Use of u2surp in the preparation of a therapeutic drug for diffuse large b-cell lymphoma

Abstract

The application belongs to the field of medicine and biotechnology, and particularly relates to application of U2SURP in preparation of a diffuse large B-cell lymphoma (DLBCL) treatment drug. The application takes U2SURP protein as a molecular marker of DLBCL, detects U2SURP expression in DLBCL tissues through immunohistochemistry (IHC), and establishes a scoring system combining staining intensity and positive cell proportion to evaluate the prognosis of patients. Experimental results show that U2SURP knockdown can effectively inhibit DLBCL cell proliferation, induce apoptosis and cause G2 / M phase cell cycle arrest. The application has dual values of prognosis evaluation and precise treatment target, not only provides a new prognosis evaluation scheme based on IHC for DLBCL, but also provides an efficient and safe treatment strategy based on variable splicing factor targeting intervention, and has a good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medical biotechnology, and particularly relates to application of U2SURP in preparation of a diffuse large B-cell lymphoma treatment drug. BACKGROUND

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive non-Hodgkin lymphoma in adults. DLBCL has great heterogeneity in clinical and biological aspects. In the past, it was classified into two molecular subtypes, GCB (germinal center B-cell-like) and ABC (activated B-cell-like), but there are still significant differences in prognosis within the subtypes. How to accurately assess prognosis and explore new targeted treatment strategies has become a key challenge to improve the treatment effect of DLBCL.

[0003] U2SURP (U2 snRNP-associated SURP motif-containing protein) is one of the core components of the U2 small nuclear ribonucleoprotein (U2 snRNP) complex, and participates in the regulation of alternative splicing of precursor mRNA through specific SURP domains. In various solid tumors and hematological tumors, abnormalities in alternative splicing have been shown to be closely related to tumorigenesis, progression and drug resistance. However, current research on the expression pattern and function of U2SURP in tumors is still in its infancy, and the mechanism of action in DLBCL has not been systematically elucidated. SUMMARY

[0004] In view of the problems and deficiencies in the prior art, the application aims to provide the application of U2SURP in the preparation of a diffuse large B-cell lymphoma treatment drug. The U2SURP inhibition strategy provided by the application has good effect and safety in the treatment of DLBCL, and provides a new direction for the prognosis evaluation and variable splicing targeted treatment of diffuse large B-cell lymphoma.

[0005] The application is achieved by the following technical solutions:

[0006] Application of U2SURP in the preparation of a diffuse large B-cell lymphoma treatment drug.

[0007] The amino acid sequence of the U2SURP is:

[0008]

[0009] Further, the drug inhibits tumor growth by inhibiting U2SURP expression.

[0010] Further, the drug inhibits diffuse large B-cell lymphoproliferation and induces apoptosis by inhibiting U2SURP expression.

[0011] Further, the drug inhibits tumor cell cycle G2 / M arrest by inhibiting U2SURP expression.

[0012] The present application also provides a kit for evaluating the prognosis of diffuse large B-cell lymphoma, which comprises a reagent for detecting U2SURP expression.

[0013] Further, the kit further comprises rabbit-derived polyclonal anti-U2SURP antibody, HRP-labeled goat anti-rabbit secondary antibody and DAB color developing reagent.

[0014] The present application also provides a drug for treating diffuse large B-cell lymphoma, which comprises a substance for inhibiting U2SURP expression and / or function.

[0015] Further, the substance for inhibiting U2SURP expression and / or function comprises siRNA specifically targeting U2SURP gene or shRNA specifically targeting U2SURP gene.

[0016] Further, the sequence of the siRNA specifically targeting U2SURP gene is shown in SEQ ID No. 2~5.

[0017] Further, the core recognition sequence of the shRNA specifically targeting U2SURP gene is shown in SEQ ID No. 12.

[0018] Beneficial effects

[0019] The present application detects the expression of U2SURP protein in tumor tissues of 121 clinical DLBCL patients by IHC, finds that U2SURP is highly expressed in DLBCL tissues, and the expression level is significantly related to the prognosis of patients. The prognosis of patients with high expression of U2SURP is significantly poorer than that of patients with low expression, which indicates that U2SURP has potential prognostic evaluation value. By establishing a scoring standard by IHC, U2SURP can be used as a new prognostic evaluation marker; at the same time, knocking down U2SURP expression by siRNA can inhibit DLBCL cell proliferation, induce apoptosis and cause G2 / M phase arrest, and in the in vivo tumor transplantation model, U2SURP knockdown significantly inhibits tumor growth without affecting normal tissues of mice.

[0020] The application firstly discovers that U2SURP protein is highly expressed in DLBCL tumor tissues and is related to the clinical prognosis of patients, proposes a U2SURP typing method based on an IHC scoring system, and fills the gap in the field of DLBCL hierarchical evaluation; the application effectively knocks down the expression of U2SURP by using siRNA and shRNA technology, significantly inhibits the proliferation of DLBCL cells, induces apoptosis and cell cycle arrest, and verifies the therapeutic potential in cells and animal models.

[0021] The U2SURP inhibition strategy provided by the application has good effect and safety in the treatment of DLBCL, and U2SURP has the dual values of a prognosis evaluation marker and a precise treatment target, provides a new direction for the prognosis evaluation and variable splicing targeted treatment of diffuse large B-cell lymphoma, and has a broad clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a U2SURP gene analysis result graph of tumor tissues and normal tissues in the application by TCGA and GTEx databases;

[0023] Figure 2 is a survival curve graph of the relationship between U2SURP expression and overall survival rate of DLBCL patients in the application by GEO database (GSE10846);

[0024] Figure 3 is a tissue chip graph of IHC detection of U2SURP protein expression in the application;

[0025] Figure 4 is a survival curve graph of the relationship between U2SURP expression and overall survival rate of DLBCL patients in the application according to the total score of IHC staining;

[0026] Figure 5 is a tissue chip graph of IHC detection of Ki67 protein expression in the application;

[0027] Figure 6 is a curve graph of the relationship between U2SURP expression and Ki67 expression in the application according to the total score of IHC staining;

[0028] Figure 7 is a result graph of real-time fluorescent quantitative polymerase chain reaction for detecting the mRNA expression level of U2SURP after DLBCL cell knockdown in the application;

[0029] Figure 8 is a result graph of Western blotting for detecting the protein expression level of U2SURP after DLBCL cell knockdown in the application;

[0030] Figure 9is a graph of the CCK-8 experiment in the present application for detecting the influence of U2SURP knockdown on the proliferation ability of DLBCL cells;

[0031] Figure 10 is a flow cytometry graph and statistical analysis column chart of the influence of U2SURP knockdown on the apoptosis induction of DLBCL cells in the present application;

[0032] Figure 11 is a flow cytometry graph and statistical analysis column chart of the influence of U2SURP knockdown on the cell cycle distribution of DLBCL cells in the present application;

[0033] Figure 12 is a result graph of the real-time fluorescent quantitative polymerase chain reaction for detecting the mRNA expression level of U2SURP after transfection of the U2SURP shRNA recombinant lentiviral vector in the present application;

[0034] Figure 13 is a result graph of the Western blot for detecting the protein expression level of U2SURP after transfection of the U2SURP shRNA recombinant lentiviral vector in the present application;

[0035] Figure 14 is a result graph of the subcutaneous transplantation tumor of NSG mice in different groups in the present application;

[0036] Figure 15 is a growth curve graph of the transplantation tumor of NSG mice in different groups in the present application;

[0037] Figure 16 is a body weight curve graph of NSG mice in different groups in the present application;

[0038] Figure 17 is a hematoxylin-eosin staining graph of the whole body organs of NSG mice in the present application. DETAILED DESCRIPTION

[0039] The present application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not intended to limit the present application.

[0040] Unless otherwise specified, the following examples and comparative examples are parallel tests, and the same processing steps and parameters are used.

[0041] Example 1

[0042] Evaluation of the value of U2SURP in the prognosis of DLBCL based on bioinformatics analysis

[0043] (1) Data sources: Transcriptome sequencing (RNA-Seq) data and corresponding clinical information of DLBCL patients were downloaded from the TCGA database of the Cancer Genomics Data Analysis Platform (UCSC Xena platform). RNA-Seq data of various tissues of normal populations were downloaded from the GTEx (Genotype-TissueExpression) database. The GSE10846 dataset, which contains gene expression matrices and follow-up survival information of 388 DLBCL patients, was downloaded from the GEO database.

[0044] (2) Data preprocessing and differential expression analysis: The expression matrices of TCGA and GTEx were read and annotated using R language (R version 4.4.3) (annotated according to Ensembl gene ID). TCGA tumor samples and GTEx normal samples were merged, and normalization and differential expression analysis were performed using the DESeq package. The expression values ​​of the U2SURP gene in the tumor group and normal group were extracted, and the expression difference was evaluated using the Wilcoxon rank test.

[0045] (3) Survival analysis: Survival analysis was performed on the GSE10846 dataset using R language (survival and survminer packages). The 388 patients were divided into a "high expression group" and a "low expression group" based on the median U2SURP expression. Survival curves (Kaplan-Meier) were constructed, and the overall survival (OS) of the two groups was compared using the log-rank test. Hazard ratios (HR) and 95% confidence intervals were calculated, and p-values ​​were extracted to evaluate the correlation between U2SURP expression level and prognosis.

[0046] Figure 1 A graph showing the differential expression of U2SURP gene between DLBCL tumor tissues and normal tissues in the TCGA / GTEx database; Figure 2 The image shows the Kaplan-Meier survival curves (U2SURP expression versus overall survival (OS) in the GSE10846 cohort. Figure 1 As shown, U2SURP expression was significantly upregulated in DLBCL tissues (P<0.001), indicating that it is a differentially expressed gene. Figure 2 The results showed that after grouping by median as the threshold, the overall survival (OS) of patients in the high expression group was significantly lower than that in the low expression group (P=0.046), leading to the conclusion that U2SURP has prognostic predictive value.

[0047] Example 2

[0048] Evaluation of the prognostic significance of U2SURP in clinical samples of DLBCL using tissue microarray-based IHC assay

[0049] (1) Sample source and tissue chip preparation: Paraffin-embedded tissue specimens from 121 patients with DLBCL diagnosed by the Department of Pathology of the Second Affiliated Hospital of Suzhou University from 2017 to 2021 were selected. All patients were not treated with any systemic therapy at the time of diagnosis. After the above 121 paraffin tissues were routinely dewaxed and alcohol gradient dehydrated, tissue microarray was prepared according to the tissue chip technology, the sampling round core diameter was 1.5 mm, and it was ensured that each sample contained at least two repeated cores.

[0050] (2) IHC staining and marker detection:

[0051] U2SURP protein detection: The expression of U2SURP protein in DLBCL tissue samples was detected by immunohistochemistry (IHC) for the prognosis evaluation of DLBCL. The known optimal conditions were used (primary antibody: rabbit polyclonal anti-U2SURP antibody from Invitrogen, 1:2000 dilution; secondary antibody: HRP-labeled goat anti-rabbit antibody from Proteintech, 1:500 dilution), and the steps of dewaxing, antigen repair, blocking, primary antibody incubation (4°C overnight), secondary antibody incubation (37°C, 45 min), DAB color development and re-staining were performed for staining. U2SURP staining was mainly located in the nuclei of tumor cells.

[0052] The kit used for immunohistochemical detection includes:

[0053] (1) Rabbit polyclonal anti-U2SURP antibody;

[0054] (2) HRP-labeled goat anti-rabbit secondary antibody;

[0055] (3) DAB color reagent.

[0056] The immunohistochemical detection method includes the following steps:

[0057] Dewaxing: sequentially immerse the sections in xylene (10 min), absolute ethanol (5 min), 95% ethanol (5 min), 70% ethanol (5 min); antigen retrieval: place the sections in Tris-EDTA antigen retrieval solution at pH 9.0, heat in a water bath at 95 °C for 15 min; blocking: wash with PBS for 3 times (5 min each time), add 1% BSA and incubate at room temperature for 2 h; primary antibody incubation: add U2SURP antibody (1:2000 dilution, 50 μL), incubate overnight at 4 °C, take out the sections on the next day and incubate at room temperature for 45 min; secondary antibody incubation: wash with PBS for 3 times, add secondary antibody (1:500 dilution, 50 μL), incubate at 37 °C for 45 min; color development and counterstaining: wash with PBS for 3 times, incubate with 1×DAB color developing solution at room temperature for 10 min, wash with PBS for 3 times, sequentially perform hematoxylin counterstaining, hydrochloric acid ethanol differentiation, blueing, transparency, and mounting, air dry at room temperature overnight, and take a photo on the next day.

[0058] Ki67 protein detection: perform staining according to the same IHC procedure using Ki67 monoclonal antibody (1:500 dilution) to evaluate cell proliferation activity.

[0059] (3) Scoring method and statistical analysis: staining intensity score: 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong); positive cell proportion score: 0 (<10%), 1 (10-25%), 2 (26-50%), 3 (51-75%), and 4 (76-100%); add the two scores to obtain the final IHC score: score <6 is defined as the U2SURP low expression group, and ≥6 is defined as the U2SURP high expression group. Use Log-rank test (Mantel-Cox) to compare the overall survival (OS) difference between the U2SURP high and low expression groups. Use Spearman rank correlation coefficient method to evaluate the correlation between the U2SURP score and the Ki67 positive rate in the same sample.

[0060] Figure 3 Figure for the IHC staining results of U2SURP protein in the tissue chip of 121 cases of DLBCL; Figure 4 Survival curve after grouping based on IHC score (staining intensity x positive proportion). As shown in Figure 3 , U2SURP mainly showed strong or moderate positive staining with nuclear localization; Figure 4 Among them, the survival of patients in the high expression group with an IHC score ≥6 was significantly poorer than that in the low expression group with a score <6 (P<0.0001), and it was concluded that U2SURP typing based on IHC could effectively distinguish prognosis.

[0061] Figure 5IHC staining plot of Ki67 proliferation marker in the same tissue chip; Figure 6 Correlation scatter plot of U2SURP IHC score and Ki67 positive rate. As shown in Figure 5 Ki67 positive cells increased significantly in the high U2SURP expression group; Figure 6 showed a significant positive correlation (P<0.0001), leading to the conclusion that U2SURP expression level was positively correlated with tumor proliferation activity.

[0062] Example 3

[0063] Knockdown of U2SURP based on siRNA transient transfection and detection of knockdown efficiency

[0064] (1) Cell collection and electroporation transfection: logarithmic growth phase SU-DHL-2, SU-DHL-8 and Farage human B lymphoma cells (Farage cells) were transferred to centrifuge tubes, centrifuged at 1000 rpm for 3 min at 4 ℃, and the supernatant was discarded. Resuspend the cells in 1 mL of pre-cooled PBS, centrifuge again at 1000 rpm for 3 min at 4 ℃, repeat the washing once and discard the supernatant. Add 90 µL of electroporation resuspension buffer and 10 µL of siRNA working solution to the cell pellet, mix gently. Transfer the mixture to an electroporation-specific microtube, and use an electroporation instrument (parameters: 1200 V, 30 ms, 2 pulses) to perform electroporation on each cell line. Immediately after transfection, add 500 µL of complete medium, stand for 10 min, then transfer the cells to a 6-well plate for culture and incubation at 37 ℃, 5 % CO2. The siRNA sequences and control siRNA used are as follows:

[0065] siRNA-1: sense strand 5'-GAGCCAAACUUCGUGAAAUTT-3' (SEQ ID No. 2), antisense strand 5'-AUUUCACGAAGUUUGGCUCTT-3' (SEQ ID No. 3);

[0066] siRNA-2: sense strand 5'-GGGAGAUUCUCCAACUAAATT-3' (SEQ ID No. 4), antisense strand 5'-UUUAGUUGGAGAAUCUCCCTT-3' (SEQ ID No. 5);

[0067] Control siRNA: sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID No. 6);

[0068] antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID No. 7).

[0069] (2) Total RNA extraction: 48 h after transfection, 1-5 x 10 7 2+

[0070] (3) RNA reverse transcription: configure the reverse transcription reaction system on ice, RNA sample 1 μg, 5 x g DNA digestion mixture (DNA Digester Mix) 3 μL, and anhydrous ribozyme water to make up to 15 μL. Incubate at 42°C for 2 min to remove gDNA. Add reverse transcription premix (containing heat-stable reverse transcriptase, reaction buffer, dNTP and Mg 2+

[0071] (4) Real-time fluorescent quantitative PCR (qRT-PCR): configure a 20 μL reaction system on ice, forward primer (10 μM) 0.4 μL, reverse primer (10 μM) 0.4 μL, cDNA template 1 μL, premix (SYBR Green Mix (2 x)) 10 μL, and anhydrous ribozyme water to make up to 20 μL. Program setting: 95°C for 30 s; cycle 40 times: 95°C for 5 s, 60°C for 30 s; melting curve stage. Each group is set in triplicate, and the relative expression is calculated by the 2ΔΔCt method, with GAPDH as the internal reference.

[0072] The primers involved in the present application are as follows:

[0073] U2SURP F: 5'-TTCAAGAGGAACGTGATGAGAGA-3' (SEQ ID No. 8);

[0074] ​U2SURP R: 5'-CGTCCATAGAACGACGCTG-3' (SEQ ID No. 9);

[0075] GAPDH F: 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID No. 10);

[0076] GAPDH R: 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID No. 11).

[0077] (5) Protein extraction: After 48 h of transfection, the cell suspension was collected and centrifuged at 1000 rpm at 4°C for 3 min, and the culture medium was discarded. The cells were washed with 4°C pre-cooled PBS for 3 times, and the supernatant was discarded. RIPA lysis buffer containing 10 μL protease inhibitor cocktail was added to 1 mL, and after mixing well, the cells were lysed on ice for 30 min, and mixed well every 10 min. The supernatant was collected by ultracentrifugation at 12 000 rpm at 4°C for 10 min, and stored at -80°C for use.

[0078] (6) Western Blot detection: After mixing the protein sample with an equal volume of loading buffer and boiling at 95°C for 5 min, the prepared gel plate and glass plate were vertically mounted on the electrophoresis frame, and fresh electrophoresis buffer was added to the electrophoresis tank, and the sample was slowly added to the wall with a pipette gun. First, electrophoresis at 80 V constant voltage until the protein band enters the separation gel, and then continue electrophoresis at 120 V. Immediately after the end, transfer the membrane. Take out the protein gel, place the gel and 0.45 μm PVDF membrane between the buffer soaked filter paper, make a transfer sandwich and put it into the transfer tank, add transfer buffer and put it in the ice box, transfer at a constant current of 200 mA for 120 min. After the end of the transfer, add an appropriate amount of 5% skim milk powder to the PVDF membrane, shake slowly at room temperature for 1 h. Then, the preferred rabbit polyclonal anti-U2SURP antibody was diluted at a dilution ratio of 1:2000, and incubated overnight at 4°C on a shaker. The next day, wash with TBST for 3 times, 5 min each time. Incubate the secondary antibody at room temperature for 1 h. Wash with TBST for 3 times, 5 min each time. Finally, expose to ECL luminescence instrument and collect the image.

[0079] Figure 7 qRT-PCR column chart of U2SURP mRNA level after transfection of siRNA-1 and siRNA-2; Figure 8 The corresponding Western blot detection results of U2SURP protein expression are shown in FIG. 2B. As shown in FIG. 2B, siRNA-1 and siRNA-2 can significantly knock down the expression of U2SURP mRNA (P<0.001); Figure 7 Figure 8 ​In the meantime, the protein level also decreased accordingly, and it was concluded that the selected siRNA could efficiently inhibit the expression of U2SURP.

[0080] Example 4

[0081] Effect of U2SURP knockdown on the function of DLBCL cells based on CCK-8 method and flow cytometry

[0082] (1) CCK-8 method for detecting cell proliferation activity: Take the DLBCL cells and U2SURP knockdown strain in the logarithmic growth phase, blow them thoroughly, and then centrifuge at 4°C, 1000 rpm for 5 min, and discard the supernatant. Wash twice with PBS, resuspend with complete medium, and adjust the cell density to 1×10 5 cells / mL. Inoculate 100 μL of cell suspension into a 96-well plate; add 100 μL of pure culture medium to the blank wells. Incubate at 37°C, 5% CO2 for 24, 48 and 72 h, take out the 96-well plate, and add 10 μL of CCK-8 reagent to each well under light-free conditions for 2 h. Use the enzyme label instrument to measure the absorbance of each well at 450 nm wavelength, and calculate the cell survival rate.

[0083] (2) Flow cytometry for detecting cell apoptosis: Take the cells in the logarithmic growth phase, centrifuge to remove the culture medium, and then wash the cells with pre-cooled PBS. Add an appropriate amount of 1×binding buffer (Binding Buffer), and adjust the cell density to 1×10 6 cells / mL. Take 200 μL of cell suspension (2×10 5 cells) in a flow tube, add an appropriate amount of phospholipid-binding protein V (Annexin V) labeled with fluorescent dye and an appropriate amount of 7-AAD, mix gently, and incubate at room temperature for 15-20 min in the dark. Add 300 μL of PBS to resuspend the cells, and detect them on the flow cytometer within 1 h. The FITC excitation wavelength is 488 nm, and the maximum emission wavelength is 525 nm; the PI excitation wavelength is 488 nm, and the maximum emission wavelength is 617 nm; the APC excitation wavelength is 633 nm, and the maximum emission wavelength is 660 nm; the 7-AAD excitation wavelength is 488 nm, and the maximum emission wavelength is 647 nm.

[0084] (3) Flow cytometry detection of cell cycle distribution: Take the logarithmic growth period cells, centrifugal to remove the culture medium, and then wash the cells with pre-cooled PBS. Resuspend the precipitate with 200 μL of PBS, and then add the cells dropwise to 1 mL of 70% ethanol pre-cooled in an ice bath, mix gently, and fix at 20°C overnight. The next day, centrifuge at 400 x g for 5 min, discard the supernatant, add 1 mL of pre-cooled PBS, resuspend the cells, centrifuge at 400 x g for 5 min, discard the supernatant, and disperse the cells to avoid cell aggregation. Stain with PI staining solution (50 μg / mL PI + 50 μg / mL RNAase) in a 500 μL system, incubate at 37°C for 30 min in the dark, and mix once during the staining process. After staining, store at 4°C or on ice in the dark. The PI excitation wavelength is 488 nm, and the maximum emission wavelength is 617 nm.

[0085] Figure 9 Figure 4 is a graph showing the CCK-8 method for detecting the proliferation activity of U2SURP knockdown cells; Figure 10 Figure 5 is a graph showing the Annexin V / 7-AAD flow cytometry for detecting apoptotic cells. As shown in Figure 5, the cell survival rate after siRNA-1 and siRNA-2 treatment for 48 h was significantly lower than that of the control group (P < 0.001); Figure 9 Figure 10 Figure 6 shows that the apoptosis rate of the two groups of cells was significantly increased (P < 0.001), and it was concluded that U2SURP knockdown can effectively inhibit DLBCL cell proliferation and induce apoptosis.

[0086] Figure 11 Figure 7 is a flow cytometry graph showing the cell cycle distribution of U2SURP knockdown cells. As shown in Figure 7, the G2 / M phase of the knockdown group cells was significantly accumulated; Figure 11 Figure 12 Figure 8 shows that the G2 / M ratio was significantly increased (P < 0.001), and it was concluded that U2SURP inhibition can cause G2 / M arrest of the cell cycle. The application of siRNA-1 or siRNA-2 to DLBCL cells can significantly reduce U2SURP expression at the mRNA and protein levels, thereby inhibiting cell proliferation and inducing cell apoptosis and G2 / M arrest.

[0087] Example 5

[0088] In vivo xenograft model verification based on shRNA lentivirus-mediated knockdown of U2SURP

[0089] ​​(1) Lentivirus vector construction and transfection: The shRNA recognition sequence of the lentivirus vector (pRRLSIN-cPPT-U6-shRNA-SFFV-EGFP-SV40-puromycin) is 5'-GGGAGATTCTCCAACTAAA-3' (SEQ ID No. 12). The packaging plasmid (pMD2.G, psPAX2) and the recombinant shU2SURP vector are co-transfected in 293T cells, and the supernatant is collected at 48 h and 72 h, concentrated by ultracentrifugation, and the virus titer is ≥1.0×10 8 TU / mL. The logarithmic phase SU-DHL-2, SU-DHL-8, and Farage human B lymphoma cells are inoculated in a 6-well plate. The concentrated lentivirus with a MOI of 50 and a special transfection enhancer are added in turn, and incubated at 37°C, 5% CO2 for 72 h. The GFP expression is observed under a fluorescence microscope, and the positive rate is recorded to evaluate the transfection efficiency (≥90%).

[0090] (2) Knockdown efficiency verification: After 72 h of transfection, the U2SURP mRNA level is determined by qRT-PCR. The total protein of the cells is extracted at the same time, and the U2SURP protein expression is detected by Western blotting, and compared with GAPDH as an internal reference.

[0091] (3) Mouse xenograft experiment: 6-8 week old female NSG mice are selected and randomly divided into two groups: empty vector control group (EV group, n=6) and knockdown group (shU2SURP group, n=6). SU-DHL-8 cells are selected, collected and suspended in PBS, and 1×10 7 cells are injected subcutaneously into each mouse. The body weight and tumor volume of the mice are monitored every two days after inoculation, and the tumor volume is calculated according to the formula length x width 2 / 2. The mice are sacrificed on the 12th day, the weight of the transplanted tumor is weighed, and the tumor photos are taken for morphological comparison.

[0092] Figure 12 Figure for the results of real-time fluorescent quantitative polymerase chain reaction for detecting the mRNA expression level of U2SURP after transfection of the U2SURP shRNA recombinant lentivirus vector; Figure 13 Figure for the results of Western blotting for detecting the protein expression level of U2SURP after transfection. As shown in Figure 12 , shU2SURP can significantly reduce the expression of U2SURP mRNA; Figure 13 The expression of U2SURP protein is significantly decreased, and it is concluded that the U2SURP shRNA recombinant lentivirus vector can significantly inhibit the expression of U2SURP.

[0093] Figure 14 Typical photos of subcutaneous tumor transplantation in NSG mice; Figure 15 Growth curve of transplanted tumor. As shown in the figure, the tumor of the shU2SURP group was significantly smaller than that of the EV control group; Figure 14 Figure 15 It is shown that the tumor volume of the shU2SURP group increased significantly slower from the 10th day, and the average volume was reduced by more than 50% (P<0.01) by the 12th day, so it is concluded that lentivirus-mediated U2SURP knockdown can significantly inhibit the growth of DLBCL tumor in vivo.

[0094] Figure 16 The curve of the change in the body weight of the mice during the experiment; Figure 17 H&E staining of the whole body organs of the mice. As shown in the figure, there was no significant difference in the body weight of the mice in the shU2SURP group and the EV group (P>0.05); Figure 16 Figure 17 In the meantime, no pathological damage was found in the heart, liver, spleen, lung and kidney by H&E staining analysis, so it is concluded that U2SURP knockdown has good anti-tumor effect and does not affect the health of the body.

[0095] In summary, the present application provides a DLBCL and treatment method based on U2SURP protein and its application. Through bioinformatics analysis and tissue chip IHC, it is first identified that U2SURP is significantly highly expressed in DLBCL tissues, and its expression level is significantly related to the overall survival of patients, and an IHC detection kit and scoring system taking U2SURP as a marker are established, which can be used for the prognosis evaluation of DLBCL. In addition, the present application also provides siRNA and shRNA compositions and vectors for U2SURP, which can effectively inhibit the proliferation of DLBCL cells, induce apoptosis and cause G2 / M phase arrest by in vitro electroporation or lentivirus transfection to stably knock down U2SURP; in the transplanted tumor mouse model, the tumor growth of the U2SURP knockdown group is significantly inhibited and has no obvious toxicity to the body, verifying the feasibility and safety of U2SURP as a therapeutic target. Compared with the prior art, U2SURP has the dual values of a prognosis evaluation marker and a precise treatment target, not only providing a new tool for the clinical stratification and individualized treatment of DLBCL, but also opening up a new idea for the development of targeted intervention strategies based on variable splicing factors.

[0096] Obviously, the above embodiments are only examples for clearly illustrating, and are not a limitation on the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. All the embodiments do not need to be exhausted here. The obvious changes or variations derived therefrom are still within the protection scope of the present application.​​

Claims

1. The application of substances that inhibit U2SURP expression in the preparation of therapeutic drugs for diffuse large B-cell lymphoma, wherein the substances that inhibit U2SURP expression include siRNA or shRNA that specifically targets the U2SURP gene; the sequence of the siRNA that specifically targets the U2SURP gene is shown in SEQ ID No. 2-5; the core recognition sequence of the shRNA that specifically targets the U2SURP gene is shown in SEQ ID No.

12.

2. The application according to claim 1, characterized in that, The drug inhibits tumor growth by suppressing U2SURP expression.

3. The application according to claim 1, characterized in that, The drug inhibits diffuse large B-cell lymphocyte proliferation and induces apoptosis by suppressing U2SURP expression.

4. The application according to claim 1, characterized in that, The drug inhibits G2 / M cell cycle arrest in tumor cells by suppressing U2SURP expression.

5. Application of substances that detect U2SURP expression in the preparation of reagents for detecting diffuse large B-cell lymphoma.

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