Discovery and application of anti-cancer signal channel ALKBH5-LRPPRC-elF4E-XPO1

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to discovery and application of an anti-cancer signal channel ALKBH5-LRPPRC-elF4E-XPO1. According to the invention, it is found for the first time that LRPPRC and ALKBH5 have interaction in a cell nucleus, and the interaction depends on m6A modification of RNA. Meanwhile, a brand new signal channel, namely ALKBH5-LRPPRC-elF4E-XPO1, is successfully disclosed by further exploring the physiological functions of the ALKBH5-LRPPRC-elF4E-XPO1. In addition, a complex formed by the interaction is also separated out, and the complex is used as a structural basis of RNAm6A demethylation nuclear export, so that RNA epigenetics and a specific nuclear export mechanism are organically combined in a breakthrough manner. In addition, experimental concept verification is carried out aiming at whether the new signal channel can regulate the expression of the proto-oncogene or not, and a new way and possibility are provided for accurately intervening elF4E nucleoplasm shuttle regulation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the discovery and application of the anti-cancer signaling pathway ALKBH5-LRPPRC-elF4E-XPO1. Background Art

[0002] With the aging of the population and the change of lifestyle, cancer has become a global burden and the second leading cause of death after cardiovascular diseases. It is estimated that by 2040, the number of new cases globally will reach 28.4 million (a 47% increase). In recent years, the technological breakthroughs in cancer research have mainly focused on the diagnostic level, such as liquid biopsy (e.g., ctDNA analysis), AI imaging technology, etc., which have significantly improved the ability of early detection. In the field of treatment, compared with the traditional chemotherapy and radiotherapy models, various new treatment models such as immunotherapy (e.g., CAR-T cell therapy), targeted therapy (e.g., protein kinase inhibitors), gene editing (e.g., CRISPR) are developing rapidly. At present, cancer treatment faces major challenges such as tumor heterogeneity, target druggability limitations, drug resistance, off-target toxicity, immune microenvironment interference, lack of biomarkers, and drug delivery obstacles. Although the cancer research is in a period of technological explosion, the global burden is still increasing, and continuous investment in cancer research is still needed. In the future, through the development of multi-target combination therapies (such as bispecific antibodies, PROTAC technology), combined with AI to predict new targets and drug resistance mechanisms, and exploring emerging fields such as epigenetics and the microbiome, it is expected to become an effective way to deal with refractory cancers, reduce the side effects of cancer treatment, and achieve the United Nations goal of "reducing the cancer mortality rate by one-third by 2030".

[0003] m6A is the most common internal chemical modification in eukaryotic mRNA. The modification process is responsible for addition by "writers" (such as the METTL3 / METTL14 complex), removal by "erasers" (such as FTO, ALKBH5), and recognition by "readers" (such as YTHDF proteins, IGF2BP family), thereby regulating processes such as RNA splicing, nuclear export, stability, and translation. m6A modification-related factors (writers, erasers, readers) are dysregulated in various cancers and may act as oncogenes or tumor suppressor genes, affecting tumorigenesis, progression, metastasis, metabolic reprogramming, treatment resistance, and immune escape. Therefore, the m6A modification profile or the expression level of related factors is considered promising as a predictive biomarker for cancer diagnosis, prognosis assessment, and treatment response. Currently, small molecule inhibitors targeting m6A modification (such as the FTO inhibitor FB23-2, the METTL3 inhibitor STM2457) have shown antitumor activity in preclinical models, especially with great potential in overcoming chemotherapy resistance and enhancing immune therapy response. With the development of precision medicine and sequencing technologies, RNA epigenetic regulation is considered a promising important breakthrough for the next generation of anticancer therapies. Among them, ALKBH5 is an m6A demethylase and is considered a potential precision treatment target. Its small molecule inhibitors MV1035 and 20m, etc., have shown the potential to inhibit tumor stem cells and enhance chemotherapy sensitivity in preclinical studies. Moreover, the combination of these small molecule inhibitors with PD-1 antibodies can enhance the effect of immune therapy, and this phenomenon has been verified in melanoma and colorectal cancer models. However, the specific mechanism by which ALKBH5 and its small molecule inhibitors exert their effects is currently unclear. LRPPRC is an m6A-binding protein. Current research on it mainly focuses on its regulation of the stability and translation of mitochondrial mRNA, affecting cellular energy metabolism, but it is not clear what role it plays in the field of epigenetics. In recent years, with the acquisition of m6A sequencing data of various cancer samples, LRPPRC is regarded as a potential m6A-related anticancer target, but it is generally believed that the mechanism by which it promotes tumorigenesis is mainly through regulating mitochondrial energy metabolism to support tumor proliferation. Therefore, it is still urgently necessary to deeply explore what function LRPPRC undertakes in m6A epigenetics and its specific mechanism at the m6A-related anti-tumor level.

[0004] Nuclear export of RNA is a highly regulated process involving multiple pathways and factors, and its abnormalities are closely related to the occurrence and development of cancer. Studies have shown that many RNA export factors (such as XPO1, eIF4E, HuR, Nup88, etc.) are abnormally expressed in various cancers, leading to abnormal export of specific mRNAs, which in turn promotes cell proliferation, survival and metastasis. These factors have thus become potential targets for cancer treatment. The RNA export pathway is diverse: the NXF1 / TAP pathway is responsible for the bulk export of most mRNAs; the XPO1 pathway mediates the export of specific mRNAs (such as mRNAs containing ARE elements or eIF4E-dependent mRNAs). Dysregulation of these pathways leads to over-export of pro-oncogene mRNAs, thus promoting tumorigenesis. eIF4E (eukaryotic translation initiation factor 4E) is a key regulator of RNA export and translation. In the nucleus, eIF4E recruits XPO1 by recognizing the 4E-SE (eIF4E-sensitivity element) in the 3'-UTR of specific mRNAs, promoting the nuclear export of pro-oncogene mRNAs such as Pim1 (overexpressed in >50% of solid tumors and hematological malignancies) and c-Myc (abnormally activated in >70% of human cancers); in the cytoplasm, eIF4E, as a component of the translation initiation factor, promotes the translation of these mRNAs, further enhancing their pro-cancer effects. In 30% of cancers (such as acute myeloid leukemia AML, breast cancer), overexpression of eIF4E leads to increased nuclear export and translation of its target mRNAs, thus promoting cell proliferation and survival. Currently, drugs that inhibit XPO1 (such as Selinexor (KPT-330)) have been approved for multiple myeloma and lymphoma, and therapies targeting eIF4E (such as ribavirin) have also achieved preliminary results in clinical trials. However, since these two proteins, eIF4E and XPO1, are essential for normal cells, the therapeutic window is narrow, and severe toxic side effects and drug resistance are likely to occur. Therefore, some studies have proposed to develop specific nuclear eIF4E inhibitors, which may help solve the problems of selectivity and drug resistance by specifically blocking the nuclear eIF4E-XPO1 axis, opening up a new way for precision intervention based on the regulation of nucleocytoplasmic shuttling.

[0005] Although certain progress has been made in the research on the relationship between RNA nuclear export and cancer, the existing research still has the following deficiencies: (1) ALKBH5 and LRPPRC are potential m6A-related epigenetic targets predicted through the analysis of m6A profiles of multiple cancer samples, but their specific mechanisms in tumors are still unclear. As a classical m6A demethylase, ALKBH5 has been preliminarily understood for its regulatory role in epigenetics (acting through demethylation of m6A) due to its demethylase activity; while LRPPRC is an m6A-binding protein with dual localization in the nucleus and mitochondria. Currently, the research on it mainly focuses on maintaining RNA stability in mitochondria, but there is almost no research at the level of m6A epigenetic regulation. In addition, whether there is an association between ALKBH5 and LRPPRC and their potential physiological functions remain to be explored. (2) XPO1 and eIF4E are two key proteins for specific nuclear export. Although there are currently drugs targeting them, because they are essential genes for normal cells and can cause cytotoxic side effects, there is an urgent need for a more precise nuclear targeting strategy to reduce the side effects. The ribavirin drug targeting eIF4E provides a proof of concept for precise nuclear targeting therapy. However, this drug mainly affects nuclear export by structurally mimicking the 7 m7G cap and competitively inhibiting the binding of eIF4E to the RNA of oncogenes. Currently, in terms of nuclear export inhibition, there are no other principles of action and targets, and a single mechanism is difficult to meet the complex cancer treatment needs. (3) Cmyc and Pim1 are both proto-oncogenes. Among them, Cmyc is regarded as the "ultimate target" of cancer treatment but is also the "most difficult target" with problems in drug development. Currently, the mechanism of m6A epigenetic regulation of the expression of oncogenes such as Cmyc and Pim1 is still unclear, lacking in-depth and systematic research. Summary of the Invention

[0006] In order to overcome the above deficiencies of the existing technology, the present invention provides a new protein interaction ALKBH5-LRPPRC and a new signaling pathway ALKBH5-LRPPRC-eIF4E-XPO1. This signaling pathway constitutes the structural basis for m6A demethylation to regulate nuclear export, realizing the organic integration of RNA epigenetics regulation and the specific RNA nuclear export system. At the same time, the present invention verifies through experiments an anti-cancer strategy of interfering with ALKBH5-LRPPRC to regulate the expression of proto-oncogene Cmyc and Pim1 proteins, providing a new approach and possibility for precisely intervening in the regulation of eIF4E nucleocytoplasmic shuttling.

[0007] To achieve the above object, the present invention, through large-scale research on the relationship between proteins and m6A map generation features, found that the effects of ALKBH5 and LRPPRC on m6A map generation features are to a certain extent the same. Therefore, the interaction relationship between ALKBH5 and LRPPRC was first verified, and it was found that there is an interaction between ALKBH5 and LRPPRC in the nucleus, and this interaction is RNA-dependent and m6A-dependent. New protein interactions often imply new signaling pathways. During the exploration of the nuclear functions of ALKBH5 and LRPPRC, a protein complex was isolated. The new findings showed that while there is an interaction between ALKBH5 and LRPPRC, it also interacts with the nuclear export protein XPO1 and elF4E, and this interaction is RNA-dependent. Thus, a new signaling pathway, ALKBH5-LRPPRC-elF4E-XPO1, was discovered. This pathway reveals the structural basis of m6A demethylation regulating nuclear export. Given the key roles of XPO1 and elF4E in the nuclear export of RNA specificity (oncogene mRNA), this discovery also incorporates the regulation of m6A epigenetics into this specific nuclear export process. Since both XPO1 and elF4E are essential genes, and elF4E has both nuclear export and translation initiation functions (its translation initiation function is a necessary function for normal cells), it has been proposed in the field that specific nuclear intervention on elF4E to precisely block its specific nuclear transport function will greatly alleviate the toxic side effects of its related treatments. The clinical success of ribavirin provides a proof of concept for the RNA export targeted therapy strategy. The present invention first integrates RNA epigenetic regulation into this pathway and further verifies whether the specific nuclear export process mediated by elF4E-XPO1 can be blocked by precisely targeting the ALKBH5-LRPPRC interaction in the nucleus. For this purpose, the present invention uses the Crispr editing technology to simulate the intervention of the ALKBH5-LRPPRC interaction and detects the final protein expression of two important proto-oncogenes, Cmyc and Pim1. The results show that intervening in the ALKBH5-LRPPRC interaction can specifically affect the final protein content of Cmyc and Pim1, but has no effect on the protein expression of other essential genes in the cells (such as the nuclear-encoded mitochondrial essential gene COXIV, the translation initiation factor eLF4E, and the nuclear splicing-related factor SRSF3). Currently, the precise intervention targeting elF4E in the nucleus mainly relies on structurally mimicking the m 7 G-cap competitively inhibits eIF4E, thereby blocking the nuclear export mediated by it. The research findings of the present invention will provide more diverse targets and mechanisms for specifically blocking the nuclear export of elF4E in the nucleus, and complete the preliminary experimental proof of concept, opening up new ways and possibilities for precise intervention based on nucleocytoplasmic shuttle regulation.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention provides the use of a reagent for inhibiting the signal pathway ALKBH5-LRPPRC-elF4E-XPO1 in the preparation of a drug for drug resistance, wherein LRPPRC and ALKBH5 interact with each other in the nucleus depending on RNA m6A modification, and interact with the nuclear export protein XPO1 and elF4E; the reagent can inhibitively regulate the specific nuclear export process mediated by elF4E-XPO1, thereby achieving cancer treatment.

[0010] The present invention also provides the use of a reagent for inhibiting the signal pathway ALKBH5-LRPPRC-elF4E-XPO1 in the preparation of a drug for intervening in the nucleocytoplasmic shuttling of elF4E, wherein LRPPRC and ALKBH5 interact with each other in the nucleus depending on RNA m6A modification, and interact with the nuclear export protein XPO1 and elF4E; the reagent can reduce the protein expression level of elF4E-related cancer drug resistance genes or proto-oncogenes, thereby precisely intervening in the regulation of elF4E nucleocytoplasmic shuttling.

[0011] Preferably, the reagent for inhibiting the signal pathway ALKBH5-LRPPRC-elF4E-XPO1 is an sgRNA that intervenes in the expression of ALKBH5 and / or LRPPRC.

[0012] More preferably, the sgRNAs for intervening in ALKBH5 include at least one of SgRNA-1 shown in SEQ ID NO.1 and 2, SgRNA-2 shown in SEQ ID NO.3 and 4, SgRNA-3 shown in SEQ ID NO.5 and 6, SgRNA-4 shown in SEQ ID NO.7 and 8, and SgRNA-5 shown in SEQ ID NO.9 and 10; the sgRNAs for intervening in the expression of LRPPRC include at least one of SgRNA-1 shown in SEQ ID NO.11 and 12, SgRNA-2 shown in SEQ ID NO.13 and 14, SgRNA-3 shown in SEQ ID NO.15 and 16, SgRNA-4 shown in SEQ ID NO.17 and 18, and SgRNA-5 shown in SEQ ID NO.19 and 20.

[0013] More preferably, when there are two or more sgRNAs for intervening in the expression of ALKBH5 and / or LRPPRC, the sgRNAs are mixed in a ratio of 1:1.

[0014] Preferably, the elF4E-related proto-oncogenes include Cmyc and Pim1.

[0015] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0016] More preferably, the excipients include at least one of excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, antioxidants, adsorbents, filter aids, and release retardants.

[0017] More preferably, the dosage forms of the drug include tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, or suppositories.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The present invention has achieved multiple innovative breakthroughs. First, it was discovered for the first time that LRPPRC and ALKBH5 interact in the cell nucleus, and this interaction depends on the m6A modification of RNA. This interaction has not been reported before. Based on this discovery, when further exploring its physiological function, a new signaling pathway - ALKBH5-LRPPRC-elF4E-XPO1 was successfully revealed. Moreover, the complex formed by this interaction was isolated. As the structural basis for the nuclear export of RNA m6A demethylation, it has breakthroughly organically combined RNA epigenetics with the specific nuclear export mechanism, opening up a new direction for specific nuclear export targeted research and treatment. In addition, the present invention also carried out an experimental proof of concept on whether this new signaling pathway can regulate the expression of proto-oncogenes. The above achievements in multiple aspects, from the discovery of the interaction, the revelation of the new signaling pathway, the establishment of the structural basis to the verification of the regulatory function, are all reported for the first time, showing significant innovation. Description of the Drawings

[0020] Figure 1 For co-immunoprecipitation of proteins (A) and Western Blot detection (B);

[0021] Figure 2 For the results of co-immunoprecipitation and Western Blot detection of the nuclear export of the ALKBH5-LRPPRC-XPO1-eLF4E RNA m6A regulatory pathway;

[0022] Figure 3 For Western Blot detection of the ALKBH5-LRPPRC-XPO1-eLF4E pathway regulating the nuclear export of proto-oncogenes Cmyc and Pim1 and affecting protein expression. Detailed Embodiments

[0023] The following further describes the specific embodiments of the present invention. It should be noted here that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation on the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all available through conventional commercial channels unless otherwise specified. Among them, the immunoprecipitation IGG beads were purchased from Abclonal, catalog number AC044N; the Flag beads were purchased from Abclonal, catalog number AE037.

[0025] In the following examples, the genes and their Gene IDs involved include ALKBH5 (Gene ID: 54890), LRPPRC (GeneID: 10128), XPO1 (Gene ID: 7514), ElF4E (Gene ID: 1977), NXF1 (Gene ID: 10482), RCC1 (GeneID: 1104), Cmyc (Gene ID: 4609), Pim1 (Gene ID: 5292), COXIV (Gene ID: 1327), SRSF3 (GeneID: 6428). Among them, the ALKBH5-Flag sequence used to construct the transfected 293T cell line of ALKBH5-Flag is:

[0026]

[0027] The present invention first conducts a large-scale exploration on the characteristics of m6A-related proteins affecting the generation of m6A maps, and discovers that there is a certain degree of commonality between ALKBH5 and LRPPRC in the characteristics of m6A map generation, that is, LRPPRC exhibits m6A demethylation characteristics similar to those of the demethylase ALKBH5 m6A. Given that LRPPRC does not possess any enzymatic domain but exhibits m6A demethylation characteristics similar to those of ALKBH5, it is inferred that there is a certain association between ALKBH5 and LRPPRC. Example 1 demonstrates the exploration of the interaction between the two, and the results show that there is an interaction between LRPPRC and ALKBH5 in the nucleus, and this interaction is dependent on RNA and m6A. It is worth mentioning that the epigenetic research on LRPPRC in the nucleus is the first time, and previous research on this gene has mainly focused on the field of mitochondrial energy metabolism.

[0028] New protein interactions in the nucleus often indicate the revelation of new biological functions. Given that both ALKBH5 and LRPPRC exhibit the ability to regulate m6A methylation, and inspired by the mechanism that m6A-modified RNA in vivo is recognized by YTHDC1 and exported from the nucleus mediated by the NXF1-SRSF3 complex, the present invention explores how the RNA that is demethylated by m6A through the regulation of ALKBH5-LRPPRC completes the nuclear export process. The experimental results of Example 2 show that the RNA regulated by ALKBH5-LRPPRC is not exported from the nucleus by the NXF1 transport system, but unexpectedly, the ALKBH5-LRPPRC-elF4E-XPO1 complex is isolated, and it is confirmed that its interaction is RNA-dependent. The discovery of this complex reveals a brand-new signaling pathway, realizing the integration of RNA epigenetic regulation and a special nuclear export mechanism, and at the same time introducing a new regulatory target for the specific nuclear export complex XPO1-elF4E, providing more diverse theoretical bases for precisely intervening in the regulation of elF4E nucleocytoplasmic shuttling.

[0029] Given that elF4E is involved in the nuclear export pathway of oncogenes, it is further verified whether the regulation of the ALKBH5-LRPPRC-elF4E-XPO1 pathway can affect the expression of proto-oncogenes and thus become a new drug target. In Example 3, the interaction between ALKBH5-LRPPRC is interfered with by the CRISPR technique, and the final protein expression of the proto-oncogenes Cmyc and Pim1 is detected. The experimental results show that interfering with ALKBH5-LRPPRC can specifically reduce the protein expression levels of the elF4E target genes Cmyc and Pim1, and has almost no effect on the expression of other essential genes, which provides a brand-new strategy for precisely intervening in the regulation of elF4E nucleocytoplasmic shuttling.

[0030] To comprehensively and clearly present the technical solution of the present invention and its remarkable advantages, the present invention will be elaborated in depth with reference to detailed specific embodiments below.

[0031] Example 1: Verification of the RNA- and m6A-dependent interaction between ALKBH5 and LRPPRC

[0032] 1. The experimental operations are as follows:

[0033] (1) Plate approximately 1×10 8 293T cell lines transfected with ALKBH5-Flag (the 293T cells were purchased from the ATCC cell bank, and Aiji Biotech was commissioned to construct the 293T cell line transfected with ALKBH5-Flag) using DMEM medium (containing 10% fetal bovine serum), and treat the cells with 5 μM STM2457 drug for 24 h. For the corresponding control group, add an equal volume of the drug solvent DMSO to treat the cells for 24 h.

[0034] (2) After collecting the cells, perform cell counting, then wash the cell line twice with PBS, centrifuge at 2000×g for 5 minutes, and discard the supernatant.

[0035] (3) Resuspend the cell pellet in 2 - 5 volumes of Buffer A (10 mM HEPES pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, approximately 3 mL of lysis buffer per 1×10 8 cells), vortex to mix well, and incubate on ice for 15 minutes. Then add IGEPAL-CA630 to a final concentration of 0.25% (v / v), vortex and incubate in an ice bath for 5 minutes. Subsequently, centrifuge at 2000×g at 4°C for 5 minutes, and retain the supernatant, which is the cytoplasmic fraction.

[0036] (4) Resuspend the nuclear pellet in 2 - 5 volumes of Buffer B (20 mM HEPES pH 7.5, 0.42 M KCl, 4 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 10% glycerol, approximately 3 mL of lysis buffer per 1×10 8 cells), vortex to mix well and incubate on ice for 1 hour. Subsequently, centrifuge at 15,000×g at 4°C for 15 minutes, and this supernatant is the nuclear fraction.

[0037] (5) Retain 1 / 10 volume of the cytoplasmic fraction or nuclear fraction as the Input for the immunoprecipitation experiment. Divide the remaining lysate supernatant into three equal parts. Incubate one part with IGG beads, and the other two parts with Flag beads, and rotate and incubate at 4°C overnight.

[0038] (6) Add 1 μg / mL RNase A to one of the Flag beads incubation samples and incubate at 37 °C for 30 minutes.

[0039] (7) Wash the magnetic beads of the 3 incubation samples 4 times with 500 μL Buffer C (50 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 0.1% IGEPAL-CA630, 10 mM β-ME, 10% glycerol).

[0040] (8) After preparing the sample by adding protein loading buffer, perform Western Blot detection. Among them, the Western Blot antibodies include: LRPPRC (abclonal, A3365, 1:1000); Flag (abmart, M20018, 1:2000); GAPDH (abclonal, AC002, 1:5000); RCC1 (abclonal, A0662, 1:1000); anti-mouse secondary antibody (abclonal, AS003, 1:10000); anti-rabbit secondary antibody (abclonal, AS014, 1:10000).

[0041] 2. Experimental results:

[0042] As Figure 1 shown in B, the results of the protein cytoplasmic / nuclear fractionation experiment are good. The cytoplasmic protein GAPDH is located in the cytoplasmic fraction, the nuclear protein RCC1 is located in the nuclear fraction, and there is basically no crosstalk between the two fractions. The target proteins LRPPRC and ALKBH5 are distributed in both the cytoplasmic and nuclear fractions, and there is no change in protein expression level due to the treatment with the m6A inhibitor (STM2457). As Figure 1 shown in the immunoprecipitation results of A, there is an interaction between ALKBH5 and LRPPRC in the nucleus. At the same time, under the condition of RNase A treatment, this interaction weakens or even disappears, proving that the interaction between ALKBH5 and LRPPRC is RNA-dependent. The treatment with the m6A inhibitor STM2457 can reduce the m6A modification content of RNA. From the results, it can be seen that the interaction between ALKBH5 and LRPPRC disappears after the treatment with the drug STM2457, proving that the interaction between the two depends on the m6A modification of RNA. Thus, based on the above protein immunoprecipitation experiments, it is first proved that there is an interaction between ALKBH5 and LRPPRC, and this interaction depends on RNA and its m6A modification.

[0043] Example 2: Verifying the Structural Basis of Nuclear Export of the ALKBH5-LRPPRC-XPO1-eIF4E RNA m6A Regulatory Pathway

[0044] 1. The experimental operations are as follows:

[0045] (1) Plate approximately 5×10 7 293T cell lines transfected with ALKBH5-Flag using DMEM medium (containing 10% fetal bovine serum), collect the cells after culturing for 48 hours, and count them.

[0046] (2) Wash the cell line twice with PBS, centrifuge at 2000×g for 5 minutes, and discard the supernatant.

[0047] (3) Resuspend the cell pellet in 2 - 5 volumes of Buffer A (10 mM HEPES pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, approximately 3 mL lysis buffer / 1×10 8 cells), vortex to mix well, and incubate on ice for 15 minutes. Then add IGEPAL-CA630 to a final concentration of 0.25% (v / v), vortex and incubate in an ice bath for 5 minutes. Subsequently, centrifuge at 2000×g for 5 minutes at 4°C, and retain the supernatant, which is the cytoplasmic fraction.

[0048] (4) Resuspend the nuclear pellet in 2 - 5 volumes of Buffer B (20 mM HEPES pH 7.5, 0.42 M KCl, 4 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 10% glycerol, approximately 3 mL lysis buffer / 1×10 8 cells), vortex to mix well, and incubate on ice for 1 hour. Subsequently, centrifuge at 15,000×g for 15 minutes at 4°C, and this supernatant is the nuclear fraction.

[0049] (5) Retain 1 / 10 volume of the cytoplasmic fraction or nuclear fraction as the Input for the immunoprecipitation experiment, and divide the remaining lysate supernatant into three equal parts. Incubate one part with IGG beads, and the other two parts with Flag beads, and rotate and incubate overnight at 4°C.

[0050] (6) Add RNase A at a concentration of 1 μg / mL to one of the Flag bead incubation samples and incubate at 37°C for 30 minutes.

[0051] (7) Wash the magnetic beads of the three incubation samples 4 times with 500 μL of Buffer C (50 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 0.1% IGEPAL-CA630, 10 mM β-ME, 10% glycerol).

[0052] (8) Samples were prepared by adding protein loading buffer for Western Blot detection. Among them, the Western Blot antibodies include: LRPPRC (abclonal, A3365, 1:1000); Flag (abmart, M20018, 1:2000); GAPDH (abclonal, AC002, 1:5000); RCC1 (abclonal, A0662, 1:1000); NXF1 (abclonal, A3488, 1:1000); XPO1 (abclonal, A0299, 1:1000); elF4E (abclonal, A25608, 1:1000); anti-mouse secondary antibody (abclonal, AS003, 1:10000); anti-rabbit secondary antibody (abclonal, AS014, 1:10000).

[0053] 2. Experimental results:

[0054] From Figure 2From the results of B, it can be seen that the protein cytoplasmic fraction and nuclear fraction are well separated. As an RNA nuclear export structural protein, NXF1 is mainly distributed in the nuclear fraction; elF4E is an initiation factor for protein translation and is mainly distributed in the cytoplasmic part. At the same time, a study (Siddiqui, Nadeem, and Katherine LB Borden. "mRNA export and cancer." Wiley Interdisciplinary Reviews: RNA 3.1 (2012): 13 - 25.) has shown that the elF4E protein is related to the nuclear export of specific RNAs (proto - oncogenes, genes closely related to the cell cycle, etc.), and the component separation results of this example also show that it is distributed in the nuclear fraction, which is consistent with previous studies. Since ALKBH5 is an RNA m6A demethylase and LRPPRC is an RNA m6A - binding protein, their interaction plays an m6A demethylation role in the nucleus. It is known that the m6A modification of RNA in the nucleus is recognized by YTHDC1 and exported from the nucleus through the NXF1 - SRSF3 nuclear export complex. However, the experimental results of this example show that there is no interaction between ALKBH5 and NXF1, indicating that there is a nuclear export pathway for RNA with m6A removed different from that of RNA with m6A added. At the same time, by detecting another nuclear export protein XPO1, it is found that there is an RNA - dependent interaction between ALKBH5 and XPO1. Since the interaction between XPO1 and elF4E is related to the selective nuclear export of some proto - oncogenes and drug - resistance gene RNAs, we further detected the interaction between ALKBH5 and elF4E and found that there is also an RNA - dependent interaction between ALKBH5 and elF4E, as shown in Figure 2 Figure A. The above experiment first isolated the nuclear export complex ALKBH5 - LRPPRC - XPO1 - elF4E for RNA with m6A removed, laying a protein structural foundation for the nuclear export of RNA after m6A regulation. Given that it has been previously reported that LRPPRC is involved in the nuclear export of elF4E and m6A modification can regulate the stability of RNA, the interaction between ALKBH5 and LRPPRC may integrate m6A epigenetic regulation into the nuclear export of specific RNAs and amplify the role of specific RNA nuclear export. Therefore, this discovery may provide new ideas for the treatment pathway of elF4E - related specific nuclear export (such as the nuclear export of cancer drug - resistance genes and proto - oncogenes) and disease treatment targets.

[0055] Example 3: Concept verification that the ALKBH5 - LRPPRC - XPO1 - eLF4E pathway regulates the nuclear export of proto - oncogenes Cmyc and Pim1 and thus affects protein expression

[0056] 1. The Target sequences of the genes used are shown in Tables 1 and 2.

[0057] Table 1 Gene editing sequences of ALKBH5

[0058]

[0059] Table 2 Gene editing sequences of LRPPRC

[0060]

[0061] 2. The experimental operations are as follows:

[0062] (1) Synthesize the gene editing sequences shown in Table 1 and Table 2, and then construct them into the LentiCRISPRv2 vector (addgene, Plasmid#52961); where the sgmix is composed of 5 gene editing vectors mixed in a ratio of 1:1:1:1:1.

[0063] (2) Plate the normal 293T cell line with DMEM medium (containing 10% fetal bovine serum) 24 hours before transfection. Digest the cells in the logarithmic growth phase, and adjust the cell density to 2×10 5 cells / mL with complete medium. Then add 500 μL of cell suspension (24-well plate) to each well and incubate overnight in a 37°C, 5% CO2 incubator to ensure that the cell confluence reaches 70–90% at the time of transfection.

[0064] (3) Add 50 μL of Opti-MEM to a 1.5 mL EP tube, and then add 0.5 μg of the plasmid DNA constructed in step (1) (final volume ≤ 50 μL), and gently mix (if it is LRPPRC knockout + ALKBH5 overexpression, then add 0.25 μg of the knockout plasmid + 0.25 μg of the pcDNA3.1-ALKBH5-Flag plasmid (purchased from Miaoling Biology)).

[0065] (4) Take another EP tube, add 50 μL of Opti-MEM, and add 1.5 μL of Lipofectamine 2000 (ThermoFisher, 11668030; DNA μg: Lipo2000 μL = 1:3), gently mix, and let stand at room temperature for 5 minutes.

[0066] (5) Mix the DNA diluted in step (3) with the Lipofectamine 2000 in step (4) (total volume 100 μL), gently vortex, and let stand at room temperature for 15–20 minutes.

[0067] (6) Add 100 μL of the transfection complex dropwise into the wells, and gently shake the culture plate to mix. After incubating in a 37°C incubator for 4–6 hours, replace the complete medium (containing serum).

[0068] (7) 24 hours after transfection, 3 μg / mL puromycin was added for screening for 48 hours.

[0069] (8) The culture was switched to antibiotic-free complete medium (DMEM + 10% fetal bovine serum) and cultured for recovery for 48 hours.

[0070] (9) Cells were collected, protein loading buffer was added to prepare samples, and Western Blot detection was performed. Among them, the Western Blot antibodies included: LRPPRC (abclonal, A3365, 1:1000); ALKBH5 (abmart, A11684, 1:1000); GAPDH (abclonal, AC002, 1:5000); COXⅣ (abclonal, A22871, 1:1000); Pim1 (abclonal, A19695, 1:1000); Cmyc (abmart, T55150, 1:1000); SRSF3 (abclonal, A6067, 1:1000); elF4E (abclonal, A25608, 1:1000); anti-mouse secondary antibody (abclonal, AS003, 1:10000); anti-rabbit secondary antibody (abclonal, AS014, 1:10000).

[0071] 3. Experimental results:

[0072] We speculate that the interaction between ALKBH5 and LRPPRC may participate in and regulate the elF4E-related nuclear export process. Based on this, a proof-of-concept study was carried out to explore whether the ALKBH5-LRPPRC-XPO1-eLF4E pathway could become a new target for cancer treatment by regulating nuclear export. Cmyc and Pim1, as proto-oncogenes, are classical molecules for elF4E-mediated selective nuclear export of RNA. If the RNA epigenetic regulation represented by ALKBH5 and LRPPRC participates in the elF4E-related nuclear export process, then interfering with the expression of ALKBH5 or LRPPRC and their interaction will affect the nuclear export of proto-oncogenes Cmyc and Pim1, and ultimately result in a decrease in the protein expression level of proto-oncogenes. By Figure 3From the experimental results, it can be seen that separately affecting LRPPRC (lanes 2 and 6), ALKBH5 (lanes 3 and 7), and simultaneously affecting LRPPRC and ALKBH5 (lanes 4 and 8) can specifically affect the protein levels of Cmyc and Pim1. At the same time, it was found that overexpressing ALKBH5 while knocking out LRPPRC could increase the expression level of LRPPRC (lanes 5 and 9), which not only indirectly verified the interaction between ALKBH5 and LRPPRC in Example 1, but also indicated that the interaction between the two may have important physiological functions. Using this phenomenon to explore whether elevated LRPPRC can rescue the expression of Cmyc and Pim1, it was found that the sgmix knockout system may cause multiple damages to the protein function of LRPPRC, and overexpression cannot rescue the protein expression of Cmyc and Pim1 (lane 5), while knocking out the elevated LRPPRC amount at a single sgRNA site can rescue the protein expression of Cmyc and Pim1 (lane 9). These results further verified that the ALKBH5-LRPPRC-XPO1-eLF4E pathway can affect the protein expression of the proto-oncogenes Cmyc and Pim1 through nuclear transport, providing a proof of concept for this new signaling pathway to become an anti-cancer treatment target. At the same time, by verifying the expression of other essential genes in the cells, it was found that affecting ALKBH5-LRPPRC did not affect the protein expression levels of other essential genes (nuclear-encoded mitochondrial gene COXIV, translation initiation factor eLF4E, nuclear splicing-related factor SRSF3).

[0073] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations made to these embodiments still fall within the protection scope of the present invention.

Claims

1. Use of a reagent for inhibiting the signal pathway ALKBH5-LRPPRC-elF4E-XPO1 in the preparation of a drug for drug resistance, characterized in that, The LRPPRC and ALKBH5 interact with each other in an RNA-dependent m6A modification in the cell nucleus, and interact with the nuclear export protein XPO1 and elF4E; the reagent can inhibitively regulate the specific nuclear export process mediated by elF4E-XPO1, thereby achieving cancer treatment.

2. Use of a reagent for inhibiting the signal pathway ALKBH5-LRPPRC-elF4E-XPO1 in the preparation of a drug for intervening in the nucleocytoplasmic shuttle of elF4E, characterized in that, The LRPPRC and ALKBH5 interact with each other in an RNA-dependent m6A modification in the cell nucleus, and interact with the nuclear export protein XPO1 and elF4E; the reagent can reduce the protein expression level of elF4E-related cancer drug resistance genes or proto-oncogenes, thereby precisely intervening in the regulation of elF4E nucleocytoplasmic shuttling.

3. The application according to claim 1 or 2, characterized in that The reagent that inhibits the signal pathway ALKBH5-LRPPRC-elF4E-XPO1 is an sgRNA that intervenes in the expression of ALKBH5 and / or LRPPRC.

4. The application according to claim 3, wherein The sgRNAs that intervene in ALKBH5 include at least one of SgRNA-1 shown in SEQ ID NO.1 and 2, SgRNA-2 shown in SEQ ID NO.3 and 4, SgRNA-3 shown in SEQ ID NO.5 and 6, SgRNA-4 shown in SEQ ID NO.7 and 8, and SgRNA-5 shown in SEQ ID NO.9 and 10; the sgRNAs that intervene in the expression of LRPPRC include at least one of SgRNA-1 shown in SEQ ID NO.11 and 12, SgRNA-2 shown in SEQ ID NO.13 and 14, SgRNA-3 shown in SEQ ID NO.15 and 16, SgRNA-4 shown in SEQ ID NO.17 and 18, and SgRNA-5 shown in SEQ ID NO.19 and 20.

5. The application according to claim 4, wherein When there are more than two sgRNAs that intervene in the expression of ALKBH5 and / or LRPPRC, the sgRNAs are mixed in a ratio of 1:

1.

6. The application according to claim 2, wherein The elF4E-related proto-oncogenes include Cmyc and Pim1.

7. The application according to claim 1 or 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. The application according to claim 7, wherein The excipients include at least one of excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, antioxidants, adsorbents, filter aids, and release retardants.

9. The application according to claim 7, wherein The dosage forms of the drug include tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, or suppositories.

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