MYC mRNA through METTL3

[0241] METTL3 has been reported to promote RNA methylation. To investigate whether NCBP1 enhances c-MYC RNA m6A methylation levels by increasing METTL3 expression, this invention performed an m6Adot blot experiment. The results showed that NCBP1 expression levels were correlated with c-MYC m6A methylation levels in SU-DHL-4 cells. Figure 6 A). Then, this invention utilizes the m6A sites recorded in the RMBasev2.0 database (RNA modification Base, http: / / rna.sysu.edu.cn / rmbase / ) to analyze the modification of m6A to obtain the DRACH motif ( Figure 6 B). In addition, RNA fragments near the m6A site were summarized, and three regions with high levels of m6A modification were selected from the CDS and 3'UTR near the 5'UTR and 3'UTR of the c-MYC mRNA transcript. Figure 6 C). Finally, three pairs of primers were designed for the three regions to perform MeRIP-qPCR.

[0242] The results showed that in SU-DHL-4 cells, when NCBP1 was overexpressed / knocked out, the number of m6A modifications in the CDS near the c-MYC mRNA3'UTR was significantly increased / decreased. Figure 6 D) indicates that NCBP1 modifies the CDS region near the 3'UTR of c-MYC mRNA through the RNA methylation function of METTL3, thereby promoting tumor proliferation.

[0243] IV. Conclusion

[0244] In summary, this invention elucidates the crucial role of NCBP1 in DLBCL tumor proliferation, suggesting it may be a novel diagnostic and prognostic biomarker for DLBCL and a potential therapeutic target. Furthermore, this invention reveals the regulatory mechanism of METTL3 in DLBCL, enriching our understanding of the m6A modification system in DLBCL and possessing significant clinical application value.

[0245] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of quantitative detection reagents for NCBP1 in the preparation of reagents for auxiliary diagnosis and / or prognosis of diffuse large B-cell lymphoma.

2. The application as described in claim 1, characterized in that, The test samples are lesion tissue samples or suspected lesion tissue samples from patients.

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