CN112007159A

CN112007159A - Application of PC4 inhibitor in preparation of products for preventing and treating diffuse large B cell lymphoma

Info

Publication number: CN112007159A
Application number: CN202010797656.6A
Authority: CN
Inventors: 史春梦; 陈洁平; 马乐; 宫蔷; 罗鹏
Original assignee: Army Medical University
Current assignee: Army Medical University
Priority date: 2020-08-10
Filing date: 2020-08-10
Publication date: 2020-12-01

Abstract

The invention relates to application of a substance with an inhibiting effect on transcription co-cofactor PC4 in preparation of a product for preventing and/or treating Diffuse Large B Cell Lymphoma (DLBCL), in particular to c-Myc high-expression diffuse large B cell lymphoma, and provides a new treatment target and direction for preventing and/or treating diffuse large B cell lymphoma. Experiments prove that after PC4 is knocked out, the expression of PC4 is inhibited, a large number of autophagosomes and autophagosomes appear in cytoplasm, LC3 is greatly increased, and P62 is reduced; pretreatment of the PC4 knockout group with the autophagy inhibitor 3-MA showed a reduction in cell death caused by the knockout of PC 4. Protein level assays also found that knock-out of PC4 resulted in activation of the AMPK signaling pathway and inhibition of the mTOR signaling pathway. All results indicate that the knock-out PC4 can induce autophagic death of c-Myc high-expression DLBCL cells.

Description

Application of PC4 inhibitor in preparation of products for preventing and treating diffuse large B cell lymphoma

Technical Field

The invention belongs to the technical field of biotechnology and medicine, and particularly relates to application of a substance with an inhibitory effect on transcription co-cofactor PC4 in preparation of a product for preventing and/or treating diffuse large B cell lymphoma.

Background

Diffuse large B-cell lymphoma (DLBCL) is a group of malignant tumors with great heterogeneity in many aspects such as clinical manifestations, tissue morphology and prognosis, and is the most common type of adult non-Hodgkin lymphoma (NHL), and the incidence rate of DLBCL in europe and america accounts for about 31% of NHL, and the incidence rate of DLBCL in asia accounts for about more than 40% of NHL. R-CHOP regimen based on Rituximab (Rituximab, R) is the standard therapy for DLBCL, but about 40% of cases remain ineffective or relapse with a poor prognosis. Among them, c-Myc high expression DLBCL is one of refractory lymphomas, and some patients with c-Myc high expression have poor chemotherapy effect and rapid disease progression, so the pathogenesis of the c-Myc high expression DLBCL is not clear at present, and an effective treatment means is lacked, and especially the resistance to an R-CHOP chemotherapy scheme is an important problem to be solved urgently. As a class of classical oncogenes, c-Myc not only regulates tumor growth and differentiation, but also regulates key genes of tumor energy metabolism, and is an important therapeutic target. However, c-Myc plays an important regulatory role in normal cell survival, and is a natural disordered protein, and lacks available drug recognition sites, so that the anti-tumor drug targeting c-Myc has been developed slowly for a long time. Therefore, the mechanism of the generation and development of the c-Myc high-expression DLBCL is deeply researched, the tumor specific target spot related to the c-Myc is searched, and the method has important significance for individual accurate treatment and improvement of clinical outcome of patients.

The transcription co-cofactor (human positive cofactor 4, PC4), also known as p15/Subl (in yeast cells), belongs to a transcription cofactor family member, and plays an important role in transcription, replication, and DNA repair. The current research shows that PC4 participates in the development of various tumors, such as lung cancer, astrocytoma, bladder cancer, esophageal squamous carcinoma, breast cancer, etc., by influencing the proliferation, apoptosis and invasion of tumor cells. However, the specific molecular mechanism of how PC4 participates in tumor development is not clear, and no research report about the involvement of PC4 in the development of hematologic malignancy exists at present.

Disclosure of Invention

In view of the above, the present invention finds the effect of the transcription co-cofactor PC4 on diffuse large B-cell lymphoma (DLBCL), and thus provides an application and a product of a substance having an inhibitory effect on the transcription co-cofactor PC4 in the preparation of a product for preventing and/or treating diffuse large B-cell lymphoma.

In order to achieve the purpose, the invention provides the following technical scheme:

1. application of substance with inhibitory effect on transcription co-cofactor PC4 in preparation of product for preventing and/or treating diffuse large B cell lymphoma.

In some embodiments, the agent is an agent that inhibits expression and/or function of PC 4.

In some embodiments, the agent is an agent for knocking-out or knocking-down the expression of PC 4.

In some embodiments, the substance is selected from: an antibody against PC4, an interfering rna (sirna) against PC4, an antisense oligonucleotide against PC4, or a small molecule inhibitor.

In some embodiments, the small molecule inhibitor is AG-1031(Wang Y, Wang H, Ge H, et al AG-1031induced autophagic cell death and apoptosis in C6 gliomas cells associated with Notch-1signaling pathway [ J ] Journal of Cellular Biochemistry, 2018.).

Preferably, the anti-PC 4 antibody is an anti-PC 4 monoclonal antibody.

Preferably, the interfering rna (siRNA) against PC4, e.g. siRNA sequences selected against different sites of PC4 gene mRNA, more preferably siRNA selected from the group consisting of: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No. 4.

More preferably, the diffuse large B-cell lymphoma is c-Myc high expression diffuse large B-cell lymphoma.

2. A product for preventing and/or treating diffuse large B-cell lymphoma comprising a substance having an inhibitory effect on the transcription co-cofactor PC 4.

In some embodiments, the product is in a form suitable for a route of administration selected from the group consisting of: the administration may be parenteral, e.g., intravenous, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, transdermal (e.g., iontophoresis), etc.

The product for the prevention and/or treatment of diffuse large B-cell lymphoma comprising a substance having an inhibitory effect on the transcription co-cofactor PC4 can be used in combination with products known in the art for the prevention and/or treatment of diffuse large B-cell lymphoma. The combination comprises: the substances or products of the invention and other known products are administered simultaneously, sequentially, separately or separately.

The invention has the beneficial effects that: provides a new target spot for preventing and/or treating diffuse large B cell lymphoma, provides the effect of transcription co-cofactor PC4 on Diffuse Large B Cell Lymphoma (DLBCL), and especially aims at c-Myc high expression diffuse large B cell lymphoma. Experiments prove that after PC4 is knocked out, the expression of PC4 is inhibited, the autophagy level of each group is detected by an electron microscope and WB, a large number of autophagosomes and autophagosomes appear in cytoplasm under the electron microscope, and the protein level is also found to be increased greatly by LC3 and reduced by P62; after the PC4 knockout group is further pretreated by using an autophagy inhibitor 3-MA, the change of autophagy flow is detected by immunofluorescence, and the apoptosis level of flow and WB detection shows that cell death caused by knockout of PC4 is reduced. Protein level assays also found that knock-out of PC4 resulted in activation of the AMPK signaling pathway and inhibition of the mTOR signaling pathway. All results indicate that the knock-out PC4 can induce autophagic death of c-Myc high-expression DLBCL cells.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a scatter plot of the expression levels of PC4 in each sample;

FIG. 2 is a graph comparing the expression levels of sample PC4 in a database;

FIG. 3 is a graph comparing the expression levels of PC4 in different cell lines;

FIG. 4 is a diagram of protein WB after knockout of PC4 by a cell;

FIG. 5 is a flow chart of cells after knock-out of PC 4;

FIG. 6 is an electron micrograph of a cell after knock-out of PC 4;

FIG. 7 is a graph comparing the activity of cells pretreated with autophagy inhibitor 3-MA for PC4 knockdown;

FIG. 8 is a flow comparison graph comparing pretreatment of PC4 knockouts with autophagy inhibitor 3-MA;

FIG. 9 is a graph comparing protein WB pretreatment of PC4 knockdown with autophagy inhibitor 3-MA.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

Example 1

The relevance of PC4, c-Myc and DLBCL is found in clinical specimens

In the early stage, q-PCR detection is carried out on 24 initial diagnosis DLBCL patients and 24 normal bone marrow specimens, and the abnormal high expression of PC4 is found in the DLBCL patients compared with normal controls, as shown in A in figure 1; meanwhile, the C-Myc (+) phase III-IV patient showed abnormally high expression of PC4, as shown in B of FIG. 1, compared with the C-Myc (-) phase I-II patient.

FIG. 1 shows a scatter plot of PC4 mRNA level expression levels of C-MYC (+) and anti III-IV stage DLBCL (1-4) and C-MYC (-) and anti I-II stage DLBCL in DLBCL, where overexpression of PC4 is associated with poor prognosis; b is C-MYC (+) and C-MYC (-) and Anbor III-IV DLBCL (H1-9) and C-MYC (-) and Anbor I-II DLBCL (L1-10) expression level of PC4 mRNA scatter plot. The results show that the expression levels of c-Myc and PC4 in DLBCL are in positive correlation, namely PC4 of a c-Myc high expression patient is also in high expression (P < 0.01). The above results suggest that highly expressed PC4 may play an important biological role in DLBCL and be closely related to c-Myc.

Example 2

Further verifying the pre-post relationship between PC4 and DLBCL by using biological information data

The expression of PC4 in various common tumors was retrieved in the TCGA visualization database GEPIA and the results indicated that there was abnormally high expression of PC4 in DLBCL, as shown in a in fig. 2.

And PC4 was found to be positively correlated with Myc expression in lymphomas (R ═ 0.46, P ═ 0), as shown in B in fig. 2.

Relevant DLBCL clinical trials GSE4475 were retrieved in Europe PMC, and 159 patients at first visit DLBCL were enrolled and whole genome sequencing was performed on all patients. Through the analysis of the disclosed gene sequencing data, the high expression of PC4 is closely related to poor prognosis of DLBCL, and the expression level of PC4 is obviously increased in the DLBCL high-risk patients, as shown in C in figure 2 and D in figure 2.

FIG. 2 shows that high expression of PC4 is closely related to poor prognosis of DLBCL, and that PC4 is positively related to c-Myc expression. Wherein A is a histogram of PC4 expression in various tumors (note: the abscissa is the abbreviation of various common tumors, wherein DLBC diffuses large B cell lymphoma); b is a scatter diagram of PC4 and MYC; c is a survival curve of DLBCL related to PC 4; d is the risk map of PC4 in DLBCL. In the figure, SUB1 is PC 4.

Example 3

4 common DLBCL cell lines are screened, PC4 high-expression DLBCL cell lines (TMD8 and HBL-1) are selected, the screening result is shown in figure 3, and PC4 is abnormally high-expression in the DLBCL cell lines, wherein A shows the protein level expression of PC4 and c-Myc in a normal human lymphocyte cell line (CCRF-SD) and different DLBCL cell lines (DOHH2, DCL-LY10, HBL-1 and TMD 8); b shows the mRNA level expression of PC4 in a normal human lymphocyte cell line (CCRF-SD) and different DLBCL cell lines (DOHH2, DCL-LY10, HBL-1, TMD 8); c showed immunofluorescent staining of PC4 in a normal human lymphocyte cell line (CCRF-SD) as well as in different DLBCL cell lines (DOHH2, DCL-LY10, HBL-1, TMD 8).

Synthesis of PC by Co4 low-expression lentivirus (shPC4-1 and shPC4-2) interferes the expression of PC4 and a control group, after cells are transfected for 24 hours, puromycin medicine with the concentration of 1.0ug/ml is used for screening for 2 weeks, Western blot is adopted to detect the expression level of PC4, namely transfection efficiency, and cell strains of TMD8 and HBL-1 stably knocked out by PC4 are obtained. The cells of each group were digested, counted and made 2X 10⁴The cell suspension with the density of/ml is added into each well of a 96-well plate by 100 mu l, each group is provided with 6 multiple wells, and the cell viability is detected by CCK-8. Add 100 μ l to each well of 6-well plate, then add 2ml culture medium, each group sets 3 multiple wells, use colony experiment to test cell clone forming ability. Detecting apoptosis and cell cycle level of each group by flow cytometry and double staining with 3 mul annexin V and 5 mul PI or single staining with PI in each tube; the results are shown in FIG. 5. The sequence of each siRNA is shown in table 1 below:

TABLE 1 sequences of the siRNAs

Collecting each group of cells (control group, shPC4-1 group and shPC4-2 group), washing with PBS, lysing the cells with RIPA buffer solution, determining protein concentration by BCA method, balancing and adding 6 × loading buffer for high temperature denaturation, preparing protein samples, performing SDS-PAGE electrophoresis, performing membrane transfer (wet transfer method), blocking, incubating primary antibody and secondary antibody, performing chemiluminescence development, and detecting the level of autophagic protein (LC3, P62 and the like). Placing the sterilized cell slide in a 24-well plate, sequentially adding each group of cells prepared into cell suspension dropwise into the plate, placing the plate in a cell incubator for culture, washing the plate with PBS, fixing the plate with 4% paraformaldehyde at room temperature for 20min, perforating the cells with Triton X-100, sealing, incubating primary antibody and secondary antibody, performing DAPI counterstaining, and observing the change of autophagy proteins (LC3, P62 and the like) under a fluorescence microscope. The morphology of each group of cells is observed by a transmission electron microscope, and autophagosome, autophagososome and the like appear.

Pretreating a PC4 knockout group (experimental grouping is a control group, a PC4 knockout group, a PC4 knockout +3-MA group or a siATG7 group) by using autophagy inhibitor 3-MA or transfected ATG7 gene siRNA, and detecting the change of the cell activity, autophagy and apoptosis level of each group by using CCK-8, Westernblot, flow and the like;

taking TMD8 cells of a well-grown control group, a PC4 knockout group and a PC4 knockout +3-MA group as research models, digesting, counting and preparing the cells of each group into a density of 2 multiplied by 10⁷The cell suspension in/ml is put in an ice box for standby. Male nude mice 5-6 weeks old were purchased and divided into 3 groups of 6 mice each. The dorsal part of the nude mice was inoculated with 150. mu.l of the cell suspension, i.e., 3X 10 cells per nude mouse⁶Individual breast cancer cells. The method comprises the following steps of (1) breeding a nude mouse in an SPF animal room, observing the growth condition of tumor-bearing and measuring the size of the tumor (the tumor-bearing volume is (length multiplied by width 2)/2) every 2 days, killing the nude mouse at the end point of an experiment, taking out the tumor, taking a picture and weighing, freezing one part of the tumor at-80 ℃, and putting the other part of the tumor in 4% paraformaldehyde for fixation, wherein specific observation indexes comprise a growth curve, a large tumor-bearing photo, weight and weight change of each group of mice, and the autophagy and apoptosis level change of each group is detected by an electron microscope, Western blot and immunohistochemistry;

FIGS. 4-9 are graphs showing the results of the above experiments, and show that the knock-out of PC4 can induce c-Myc high-expression DLBCL cells to undergo autophagic death. FIG. 4A shows that C-Myc highly expressed DLBCL cell lines TMD8 and HBL-1 were selected, and PC4 gene was knocked out by lentivirus transfection with almost no PC4 expression; FIGS. 4B and 5A, B show that after the PC4 gene is knocked out, the level of apoptosis of each group is detected by flow and WB, and a large number of cells are dead and vacuoles appear in cytoplasm; FIG. 6 shows that after knocking out PC4 gene, the level of autophagy in each group was detected by electron microscopy and WB, and a large number of autophagosomes and autophagosomes were found in cytoplasm under electron microscopy (A in FIG. 6), and the protein level was also found to be greatly increased by LC3 and decreased by P62 (B in FIG. 6); fig. 7 and 8 show the changes in the flow of autophagy, detected by immunofluorescence after pretreatment of the PC4 knockout with the autophagy inhibitor 3-MA, and the changes in the level of apoptosis, detected by flow and WB (. P <0.05,. P <0.01,. P < 0.001). It was shown that pre-treatment with the autophagy inhibitor 3-MA reduced cell death caused by knock-out of PC 4. Figure 9 shows that protein level detection also found that knock-out of PC4 resulted in activation of the AMPK signaling pathway and inhibition of the mTOR signaling pathway. The results show that the knock-out PC4 can induce the autophagic death of c-Myc high-expression DLBCL cells.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

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Claims

2. The use according to claim 1, wherein the substance is a substance that inhibits the expression and/or function of PC 4.

3. The use of claim 1, wherein the agent is an agent for knocking-out or knocking-down the expression of PC 4.

4. Use according to claim 1, wherein said substance is selected from: an antibody against PC4, an interfering rna (sirna) against PC4, an antisense oligonucleotide against PC4, or a small molecule inhibitor.

5. The use according to claim 4, wherein the small molecule inhibitor is AG-1031.

6. The use according to claim 1, wherein the substance is a monoclonal antibody against PC 4.

7. The use according to claim 1, wherein the agent is an siRNA selected from the group consisting of: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No. 4.

8. The use according to any one of claims 1 to 7, wherein the diffuse large B-cell lymphoma is c-Myc high expression diffuse large B-cell lymphoma.

9. Product for the prevention and/or treatment of diffuse large B-cell lymphoma according to the use of any one of claims 1 to 8, characterized in that it comprises a substance having an inhibitory effect on the transcription co-cofactor PC 4.

10. The product of claim 9, wherein the agent is an siRNA selected from the group consisting of: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No. 4.