Application of sunitinib in preparation of medicine for preventing and / or treating ALT tumor

Abstract

The invention relates to the field of biological medicine, in particular to application of sunitinib in preparation of medicine for preventing and / or treating ALT tumor. Experiments discover that sunitinib can selectively inhibit proliferation of ALT tumors in vitro and in vivo, cause damage accumulation at the telomere of the ALT tumors and interfere positioning of proteins related to telomere maintenance functions at the telomere, and analysis of gene expression profiles of ALT and non-ALT tumor cell lines treated by sunitinib shows that the sunitinib can be used for inhibiting the proliferation of the ALT tumors in vitro and in vivo. The invention proposes that the transmethylase coding gene EZH2 is a main action target of sunitinib for inhibiting ALT tumors, and proposes that the EZH2 gene or EZH2 protein treatment target has important potentials in the aspects of ALT tumor treatment, drug screening and the like, and also proposes the application of sunitinib in treatment of ALT tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of sunitinib in preparing a medicine for preventing and / or treating ALT tumors. Background Art

[0002] In order to maintain their highly proliferative state, tumor cells usually activate telomere maintenance mechanisms to solve the "end replication problem". There are currently two known telomere maintenance mechanisms: the telomerase reactivation mechanism present in most tumors, and the alternative telomere lengthening mechanism (ALT) that is independent of telomerase. ALT is a telomere lengthening mechanism mediated by the abnormally activated homologous recombination repair pathway (HR). It achieves telomere extension by synthesizing DNA under the action of DNA polymerase using longer or adjacent telomere sequences as templates. In addition to the lack of telomerase activity, the currently recognized characteristics of ALT tumor cells also include: the presence of C-rich extrachromosomal telomeric repeat sequences (C-Circle), APBs; Telomere sister-chromatid exchange (T-SCE) and the loss of chromatin factors ATRX / DAXX. The types of tumors associated with ALT are very wide. Most sarcoma tumor cells originating from mesenchymal tissues, according to statistics, about 50% of osteosarcoma, 30% of soft tissue sarcoma, 25% of glioblastoma multiforme, 10% of neuroblastoma, and lower rates of undifferentiated pleomorphic sarcoma cells, leiomyosarcoma cells, astrocytic tumors grades 2 and 3, pancreatic neuroendocrine tumor cells, etc. have ALT activity. In epithelial cancers with a lower incidence of ALT, ALT activity has also been widely reported in cancer cells such as bladder, cervix, endometrium, esophagus, gallbladder, breast, kidney, liver and lung. Because the molecular mechanism of ALT production is still unclear, the research on targets and targeted drugs for ALT tumors has been in a relatively slow state for a long time. Currently, there is an urgent need to find new targets and targeted therapeutic drugs for ALT tumors.

[0003] Sunitinib is an oral small molecule drug that can inhibit receptor tyrosine kinases at multiple targets. However, the inhibitory effect and target of sunitinib on ALT tumors have not been reported. Summary of the invention

[0004] The purpose of the present invention is to provide the use of sunitinib in the preparation of a drug for preventing and / or treating ALT tumors, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of sunitinib in preparing a medicine for preventing and / or treating ALT tumors.

[0007] Preferably, the drug prevents and / or treats ALT tumors by inhibiting the activity and proliferation of ALT tumor cells, increasing the DNA damage level and replication pressure at the telomeres of ALT tumor cells, and inhibiting the expression of methyltransferases EZH2 and H3K27me3.

[0008] Further preferably, the ALT tumor cells are telomerase negative and maintain telomere length by the ALT mechanism.

[0009] The present invention provides a medicine for preventing and / or treating ALT tumors, comprising sunitinib and pharmaceutically acceptable auxiliary materials.

[0010] The present invention provides the use of EZH2 protein or EZH2 gene as a drug target in the preparation of a drug for preventing and / or treating ALT tumors.

[0011] As an additional embodiment, the present invention provides the use of EZH2 protein or EZH2 gene as a drug target in response to the sensitivity of ALT tumors to sunitinib in in vitro and in vivo environments.

[0012] The present invention provides the use of an inhibitor of EZH2 gene in preparing a medicine for preventing and / or treating ALT tumor.

[0013] Preferably, the inhibitor comprises GSK-126.

[0014] The present invention provides a medicine for preventing and / or treating ALT tumors, and the medicine comprises an inhibitor of EZH2 gene.

[0015] Preferably, the inhibitor comprises GSK-126.

[0016] The present invention provides the use of EZH2 protein or EZH2 gene as a drug target in screening drugs for preventing and / or treating ALT tumors.

[0017] As an additional embodiment, the present invention provides a method for screening drugs for preventing and / or treating ALT tumors, the method comprising the step of detecting the expression level of EZH2 protein or EZH2 gene in a subject before and after administration.

[0018] As an additional solution, the present invention provides the use of a reagent for detecting the expression level of EZH2 protein or EZH2 gene in preparing a kit for determining a personalized treatment plan for ALT tumors.

[0019] As an additional embodiment, the present invention provides a drug for preventing and / or treating ALT tumors, wherein the drug comprises an antibody against EZH2 protein and / or a small molecule compound that inhibits the expression of EZH2 gene.

[0020] As an additional solution, the present invention provides a molecular marker for diagnosing the sensitivity of ALT tumors to sunitinib, wherein the molecular marker includes EZH2 protein or EZH2 gene. In a specific embodiment of the present invention, inhibiting EZH2 can significantly inhibit the proliferation of ALT tumors.

[0021] The present invention discloses the following technical effects:

[0022] The present invention experiments found that sunitinib can selectively inhibit the proliferation of ALT tumors in vitro and in vivo, and cause damage accumulation at the telomeres of ALT tumors and interfere with the localization of proteins related to telomere maintenance function at the telomeres. Analysis of the gene expression profiles of ALT and non-ALT tumor cell lines after sunitinib treatment suggested that the methyltransferase encoding gene EZH2 is the main target of sunitinib in inhibiting ALT tumors, and proposed that the EZH2 gene or EZH2 protein therapeutic target has great potential in ALT tumor treatment and drug screening, as well as the application of sunitinib in the treatment of ALT tumors, which is specifically verified by the following experiments:

[0023] First, we established ALT transplant tumor models in vitro and in vivo to confirm that sunitinib has specific inhibitory activity against ALT tumors. Second, we treated ALT cells: U2OS, WI38 / VA13, and U251 with sunitinib. shATRX Immunofluorescence staining experiments have shown that sunitinib can upregulate the number of APBs in ALT cells, and the co-localization levels of γ-H2AX and PCNA with telomeres are significantly increased, indicating that sunitinib treatment can change the phenotype of ALT cells. shATRXCells were screened for differentially expressed genes, and it was found that the gene EZH2 was downregulated by sunitinib in all three ALT cells. EZH2 can be used as a target for sunitinib to act on ALT tumors. Finally, the present invention found that sunitinib can inhibit the proliferation of ALT tumors in vivo and in vitro, and the main targets are EZH2 protein and EZH2 gene. Therefore, EZH2 protein and EZH2 gene can be used as new targets for ALT tumor treatment, and sunitinib and EZH2 protein / EZH2 gene inhibitors have great application prospects in the preparation of drugs for preventing and / or treating ALT tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 CCK8 detected the inhibition of sunitinib on the proliferation activity of ALT cells and non-ALT cells; A was the survival rate of ALT tumor cell line U2OS and non-ALT tumor cell line Hela after treatment with different concentrations of sunitinib detected by CCK8; B was the difference in the survival rate of U2OS and Hela cells after treatment with 10μM sunitinib detected by CCK8, **** indicates P<0.0001;

[0026] Figure 2 Immunofluorescence experiments prove that sunitinib treatment increases the level of DNA damage at the telomeres of ALT cells; A is the immunofluorescence detection of the changes in APBs levels in cells after 24 hours of treatment with 10 μM sunitinib, the green signal is the telomere, and the red signal is the γH2AX protein; B is the statistical chart of the percentage of cells with ≥3 γH2AX co-localized at the telomeres in A, * indicates P < 0.05;

[0027] Figure 3 Immunofluorescence experiments prove that sunitinib treatment increases replication pressure at the telomeres of ALT cells; A is the immunofluorescence detection of the changes in APBs levels in cells after 24 hours of treatment with 10 μM sunitinib, the green signal is the telomere, and the red signal is the PCNA protein; B is the statistical chart of the percentage of cells with ≥3 PCNA co-localized at the telomeres in A, * indicates P < 0.05;

[0028] Figure 4The pathways and targets regulated by sunitinib treatment were discovered based on RNA-seq data analysis; A is a Venn diagram showing the genes that are commonly downregulated (left) and upregulated (right) in three ALT tumor cells after sunitinib treatment; B is a KEGG pathway enrichment analysis display of genes commonly downregulated in ALT tumor cells after sunitinib treatment; C is a PPI analysis diagram of genes commonly downregulated in ALT tumor cells after sunitinib treatment, in which the genes encircled by dotted lines are in the same pathway, the links between proteins represent possible interactions, and different line colors indicate the type of evidence for the interaction;

[0029] Figure 5 Sunitinib inhibits the proliferation of ALT cells by downregulating the expression of methyltransferase EZH2; A is the Realtime qPCR experimental result of EZH2 gene mRNA in different cells after sunitinib treatment; B is the protein immunoblotting experimental result of EZH2 protein in different cells after sunitinib treatment; C is the protein immunoblotting experimental result of H3K27me3 protein methylation level in different cells after sunitinib treatment; D is the detection result of EZH2 protein and its substrate H3K27me3 level after U2OS cells were treated with 2.5μM and 5μM EZH2 inhibitor GSK-126; E is the CCK8 experiment to detect the inhibitory activity of EZH2 inhibitor GSK-126 on ALT cells and non-ALT cells;

[0030] Figure 6 The mechanism of sunitinib's anti-ALT tumor effect. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Experimental equipment and materials used in the examples:

[0037] Fetal bovine serum (OPCEL), DMEM medium (Gibco), trypsin (Solarbio), 10 cm cell culture dish (BIOFIL), 96-well cell culture plate (servicebio), CCK-8 (Abbkine), microplate reader (BioTek), human osteosarcoma cells (U2OS cells, purchased from China Cell Resource Bank), human cervical cancer cells (Hela cells, purchased from China Cell Resource Bank), human SV-40 transformed lung fibroblasts (WI38 / VA13, purchased from Shanghai Bohu Biotechnology Co., Ltd.), human glioma cells (U251 cells, purchased from Wuhan Punosai Life Science Co., Ltd. Lentiviral shRNA targeting ATRX (shATRX) and its control (shNC) were purchased from Qingke Biotechnology. Polyethylenimine Linear (PEI) MW40000 (yeasen) was used to transfect it into U251 cells to obtain U251 shATRX Cells. Target sequence: ATRX 5'-CGACAGAAACTAACCCTGTAA-3', SEQ ID NO.5), inverted microscope (Nikon Corporation, Japan), sunitinib (MCE), anti-fading agent (Vectorlabs), primary antibody (protein immunoblotting: EZH2, purchased from CST; H3K27Me3 / GAPDH, purchased from ABclonal; immunofluorescence: γ-H2AX, purchased from CST; PCNA, purchased from Bioss), secondary antibody (protein immunoblotting: AffiniPure TMGoat Anti-Mouse IgG(H+L) / AffiniPure TM Goat Anti-Rabbit IgG (H+L), purchased from Jackson ImmunoResearch, Immunofluorescence: Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 568, purchased from Invitrogen), probe (PNA-FISH: probe TelC telomere probe, purchased from PNA Bio), 10× ice-free rapid transfer buffer (servicebio), six-well plate (servicebio), DMSO (Sigma), centrifuge (Beckman Coulter, USA), chemiluminescence imager (Tannon), upright fluorescence microscope (Nikon Corporation, Japan).

[0038] The following experiments were conducted using the above test equipment to verify the mechanism of action of sunitinib in inhibiting ALT tumors. The specific verification experiments and results are as follows:

[0039] Example 1 CCK8 detection of the inhibitory activity of sunitinib on ALT cells and non-ALT cells

[0040] 1. ALT tumor cell line (human osteosarcoma cell, U2OS) and non-ALT tumor cell line (human cervical cancer cell, Hela) in logarithmic growth phase were seeded in a 96-well cell culture plate at a density of about 8000 cells / well, 80 μL of DMEM high-glucose medium containing 10% fetal bovine serum was added thereto, and the plates were incubated at 37°C, 5% CO 2 The cells were cultured in a cell culture incubator. When the cell density reached 70-80%, 20 μL of DMEM high-glucose medium containing sunitinib with a gradient final concentration of (1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) was added. Cells treated with an equal amount of DMSO served as a negative control group. Three replicate wells were set for each cell type.

[0041] 2. After 24 hours of drug treatment, the drug-containing culture medium was removed, 100 μL of CCK-8 solution (10% CCK-8) was added to each well, and incubated in a cell culture incubator for 0.5-4 hours, and in this embodiment, the incubation time was 45 minutes.

[0042] 3. After the incubation is terminated, the absorbance at 450 nm is measured with an ELISA reader to calculate the cell activity. The results are as follows: Figure 1 shown.

[0043] 4. Cell survival rate is calculated as follows:

[0044] Cell survival rate = OD value of experimental group / OD value of blank control group × 100%;

[0045] IC 50 The value is the drug concentration corresponding to the cell survival rate of 50%; data statistics are expressed as mean ± standard deviation (x ± sd) and the t test is used.

[0046] Depend on Figure 1 It can be seen that the IC of sunitinib on U2OS and Hela cells 50 The values ​​were 5.08±0.14μM and >20μM, respectively. It can be seen that sunitinib has a better killing effect on ALT tumor cells, suggesting that sunitinib has the effect of targeted killing of ALT tumor cells.

[0047] Example 2 Sunitinib treatment increases DNA damage levels and replication stress at telomeres of ALT cells

[0048] 1. Preliminary preparation for the experiment: Place a 24×24 mm cover glass in a 6-well plate, wash twice with 1× PBS, and remove the cover glass for later use.

[0049] 2. After digesting and counting the target cells, inoculate 2×10 5 cells, draw an “∞” mark and shake evenly (observe under a microscope to see whether the cells are evenly distributed), and culture at 37°C overnight.

[0050] 3. After the cells adhered, the experimental group was added with 10 μM sunitinib, and the control group was added with an equal amount of DMSO for 24 h.

[0051] 4. After treatment, remove the old culture medium, add 1 mL 1×PBS to each well and wash 3 times, 5 minutes each time, remove the waste liquid, add 1 mL fixative (2% sucrose, 2% paraformaldehyde, 1×PBS) to each well along the side wall and fix at room temperature for 10 minutes.

[0052] 5. Aspirate the waste liquid, add 1 mL of 1×PBS to each well, wash 3 times, 5 min each time, aspirate the waste liquid. Add 1 mL of 0.5% NP-40 to each well, shake well and let stand on ice for 5 min.

[0053] 6. Aspirate the waste liquid, add 1 mL of 1×PBS to each well and wash three times, each time for 5 min. Add 1 mL of PBG (0.2% fish gelatin, 0.5% bovine serum albumin, 1×PBS) to each well and block at 25°C for 2 h.

[0054] 7. Aspirate the waste liquid, dilute the primary antibody with PBG at a ratio of 1:500 (the dilution ratio can also be adjusted according to the antibody manual), dry the liquid around the coverslip and the four corners to prevent the antibody from flowing out of the coverslip due to the surface tension of the liquid, add 100 μL of antibody to each slide by the 5-point drop method, flatten it, and add an appropriate amount of ddH2O to the groove outside the 6-well plate.2 O to prevent the antibody from evaporating. Place horizontally at 4°C overnight. Antibody sources: γ-H2AX: Product No. 80312, purchased from CST; PCNA: Product No. BAM-2006M, purchased from Bioss.

[0055] 8. Add 1 mL 1×PBST to each well and wash 3 times, 5 min each time. Add fluorescent secondary antibody with a dilution ratio of 1:1000, add 100 μL antibody by 5-point drop method, spread evenly, and let stand at room temperature for 2 hours. From this step, avoid light. Add 1 mL 1×PBST to each well and wash 3 times, 5 min each time. Antibody source: AffiniPure TM Goat Anti-Mouse IgG(H+L) / AffiniPure TM Goat Anti-Rabbit IgG (H+L): Catalog number: A11004, purchased from Invitrogen.

[0056] 9. Aspirate the waste liquid, add 1 mL of 4% paraformaldehyde solution to each well and fix for 10 min at room temperature. Aspirate the waste liquid, add 1 mL of 1×PBST to each well and wash three times, 5 min each time.

[0057] 10. Aspirate the waste liquid, prepare the slide in advance and drop 30μL PNA-FISH probe on it, pick up the cover glass with cells and cover it on the slide, place it at room temperature for 10 minutes, then place the slide in an 85℃ metal bath for 5 minutes for denaturation and hybridization. Place it in a light-proof incubation box at room temperature for 2 hours. PNA-FISH: Product No. F1001, purchased from PNABio.

[0058] 11. Remove the coverslip and put it back into the 6-well plate. Add 1 mL of Wash Solution I to each well and wash twice, 15 minutes each time.

[0059] 12. Aspirate the waste liquid, add 1 mL of Wash Solution II to each well, and wash three times, 5 minutes each time.

[0060] 13. Covering: Add anti-fading agent containing DAPI to the marked slide, pick up the cover glass with a needle, and slowly cover the slide with the cells facing down to avoid bubbles. Store in a light-proof humid box at -20℃. Take pictures with a fluorescence microscope and count the results. Figure 2 and Figure 3 shown.

[0061] Depend on Figure 3 It can be seen that after treatment with sunitinib, ALT cell lines U2OS, WI38 / VA13 and U251 shATRXThe co-localization of γ-H2AX and telomeres in ALT cells increased significantly, while the level of telomere damage in non-ALT Hela cells did not change significantly, indicating that sunitinib treatment specifically damaged telomere sequences in ALT cells.

[0062] Depend on Figure 2 and Figure 3 It can be seen that after treatment with sunitinib, ALT cell lines U2OS, WI38 / VA13 and U251 shATRX The co-localization of γH2AX or PCNA with telomeres in ALT cells was significantly increased, while the aggregation of γH2AX or PCNA at telomeres in non-ALT Hela cells did not change significantly. The results suggest that sunitinib treatment specifically causes the accumulation of telomere DNA damage and replication stress in ALT cells.

[0063] Example 3 Sunitinib downregulates the expression of methyltransferase EZH2

[0064] In U2OS, WI38 / VA13 cells and U251 shATRX The cells in the experimental group were treated with 10 μM sunitinib, and the cells in the control group were treated with an equal amount of DMSO for 48 h. The cells were then collected and RNA was extracted. RNA library construction and bioinformatics and network analysis of RNA-seq data were performed by Biomarker Biotech (www.biomarker.com.cn).

[0065] In order to obtain a fair and correct understanding of sunitinib-induced ALT cells, the present invention performed RNA-seq analysis on ALT cells treated with sunitinib for 48 hours. Figure 4 The results showed that compared with the DMSO control, 3337 genes were downregulated in U2OS cells, 4101 genes were downregulated in WI38 / VA13 cells, and 4101 genes were downregulated in U251 cells after sunitinib. shATRX 1728 genes were downregulated in the cells. Bioinformatics analysis showed that there were overlapping differentially expressed genes (DEGs) between the three ALT cells. Compared with the DMSO control, 119 common genes were downregulated and 96 common genes were upregulated after sunitinib treatment. Afterwards, KEGG pathways in the three ALT cells were enriched and analyzed. Figure 4As shown in B. The results showed that the top 10 KEGG pathways of sunitinib-downregulated gene regulatory pathways included cell cycle (PPP2R1B, MCM4, CDC25A), Fanconi-related pathways (FANCI, FANCM, BLM, etc.), DNA replication (FEN1, POLA1, MCM4, etc.), etc. In addition, the genes downregulated by sunitinib in WI38 / VA13 cells were also enriched for tyrosine kinase inhibitor resistance mediated by EGFR, the target of sunitinib. The 119 commonly downregulated genes were analyzed through the STRING website (https: / / cn.string-db.org) to obtain the protein-protein interaction (PPI) network analysis, and the results are shown in Figure 4 As shown in C. The results showed that important pathways in the PPI network involved classical RTK pathways (such as Ras signaling pathway) and ALT-related pathways (Fanconi pathway and DNA replication). Notably, we found that enhancer of zeste homolog 2 (EZH2), a histone H3K27 methyltransferase, was associated with these signaling pathways, suggesting that sunitinib may inhibit EZH2-mediated ALT activity by downregulating the RTK pathway.

[0066] In order to further determine whether sunitinib targets EZH2 protein, this example uses Realtime qPCR technology and protein immunoblotting technology to detect the expression levels of EZH2 mRNA and protein in cells after sunitinib treatment, respectively. The specific implementation steps are as follows:

[0067] 1. Realtime qPCR technical steps

[0068] 1. Preparation in advance: pre-cool chloroform, isopropanol, 75% ethanol, and 95% ethanol reagents for extraction.

[0069] 2. Collect cell pellets: Place the cell pellet in a 1.5 mL EP tube, add 1 mL Trizol, pipette to resuspend, and place on ice until the solution becomes viscous and transparent.

[0070] 3. Layering: Add 200 μL of chloroform, gently invert to mix, let stand on ice for 10 minutes, then centrifuge at 4°C, 13,000 rpm for 8 minutes, and remove the RNA phase after layering.

[0071] 4. Precipitate RNA: Add 80% volume of isopropanol, mix thoroughly by inversion, and let stand at -20°C for 30 min. Centrifuge at 4°C, 12,000 rpm for 10 min and discard the supernatant.

[0072] 5. Wash RNA: Add 1 mL of 75% ethanol, invert to resuspend, and place on ice for 1 min. Centrifuge at 4°C, 12,000 rpm for 5 min and discard the supernatant.

[0073] 6. Repeat washing: Add 1 mL of 95% ethanol, repeat step 5, and discard the supernatant.

[0074] 7. Drying and dissolving RNA: Turn the EP tube upside down to absorb the ethanol. When the precipitate becomes translucent, add sterile water in proportion, turn it upside down to dissolve the RNA, let it stand on ice, and finally test the RNA quality and concentration.

[0075] 8. Reverse transcription and cDNA synthesis: Use the Yisheng Biotech kit to reverse transcribe RNA into cDNA according to the instructions. cDNA can be stored at -20℃ for short-term storage. For long-term storage, it needs to be packaged in EP tubes and stored at -80℃.

[0076] 9. Real-time fluorescence quantitative PCR detection: set up three replicate wells, 10 μL reaction system in each well, seal with RT sealing film after adding sample and centrifuge instantly, put into 96-well plate, and then test on the machine. The reaction system is shown in Table 1, the primer sequence is shown in Table 2, and the machine detection procedure is shown in Table 3.

[0077] Table 1 Reaction system

[0078] Reagent name Dosage cDNA (1000ng / μL) 1μL Forward Primer (10 μM) 0.5μL Reverse Primer (10 μM) 0.5μL 2×SYBR Green 5μL <![CDATA[ddH 2 The]]> 3μL

[0079] Table 2 Primer sequences

[0080]

[0081] Table 3 On-machine testing procedures

[0082]

[0083] 10. Analysis: Save data, calculate the expression of target genes, analyze the changes in expression levels and perform indexation processing, and draw graphs using GraphPad Prism software.

[0084] 2. Protein immunoblotting technique steps

[0085] 1. Cell treatment conditions: ALT cells and non-ALT cell lines were treated with 10 μM sunitinib for 24 h.

[0086] 2. Extraction of total cell protein: Collect cells in EP tubes, wash with 1×PBS and place on ice. Add 100-300μL RIPA lysis buffer (containing PMSF) according to the amount of cell pellet, and gently disperse by blowing. Lyse on ice for 30-40min, and ultrasonicate (10s ultrasonication / 5s pause, 10 times) until lysis is complete. Centrifuge (4℃, 13000rpm, 20min), take the supernatant to a new EP tube, and mark the information.

[0087] 3. Protein quantification and protein loading system configuration: Set the standard curve according to the instructions of the BCA kit and prepare the BCA working solution at a ratio of 50:1. Add 200 μL of working solution and 4 μL of protein sample to a 96-well plate and incubate at 37°C for 30 min (avoid bubbles). Detect with an enzyme reader and calculate the protein concentration. According to the system configuration, add 5× concentrated loading dye and 1× PBS buffer, mix and centrifuge, heat at 100°C for 10 min to denature, centrifuge again and store on ice.

[0088] 4. Gradient glue preparation: Assemble the glue plate device: bottom plastic plate - glue plate (cover plate facing down) - plastic plate - glue plate - plastic plate - thick glass sheet - plastic plate, clamp. Prepare the reaction reagent (gradient glue) according to Table 4, vortex mix and pour into the glue tank, add a rotor (speed 4-5) in the 4% glue tank. The glue filling speed is 17-18, insert a 10 or 15-hole glue comb, and let it stand for 2-3 hours to solidify.

[0089] Table 4 Gradient gel preparation

[0090] Reagents 4% gradient glue 20% gradient glue <![CDATA[ddH 2 The]]> 12.67mL 1.47mL Acrylamide 2.8mL 14mL 1.5MTris-HCl(pH8.8) 5.25mL 5.25mL 10% SDS 210μL 210μL 10% APS 70μL 70μL TEMED 25μL 8μL

[0091] 5. Electrophoresis: Assemble the electrophoresis tank and pour in the electrophoresis solution. Remove the gel comb and add the protein marker and sample in order. Add 4 μL 5× loading dye to the blank well. Set the electrophoresis parameters to 140V, 200mA, and continue for 105min (1 hour and 45 minutes). Stop when the bromophenol blue reaches the bottom of the gel.

[0092] 6. Transfer: Dilute the 10× transfer solution to 1× transfer solution according to the instructions, soak the sponge and filter paper in the 1× transfer solution, and activate the PVDF membrane with methanol for 5 minutes. Take out the gel and cut it, and assemble the transfer device in order: positive electrode, sponge, filter paper, PVDF membrane, gel, filter paper, sponge, negative electrode. The transfer conditions are 140V, 200mA, 30-50min, and the electrotransfer in this example is 45min.

[0093] 7. Blocking: Wash the PVDF membrane with 1×TBST (90 rpm, 3 times, 5 min each time). Block the membrane in 5% skim milk or 2% BSA at room temperature for 1-2 hours.

[0094] 8. Primary antibody incubation: Wash the membrane with 1×TBST (90rpm, 3 times, 5min each time). Cut the corresponding part of the membrane, add the primary antibody and seal it, and incubate it on a shaker at 4℃ for 8h. Antibody sources: EZH2: Catalog No. 5246, purchased from CST; H3K27Me3: Catalog No. A2363, purchased from ABclonal; GAPDH: Catalog No. AC001, purchased from ABclonal.

[0095] 9. Secondary antibody incubation: Wash the membrane with 1×TBST (90rpm, 3 times, 5min each time). Dilute the secondary antibody at a ratio of 1:10000 and incubate on a shaker at room temperature for 2-3h. Antibody source: AffiniPure TM Goat Anti-Mouse IgG(H+L): Catalog No. 115-035-003, AffiniPure TM Goat Anti-Rabbit IgG (H+L): Catalog No. 111-005-003, both purchased from Jackson ImmunoResearch.

[0096] 10. Development: Wash the membrane with 1×TBST (90rpm, 3 times, 5 min each time). Precool the developer in advance and prepare the developer in a 1:1 ratio. Add 200μL of developer on the membrane, shake gently 6-7 times, put it into the developer for development and take pictures for preservation.

[0097] The results are as follows Figure 5 As shown in A and B, the results showed that sunitinib treatment significantly inhibited the proliferation of ALT cell lines U2OS, WI38 / VA13 and U251 shATRX In addition, it has been reported that EZH2 can mediate the trimethylation modification of lysine 27 of histone 3 (H3K27me3). In the present invention, protein immunoblotting technology was used to detect the level of H3K27me3 in cells after sunitinib treatment. Figure 5 As shown in Figure C, sunitinib treatment significantly downregulated the level of H3K27me3 in ALT cell lines U2OS and WI38 / VA13, but had no effect on the level of H3K27me3 in Hela cells. In summary, this experiment verified the downregulation of EZH2 mRNA level after sunitinib treatment by Realtime qPCR technology, and verified the downregulation of EZH2 protein level after sunitinib treatment by protein immunoblotting technology, proving that sunitinib treatment significantly inhibited the ALT cell lines U2OS, WI38 / VA13 and U251 shATRXThe expression of EZH2 mRNA and protein in ALT cells was verified by western blot technology, which confirmed that the downstream substrate H3K27me3 level was downregulated after sunitinib treatment, further proving that EZH2 is the only target of sunitinib in ALT cells.

[0098] The mechanism of sunitinib against ALT tumors is as follows Figure 6 As shown. Figure 6 It can be seen that sunitinib downregulates EZH2 expression by inhibiting RTK and downstream signaling pathways, thereby changing the telomeric heterochromatin state, inducing replication stress and DNA damage, and inhibiting ALT tumor proliferation.

[0099] In this example, the cells were treated with GSK-126, an inhibitor of EZH2 methyltransferase activity, and the changes in EZH2 and H3K27me3 protein levels were detected by Western blotting, and cell survival was detected by CCK8. The specific implementation steps are as follows:

[0100] 1. Western Blot: U2OS cells were treated with 2.5μM and 5μM sunitinib for 24h, and then the cell pellets were collected and lysed for subsequent experiments.

[0101] 2. CCK8: ALT tumor cell line (human osteosarcoma cell, U2OS) and non-ALT tumor cell line (human cervical cancer cell, Hela) in logarithmic growth phase were seeded in a 96-well cell culture plate at a density of about 8000 cells / well, and 80 μL of DMEM high-glucose medium containing 10% fetal bovine serum was added thereto and incubated at 37°C, 5% CO 2 The cells were cultured in a cell culture incubator. When the cell density reached 70-80%, 20 μL of DMEM high-glucose medium containing GSK-126 with a gradient final concentration of (1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) was added. The cells treated with an equal amount of DMSO were used as the negative control group, and three replicate wells were set for each cell type.

[0102] 3. The remaining steps are consistent with the protein immunoblotting technical steps in Example 3 and the CCK8 experimental steps in Example 1.

[0103] The results are as follows Figure 5 As shown in D and E. Figure 5 As shown in D, after U2OS cells were treated with 2.5 μM and 5 μM of the EZH2 inhibitor GSK-126, the levels of EZH2 protein and its substrate H3K27me3 were downregulated. Figure 5 As shown in E, GSK-126 has an IC of 1.5 for U2OS, WI38 / VA13 and Hela cells. 50The values ​​were 11.98±0.19μM, 12.10±0.14μM and >16.83±1.26μM, respectively. This further proves that EZH2 is an important target for inhibiting ALT cell proliferation.

[0104] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of sunitinib in the preparation of a drug for preventing and / or treating ALT tumors.

2. The use according to claim 1, characterized in that: The drug inhibits the activity and proliferation of ALT tumor cells, increases the DNA damage level and replication pressure at the telomeres of ALT tumor cells, and inhibits the expression of methyltransferase EZH2 and H3K27me3, thereby preventing and / or treating ALT tumors.

3. A drug for preventing and / or treating ALT tumors, characterized in that: The medicine comprises sunitinib and pharmaceutically acceptable auxiliary materials.

4. Use of EZH2 protein or EZH2 gene as a drug target in the preparation of drugs for preventing and / or treating ALT tumors.

5. Use of an inhibitor of the EZH2 gene in the preparation of a drug for preventing and / or treating ALT tumors.

6. The use according to claim 5, characterized in that: Such inhibitors include GSK-126.

7. A drug for preventing and / or treating ALT tumors, characterized in that: The drugs include inhibitors of the EZH2 gene.

8. The drug according to claim 7, characterized in that Such inhibitors include GSK-126.

9. Use of EZH2 protein or EZH2 gene as a drug target in screening drugs for preventing and / or treating ALT tumors.

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