Antisense oligonucleotide for regulating selective splicing of PKM gene and application of antisense oligonucleotide

Abstract

The invention discloses an antisense oligonucleotide for regulating and controlling selective splicing of a PKM gene and application of the antisense oligonucleotide. The antisense oligonucleotide comprises the following sequence: 5 '-CGC CAG GCG GCG GAG TTC CTC AAA TAA T-3' (SEQ ID NO.1). The antisense oligonucleotide for regulating and controlling selective splicing of the PKM gene can be used for preparing a medicine for treating chronic myeloid leukemia or tumors with abnormal PKM splicing. The compound is especially used for chemosensitization of chronic myeloid leukemia, prevention of progress of chronic myeloid leukemia or inhibition of drug resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology and relates to an antisense oligonucleotide, specifically to an antisense oligonucleotide for regulating the selective splicing of the PKM gene and its application in preparing drugs for enhancing chemotherapy sensitization of chronic myeloid leukemia, preventing the progression of chronic myeloid leukemia and inhibiting drug resistance, and in preparing drugs for treating tumors with abnormal PKM splicing. Background Art

[0002] Alternative splicing refers to the generation of different mRNA splicing isoforms in a messenger RNA (mRNA) precursor through different splicing modes (selecting different splicing site combinations), so that the final protein product will exhibit different or antagonistic functional and structural characteristics. The result of alternative splicing is that a gene can form multiple different mature mRNAs through this mechanism, and then encode different proteins, mediating that about one-third of the genes in the human genome can encode different transcripts. More and more studies have found that the phenomenon of alternative splicing of transcripts is closely related to the occurrence and development of tumors: it is currently believed that tumor-specific splicing changes are caused by mutations, which destroy splicing regulatory elements within genes and impair splicing recognition. In addition, alternative splicing can also be used as an important entry point for anti-tumor drug screening, making alternative splicing a hot topic in the field of tumor research in recent years. Alternative splicing is now considered to be a key molecular mechanism of cancer hallmarks, which is associated with the expression of different subtypes during disease onset and progression.

[0003] Abnormal glucose metabolism is one of the important characteristics of tumors, which is specifically manifested in the Warburg effect proposed by German biologist Warburg in 1956 - even in the presence of sufficient oxygen, tumor cells still take up a large amount of glucose to produce lactic acid (aerobic glycolysis). Pyruvate kinase (PK) is a key enzyme that regulates the last step of glycolysis, catalyzing the reaction of phosphoenolpyruvate (PEP) and ADP to produce pyruvate and ATP. Pyruvate kinase includes erythrocyte / liver pyruvate kinase (PKLR) and muscle pyruvate kinase (PKM). PKM1 and PKM2 are two different subtypes formed by selective splicing of the PKM gene. In recent years, there have been a lot of studies on the role of PKM1 and PKM2 in tumors.

[0004] Leukemia is a type of malignant proliferative blood disease that seriously threatens human health. Currently, the main treatments for leukemia include chemotherapy and hematopoietic stem cell transplantation, but leukemia is difficult to cure and has a high relapse rate. Chronic myeloid leukemia (CML), as a malignant myeloproliferative disease originating from hematopoietic stem cells, also faces the problems of chemotherapy resistance and drug resistance. Imatinib (IM) resistance is one of the main reasons for the failure of CML treatment. Its resistance mechanism is very complex, mainly including the upregulation of bcr-abl fusion gene expression and pharmacological changes.

[0005] As early as the 1870s, the concept of antisense nucleotides (AONs) was proposed by scholars. It refers to a single-stranded DNA or RNA sequence that is complementary to the target gene mRNA, usually composed of a dozen to dozens of bases. Through the principle of base complementarity, it interferes with gene replication, transcription, mRNA splicing, processing, output and translation, and other links, thereby regulating cell growth and differentiation. To date, antisense nucleotide drugs have become a hot topic in scientific research and have also provided new ideas for antiviral and anti-tumor treatment. However, it also has natural disadvantages. Taking the third-generation antisense nucleotide, antisense morpholino oligomer (AMO), as an example, its biggest defect is that its special molecular structure causes it to have no charge, making AMO unable to be recognized by any receptor on the cell surface, and it cannot be introduced into the cell by transfection. Therefore, AMO molecules can only be introduced into cells by physically damaging the cell membrane. This defect greatly limits the application of AMO. To this end, Gene Tools has developed a specific delivery vector, called the "triazine octazidine structure tree", the core of which is a triazine core with three N-termini. The two opposite N-termini are respectively bound to four guanidine head structures, and the third N-terminus is conjugated to AMO. The eight guanidine head structures in the vector can bind to the phospholipids in the cell membrane structure through electrostatic attraction and hydrogen bonds, and efficiently carry AMO across the cell membrane structure to the cytoplasm and nucleus. This vector is conjugated with AMO to form a whole with transport function and biological effect, namely Vivo-Morpholino Antisense Oligomer (vMO) (see US patent No. 7935816 and Fig.23 ).

[0006] There are three main regulatory mechanisms for alternative splicing of human genes, namely splicing enhancement, inhibition and comprehensive regulation. Among them, splicing enhancement regulation is mainly completed by exonic splicing enhancers (ESE) and intronic splicing enhancers (ISE). ESE and ISE can enhance the splicing of 5′ or 3′ splicing sites by binding to various splicing regulatory proteins, thereby increasing the proportion of mRNA containing these exons. At present, there are no reports that antisense oligonucleotides that regulate alternative splicing of PKM gene are effective in treating CML and related chemotherapeutic drug sensitization. Research and development of such antisense oligonucleotides to prevent the progression of CML and enhance its sensitivity to chemotherapeutic drugs such as TKIs is an urgent need for clinical treatment. Summary of the invention

[0007] The purpose of the present invention is to solve the problems of chemotherapy resistance and drug resistance in the existing CML treatment, and to provide an antisense oligonucleotide for regulating the selective splicing of the PKM gene. The antisense oligonucleotide can target and regulate PKM shearing to convert PKM2 to PKM1, so that PKM2 is downregulated while PKM1 is upregulated, thereby effectively eliminating the molecular basis for CML progression and drug resistance, thereby preventing CML progression and enhancing its sensitivity to chemotherapy drugs such as TKIs. It can also be used to treat tumors with abnormal PKM splicing.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides an antisense oligonucleotide for regulating the selective splicing of a PKM gene, characterized in that the nucleotide comprises the following sequence: 5'-CGC CAG GCG GCG GAG TTC CTC AAA TAA T-3' (SEQ ID NO.1).

[0010] The antisense oligonucleotide is a morpholino antisense oligonucleotide modified as follows:

[0011]

[0012] wherein B is independently a base pairing moiety at each occurrence.

[0013] The above structure is based on Vivo-Morpholinos technology, which conjugates AMO with a specific carrier to form a whole with transport function and biological effect. The core of the specific carrier is a triazine core, which has three N-termini. The two opposite N-termini are respectively combined with four guanidine head structures, and the third N-terminus is conjugated with AMO (see US patent No. 7935816 and Fig.23). The eight guanidine head structures in the carrier can bind to the phospholipids in the cell membrane structure through electrostatic attraction and hydrogen bonds, and efficiently carry AMO across the cell membrane structure to the cytoplasm and nucleus.

[0014] The PKM gene sequence acted upon by the antisense oligonucleotide of the present invention is: att att TGAGGAACT CCG CCGCCT GGC G, wherein capital letters represent exon sequences, and lowercase letters represent intron sequences.

[0015] The antisense oligonucleotide of the present invention is used in preparing medicine for treating chronic myeloid leukemia.

[0016] The present invention also provides a pharmaceutical composition for treating chronic myeloid leukemia, the pharmaceutical composition comprising a pharmacologically effective dose of the antisense oligonucleotide for regulating the selective splicing of the PKM gene of the present invention and a pharmacologically acceptable carrier. The antisense oligonucleotide and the carrier of the present invention can be prepared into dosage forms such as liposomes, nanomaterials, microemulsions, microspheres, gels or vesicles, the preparation methods of these dosage forms and the excipients used are all prior art, and the administration method can be intravenous, intraperitoneal injection, etc.

[0017] Furthermore, the treatment described in the present invention refers to chemosensitizing chronic myeloid leukemia, preventing the progression of chronic myeloid leukemia or inhibiting drug resistance.

[0018] As a preferred embodiment of the present invention, the pharmaceutical composition for treating chronic myeloid leukemia of the present invention further contains a tyrosine kinase inhibitor in addition to the antisense oligonucleotide for regulating the selective splicing of the PKM gene of the present invention.

[0019] Tyrosine kinase inhibitors (TKIs) are a class of compounds that can inhibit the activity of tyrosine kinases, including the first-generation drug imatinib, and the second-generation drugs dasatinib and nilotinib. Tyrosine kinase inhibitors mainly exert their anti-leukemia effects by inhibiting BCR-ABL fusion protein.

[0020] The present invention also provides a pharmaceutical composition for treating tumors with abnormal PKM splicing, comprising a pharmacologically effective dose of an antisense oligonucleotide for regulating the selective splicing of the PKM gene and a pharmacologically acceptable carrier. The antisense oligonucleotide and the carrier described in the present invention can be prepared into dosage forms such as liposomes, nanomaterials, microemulsions, microspheres, gels or vesicles, and the preparation methods and excipients used in these dosage forms are all prior art, and the administration method can be intravenous or intraperitoneal injection.

[0021] As a preferred embodiment of the present invention, the pharmaceutical composition for treating tumors with abnormal PKM splicing of the present invention contains, in addition to the antisense oligonucleotide for regulating the selective splicing of the PKM gene of the present invention, a chemotherapeutic drug.

[0022] In summary, the beneficial effects of the present invention are:

[0023] The antisense oligonucleotides of the present invention can directly regulate the abnormal selective splicing of the PKM gene. Especially after combining with the Vivo-Morpholinos technology, the disadvantages of antisense nucleotides can be avoided, and the advantages of antisense nucleotides in regulating the abnormal selective splicing of the PKM gene can be brought into play. PKM shearing is targeted and regulated to convert PKM2 into PKM1, so that PKM2 is downregulated and PKM1 is upregulated, effectively eliminating the molecular basis for CML progression and drug resistance, thereby enhancing the sensitivity to chemotherapy drugs such as TKIs and preventing CML progression. The drug of the present invention can be used alone or in combination with chemotherapy drugs for the treatment of CML and tumors with abnormal PKM splicing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram of PKM gene changes during CML progression in Example 1 of the present invention, Figure A shows the difference in PKM1 and PKM2 gene expression between CML-CP and CML-BC samples, and Figure B shows the difference in PKM1 and PKM2 gene expression ratios between CML-CP and CML-BC samples.

[0025] Figure 2 Example 2 of the present invention shows that imatinib (IM) is effective against K562 ( Figure 2 A) and K562 / G01( Figure 2 B) Cell growth inhibition graph.

[0026] Figure 3 Schematic diagram of the selective splicing of the PKM gene and the vMO binding position in Example 2 of the present invention.

[0027] Figure 4 A is Example 2 of the present invention, K562 / G01 cells were treated with 10 μmol / l vMO for 48 h, and the cell fluorescence intensity was detected by flow cytometry. Figure 4 B is a graph showing the transfection efficiency of K562 / G01 cells treated with 2, 4, 6, 8, 10, and 12 μmol / l vMO for 48 h, respectively, and examined by flow cytometry.

[0028] Figure 5 A is a graph showing the changes in PKM2 mRNA expression after K562 / G01 cells were treated with 10 μmol / l vMO using PCR analysis in Example 2 of the present invention. Figure 5B is a graph showing the protein expression changes of PKM1 and PKM2 analyzed by western blot, RS-Vmo: random sequence vMO (RS-vMO).

[0029] Figure 6 In the embodiment 2 of the present invention, K562 ( Figure 6 A) and K562 / G01( Figure 6 B) The IC50 value of IM was calculated by adding 10 μmol / l vMO to the cells.

[0030] Figure 7 A is a graph showing the expression of PKM1 and PKM2 proteins in K562 cells after IM (0.2 μmol / l), vMO (10 μmol / l) or the combination of the two drugs transfected in Example 3 of the present invention. Figure 7 B is the expression graph of PKM1 and PKM2 proteins in K562 / G01 cells after transfection with IM (2 μmol / l), vMO (10 μmol / l) or the combination of the two drugs.

[0031] Figure 8 This is a flow cytometry histogram of K562 cells after treatment with different groups of IM, vMO and IM+vMO in Example 3 of the present invention.

[0032] Fig. 9 This is a bar graph of cell cycle distribution of K562 cells after treatment with different groups of IM, vMO and IM+vMO in Example 3 of the present invention.

[0033] Fig.10 This is a flow cytometry histogram of K562 / G01 cells after treatment with different groups of IM, vMO and IM+vMO in Example 3 of the present invention.

[0034] Fig.11 This is a bar graph of cell cycle distribution of K562 / G01 cells after treatment with different groups of IM, vMO and IM+vMO in Example 3 of the present invention.

[0035] Fig.12 This is a scatter plot of cell apoptosis in K562 cells after treatment with different groups of IM, vMO and IM+vMO in Example 3 of the present invention.

[0036] Fig.13 This is a bar graph of cell apoptosis in K562 cells after treatment with different groups of IM, vMO and IM+vMO in Example 3 of the present invention.

[0037] Fig.14 This is a scatter plot of cell apoptosis in K562 / G01 cells after treatment with different groups of IM, vMO and IM+vMO in Example 3 of the present invention.

[0038] Fig.15 This is a bar graph of cell apoptosis in K562 / G01 cells after treatment with different groups of IM, vMO and IM+vMO according to Example 3 of the present invention.

[0039] Fig.16 K562 cells were treated with different groups of IM, vMO and IM+vMO in Example 3 of the present invention ( Fig.16 A) and K562 / G01 cells ( Fig.16 B) Line graph of cell viability after 4 h.

[0040] Fig.17 This is a comparison chart of the tumor sizes of the CML xenograft model in each treatment group of Example 4 of the present invention.

[0041] Fig.18 This is a scatter plot of the tumor volume of the CML xenograft model in each treatment group of Example 4 of the present invention.

[0042] Fig.19 The figures are H&E stained sections showing the local characteristics of the tumors in the treatment groups of Example 4 of the present invention, wherein ① is the control group, ② is the IM (50 μmol / L) group, ③ is the IM (100 μmol / L) group, ④ is the IM (200 μmol / L) group, ⑤ is the RS-vMO group, ⑥ is the vMO group, ⑦ is the IM (50 μmol / L) + vMO group, ⑧ is the IM (100 μmol / L) + vMO group, ⑨ is the IM (200 μmol / L) + vMO group, and ⑩ is the IM (100 μmol / L) + vMO (IV) group.

[0043] Fig. 20 This is a graph of Ki-67 levels detected by immunohistochemistry of tumors in each treatment group of Example 4 of the present invention, wherein ① is the control group, ② is the IM (50 μmol / L) group, ③ is the IM (100 μmol / L) group, ④ is the IM (200 μmol / L) group, ⑤ is the RS-vMO group, ⑥ is the vMO group, ⑦ is the IM (50 μmol / L) + vMO group, ⑧ is the IM (100 μmol / L) + vMO group, ⑨ is the IM (200 μmol / L) + vMO group, and ⑩ is the IM (100 μmol / L) + vMO (IV) group.

[0044] Fig.21 The protein expression changes of PKM1 and PKM2 in each treatment group in Example 4 of the present invention are shown. IV means intravenous injection.

[0045] Fig. 22 This is a graph showing the changes in the protein expression ratio of PKM1 and PKM2 in each treatment group in Example 4 of the present invention.

[0046] Fig.23 Figure 2 is the structure diagram of morpholino antisense oligonucleotide. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific examples. However, this should not be understood as the scope of the above-mentioned subject matter of the present invention being limited to the following examples, and all technologies implemented based on the content of the present invention belong to the scope of the present invention. Unless otherwise specified, the technical means and operating methods used in the following examples are conventional means and methods well known to those skilled in the art, and the raw materials used are all commercially available products.

[0048] Example 1 Expression of PKM2 in CML Blast Phase

[0049] According to the Expert Consensus on the Treatment of Chronic Myeloid Leukemia (2010 Edition), the diagnostic criteria for IM resistance include failure to achieve complete hematological remission after 3 months of IM treatment, failure to achieve cytogenetic remission after 6 months of treatment, or failure to achieve major cytogenetic remission after 12 months of treatment, loss of complete hematological remission or cytogenetic remission, disease progression, or the emergence of resistant Bcr-Abl kinase mutations. Patients with CML diagnosed in the Second Affiliated Hospital of Nanchang University from June 2022 to December 2022 were selected, and clinical peripheral blood samples from 20 patients with CML chronic phase (CP) (imatinib sensitive) and 7 patients with blast phase (BC) (imatinib resistant) were collected. Total RNA was extracted from cells using TRIzol reagent (Invitrogen, California, USA) according to the manufacturer's instructions.

[0050] 1. In this experiment, total RNA was extracted using the Trizol lysis method.

[0051] (1) Collect cells and centrifuge at 1000 rpm, remove the supernatant, and wash once with PBS.

[0052] (2) Every 5×10 6 Add 500 μl Trizol to each cell, mix thoroughly with a pipette, and let stand for 10 min.

[0053] (3) Add chloroform in a volume ratio of Trizol:chloroform = 5:1, oscillate on an oscillator for 5 s / time, repeat three times, and leave at room temperature for 10 min.

[0054] (4) Place in a centrifuge at 4°C, 12,000 rpm for 10 min. After centrifugation, the sample is separated into three layers. The RNA is concentrated in the upper aqueous phase. Pipette the aqueous phase into a new centrifuge tube.

[0055] (5) Add an equal volume of isopropanol to the aqueous solution, invert several times to mix, and place at -20°C for 30 min.

[0056] (6) Centrifuge at 4°C, 12,000 rpm for 10 min. Remove the supernatant and the precipitate at the bottom of the tube is the RNA precipitate.

[0057] (7) Prepare 75% ethanol with a volume ratio of anhydrous ethanol: nuclease-free water = 3:1. Add 500 μl of 75% ethanol to each tube to wash the precipitate and gently invert to mix (do not shake).

[0058] (8) Centrifuge at 8000 rpm at 4°C for 5 min, discard the supernatant, and let it dry at room temperature for about 5 min.

[0059] (9) Add 10 μl-100 μl of nuclease-free water and pipette to mix thoroughly to dissolve the RNA.

[0060] (10) Use a nucleic acid concentration detector to detect the RNA concentration in the sample. If the OD 260 / 280 value is between 1.8 and 2.0, it means that the RNA purity meets the requirements. Record the RNA concentration of each sample for later cDNA preparation experiments.

[0061] 2. Reverse Transcription cDNA Preparation

[0062] According to the instructions of the Takara kit, the two-step method was used to remove the DNA in the genome. The reagents were added to a 0.2 ml reaction tube and the reaction system was prepared as shown in the following table:

[0063]

[0064] Note: The total RNA loading volume is calculated based on its concentration, and the remaining volume is supplemented with ddH2O. The total volume of TotalRNA and ddH2O is 7.0μl. After the first step of loading, place the sample solution in a PCR instrument, treat it at 42℃ for 2min, and then quickly cool it at 4℃ for use.

[0065] 3. Reverse transcription of RNA into cDNA.

[0066] Add the reagents to the reaction tube that completed the first step and prepare the reaction system as shown in the following table:

[0067]

[0068] After the second step of adding samples, place the sample solution in a PCR instrument and react under the following conditions: 37°C for 15 min, 85°C for 5 s, and finally quickly cool to 4°C to complete the preparation.

[0069] 4.RT-qPCR

[0070] The Takara kit was used to detect the SYBR Green method. The reaction solution was prepared according to the kit instructions and added to the eight-tube reaction tube. The prepared reaction system is as follows:

[0071]

[0072]

[0073] After the sample addition was completed, the sample was placed in the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, USA) and the reaction was carried out according to the following procedure:

[0074]

[0075] After the reaction is completed, observe the melting and amplification curves to ensure that the RNA is specifically amplified and presents a "single peak" curve.

[0076] Each sample was tested in triplicate, and the average value was taken. The GAPDH gene was used as the internal reference gene, and the relative expression of the target gene in the sample was calculated using the 2-△△Ct method. △△Ct = (average Ct value of the target gene in the experimental group - average Ct value of the internal reference gene in the experimental group) - (average Ct value of the target gene in the control group - average Ct value of the internal reference gene in the control group).

[0077] The primer information of each related gene is shown in the table below:

[0078]

[0079] The analysis results showed that PKM2 expression was significantly upregulated in CML-BC samples compared with CML-CP samples ( Figure 1 A), and the PKM2 / PKM1 expression ratio was significantly increased ( Figure 1 B).

[0080] Example 2 vMO transfection efficiency and stability test and analysis of its ability to regulate PKM splicing

[0081] K562 (imatinib-sensitive) and K562 / G01 (imatinib-resistant) cell lines were derived from the Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China, and cultured in RMPI 1640 medium (Bioind, Israel) supplemented with 10% fetal bovine serum (FBS, Bioind), 100 U / ml penicillin and streptomycin. Afterwards, the cells were incubated in a humidified incubator at 37°C and 5% CO2 concentration. Imatinib (IM) was purchased from Sigma-Aldrich (St. Louis, USA). The IC50 of IM treatment of K562 and K562 / G01 cells was approximately 0.2 μmol / l and 2 μmol / l, respectively, among which the IM resistance of K562 / G01 cells was approximately 10 times that of K562 cells ( Figure 2 A is the growth inhibition graph of K562 cells by IM. Figure 2 B is a graph showing the growth inhibition of K562 / G01 by IM).

[0082] Based on the abnormal splicing pattern of PKM precursor mRNA, the present invention designs vMO to target and regulate PKM splicing to convert PKM2 to PKM1 ( Figure 3 ), and commissioned Gene Tools, Inc., USA, to synthesize a specific vMO sequence (5'-CGC CAG GCG GCG GAG TTC CTC AAATAAT-3') labeled with fluorescein FAM, which is designed to target the splicing site at the beginning of exon 10 of PKM pre-mRNA. To further optimize the reaction conditions, K562 / G01 cells were incubated with vMO concentrations ranging from 2 to 12 μmol / l (equivalent to adding 2 to 12 μl vMO per milliliter of culture medium) for comprehensive evaluation. The co-cultured cells were washed with PBS until there was no fluorescent signal, and then the fluorescence activity level was measured using flow cytometry. Endo-Porter is known for its unique delivery capabilities, with minimal toxicity and optimal delivery effect when evaluating antisense activity at 48 hours at a concentration of 6 μmol / l (equivalent to 6 μl / ml culture medium) recommended by Gene Tools. Flow cytometry detection found that the fluorescence of K562 / G01 cells when vMO was transfected was strongest at 48 hours ( Figure 4 A, K562 / G01 cells were treated with 10μmol / l vMO for 48h, and the cell fluorescence intensity was detected by flow cytometry. K562 / G01 cells were co-transfected with different concentrations of vMO for 48h. When the vMO concentration was 2, 4, 6, 8, 10 and 12μmol / l, the transfection efficiency of 10μmol / l vMO was the highest at 48h ( Figure 4 B). Compared with the random sequence vMO (RS-vMO) group and the untreated group (control), 10 μmol / l vMO could effectively reduce the expression of PKM2 ( Figure 5 A), and increased PKM1 expression ( Figure 5 B). In both K562 and K562 / G01 cells, it was found that 10 μmol / l vMO could effectively reduce the IC50 of IM ( Figure 6 A and Figure 6 B).

[0083] Example 3 vMO inhibits CML cell growth and reduces drug resistance

[0084] The changes of cell cycle, apoptosis and cell viability were analyzed to evaluate whether the application of vMO could inhibit CML cells and enhance the cytotoxic effect induced by IM.

[0085] IM (0.2 μmol / l for K562 and 10 μmol / l for K562 / G01), vMO (10 μmol / l), and IM+vMO (IM dose of K562 was 0.2 μmol / l, K562 / G01 was 10 μmol / l, and vMO dose was 10 μmol / l) as well as untreated control groups were co-cultured with K562 or K562 / G01 cells for 48 h. First, the protein expression changes of PKM1 and PMK2 in each group were analyzed. After the treated cells in each group were collected, the total protein was extracted, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride (PVDF) membrane. To block the PVDF membrane, it was incubated with 5% skim milk in PBS for 2 h. Subsequently, the membrane was incubated with anti-PKM1 / 2 antibody and anti-β-actin antibody (1:1000 dilution; Cell Signaling Technology, USA) at room temperature overnight. After washing three times with TBST buffer, the membrane was exposed to horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000 dilution; Proteintech, USA) and incubated at room temperature for 1 hour. After washing again, the protein bands on the strips were visualized using a chemiluminescence kit (Beyotime, China). The resulting images of protein bands were captured using a ChemiScope series device (ClinxScience Instrument Co., China). The results showed that IM+vMO treatment could also effectively induce downregulation of PKM2 expression and upregulation of PKM1 expression in K562 cells and K562 / G01 cells ( Figure 7 A,7B).

[0086] For cell cycle detection, (2-3)×105 cells were inoculated into each well of a 6-well cell culture plate. The treated cells were collected and washed once with PBS and then centrifuged at 1500rpm for 5 minutes. The supernatant was discarded and 1ml of -20℃ pre-cooled 75% ethanol was slowly added to the precipitate to resuspend the cells and incubate at 4℃ overnight. Wash once more with PBS and discard the supernatant. Add 100μl PBS to resuspend the cells. Add 2μl of 10mg / ml RNaseA to each tube of sample and bathe at 37℃ for 30min. Add 100μl of 100μg / ml PI staining solution and stain for 10min away from light. Detect by flow cytometry within 1h and analyze the results with Modfit software. Each experiment was repeated three times and the data obtained were averaged. The results showed that compared with other groups, IM+vMO treatment could induce G1 phase arrest of the cell cycle to a greater extent ( Figure 8 , Fig. 9 , Fig.10 and Fig.11 ).

[0087] For apoptosis detection, apoptotic cells were assessed using Annexin V-FITC / PI (for K562) or Annexin V PE / 7-AAD (for K56 / G01) apoptosis detection kits (Solarbio, Beijing, China) according to the manufacturer's guidelines. Cells were seeded at a density of 106 cells per well, and the cells were collected 48 hours after treatment and incubated at room temperature for 15 minutes according to the manufacturer's instructions. The proportion of early apoptotic and late apoptotic / dead cells was analyzed using a Beckman Coulter CytoFLEX S flow cytometer (Beckman, USA). Each experiment was repeated three times, and the data obtained were averaged. The results showed that vMO treatment alone could induce cell apoptosis, and IM+vMO could further enhance the pro-apoptotic effect on the basis of single IM ( Fig.12 , Fig.13 , Fig.14 and Fig.15 ).

[0088] For cell viability detection, 20,000 cells / well were first seeded in a 96-well plate. After 48 h of treatment, 10 μl of CCK-8 reagent was added to each well of the treatment group and incubated for 2 h. The absorbance at 450 nm was measured using an ELISA reader. Each experiment was repeated three times, and the data obtained were averaged.

[0089] The inhibition rate was calculated as follows: 1-(OD after treatment-OD of blank) / (OD of control-OD of blank).

[0090] The results showed that the combined treatment of IM+vMO maximally inhibited the activity of K562 cells and K562 / G01 cells ( Fig.16 A and Fig.16 B).

[0091] Example 4 Xenograft Model Confirms the In Vivo Therapeutic Effect and Resistance Reversal Value of vMO

[0092] To evaluate the in vivo therapeutic effect of vMO and whether it can enhance the activity of IM, a K562 / G01 cell xenograft model was established.

[0093] To induce local tumor formation, 1×10 7 K562 / G01 cells were subcutaneously injected into 5-6 week old female BALB / c-nu mice (n=4 per group). The mice were randomly divided into 10 groups and given drugs two weeks after transplantation. The grouping and drug administration were as follows:

[0094] Control group (Control): Orally administered with the same volume of normal saline as other groups once a day.

[0095] IM (50 μmol / L) group: Orally administered once a day, dosage 50 mg / kg / day.

[0096] IM (100 μmol / L) group: Orally administered once a day, dosage 100 mg / kg / day.

[0097] IM (200 μmol / L) group: Orally administered once a day, dosage 200 mg / kg / day.

[0098] RS+vMO group: RS-vMO (8.4 mg / kg / day) was injected intratumorally every other day.

[0099] vMO group: vMO (8.4 mg / kg / day) was injected intratumorally every other day.

[0100] IM (50 μmol / L) + vMO group: IM was taken orally once a day at a dosage of 50 mg / kg / day, and vMO was injected intratumorally every other day at a dosage of 8.4 mg / kg / day.

[0101] IM (100 μmol / L) + vMO group: IM was administered orally once a day at a dosage of 100 mg / kg / day, and vMO was injected intratumorally every other day at a dosage of 8.4 mg / kg / day.

[0102] IM (200 μmol / L) + vMO group: IM was administered orally once a day at a dosage of 200 mg / kg / day, and vMO was injected intratumorally every other day at a dosage of 8.4 mg / kg / day.

[0103] IM (200 μmol / L) + vMO (IV) group: IM was administered orally once a day at a dose of 200 mg / kg / day, and vMO was injected intravenously every other day at a dose of 8.4 mg / kg / day.

[0104] The above administration lasted for two weeks. Tumor growth was monitored regularly, and tumor size was measured with a caliper every other day. The tumor volume was calculated using the following formula: volume = 1 / 2 × length × width^2.

[0105] Paraffin-embedded tissue specimens were prepared for immunohistochemical staining and hematoxylin-eosin staining (HE staining). Immunohistochemical staining of Ki-67 was used to reflect the proliferation activity of tumor cells. Paraffin-embedded tissue specimens were sliced, dewaxed and hydrated, antigen repaired, inactivated, blocked, incubated with primary antibodies, incubated with secondary antibodies, developed, counterstained, and sealed for observation in 10 sections. For HE staining, paraffin sections were sequentially placed in xylene, anhydrous ethanol, 95% alcohol, 90% alcohol, 80% alcohol, 70% alcohol, and distilled water for washing, dewaxed to water, and sections were sequentially stained with hematoxylin and eosin, then dehydrated, sealed, observed under a microscope, and photographed.

[0106] The analysis results showed that mice in the high-level IM group (100 μmol / L, 200 μmol / L) showed smaller tumor volumes compared with the low-level IM group (50 μmol / L). Fig.17 and Fig.18 ), revealing that IM can inhibit tumor growth in a dose-dependent manner. At the same time, vMO treatment alone can also significantly reduce tumor volume compared with the RS-vMO treatment group ( Fig.17 and Fig.18 When IM and vMO were combined, vMO either intratumorally or intravenously (IV group) resulted in a further reduction in tumor volume ( Fig.17 and Fig.18 HE staining showed that after combined treatment with IM and vMO, tumor cells were loosely distributed and there were more necrotic areas ( Fig.19 ), confirming that drug treatment was more effective and that Ki-67 signal was weaker ( Fig. 20 ), reflecting slower cell proliferation. Consistent with the expression trend of cell experiments, the expression level of PKM2 was significantly reduced after combined treatment ( Fig.21 and Fig. 22 ).

[0107] In summary, our results suggest that CML xenograft models are more sensitive to IM treatment in the presence of vMO.

Claims

1. An antisense oligonucleotide for regulating the selective splicing of the PKM gene, characterized in that: The nucleotide comprises the following sequence: 5'-CGC CAG GCG GCG GAG TTC CTC AAA TAA T-3' (SEQ ID NO. 1).

2. The antisense oligonucleotide for regulating selective splicing of the PKM gene according to claim 1, characterized in that The antisense oligonucleotide is a morpholino antisense oligonucleotide modified as follows: wherein B is independently a base pairing moiety at each occurrence.

3. The antisense oligonucleotide for regulating selective splicing of the PKM gene according to claim 1 or 2, characterized in that The PKM gene sequence acted by the antisense oligonucleotide is: att att TGAGGAACT CCG CCG CCT GGC G (SEQ ID NO. 2), wherein capital letters represent exon sequences, and lowercase letters represent intron sequences.

4. Use of the antisense oligonucleotide for regulating the selective splicing of the PKM gene according to claim 1 or 2 in the preparation of a drug for treating chronic myeloid leukemia.

5. A pharmaceutical composition for treating chronic myeloid leukemia, characterized in that The invention comprises a pharmacologically effective dose of the antisense oligonucleotide for regulating the selective splicing of the PKM gene according to claim 1 or 2 and a pharmacologically acceptable carrier.

6. The pharmaceutical composition for treating chronic myeloid leukemia according to claim 5, characterized in that The treatment refers to chemotherapy sensitization of chronic myeloid leukemia, prevention of chronic myeloid leukemia progression or inhibition of drug resistance.

7. The pharmaceutical composition for treating chronic myeloid leukemia according to claim 5, characterized in that The pharmaceutical composition also contains a tyrosine kinase inhibitor.

8. A pharmaceutical composition for treating tumors with abnormal PKM splicing, characterized in that The invention comprises a pharmacologically effective dose of the antisense oligonucleotide for regulating the selective splicing of the PKM gene according to claim 1 or 2 and a pharmacologically acceptable carrier.

9. The pharmaceutical composition for treating tumors with abnormal PKM splicing according to claim 8, characterized in that The pharmaceutical composition also contains a chemotherapeutic drug.

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