Antisense oligonucleotide based on tumor splicing conversion therapy and application thereof

Abstract

The invention discloses antisense oligonucleotide based on tumor splicing conversion treatment and application of the antisense oligonucleotide, the antisense oligonucleotide is designed based on targeted tumor specific abnormal splicing events (such as PKM, MCL1 and BCLX), and the antisense oligonucleotide is used for inhibiting tumor growth by inducing splicing conversion. An abnormal selective splicing event gradually becomes a marker and a potential therapeutic target of a cancer, and by designing splicing conversion oligonucleotide for recurrent tumor specific splicing event genes (such as PKM, MCL1 and BCLX), regulation and control of the abnormal splicing event in the tumor are successfully realized, growth of the tumor is remarkably inhibited, and the tumor has a good clinical application prospect. And verification is carried out in various tumor cell lines. Besides, the invention further optimizes the design of splicing conversion oligonucleotides and explores the strategy of bifunctional oligonucleotides, so as to enhance the effect of the tumor splicing conversion therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to an antisense oligonucleotide based on tumor splicing conversion therapy and its application, and specifically to an antisense oligonucleotide for selective splicing regulation of PKM, MCL1, and BCLX genes, a bifunctional oligonucleotide design and its application in tumor growth inhibition. Background Art

[0002] Dysregulation of RNA alternative splicing is a prominent feature of almost all cancer types (Bradley and Anczuków, 2023). Tumor cells often undergo multiple genetic and epigenetic changes during carcinogenesis, among which abnormal changes in RNA splicing have an important impact on the occurrence, development and treatment resistance of tumors (Bradley and Anczuków, 2023; Sheng et al., 2018; Sotillo et al., 2015). As cells acquire the ability to proliferate, angiogenesis, invade, and resist apoptosis during tumorigenesis, the splicing patterns of their specific genes will also change. These splicing variations play a key role in the biological behavior of tumor cells, directly affecting the proliferation, migration, invasion and drug resistance of tumor cells. Therefore, tumor-specific splicing events are gradually becoming a hallmark of cancer and a potential therapeutic target (Anczukow et al., 2024).

[0003] In tumor cells, many tumor-related genes show dysregulated alternative splicing, which is accompanied by the generation of tumor-specific splicing isoforms, which are closely related to various characteristics of tumors (Bradley and Anczuków, 2023). For example, abnormal splicing of genes such as PKM (pyruvate kinase M), MCL1 (MCL1 apoptosis regulator, BCL2 family protein member), and BCLX (BCL2 family protein member) has been shown to play an important role in the occurrence and development of various types of cancer (Delbridge et al., 2016). The PKM gene can generate two subtypes, PKM1 and PKM2, through mutually exclusive alternative splicing, and their expression differences are closely related to cancer. PKM2 is upregulated in most cancers, regulates the Warburg effect and promotes tumor metabolic reprogramming (Christofk et al., 2008), while PKM1 is mainly expressed in terminally differentiated non-proliferating cells and has a certain tumor suppressor effect (Lunt et al., 2015). Therefore, simultaneously inhibiting PKM2 expression and upregulating PKM1 is a potential and efficient anti-cancer strategy. BCL-2 gene family members such as MCL1 and BCLX play a key role in regulating programmed cell death by controlling pro-apoptotic and anti-apoptotic intracellular signals. In cancer, apoptosis escape caused by dysregulated splicing of BCL-2 family genes is a recurring event (Delbridge et al., 2016). BCLX and MCL1 nascent transcripts produce two mutually antagonistic isoforms through alternative splicing. Among them, BCLX produces a long isoform Bcl-xL that inhibits apoptosis by splicing using the distal 5' splice site. Bcl-xL inhibits apoptotic signals by antagonizing pro-apoptotic proteins of the BCL2 family; while the short isoform Bcl-xS using the proximal splice site promotes apoptosis (Dou et al., 2021). Similarly, alternative splicing of MCL1 also produces a long isoform MCL1 (L) that inhibits apoptotic signals and a short isoform MCL1 (S) that promotes apoptosis. Bclx(L) and MCL1(L)-mediated apoptosis inhibition is also the main reason for tumor resistance to chemotherapy and radiotherapy (Dou et al., 2024). Therefore, regulating the splicing patterns of these specific genes can also affect the biological characteristics of tumor cells, thereby providing new strategies for cancer treatment.

[0004] At present, intervention methods for RNA splicing are mainly concentrated in two types of technologies: small molecule splicing regulators and splicing conversion antisense oligonucleotides (Biswas et al., 2024). Small molecule splicing regulators adjust the splicing selection of specific genes by intervening in the interaction between splicing factors and RNA (Seiler et al., 2018). However, the targeting and specificity of small molecule drugs still face certain challenges, and the side effects are more obvious. In contrast, antisense oligonucleotides, as an efficient and specific splicing regulation tool, bind to specific sites of target RNA with high affinity, redirect the splicing selection of target pre-mRNA by spatial blocking, and have shown good prospects in basic research and clinical applications (Egli and Manoharan, 2023).

[0005] Therefore, by specifically regulating abnormal splicing events with pan-cancer characteristics in tumor cells (such as tumor-related genes such as PKM, MCL1, BCLX, etc.) through antisense oligonucleotides, tumor cell proliferation and growth can be effectively inhibited, and even tumor resistance can be reversed, which provides a promising strategy for tumor treatment. However, how to further improve the effect of oligonucleotide splicing conversion on tumor treatment remains an urgent problem to be solved. Traditional splicing conversion antisense oligonucleotides have certain limitations, such as low splicing conversion efficiency and unsatisfactory tumor killing effect. To this end, this study further proposed a bifunctional antisense oligonucleotide design strategy to enhance the effect of selective splicing conversion by assisting in the recruitment or interference of splicing regulatory proteins and regulating the splicing signal intensity. Summary of the invention

[0006] The purpose of the present invention is to address the deficiencies of the prior art and to propose an antisense oligonucleotide based on tumor splicing conversion therapy and its application.

[0007] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: as a first aspect, an antisense oligonucleotide for regulating abnormal splicing of PKM, MCL1 and BCLX genes in tumor cells is provided, wherein the antisense oligonucleotides target the pan-cancer abnormal splicing events PKM, MCL1 and BCLX genes respectively, and have a complementarity with the target sequence of at least 96%, thereby regulating abnormal splicing of tumors; the oligonucleotide sequence targeting the PKM gene is shown in SEQ ID NO.1, the oligonucleotide sequence targeting the MCL1 gene is shown in SEQ ID NO.2, and the oligonucleotide sequence targeting the BCLX gene is shown in SEQ ID NO.3.

[0008] The complementarity between the antisense oligonucleotide and the target sequence is at least 96%, 97%, 98%, 99%, and optimally 100% complete complementarity.

[0009] Among them, the oligonucleotide sequence targeting the PKM gene (SEQ ID NO.1):

[0010] 5′-CCAGGCGGCGGAGTTCCTCA-3′;

[0011] Oligonucleotide sequence targeting MCL1 gene (SEQ ID NO.2):

[0012] 5'-AACGTCTGTGATACTTTCTGCTAAT-3';

[0013] Oligonucleotide sequence targeting BCLX gene (SEQ ID NO.3):

[0014] 5′-TGGTTCTTACCCAGCCGCCG-3′.

[0015] Preferably, the antisense oligonucleotides of the present invention are all modified with 2'-O-methoxyethyl (2'MOE) bases, and all nucleotide backbones are modified with phosphorothioate (PS) to improve their stability, affinity and intracellular transduction efficiency.

[0016] Further, the present invention adopts Invitrogen's 3000 transfection reagent to deliver the antisense oligonucleotide. The antisense oligonucleotide forms a complex with the cationic polymer of 3000, enters the tumor cell by means of mechanisms such as endocytosis, crosses the cell membrane and nuclear membrane, and finally reaches the cell nucleus to achieve splicing regulation of the target transcript.

[0017] As a second aspect, there is provided a use of the antisense oligonucleotide in the preparation of a drug for inhibiting tumor cell proliferation, promoting tumor cell apoptosis and / or treating cancer.

[0018] Furthermore, the cancer includes at least: liver cancer, colon cancer, and cervical cancer.

[0019] Furthermore, the application is manifested as:

[0020] (1) In a variety of tumor cell lines (including human liver cancer cell line HepG2, human colon cancer cell line HT29, and human cervical cancer cell line Hela), it successfully promoted the transformation of the splicing pattern of the PKM gene, inhibited the splicing of the PKM2 isoform, and promoted the splicing of the PKM1 isoform. The splicing conversion effect was positively correlated with the oligonucleotide dose.

[0021] (2) In multiple tumor cell lines (including human liver cancer cell line HepG2 and human cervical cancer cell line Hela), the splicing conversion of the MCL1 gene was successfully promoted, the splicing of the MCL1(L) isoform was inhibited, and the splicing of the MCL1(S) isoform was promoted. The splicing conversion effect was positively correlated with the oligonucleotide dose. However, the human colon cancer cell line HT29 showed a certain degree of resistance to this splicing conversion strategy.

[0022] (3) In a variety of tumor cell lines (including the human liver cancer cell line HepG2 and the human cervical cancer cell line Hela), the splicing conversion of the BCLX gene was successfully promoted, the splicing of the BCLX(L) isoform was inhibited, and the splicing of the BCLX(S) isoform was promoted. The splicing conversion effect was positively correlated with the oligonucleotide dose. Similarly, the human colon cancer cell line HT29 also showed a certain degree of resistance to this splicing conversion strategy.

[0023] (4) Multi-target combined splicing conversion shows a more significant effect in inhibiting tumor cell growth. On the one hand, the abnormal proliferation of tumor cells is usually the result of the synergistic action of multiple genes and multiple pathways. By targeting multiple abnormal splicing events, the multi-target combined strategy can cover more key factors related to tumor growth, thereby significantly enhancing the inhibitory effect; on the other hand, by targeting the abnormal splicing forms of multiple tumor-related genes, this strategy can block the compensatory response of tumor cells to a single intervention mechanism and reduce their tolerance to treatment.

[0024] As a third aspect, a method for inhibiting tumor growth based on multi-target combined splicing conversion is provided, using at least two of the antisense oligonucleotides to target abnormal splicing events of different tumor-related genes, respectively, to simultaneously regulate multiple key genes closely related to tumor growth, and significantly enhance the effect of inhibiting tumor growth. The multi-target combined splicing conversion strategy can effectively block the compensatory response of tumor cells to a single intervention mechanism and reduce their tolerance to treatment.

[0025] As a fourth aspect, as a design optimization strategy for tumor splicing conversion oligonucleotides, the present invention combines the design of bifunctional oligonucleotides, proposes three different tail domain design strategies, and further verifies them on the two important splicing conversion targets mentioned above, including tumor metabolism-related genes PKM and anti-apoptosis gene MCL1, that is, a bifunctional oligonucleotide for regulating abnormal splicing of PKM and MCL1 genes in tumor cells, and enhancing the effect of tumor splicing conversion therapy by designing bifunctional oligonucleotides. The bifunctional oligonucleotide comprises an antisense domain and a tail domain, wherein the antisense domain is reverse complementary to the target sequence, with a complementarity of at least 96%, and is used to specifically regulate the abnormal splicing of PKM and MCL1 genes in tumor cells, and the tail domain comprises multiple splicing regulatory element motifs, which are used to provide additional splicing stimulation or inhibition signals to further enhance the splicing regulation effect. The bifunctional oligonucleotide is used to improve the splicing conversion efficiency and tumor killing effect.

[0026] Among them, the complementarity with the target sequence is at least 96%, 97%, 98%, 99%, and preferably 100% complete complementarity.

[0027] Preferably, all nucleotides in the antisense domain are modified with 2'-O-methoxyethyl, and the nucleotide backbones are all modified with phosphorothioate; the tail domain is not chemically modified except for the last five nucleotides at the 3' end of the tail; the splicing regulatory element motif includes: PTBP1 binding motif, hnRNPA1 binding motif or a mixed combination of the above two.

[0028] Preferably, the last five nucleotides at the 3' end may be chemically modified to improve the overall stability of the oligonucleotide.

[0029] Preferably, the bifunctional oligonucleotide targeting the PKM gene is shown as SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6; the bifunctional oligonucleotide targeting the MCL1 gene is shown as SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.

[0030] in:

[0031] Bifunctional oligonucleotide targeting PKM gene:

[0032] SEQ ID NO.4: 5'-GAGGACGAUUAUGGCCucuucucuucucuuc-3'

[0033] SEQ ID NO.5: 5'-GAGGACGAUUAUGGCCuagguuagguuaggu-3'

[0034] SEQ ID NO.6: 5'-GAGGACGAUUAUGGCCuagguuagguuaggu-3'

[0035] Bifunctional oligonucleotide targeting the MCL1 gene:

[0036] SEQ ID NO.7: 5'-AACGUCUGUGAUACUUUCUGCUAAUucuucucuucucuuc-3'

[0037] SEQ ID NO.8: 5'-AACGUCUGUGAUACUUUCUGCUAAUuagguuagguuaggu-3'

[0038] SEQ ID NO.9: 5'-AACGUCUGUGAUACUUUCUGCUAAUucuucuaagguucuuc-3'

[0039] Preferably, Invitrogen's 3000 transfection reagent to deliver bifunctional oligonucleotides. Due to the cationic polymer properties of 3000, oligonucleotides form complexes with it and effectively enter tumor cells through mechanisms such as endocytosis, cross the cell membrane and nuclear membrane, and finally reach the cell nucleus to achieve splicing regulation of target transcripts.

[0040] As a fifth aspect, a use of the bifunctional oligonucleotide in the preparation of a drug for inhibiting tumor cell proliferation, promoting tumor cell apoptosis and / or treating cancer.

[0041] Furthermore, the cancer at least includes: cervical cancer.

[0042] Furthermore, the application is manifested as:

[0043] (1) PKM and MCL1 oligonucleotides and bifunctional oligonucleotides significantly inhibited the proliferation of human cervical cancer cells and promoted the apoptosis of human cervical cancer cells. The apoptotic effect was enhanced with the increase of oligonucleotide concentration.

[0044] (2) The effects of bifunctional oligonucleotides on different splicing regulatory targets vary. In the splicing switching regulation of PKM, the introduction of the hnRNPA1 competitive motif site enhanced the inhibitory effect of ASO on tumor growth, while the design of the PTBP1 competitive motif site weakened the inhibitory effect of ASO on tumor growth to a certain extent; in the splicing switching regulation of the MCL1 gene, the introduction of the PTBP1 and hnRNPA1 competitive motif sites did not have a significant effect on the inhibitory effect on tumor growth.

[0045] (3) PKM and MCL1 oligonucleotides and bifunctional oligonucleotides successfully induced tumor cell splicing switching. Specifically, in the PKM gene, both oligonucleotides and bifunctional oligonucleotides induced the inhibition of PKM2 splicing and promoted the splicing of PKM1, significantly upregulating the ratio of PKM1 to PKM2; in the MCL1 gene, both oligonucleotides and bifunctional oligonucleotides inhibited the formation of the MCL(L) splicing isoform and promoted the formation of the MCL(S) splicing isoform.

[0046] The beneficial effects of the present invention are as follows:

[0047] The present invention proposes a broad-spectrum tumor treatment strategy based on splicing conversion antisense oligonucleotides and bifunctional oligonucleotides. By targeting the abnormal splicing of PKM, MCL1 and BCLX genes, tumor cell proliferation is inhibited and apoptosis is promoted. At the same time, a set of bifunctional oligonucleotide design strategies are proposed, which regulate the overexpressed splicing regulatory factors in tumor cells by introducing competitive binding sites of specific splicing regulatory factors, thereby further enhancing the splicing conversion effect. At the same time, it also further verifies the advantages of multi-target collaborative splicing conversion strategies in improving the efficacy of tumor treatment, which can effectively overcome the drug resistance of tumor cells and provide a new and efficient treatment method for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the oligonucleotides designed for the present invention inducing abnormal splicing switching of PKM, MCL1, and BCLX in three different tumor cell lines;

[0049] Figure 2 Schematic diagram of the oligonucleotides designed for the present invention inducing abnormal splicing switching of PKM in three different tumor cell lines, wherein Figure 2 A is the electrophoresis diagram of PKM abnormal splicing conversion induced by 100 nM oligonucleotide administration concentration in three different tumor cell lines. Figure 2 B is the effect of different drug concentrations on splicing conversion efficiency;

[0050] Figure 3Schematic diagram of the oligonucleotides designed for the present invention inducing abnormal splicing switching of MCL1 in three different tumor cell lines, wherein Figure 3 A is the electrophoresis diagram of abnormal splicing conversion of MCL1 induced in three different tumor cell lines at a concentration of 100 nM oligonucleotide. Figure 3 B is the effect of different drug concentrations on splicing conversion efficiency;

[0051] Figure 4 Schematic diagram of the oligonucleotides designed for the present invention inducing abnormal splicing switching of BCLX in three different tumor cell lines, wherein Figure 4 A is the electrophoresis diagram of abnormal splicing conversion of BCLX induced by 100 nM oligonucleotide administration concentration in three different tumor cell lines. Figure 4 B is the effect of different drug concentrations on splicing conversion efficiency;

[0052] Figure 5 The effect of the oligonucleotides designed for the present invention on the inhibition of tumor cell growth by multi-target combined splicing switching at the same administration concentration;

[0053] Figure 6 Representative images of tumor cell growth status after treatment with the oligonucleotides and bifunctional oligonucleotide drugs designed for the present invention;

[0054] Figure 7 Schematic diagram of the oligonucleotides and bifunctional oligonucleotides designed for the present invention inducing abnormal splicing switching of PKM and MCL1 in cervical cancer cell lines, and showing the effects of different oligonucleotide drugs on splicing switching efficiency;

[0055] Figure 8 Figure 2 shows the cell viability detection after the oligonucleotides and bifunctional oligonucleotide drugs designed for targeting abnormal PKM splicing of the present invention enter tumor cells;

[0056] Fig. 9 Figure 2 shows the cell viability detection of the oligonucleotides and bifunctional oligonucleotide drugs designed for targeting abnormal splicing of MCL1 after entering tumor cells;

[0057] Fig.10 Diagram of the mechanism model by which oligonucleotides and bifunctional oligonucleotide drugs enter tumor cells to induce splicing switching. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0059] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0060] The present invention proposes a broad-spectrum tumor treatment strategy based on splicing conversion antisense oligonucleotides, which effectively inhibits the proliferation and growth of tumor cells by specifically regulating abnormal splicing events with pan-cancer characteristics in tumor cells (such as abnormal splicing of genes such as PKM, MCL1, and BCLX). And by optimizing the sequence design of oligonucleotides, a design strategy for bifunctional oligonucleotides is proposed, which aims to further assist in recruiting or interfering with splicing regulatory proteins and regulating the intensity of splicing signals, thereby improving splicing conversion efficiency and tumor killing effects, providing new potential targets and more efficient treatment methods for cancer treatment.

[0061] Example 1: Antisense oligonucleotides successfully induced PKM, MCL1, and BCLX gene splicing switching in various tumor cell lines

[0062] The present invention provides three modified oligonucleotides for practical application in regulating tumor splicing conversion, targeting PKM, MCL1, and BCLX respectively, with complementarity to the target sequence reaching at least 96%, 97%, 98%, and 99%, and optimally 100% complete complementarity, effectively reversing the abnormal splicing pattern in tumor cells.

[0063] The nucleotide sequence length of the antisense oligonucleotide is 20-25 nucleotides.

[0064] The oligonucleotide sequences designed by the present invention for targeting and regulating the selective splicing of PKM, MCL1 and BCLX genes are as follows: the oligonucleotide sequence targeting the PKM gene (SEQ ID NO.1): 5'-CCAGGCGGCGGAGTTCCTCA-3'; the oligonucleotide sequence targeting the MCL1 gene (SEQ ID NO.2): 5'-AACGTCTGTGATACTTTCTGCTAAT-3'; the oligonucleotide sequence targeting the BCLX gene (SEQ ID NO.3): 5'-TGGTTCTTACCCAGCCGCCG-3'; the negative control random oligonucleotide sequence (SEQ ID NO.10): 5'-CCTCTTACCTCAGTTACAATTTATA-3'.

[0065] The oligonucleotide sequence of the present invention was purchased from Huzhou Hippo Biotechnology Co., Ltd. (HIPPOBIO). The antisense oligonucleotide was modified with all bases 2'-O-methoxyethyl (2'MOE), and all nucleotide backbones were modified with phosphorothioate (PS).

[0066] In the experiment, human liver cancer cell line HepG2, colon cancer cell line HT29 and cervical cancer cell line HeLa were cultured in DMEM containing 10% FBS, and the cells were placed in an incubator with 5% CO2 and 37°C. One day before transfection, cells were digested with trypsin and counted, and seeded into 6-well plates with a cell density of 2×10 per well. 5 After inoculation, the cells were cultured in a CO2 incubator overnight and transfected the next day. The solution preparation method of the transfection system was as follows: the oligonucleotide powder was diluted with nuclease-free water to a 100 nM storage solution and stored at -20°C. Invitrogen 3000 transfection reagent was used according to the instructions, and the transfection concentrations were set to 50, 100, and 200 nmol / L.

[0067] 48 hours after transfection, total RNA was extracted. 1000 ng RNA was used to synthesize cDNA using oligo(dT) as reverse transcription primer. PCR technology was used to analyze the splicing conversion efficiency of the target gene.

[0068] PKM:

[0069] Upstream primer hPKMF (SEQ ID NO. 19): 5′-AGAAACAGCCAAAGGGGACT-3′;

[0070] Downstream primer hPKMR (SEQ ID NO. 20): 5′-CATTCATGGCAAAGTTCACC-3′;

[0071] The reaction conditions were as follows: pre-denaturation at 94°C for 5 min, amplification at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 15 s, 35 cycles, and a final extension at 72°C for 10 min.

[0072] The length of the amplified fragments of PKM1 and PKM2 subtypes obtained by amplification under the above PCR conditions is 398 bp, wherein the nucleotide sequences of PKM1 and PKM2 are shown as SEQ ID NO.11 and SEQ ID NO.12, respectively (sequences are 5'-3'). Since the amplified products of PKM1 and PKM2 appear as the same bands in nucleic acid gel electrophoresis, PstI restriction endonuclease is used to specifically digest PKM2. After digestion, PKM2 will be digested to produce two small fragments of 185 bp and 218 bp (the nucleotide sequences after digestion are shown as SEQ ID NO.13 and SEQ ID NO.14, respectively), while PKM1 will retain the original 398 bp fragment. After digestion by PstI, electrophoresis can clearly distinguish the expression levels of PKM1 and PKM2, thereby accurately identifying the distribution ratio of splicing subtypes of the PKM gene.

[0073] MCL1:

[0074] Upstream primer hMCL1F (SEQ ID NO. 21): 5′-GAGGAGGAGGAGGACGAGTT-3′;

[0075] Downstream primer hMCL1R (SEQ ID NO. 22): 5′-′CAAAAGCCAGCAGCACATTC-3;

[0076] The reaction conditions were as follows: pre-denaturation at 94°C for 5 min, amplification at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 15 s, 35 cycles, and a final extension at 72°C for 10 min.

[0077] The obtained MCL1(L) and MCL1(S) subtype amplified fragments were 505 bp and 257 bp in length, respectively, and the nucleotide sequences were shown in SEQ ID NO.15 and SEQ ID NO.16, respectively.

[0078] BCLX:

[0079] Upstream primer hBCLXF (SEQ ID NO. 23): 5′-AGTAAAGCAAGCGCTGAGGGAG-3′;

[0080] Downstream primer hBCLX R (SEQ ID NO. 24): 5′-ACTGAAGAGTGAGCCCAGCAGA-3;

[0081] The reaction conditions were as follows: pre-denaturation at 94°C for 5 min, amplification at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 15 s, 35 cycles, and a final extension at 72°C for 10 min.

[0082] The BCLX(L) and BCLX(S) subtype amplified fragments were obtained, with lengths of 439 bp and 250 bp, respectively. The nucleotide sequences are shown in SEQ ID NO.17 and SEQ ID NO.18, respectively.

[0083] The results of splicing conversion showed that in a variety of tumor cell lines (including human liver cancer cell line HepG2, human colon cancer cell line HT29 and human cervical cancer cell line HeLa), antisense oligonucleotides effectively promoted the splicing conversion of PKM, MCL1 and BCLX genes, and the splicing conversion effect was positively correlated with the dose of oligonucleotides. Except for the human colon cancer cell line HT29, it showed a certain resistance to the antisense oligonucleotide splicing conversion strategy of MCL1 and BCLX ( Figure 1-4 ).

[0084] Example 2: Tumor treatment strategy based on oligonucleotide multi-target coordinated splicing conversion

[0085] Multi-target combined splicing conversion has shown significant effects in inhibiting tumor cell growth. The abnormal proliferation of tumor cells is usually caused by the synergistic effects of multiple genes and multiple signaling pathways. Therefore, the multi-target splicing conversion strategy can simultaneously regulate multiple key genes closely related to tumor growth by targeting multiple abnormal splicing events, covering more regulatory factors, thereby enhancing the therapeutic effect. At the same time, this strategy can effectively block the compensatory response of tumor cells to a single intervention mechanism, reduce their tolerance to treatment, and further improve the durability and effectiveness of treatment.

[0086] Cell culture and transfection conditions: The human cervical cancer cell line HeLa was selected as a model for experimental studies on multi-target splicing conversion. The culture medium was DMEM containing 10% FBS, and the cells were placed in an incubator with 5% CO2 and 37°C. The day before the transfection operation, HeLa cells were digested with trypsin, counted and inoculated into 96-well plates, with 5×103 cells in each well. After the cells were inoculated, they were placed in a CO2 incubator overnight and transfected the next day.

[0087] The solution preparation method of the transfection system is as follows: dilute the oligonucleotide powder with nuclease-free water to a 100 nM stock solution and store it at -20°C. The transfection was performed with 3000 transfection reagent according to the instruction manual, and the total oligonucleotide transfection concentration was set to 300nmol / L. Single-target (PKM, BCLX, MCL1, all at 300nM), dual-target (PKM+BCLX, PKM+MCL1, BCLX+MCL1, each at 150nM) and triple-target (PKM+BCLX+MCL1, each at 100nM) transfection treatments were performed to systematically evaluate the functional effects of different target combinations.

[0088] 48 hours after transfection, the CCK8 method was used to detect changes in cell viability and evaluate the effect of multi-target splicing conversion on tumor cell growth inhibition. 10 μL of CCK8 reagent was added to each well and incubated in a 37°C, 5% CO2 incubator for 1 hour. The absorbance value of each well was read at 450nm using an ELISA reader. Cell viability (%) was calculated according to the following formula:

[0089] Cell viability (%) = (absorbance value of control group - absorbance value of blank group) / (absorbance value of experimental group - absorbance value of blank group) × 100%;

[0090] The experimental results showed that multi-target combined splicing conversion significantly inhibited the proliferation of HeLa cells, and compared with the single-target transfection group, the multi-target splicing conversion group showed a stronger cell killing effect. Specifically, the cell viability of the multi-target transfection group was significantly lower than that of the single-target transfection group, and was close to less than 50% of the control group. Compared with the blank control group and the single-target transfection group, the multi-target combined splicing conversion can more effectively inhibit the growth of cervical cancer cells, showing a stronger therapeutic potential ( Figure 5 ).

[0091] Example 3. Design of bifunctional oligonucleotides and their coordinated regulation of splicing switching in tumor cells

[0092] The oligonucleotides and bifunctional oligonucleotides designed by the present invention for targeting and regulating the selective splicing of PKM and MCL1 genes are as follows: the oligonucleotide sequence SEQ ID NO.1 targeting the PKM gene and the bifunctional oligonucleotides SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6; the oligonucleotide sequence (SEQ ID NO.2) targeting the MCL1 gene, and the bifunctional oligonucleotides SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9.

[0093] The oligonucleotide sequences were all purchased from Huzhou Hippo Biotechnology Co., Ltd. (HIPPOBIO). Among them, the above-mentioned antisense oligonucleotides are modified with all bases 2'-O-methoxyethyl (2'MOE), and all nucleotide backbones are modified with phosphorothioate (PS). The bifunctional oligonucleotide is based on the traditional antisense sequence (antisense domain), and a non-hybridized chain (tail domain) containing a specific splicing regulatory factor binding site is introduced at its tail. Among them, the antisense domain specifically regulates the abnormal splicing of the target gene by reverse complementation with the target sequence, and its complementarity with the target sequence is at least 96%, 97%, 98%, 99%, preferably 100% completely complementary. In order to enhance its stability and specificity, all nucleotides in the antisense domain are modified with 2'-O-methoxyethyl (2'-MOE), and all its nucleotide backbones are modified with phosphorothioate (PS). Tail domain further enhances the splicing regulation effect by providing additional splicing stimulation or inhibition signals. The design idea of ​​the Tail domain is to take advantage of the characteristics of widespread abnormal expression of splicing regulatory factors in tumor tissues, and to competitively recruit these splicing regulatory factors to interfere with their normal functions or redistribute their binding sites, thereby achieving more efficient regulation of abnormal splicing events. The Tail domain contains multiple splicing regulatory element motifs to improve the splicing conversion effect of the oligonucleotide. The design includes a three-linked tandem PTBP1 binding motif, a three-linked tandem hnRNPA1 binding motif, and a mixed combination of the two. Except for the last five nucleotides at the 3′ end of the Tail domain, which are modified with 2'-MOE and PS (to enhance the overall stability of the oligonucleotide), the rest of the Tail domain is not chemically modified to ensure its affinity with the target splicing regulatory protein. The nucleotide sequence length of the bifunctional oligonucleotide is 35-40 nucleotides.

[0094] Human cervical cancer cell line HeLa was cultured in DMEM containing 10% FBS, and the cells were placed in an incubator with 5% CO2 and 37°C. One day before transfection, cells were digested with trypsin and counted, and seeded into 6-well plates with a cell density of 2×10 per well. 5 After inoculation, the cells were cultured in a CO2 incubator overnight and transfected the next day. The solution preparation method of the transfection system was as follows: the oligonucleotide powder was diluted with nuclease-free water to a 100 nM storage solution and stored at -20°C. Invitrogen Transfection was performed with 3000 transfection reagent according to the instruction manual, and the transfection concentration was set to 200nmol / L.

[0095] 48 hours after transfection, the cell growth status was observed and recorded using a 10x20 microscope ( Figure 6 ). Total cell RNA was then extracted. 1000 ng RNA was used and oligo(dT) was used as a reverse transcription primer to synthesize cDNA. The PCR primers described in Example 1 were used for specific amplification, and the splicing conversion efficiency of the target gene was analyzed by gel electrophoresis. The splicing conversion results showed that in the human cervical cancer cell line HeLa, both antisense oligonucleotides and bifunctional oligonucleotides effectively promoted the splicing conversion of PKM and MCL1 genes ( Figure 7 ), in the PKM gene, both oligonucleotides and bifunctional oligonucleotides induced the inhibition of PKM2 splicing, promoted the splicing of PKM1, and significantly upregulated the ratio of PKM1 to PKM2; in the MCL1 gene, both oligonucleotides and bifunctional oligonucleotides inhibited the formation of the MCL(L) splicing isoform and promoted the formation of the MCL(S) splicing isoform.

[0096] The CCK8 method was used to detect the effects of different bifunctional oligonucleotide design strategies on the cell viability of human cervical cancer HeLa cells. Cell culture and transfection conditions: The human cervical cancer cell line HeLa was selected as a model, the culture medium was DMEM containing 10% FBS, and the cells were placed in a 5% CO2, 37°C incubator. The day before the transfection operation, HeLa cells were digested with trypsin, counted and inoculated into 96-well plates, with 5×103 cells inoculated per well. After the cells were inoculated, they were placed in a CO2 incubator overnight and transfected the next day.

[0097] The solution preparation method of the transfection system is as follows: dilute the oligonucleotide powder with nuclease-free water to a 100 nM stock solution and store it at -20°C. The cells were transfected with 3000 transfection reagent according to the instruction manual, and different oligonucleotide transfection concentration gradients were set (25 nmol / l, 50 nmol / l, 100 nmol / l, 200 nmol / l, 400 nmol / l, 800 nmol / l, respectively).

[0098] 48 hours after transfection, the CCK8 method was used to detect changes in cell viability. 10 μL of CCK8 reagent was added to each well and incubated in a 37°C, 5% CO2 incubator for 1 hour. The absorbance value of each well was read at 450 nm using an ELISA reader. Cell viability (%) was calculated according to the following formula:

[0099] Cell viability (%) = (absorbance value of control group - absorbance value of blank group) / (absorbance value of experimental group - absorbance value of blank group) × 100%

[0100] The experimental results showed that oligonucleotides and bifunctional oligonucleotides of PKM and MCL1 significantly inhibited the proliferation of human cervical cancer cells and promoted cell apoptosis, and the apoptotic effect increased with the increase of oligonucleotide concentration. The effects of bifunctional oligonucleotides on different splicing regulatory targets were different. In the regulation of PKM splicing switching, the introduction of hnRNPA1 competitive motif enhanced the inhibitory effect of antisense oligonucleotides (ASO) on tumor growth, while the PTBP1 competitive motif design weakened the inhibitory effect of ASO to a certain extent. For the splicing switching regulation of the MCL1 gene, the introduction of PTBP1 and hnRNPA1 competitive motifs did not significantly change the tumor growth inhibitory effect ( Figures 8-9 ).

Claims

1. An antisense oligonucleotide for regulating abnormal splicing of PKM, MCL1 and BCLX genes in tumor cells, characterized in that: The antisense oligonucleotides respectively target the pan-cancer abnormal splicing events PKM, MCL1 and BCLX genes, and have a complementarity of at least 96% with the target sequence, thereby regulating abnormal splicing of tumors; the oligonucleotide sequence targeting the PKM gene is shown in SEQ ID NO.1, the oligonucleotide sequence targeting the MCL1 gene is shown in SEQ ID NO.2, and the oligonucleotide sequence targeting the BCLX gene is shown in SEQ ID NO.

3.

2. The antisense oligonucleotide according to claim 1, characterized in that All nucleotides in the antisense oligonucleotide are fully base 2'-O-methoxyethyl modified, and the nucleotide backbones are all phosphorothioate modified.

3. The antisense oligonucleotide according to claim 1, characterized in that pass 3000 transfection reagent to deliver the antisense oligonucleotide, the antisense oligonucleotide 3000 forms a complex, enters the tumor cell, crosses the cell membrane and nuclear membrane, and finally reaches the cell nucleus to achieve splicing regulation of the target transcript.

4. Use of the antisense oligonucleotide according to any one of claims 1 to 3 in the preparation of a medicament for inhibiting tumor cell proliferation, promoting tumor cell apoptosis and / or treating cancer.

5. A method for inhibiting tumor growth based on multi-target combined splicing conversion, characterized in that: At least two antisense oligonucleotides according to claim 1 are used to target abnormal splicing events of different tumor-related genes respectively, so as to simultaneously regulate multiple key genes closely related to tumor growth, thereby significantly enhancing the effect of inhibiting tumor growth.

6. A bifunctional oligonucleotide for regulating abnormal splicing of PKM and MCL1 genes in tumor cells, characterized in that: The bifunctional oligonucleotide comprises a traditional antisense complementary sequence antisense domain and a tail non-hybridizing sequence taildomain, wherein the antisense domain is reverse complementary to the target sequence with a complementarity of at least 96%, and is used to specifically regulate the abnormal splicing of PKM and MCL1 genes in tumor cells, and the tail domain comprises multiple splicing regulatory element motifs, which are used to provide additional splicing stimulation or inhibition signals, thereby further enhancing the splicing regulatory effect.

7. The bifunctional oligonucleotide according to claim 6, characterized in that: All nucleotides in the antisense domain are modified with 2'-O-methoxyethyl, and the nucleotide backbones are modified with phosphorothioate; the tail domain is not chemically modified except for the last five nucleotides at the 3' end of the tail; the splicing regulatory element motif includes: PTBP1 binding motif, hnRNPA1 binding motif or a mixed combination of the above two.

8. The bifunctional oligonucleotide according to claim 6, characterized in that: The bifunctional oligonucleotide targeting the PKM gene is shown as SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6; the bifunctional oligonucleotide targeting the MCL1 gene is shown as SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.

9.

9. Use of the bifunctional oligonucleotide according to any one of claims 6 to 7 in the preparation of a drug for inhibiting tumor cell proliferation, promoting tumor cell apoptosis and / or treating cancer.

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