Application of METTL2 in sensitization of digestive system tumor radiotherapy

Abstract

The invention relates to an application of METTL2 in cancer radiotherapy sensitization. The METTL2 is found to be a gene which is extremely related to the radiotherapy tolerance of the liver cancer through a large number of researches and screening, the high expression level of the METTL2 has obvious positive correlation with the radiotherapy tolerance, the METTL2 can be used for predicting the radiotherapy tolerance and the sensitivity degree of the liver cancer, and the relation of preoperative treatment of a patient is evaluated in advance. By revealing the relevance between the METTL2 gene and the liver cancer radiotherapy tolerance, the METTL2 gene has important practical significance for solving the problems of clinical curative effect difference and recession effect / prognosis evaluation blank among individuals and better realizing accurate treatment. And a new drug treatment target is provided for human to overcome liver cancer.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of METTL2 in radiotherapy sensitization of digestive system tumors. Background Art

[0002] Hepatocellular carcinoma (HCC) is currently the fourth most common malignant tumor and the second leading cause of death from cancer in my country, seriously threatening people's lives and health. The treatment of HCC mainly includes surgery, local ablation therapy, radiotherapy (RT), transcatheter arterial chemoembolization (TACE) and systemic drug therapy. Among them, new radiotherapy methods represented by three-dimensional or intensity-modulated conformal radiotherapy (3D-CRT or IMRT) and stereotactic body radiotherapy (SBRT) have gradually become an important part of HCC treatment. In recent years, domestic and foreign scholars have successively reported the unique advantages of radiotherapy in efficacy: it can improve local control rate and significantly prolong survival time (3-year survival rate can reach about 30%); it can also relieve symptoms such as pain, obstruction or bleeding caused by lymph node, lung, bone, brain or adrenal metastasis; some tumors that cannot be treated with surgery shrink or downstage after radiotherapy and can be converted to surgical resection.

[0003] Both the NCCN guidelines and the CSCO guidelines recommend radiotherapy (radical or palliative) as the standard treatment for liver cancer. However, radiotherapy for liver cancer has many constraints, such as low overall effectiveness, short duration of efficacy, and patients are more likely to relapse, which seriously limits the widespread application of radiotherapy in the field of liver cancer. It is generally believed that the high heterogeneity of liver cancer cells and the insensitivity or resistance of some cells to radiation are the main reasons for the poor efficacy of radiotherapy. Therefore, it is of great practical significance to screen and identify key targets that mediate radiotherapy resistance in liver cancer, and to develop corresponding intervention strategies based on this mechanism of action to improve the efficacy of radiotherapy for liver cancer in clinical practice. Summary of the invention

[0004] The purpose of the present invention is to solve the problem of unclear mechanism of radiotherapy tolerance of liver cancer in the prior art, so as to start from the nature of radiation (physical basis), expand other biological effect mechanisms caused by radiation on the basis of DNA damage biological effects, clarify METTL2 as a target related to radiotherapy resistance of liver cancer, and provide a more comprehensive scientific basis and solution for achieving radiotherapy sensitization of liver cancer.

[0005] In order to solve the above technical problems, the present invention is implemented through the following technical solutions.

[0006] The first aspect of the present invention provides the use of a METTL2 inhibitor in the preparation of a drug for improving the radiosensitivity of digestive system tumors.

[0007] Preferably, the METTL2 inhibitor is selected from one or more of shRNA, sgRNA, and siRNA designed based on METTL2.

[0008] Preferably, the METTL2 inhibitor is selected from sgRNA designed based on METTL2; most preferably, the sgRNA sequence designed based on METTL2 is selected from one or more of SEQ ID NO: 9 (5'-GCTGCTCCACAGG ATGCAGA-3'), SEQ ID NO: 10 (5'-CGCAGAAGCATCATCCCGCC-3'), and SEQ ID NO: 11 (5'-GTGTCTCCACAGGATGCAGA-3').

[0009] Preferably, the digestive system tumor is liver cancer.

[0010] A second aspect of the present invention provides a use of a reagent for detecting the expression level of METTL2 in the preparation of a product for detecting the radiotherapy sensitivity and / or predicting the therapeutic effect of a digestive system tumor.

[0011] Preferably, the reagent for detecting the expression level of METTL2 includes primers for detecting the expression level of METTL2 gene and / or a reagent for detecting the expression level of METTL2 protein.

[0012] Preferably, the primers for detecting the expression level of the METTL2 gene are selected from at least one of the following primer pairs:

[0013] Primer pair 1: the upstream sequence is as shown in SEQ ID NO: 1 (5'-GCAGTCCTCGCCGATAAGAG-3'), and the downstream sequence is as shown in SEQ ID NO: 2 (5'-CTTCCGACCACTCCACATTGT-3');

[0014] Primer pair 2: the upstream sequence is shown in SEQ ID NO: 3 (5'-ATGTGGAGTGGTCGGAAGAG-3'), and the downstream sequence is shown in SEQ ID NO: 4 (5'-CCAGTATTTGTGGGCATTGATCT-3');

[0015] Primer pair 3: the upstream sequence is shown in SEQ ID NO: 5 (5'-TCCTCAGCCACCTACCGAAT A-3'), and the downstream sequence is shown in SEQ ID NO: 6 (5'-CTGAATTTGTCTGGACCAGTTCT-3');

[0016] Primer pair 4: the upstream sequence is shown in SEQ ID NO: 7 (5'-GCGAGCTCATGGCCGGCTCC TACCCT-3'), and the downstream sequence is shown in SEQ ID NO: 8 (5'-GCGCGGCCGCTCAGCTGGTG CTGGACAG-3').

[0017] Preferably, the reagent for detecting the expression level of METTL2 protein is selected from METTL2 monoclonal antibody and / or METTL2 polyclonal antibody.

[0018] Preferably, the reagent for detecting the expression level of METTL2 protein is selected from ab101510 (ABcam).

[0019] Preferably, the digestive system tumor is liver cancer.

[0020] The third aspect of the present invention provides the use of a METTL2 inhibitor in the preparation of a product that promotes ferroptosis of tumor cells.

[0021] Preferably, the METTL2 inhibitor is selected from one or more of shRNA, sgRNA, and siRNA designed based on METTL2.

[0022] Preferably, the METTL2 inhibitor is selected from sgRNA designed based on METTL2; most preferably, the sgRNA sequence designed based on METTL2 is selected from one or more of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0023] Preferably, the tumor cells are selected from liver cancer cells.

[0024] The fourth aspect of the present invention provides a kit for detecting the sensitivity and / or efficacy of radiotherapy for digestive system tumors, comprising primers for detecting the expression level of the METTL2 gene and / or reagents for detecting the content of the METTL2 protein; and one or more of PCR enzyme, PCR buffer, dNTPs, and fluorescent substrate.

[0025] Preferably, the primers for detecting the expression level of the METTL2 gene are selected from at least one of the following primer pairs:

[0026] Primer pair 1: the upstream sequence is shown in SEQ ID NO: 1, and the downstream sequence is shown in SEQ ID NO: 2;

[0027] Primer pair 2: the upstream sequence is shown in SEQ ID NO: 3, and the downstream sequence is shown in SEQ ID NO: 4;

[0028] Primer pair 3: the upstream sequence is shown in SEQ ID NO: 5, and the downstream sequence is shown in SEQ ID NO: 6;

[0029] Primer pair 4: the upstream sequence is shown as SEQ ID NO: 7, and the downstream sequence is shown as SEQ ID NO: 8.

[0030] Preferably, the reagent for detecting the expression level of METTL2 protein is selected from METTL2 monoclonal antibody and / or METTL2 polyclonal antibody.

[0031] Preferably, the reagent for detecting the expression level of METTL2 protein is selected from ab101510 (ABcam).

[0032] Preferably, the fluorescent substrate is selected from Syber Green or a fluorescently labeled probe.

[0033] Preferably, the digestive system tumor is liver cancer.

[0034] A fifth aspect of the present invention provides a pharmaceutical composition for improving the radiosensitivity of digestive system tumors, comprising a METTL2 inhibitor and a pharmaceutically acceptable carrier.

[0035] Preferably, the METTL2 inhibitor is selected from one or more of shRNA, sgRNA, and siRNA designed based on METTL2.

[0036] Preferably, the METTL2 inhibitor is selected from sgRNA designed based on METTL2; most preferably, the sgRNA sequence designed based on METTL2 is selected from one or more of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0037] Preferably, the digestive system tumor is liver cancer.

[0038] Preferably, the pharmaceutically acceptable carrier is selected from one or more of a filler, a binder, a lubricant, a disintegrant, a flavoring agent, a colorant, an antioxidant, an antibacterial agent, a chelating agent, a surfactant, and a solvent.

[0039] The sixth aspect of the present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for improving the radiotherapy sensitivity of digestive system tumors.

[0040] Preferably, the METTL2 inhibitor is selected from one or more of shRNA, sgRNA, and siRNA designed based on METTL2.

[0041] Preferably, the METTL2 inhibitor is selected from sgRNA designed based on METTL2; most preferably, the sgRNA sequence designed based on METTL2 is selected from one or more of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0042] Preferably, the digestive system tumor is liver cancer.

[0043] It should be understood that, unless otherwise specified, in the context of the present invention, the primers and / or primer pairs refer to PCR primers used to synthesize the cDNA chain of the METTL2 gene in PCR, thereby being used to detect the expression level of the METTL2 gene mRNA. In addition to the primers and / or primer pairs listed in the present invention, those skilled in the art are fully capable of designing corresponding primers and / or primer pairs based on the gene sequence of METTL2 using conventional methods and means in the art including but not limited to molecular biology, and screening the designed primers and / or primer pairs by conventional experimental means, as long as the specific detection of the expression level of METTL2 can be achieved. The METTL2 inhibitor refers to a substance that can specifically downregulate the expression level of METTL2 and / or the transcription level of its mature mRNA and / or the expression level or activity of METTL2 protein, for example, antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA Erasers, Target Masking and / or multi-target methods are used to downregulate the expression level and / or activity of METTL2, as long as the level and / or activity of METTL2 can be reduced.

[0044] The X (or γ) rays used in radiotherapy are high-energy rays with wave-particle duality and the properties of photons. After the X (or γ) rays collide with molecules, energy transfer and exchange occurs, causing the molecules or atoms to undergo ionizing radiation (Ionizing Radiation, IR), generating a large number of free radicals. It is generally believed that the biological effects of IR are mainly caused by free radical-mediated DNA damage. However, the active oxygen free radicals generated by the ionization of water molecules (H2O) by X (or γ) rays can not only attack the nucleic acid linking sites to cause DNA breaks, but can also accept divalent iron (Fe 2+ ) transfer electrons to become peroxyl free radicals, which provides raw materials and a reaction environment for highly reactive oxygen species (ROS) for the peroxidation of biological macromolecules such as nucleic acids, lipids or proteins. It is worth noting that lipids form lipid peroxides under the catalysis of lipoxygenase, which in turn causes membrane oxidative damage and induces cells to enter the programmed death link, that is, ferroptosis. In summary, it can be judged that radiotherapy creates conditions for ferroptosis of tumor cells.

[0045] In order to clarify the relationship between radiotherapy and ferroptosis in liver cancer, it is first necessary to know whether ferroptosis contributes to the killing effect of radiotherapy on liver cancer cells. 2+ The supply of DFO inhibited the occurrence of ferroptosis at the source, and then observed its effect on radiotherapy resistance. The results showed that the inhibitor could slightly promote radiotherapy resistance of liver cancer cells (SKHep1 / Huh7 / SNU449); at the same time, the allosteric inhibitor of acetyl-CoA carboxylase (ACCA)-TOFA was further used to block the synthesis of fatty acids. The results were similar to those of DFO, and TOFA could only slightly promote radiotherapy resistance. Based on the above results, it can be seen that blocking the material basis that causes ferroptosis at the source does not contribute much to the radiotherapy resistance effect. This may be due to the presence of factors in cells that mediate ferroptosis escape, resulting in ferroptosis contributing less to the killing effect of radiotherapy.

[0046] Lipid peroxidation is an important sign of cell ferroptosis. Therefore, by evaluating the accumulation of lipid peroxides (PUFA-PLs-OOH) in liver cancer cells after radiotherapy, it is possible to determine whether the ferroptosis escape process is mediated by "PUFA-PLs-OOH downregulation". First, multiple radiotherapy methods were set up in liver cancer cells. The results showed that only when the single irradiation dose was as high as 12Gy or more or multiple medium-dose continuous irradiations (6Gy / day, 3 or 5 consecutive days), PUFA-PLs-OOH accumulation in liver cancer cells could be observed, and the degree was low. At the same time, two radiotherapy fractionation methods were set up in the liver cancer PDX model to investigate the accumulation of 4-hydroxynonenal (4-HNE, a lipid peroxidation product) in the tumor after treatment. The results showed that the accumulation of 4-HNE in the tumor after treatment was not obvious, whether it was conventional small-fraction irradiation (2Gy / time, 1 time / day, 5 times / week, a total of 20Gy) or large-fraction irradiation (5Gy / time, 3 times / week, a total of 15Gy). Based on the results in vivo and in vitro, insufficient accumulation of lipid peroxides is the main reason why ferroptosis is difficult to trigger after radiotherapy. Through the analysis of the PUFA-PLs-OOH generation stage, it was found that radiotherapy created favorable conditions for the generation of PUFA-PLs-OOH and promoted its generation; and the study of the PUFA-PLs-OOH clearance (ferroptosis defense) link showed that after radiotherapy, the SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ10 reduction system jointly constructed ferroptosis defense for liver cancer cells, and quickly cleared lipid peroxides to mediate ferroptosis escape.

[0047] Through screening of the whole genome sgRNA library, it was verified that the tRNA methyltransferase METT L2 promoted the clearance of lipid peroxides by positively regulating the construction of the SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ10 dual defense system, mediated ferroptosis escape, and thus promoted liver cancer radiotherapy resistance in vivo and in vitro. In vitro experiments showed that METTL2 knockdown could significantly improve the radiotherapy sensitivity of liver cancer cells, and replenishing METTL2 in METTL2 knockout cells could restore the original degree of resistance; at the same time, in vivo experiments also confirmed that METT L2 knockout could enhance the function of liver cancer cell radiotherapy. This made it clear that METTL2 is a liver cancer radiotherapy tolerance factor, and knocking out or inhibiting the expression of METTL2 can effectively promote sensitivity to radiotherapy and prevent the occurrence of radiotherapy tolerance. The results of mechanism research show that METTL2 can quickly sense radiotherapy signals, catalyze the m3C modification of certain tRNAs, specifically enhance the translation efficiency of GPX4 and FSP1, increase their protein levels, promote the continuous activation of their respective defense systems, and accelerate the clearance of lipid peroxides.

[0048] Compared with the prior art, the present invention has the following technical effects:

[0049] (1) Through extensive research and screening, the present invention found that METTL2 is a gene that is extremely related to liver cancer radiotherapy tolerance. A higher expression level of METTL2 is significantly positively correlated with liver cancer radiotherapy resistance, and can be used to predict liver cancer radiotherapy tolerance and sensitivity, providing an early assessment for patients to receive preoperative treatment.

[0050] (2) The present invention reveals the correlation between the METTL2 gene and liver cancer radiotherapy tolerance, which has important practical significance for solving the problem of inter-individual differences in clinical efficacy and blanks in regression effect / prognosis assessment, and better realizing precision treatment. It provides a new drug treatment target for humans to conquer liver cancer, thereby providing a new direction for subsequent drug development, clinical treatment, etc., and has extremely high social value and market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the contribution of ferroptosis inhibitors DFO and TOFA to radioresistance of liver cancer cells.

[0052] Figure 2 This is a schematic diagram of the results of lipid peroxidation in liver cancer cells after radiotherapy.

[0053] Figure 3 Schematic diagram of the results of lipid peroxidation in the PDX model after radiotherapy.

[0054] Figure 4Schematic diagram of the effect of radiotherapy on ACSL4 activation.

[0055] Figure 5 Schematic diagram of the effects of Ferrostatin-1 on intracellular ROS levels and radiotherapy resistance after radiotherapy.

[0056] Figure 6 Schematic diagram of the effect of DFO on lipid peroxidation after radiotherapy.

[0057] Figure 7 Schematic diagram of the effects of combined radiotherapy and ferroptosis inducers on lipid peroxidation in liver cancer cells.

[0058] Figure 8 Schematic diagram of the effect of ferroptosis inducers on the radiosensitivity of liver cancer cells.

[0059] Fig. 9 GSH and CoQ in liver cancer cells before and after radiotherapy 10 Schematic diagram of H2 content determination results.

[0060] Fig.10 Schematic diagram of the whole genome sgRNAs library screening process and screening conditions.

[0061] Fig.11 Schematic diagram of the effects of radiotherapy and combination with ferroptosis inducers on lipid peroxidation in METTL2 knockout cells.

[0062] Fig.12 Schematic diagram of the effect of METTL2 knockout on the radiosensitivity of liver cancer cells.

[0063] Fig.13 Schematic diagram of the effect of METTL2 replenishment on radiosensitivity in METTL2 knockout liver cancer cells.

[0064] Fig.14 Schematic diagram of the effect of METTL2 knockout on tumor volume in tumor-bearing mice.

[0065] Fig.15 Schematic diagram of the effect of METTL2 knockout on tumor growth rate in tumor-bearing mice.

[0066] Fig.16 Schematic diagram of the effect of METTL2 on ferroptosis and the translation efficiency of defense-related factors after radiotherapy.

[0067] Fig.17 Schematic diagram of the effect of METTL2 knockout and then METTL2 complementation on the translation efficiency of GPX4 and FSP1.

[0068] Fig.18This is a schematic diagram of the effect of METTL2 on GPX4 and FSP1 protein levels before and after radiotherapy. DETAILED DESCRIPTION

[0069] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0070] Unless otherwise specified, the cell lines listed in the context of the present invention, including Huh7, SNU449 and MHC C97H, were cultured according to the prior art. All cell lines were identified by short tandem repeat analysis of the China Center for Type Culture Collection (Wuhan), and the presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai BiothriveSci), and were frozen in liquid nitrogen and used for subsequent experiments. The reagents used in the present invention were all commercially available. For the use of clinical specimens, informed consent forms were signed with the patients, and the relevant procedures and methods met the requirements of medical ethics and the quality management standards for drug clinical trials. The experimental methods used in the present invention, such as DNA extraction, whole genome sequencing, primer design, immunohistochemistry, Western blot, cell experiments, animal experiments, etc., are all conventional methods and techniques in the field. For the intracellular / in vivo METTL2 expression level, conventional methods in the art (such as PCR, Western blot, etc.) can be used for detection; and the present invention verifies the detection specificity of the relevant primer pairs (primer pair 1-primer pair 4) and antibody ab101510, etc., for the METTL2 expression level through specific experiments. In view of the fact that the detection of the expression level of a specific gene / protein belongs to the conventional means in the art and is not the main improvement direction of the present invention, the relevant detection results are not particularly presented in the present invention, and those skilled in the art can perform detection and verification according to the experimental methods described in the present invention or other conventional methods in the prior art as needed. In addition, in addition to the relevant primer pairs and antibodies listed in the present invention, those skilled in the art can also design relevant primer pairs / antibodies according to the METTL2 gene sequence and / or protein structure, or obtain commercial reagents from the market to complete the detection of the METTL2 expression level, so the specific information of the primer pairs, antibodies, etc. listed in the context of the present invention does not constitute a limitation on the actual protection scope of the present invention.

[0071] Representative results from repeated biological experiments were presented in the context figures, and the data were presented as mean ± SD and mean ± SEM as specified in the figure. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. Data were analyzed using GraphPad Prism 8.0 or SPSS22.0 software. Conventional medical statistical methods such as t-test, chi-square test, and analysis of variance were used to compare the mean differences between two or more groups. p < 0.05 was considered a significant difference.

[0072] Example 1

[0073] In order to explore the relationship between radiotherapy and ferroptosis in liver cancer, it is first necessary to clarify whether ferroptosis has a definite contribution to the killing effect of radiotherapy on liver cancer cells. 2+ The supply of ferroptosis is used to inhibit the occurrence of ferroptosis at the source, and then its effect on radiotherapy resistance is observed. The specific steps are as follows:

[0074] (1) Hepatoma cells (SKHep1 / Huh7 / SNU449) in the logarithmic growth phase were seeded in 6-well plates at 1000 cells / well.

[0075] (2) 24 hours later, the cells were irradiated with 6 Gy of X-ray and treated with the drug DFO (10 μM).

[0076] (3) After culturing for 10-14 days, discard the culture medium and add 1 mL of methanol containing 0.5% crystal violet to each well for staining for 30 min. Discard the methanol and wash away the residual methanol with water to observe cell clones.

[0077] Observe under a microscope, and only when the number of cells is greater than 50 is it considered a valid clone. Count the total number of clones in each group. The calculation is as follows:

[0078]

[0079] The results are as follows Figure 1 As shown in the results, the use of ferroptosis inhibitor DFO can slightly promote the radioresistance of liver cancer cells (SKHep1 / Huh7 / SNU449); then, the acetyl-CoA carboxylase (ACCA) allosteric inhibitor TOFA was used to block the synthesis of lipase to observe the effect of ferroptosis on radioresistance. Figure 1 The results are similar to those of DFO, that is, TOPA can only slightly promote radioresistance. The above results together indicate that blocking the material basis that triggers ferroptosis at the source does not contribute much to radioresistance, which may be due to the presence of factors in cells that mediate ferroptosis escape, making the contribution of ferroptosis to the radiotherapy killing effect less.

[0080] Subsequently, the accumulation of lipid peroxides (PUFA-PLs-OOH) in liver cancer cells after radiotherapy was evaluated to determine whether the ferroptosis escape process was mediated by the downregulation of PUFA-PLs-OOH. First, Huh7 and SNU449 cells were treated with different radiotherapy methods, and then the cells were stained with lipid peroxide dye (C11-BODIPY staining) and detected by flow cytometry. The results are shown in Figure 2. Figure 2 As shown. The results showed that only when the single irradiation dose was as high as 12Gy or more or multiple medium-dose continuous irradiations (6Gy / day, 3 or 5 consecutive days), PUFA-PLs-OOH accumulation in liver cancer cells could be observed, and the degree was low. Subsequently, two radiotherapy splitting methods were set in the liver cancer PDX model to investigate the accumulation of 4-hydroxynonenal (4-HNE, a lipid peroxidation product) in the tumor after treatment. The specific steps are as follows:

[0081] PDX tumor tissue was minced into 5-10 mm pieces in cold cell culture medium. 3 Each PDX tumor piece was then inoculated subcutaneously into the right flank of NSG mice. 3 The mice were randomly divided into three groups: no radiotherapy group, conventional fractionated radiotherapy 20Gy group (2Gy*5 times / week), and large fractionated radiotherapy 15Gy group (5Gy*3 times). The mouse flank tumor was locally irradiated during radiotherapy. The tumor volume was measured three times a week until the end point, and the volume was calculated according to the formula volume = 0.5×width 2 The tumor volume was calculated by × length. The mice were sacrificed 2-3 weeks after the last treatment (the maximum diameter of the tumor did not exceed 2 cm), the xenografts were removed, and the expression of 4-HNE was detected by immunohistochemistry.

[0082] The results are as follows Figure 3 The results showed that the accumulation of 4-HNE in the tumor after treatment was not obvious, whether it was conventional fractionated irradiation (2Gy / time, 1 time / day, 5 times / week, a total of 20Gy) or large fractionated irradiation (5Gy / time, 3 times / week, a total of 15Gy). The above results together indicate that insufficient accumulation of lipid peroxides is the main reason why ferroptosis is difficult to trigger after radiotherapy.

[0083] Furthermore, the phosphorylation level of the activation site of PUFA long-chain acyl-CoA synthetase 4 (ACSL4), a core enzyme in the lipid supply chain, was observed after radiotherapy in liver cancer cells (Huh7 and SNU449). Figure 4As shown. The results showed that radiotherapy can increase the phosphorylation level of the activation site of the core enzyme of the lipid supply chain - PUFA long-chain acyl-CoA synthetase 4 (ACSL4), indicating that radiotherapy has a promoting effect on the activation of ACSL4. Secondly, when the antioxidant Ferrostatin-1, which scavenges reactive oxygen species (ROS) free radicals, was added to the liver cancer cells during radiotherapy, it was found that it could not significantly reduce the level of ROS caused by radiotherapy, and Ferrostatin-1 did not affect the sensitivity of radiotherapy at all (see Figure 5 ), indicating that the high ROS environment after radiotherapy is not easy to be reversed. Furthermore, DFO was used for Fe 2+ The results showed that the accumulation of PUFA-PLs-OOH in cells was significantly downregulated (see Figure 6 ). In summary, radiotherapy creates favorable conditions for the formation of PUFA-PL s-OOH and promotes its formation.

[0084] In order to further study the effect of radiotherapy on PUFA-PLs-OOH clearance (ferroptosis defense), liver cancer cells (Huh7) were treated with glutathione peroxidase 4 (GPX4) inhibitor RSL3, ferroptosis inhibitory protein 1 (FSP1) inhibitor Ifsp1 or GTP cyclohydrolase 1 (GCH1) inhibitor DAHP. Figure 7 As shown. The results showed that neither the GPX4-dependent reduction pathway (RS L3) nor the GPX4-independent reduction pathway (FSP1, DAHP) could promote the accumulation of PUFA-PLs-OOH after radiotherapy, nor effectively improve radiosensitivity, which means that the three reduction pathways that inhibit ferroptosis separately cannot break the ferroptosis defense. Based on this, it is speculated that the ferroptosis defense after radiotherapy may be jointly constructed by multiple reduction pathways. In this regard, the above three ferroptosis inducers were used in combination in pairs, and it was found that when RSL3 was used in combination with iFSP1, it could significantly promote the accumulation of PUFA-PLs-OOH after radiotherapy and greatly improve radiosensitivity; when RSL3 or iFSP1 was used in combination with DAHP, no enhancement effect was seen (see Figure 8 ). Detection of GSH and CoQ in liver cancer cells before and after radiotherapy by high performance liquid chromatography-mass spectrometry (LC-MS) 10 H2 levels, and found that the above core mechanism also occurs in GSH and CoQ 10 The results of the H2 level surge were confirmed (see Fig. 9 ). In the absence of radiation treatment, even the simultaneous use of three types of ferroptosis inducers could not increase the level of PUFA-PLs-OOH to induce ferroptosis of liver cancer cells (see Figure 7), which shows that radiotherapy is a prerequisite for ferroptosis. In summary, it can be clearly seen that after radiotherapy, liver cancer cells are activated by SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ 10 The reducing system jointly builds ferroptosis defense and quickly removes lipid peroxide-mediated ferroptosis escape.

[0085] Example 2

[0086] In order to clarify the regulatory factors of the ferroptosis defense system of liver cancer cells after radiotherapy, the lipid peroxidation in liver cancer cells after radiotherapy was screened by using the human whole genome sgRNA library. The schematic diagram of the screening process is shown in the figure. Fig.10 As shown, the specific steps are as follows:

[0087] (1) In liver cancer cells, a stable cell line containing a whole-genome sgRNA library was constructed (hybrid type, ensuring that each cell contains one sgRNA).

[0088] (2) After the cells are irradiated, lipid peroxide dye is used to differentiate the cells; then, the genes corresponding to the decreased sgRNA copy number in the library cells with accumulated lipid peroxidation are analyzed.

[0089] (3) The following screening conditions were set: |Log2(Fold Change)|>4, the number of captured sgRNAs>3, and all met the criteria in three liver cancer cells (SKHep1 / Huh7 / SNU449). The results showed that ME TTL2 may have the potential to downregulate lipid peroxide levels after radiotherapy.

[0090] Since lipid peroxidation is an important sign of cell ferroptosis, we first studied the effect of METTL2 on lipid peroxidation after radiotherapy. We used BODIPY 581 / 591C11 dye to stain the peroxidized lipids (PUFA-PLs-OOH) on the Hu7 cell membrane, and then detected it by flow cytometry. Fig.11 The results showed that knocking out METTL2 did significantly increase the level of lipid peroxidation in liver cancer cells after radiotherapy, and knocking out METTL2 would lead to the failure of the GPX4 inhibitor combined with the FSP1 inhibitor to promote lipid peroxidation after radiotherapy. In addition, it was found that in METTL2 knocked-out cells, the cell survival rate was significantly decreased, and when DF O was added to block Fe 2+ The supply of can greatly improve the radioresistance of cells (not shown in the figure), indicating that the ferroptosis defense in METTL2 knockout cells is broken.

[0091] Furthermore, in vitro and in vivo experiments were performed to verify the effect of METTL2 on radiosensitivity. First, a cell clone formation experiment was performed. The specific steps are as follows:

[0092] (1) CRISPR-cas9-sgRNAs (Sg1, sequence as shown in SEQ ID NO: 9, 5'-GCTGCTCCACAGGATGCAGA-3') were used to construct METTL2 knockout stable cell lines in Huh7 and SUN449 cells.

[0093] (2) Cells in the logarithmic growth phase were seeded in 6-well plates at 1000 cells / well.

[0094] (3) 0-4 Gy radiotherapy intervention was performed after 24 hours; culture was continued for 10-14 days.

[0095] (4) Discard the culture medium and add 1 mL of methanol containing 0.5% crystal violet to each well for staining for 30 min. Discard the methanol and wash away the residual methanol with water to observe cell clones. Observe under a microscope and count a valid clone only when the number of cells is greater than 50. Count the total number of clones in each group.

[0096]

[0097] The results are as follows Fig.12 The results showed that knocking out METTL2 can significantly increase the sensitivity of liver cancer cells and liver cancer cells to radiotherapy, that is, inhibiting the expression of METTL2 in cells can effectively improve the therapeutic effect of radiotherapy and inhibit the generation of radiotherapy tolerance (**p<0.01, ***p<0.001). It proves that the existence and expression level of METTL2 are positively correlated with the degree of radiotherapy resistance of liver cancer.

[0098] Subsequently, wild-type METTL2 (WT) was introduced into METTL2-KO Huh7 and SUN449 cells, and the radiosensitivity (single radiation dose of 2Gy) was investigated. Specifically, the above-mentioned colony formation experiment was still used. The results showed that the replenishment of METTL2 could significantly improve the survival ability of cells after radiotherapy, allowing them to recover to the original level of resistance (***p < 0.001, ****p < 0.0001) (see Fig.13 ).

[0099] Furthermore, tumor-bearing mouse experiments were performed to verify the role of METTL2 in radiosensitivity in vivo. The specific steps are as follows:

[0100] (1) Five-week-old female BALB / c-nu / nu mice were divided into two groups. One group was subcutaneously injected with normal Huh7 (METTL2-WT) cells, and the other group was subcutaneously injected with METTL2 stable knockout MHCC97H (METTL2-KO) cells constructed by sgMETTL2 (Sg1, sequence as shown in SEQ ID NO: 9, 5'-GCTGCTCCACAGGATGCAGA-3') lentivirus.

[0101] (2) After tumor formation, the mice in the METTL2-WT group were randomly divided into two groups, 10 mice in each group, namely Group 1 and Group 2; the mice in the METTL2-KO group were also randomly divided into two groups, 10 mice in each group, namely Group 3 and Group 4.

[0102] (3) The mice in Group 1 and Group 3 were not given any treatment. The mice in Group 2 and Group 4 were treated with tumor area irradiation (RS2000 X-ray irradiator, with a source-skin distance of 100 cm as the standard, medium- and long-range daily average equal dose radiation; the non-tumor area was shielded with a lead container specially made for mice), 2 Gy / day, once every other day, for 5 consecutive times; the tumor volume was measured regularly during the treatment period, and the mice were killed 7 weeks after treatment, the tumors were removed, and the tumor inhibition rate was calculated by weighing.

[0103] The results are as follows Figure 14-15 The results showed that for mice without radiotherapy, the tumor growth rate and tumor volume of METTL2 knockout tumor-bearing mice were not significantly different from those of METTL2 wild-type tumor-bearing mice, and the difference between the two was not statistically significant; after radiotherapy, the tumor growth rate of METTL2 knockout tumor-bearing mice was significantly slower, the tumor volume was significantly smaller, and the tumor inhibition rate was significantly improved compared with METTL2 wild-type tumor-bearing mice, and the difference between the two was statistically significant (****p<0.0001).

[0104] Sg2 (sequence as shown in SEQ ID NO: 10, 5'-CGCAGAAGCATCATCCCGCC-3') or sg3 (sequence as shown in SEQ ID NO: 11, 5'-GTGTCTCCACAGGATGCAGA-3') was used to replace sg1, and CRISPR-cas9 technology was used to construct METTL2 knockout stable cell lines in Huh7 and SUN449 cells, and the above-mentioned cell clone formation experiment and in vivo radiotherapy sensitivity experiment were repeated. The results were similar to those of sg1 above, and therefore will not be repeated.

[0105] From the above, it can be seen that simply inhibiting the expression level of METTL2 in liver cancer cells cannot significantly inhibit the growth of tumor cells; however, during radiotherapy of liver cancer cells, the knockout of METTL2 can significantly increase the sensitivity of tumor cells to radiotherapy, reduce the growth rate and volume of tumor cells, significantly improve the tumor inhibition effect, and effectively prevent the occurrence of radiotherapy tolerance. Based on the above results, it can be clearly seen that METTL 2 is a liver cancer radiotherapy tolerance factor. Knocking out or inhibiting the expression of METTL2 can effectively promote sensitivity to radiotherapy and prevent the occurrence of radiotherapy tolerance.

[0106] Example 3

[0107] In order to study the mechanism of action of METTL2 and radioresistance, we first used the method of measuring the mRNA content in the ribosome-nascent chain complex (RNC-qPCR) to examine the types of proteins driven by METTL2 translation after radiotherapy. This method is based on the capture of the full-length mRNA being translated and bound to the ribosome, and can be used to determine the translation efficiency of the protein, as follows:

[0108] (1) The cells were treated with sgRNA (sg1) knockout or METTL2 overexpression plasmid and divided into 4 groups: METTL2-WT (non-knockdown group) and METTL2-KO (METTL2 knockdown group). The response experiment was performed by adding or not adding METTL2 overexpression plasmid after knockdown in the above two groups.

[0109] (2) After the cell samples were treated with cycloheximide for 15 minutes, they were lysed with lysis buffer at 4°C for 30 minutes. The cell lysate was centrifuged at 16000g and 4°C for 10 minutes.

[0110] (3) Extract the supernatant RNA and perform sequencing and qRT-PCR for each fund. Add 30% sucrose solution to the remaining supernatant and centrifuge at 185,000 g and 4°C for 5 hours. Extract the sediment RNA and perform sequencing and qRT-PCR using conventional methods in the art. Enrichment analysis was performed using an open source website (http: / / www.webgestalt.org). Gene expression levels were normalized using the FPKM method. The translation ratio (TR) was calculated as follows: TR = (FPKM in RNA-seq) / (FPKM in input RNA-seq).

[0111] The results showed that after radiotherapy, METTL2 knockout caused a significant decrease in the translation efficiency (TE) of glutathione peroxidase 4 (GPX4) and ferroptosis inhibitory protein 1 (FSP1) (see Fig.16 To further verify the above results, the restoration of METTL2 in the knockout cell line significantly increased the translation efficiency of GPX4 and FSP1 (see Fig.17 ).

[0112] Subsequently, verification was carried out at the protein level, as follows:

[0113] (1) In METTL2 overexpression or control cells, cells were irradiated (6 Gy) or not irradiated, and cells were collected 6 h after irradiation.

[0114] (2) Digest and collect the cells, wash them twice with PBS to remove the residual culture medium, add an appropriate amount of lysis buffer, and lyse them on ice for 1 h (or there is no need to digest the cells, discard the culture medium, wash them twice with PBS, remove the residual liquid, add lysis buffer, scrape the cells with a scraper, transfer them to a centrifuge tube, and lyse them on ice for 1 h); centrifuge at 15000×g at 4℃ for 15 min, take the supernatant for later use; determine the protein concentration using the BCA protein concentration detection kit; based on the sample with the lowest concentration, add lysis buffer to the remaining samples to adjust the concentration of all samples to the same; add loading buffer (to 1×), incubate at 95℃ for 5 min, and set aside.

[0115] (3) Taking the sample prepared in step (2) and performing protein gel electrophoresis.

[0116] (4) After electrophoresis, the protein gel was transferred to a membrane (PVDF membrane, 200 mA constant current transfer for 2 h).

[0117] (5) After the transfer is completed, remove the PVDF membrane and place it in the blocking solution. Block it at room temperature for 2 h in a vertical shaker at 10 rpm.

[0118] (6) After blocking, the PVDF membrane was cleaned and immersed in the primary antibody (GPX4 (E5Y8K) Rabbit mAb Catalog No.: 59735, Brand: Cell Signal Technology; FSP1 (F7A3L) Rabbit mAb Catalog No.: 51676, Brand: Cell Signal Technology), and incubated on a vertical shaker at 10 rpm and 4°C overnight.

[0119] (7) After the primary antibody incubation is completed, the PVDF membrane is washed and then immersed in the secondary antibody (HRP-linked anti-rabbit IgG (anti-rabbit): Catalog No.: 7074, Brand: Cell Signal Technology; HRP-linked anti-mouse IgG (anti-mouse): Catalog No.: 7076, Brand: Cell Signal Technology) and incubated on a vertical shaker at 10 rpm at room temperature for 2 h.

[0120] (8) After the secondary antibody incubation is completed, the PVDF membrane is cleaned and the target protein is detected using a chemiluminescence instrument.

[0121] The results are as follows Fig.18 As shown, the expression levels of GPX4 and FSP1 were low without radiation intervention; only in the presence of METTL2, their protein levels were significantly upregulated after radiotherapy.

[0122] Furthermore, tRNA chip (ArraystarnStar TM tRNA PCR chip) detection and analysis found that after radiotherapy, the content of nine types of tRNA in METTL2 knockout liver cancer cells was significantly lower than that in control cells, including tRNA Ser , tRNA Ala tRNA Leu tRNA Val , tRNA Ile tRNA Gln , tRNA Thr , tRNA Glu and tRNA Arg(ACG / CCG) (See Table 1). The frequency of occurrence of these nine types of tRNA anticodons in the mRNA of ferroptosis-related genes was analyzed and it was found that the frequency of occurrence in GPX4 and FSP1-mRNA was much higher than that in other genes. Based on the above results, it can be seen that the decisive factor for METTL2 to specifically regulate the translation of GPX4 and FSP1 may be the mRNA regulation of these nine types of tRNA. 3 C methylation modification.

[0123] Table 1 Changes in tRNA copy number after radiotherapy in the METTL2 knockout group compared with the control group

[0124]

[0125]

[0126] After radiotherapy, liver cancer cells escape ferroptosis due to the rapid clearance of lipid peroxidation, which makes ferroptosis contribute less to the killing effect caused by radiotherapy. Thus, in order to explore the regulatory mechanism of ferroptosis defense construction, the present invention screened through the whole genome sgRNA library, and then verified that tRNA methyltransferase METTL2 promotes the clearance of lipid peroxides by positively regulating the construction of SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ10 dual defense system, mediates ferroptosis escape and promotes liver cancer radiotherapy resistance in vivo and in vitro, and clarifies that METTL2 is a liver cancer radiotherapy tolerance factor. By knocking out or inhibiting the expression of METTL2, it can effectively promote sensitivity to radiotherapy and prevent the occurrence of radiotherapy tolerance. The results of the mechanism study show that METTL2 can quickly sense radiotherapy signals, catalyze m3C modification of certain tRNAs, improve the translation efficiency of GPX4 and FSP1 in a targeted manner, increase their protein levels, promote the continuous activation of their respective dominant defense systems, and accelerate the removal of lipid peroxides. It can be clearly seen from the above that METTL2 is a gene that is extremely related to liver cancer radiotherapy tolerance. The higher expression level of METTL2 is significantly positively correlated with liver cancer radiotherapy tolerance, which can be used to predict liver cancer radiotherapy tolerance and sensitivity, and provide an early assessment of the connection between patients receiving preoperative treatment. At the same time, the present invention, by revealing the correlation between the METTL2 gene and liver cancer radiotherapy tolerance, has important practical significance for solving the problem of clinical efficacy differences between individuals and the blank of retreat effect / prognosis evaluation, and better realizing precision treatment. It provides a new drug treatment target for humans to conquer liver cancer, thereby providing a new direction for subsequent drug development, clinical treatment, etc., and has extremely high social value and market application prospects.

[0127] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. Application of METTL2 inhibitors in the preparation of drugs for improving the radiosensitivity of digestive system tumors.

2. The use according to claim 1, characterized in that: The METTL2 inhibitor is selected from one or more of shRNA, sgRNA, and siRNA designed based on METTL2.

3. The use according to claim 1, characterized in that: The digestive system tumor is liver cancer.

4. Use of a reagent for detecting the expression level of METTL2 in the preparation of a product for detecting the radiotherapy sensitivity and / or predicting the efficacy of digestive system tumors.

5. The use according to claim 4, characterized in that: The reagent for detecting the expression level of METTL2 includes primers for detecting the expression level of METTL2 gene and / or a reagent for detecting the expression level of METTL2 protein.

6. The use according to claim 4, characterized in that: The digestive system tumor is liver cancer.

7. Application of METTL2 inhibitors in the preparation of products that promote ferroptosis of tumor cells.

8. The use according to claim 1, characterized in that: The METTL2 inhibitor is selected from one or more of shRNA, sgRNA, and siRNA designed based on METTL2.

9. The use according to claim 7, characterized in that: The tumor cells are selected from liver cancer cells.

10. A pharmaceutical composition for improving the radiotherapy sensitivity of digestive system tumors, characterized in that: It includes a METTL2 inhibitor and a pharmaceutically acceptable carrier.

Images