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 present invention belongs to the field of biomedicine and relates to the application of METTL2 in radiosensitization of digestive system tumors. Background Art

[0002] Hepatocellular carcinoma (referred to as liver cancer) is currently the fourth most common malignant tumor and the second leading cause of cancer death in China, seriously threatening people's lives and health. The treatment of liver cancer mainly includes various means such as surgery, local ablation therapy, radiotherapy (RT), transcatheter arterial chemoembolization (TACE), and systemic drug therapy. Among them, new technology radiotherapy means 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 liver cancer treatment. In recent years, scholars at home and abroad have successively reported the unique advantages of radiotherapy in terms of efficacy: it can improve the local control rate and significantly prolong the survival time (the 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; after radiotherapy, some tumors that cannot be surgically treated shrink or downstage 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 plan for liver cancer. However, there are many restrictive factors in liver cancer radiotherapy, such as low overall effectiveness, short duration of efficacy, and easy recurrence of patients, which seriously limit the wide application of radiotherapy in the field of liver cancer. It is generally believed that the high heterogeneity of liver cancer cells and the radioresistance or resistance effect of some cells to radiation are the main reasons for the poor efficacy of radiotherapy. Therefore, screening and identifying the key targets mediating liver cancer radiotherapy resistance and developing corresponding intervention strategies based on this mechanism of action for clinical use to improve the efficacy of liver cancer radiotherapy have very important practical significance. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem that the mechanism of radiotherapy tolerance in liver cancer in the prior art is not clear. Therefore, starting from the essence (physical basis) of radiation and on the basis of the biological effect of DNA damage, other biological effect mechanisms caused by radiation are expanded, and METTL2 is identified as a target related to liver cancer radiotherapy resistance, providing a more comprehensive scientific basis and solution for realizing radiosensitization of liver cancer radiotherapy.

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

[0006] The first aspect of the present invention provides the application of METTL2 inhibitors in the preparation of drugs 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'-GCTGCTCCACAGGATGCAGA-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] The second aspect of the present invention provides an application of a reagent for detecting the expression level of METTL2 in the preparation of a product for detecting the radiosensitivity and / or predicting the efficacy of digestive system tumors.

[0011] Preferably, the reagent for detecting the expression level of METTL2 includes primers for detecting the expression level of the METTL2 gene and / or a reagent for detecting the expression level of the 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 as shown in SEQ ID NO: 3 (5'-ATGTGGAGTGGTCGGAAGAG-3'), and the downstream sequence is as shown in SEQ ID NO: 4 (5'-CCAGTATTTGTGGGCATTGATCT-3');

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

[0016] Primer pair 4: The upstream sequence is as shown in SEQ ID NO: 7 (5’-GCGAGCTCAT GGCCGGCTCCTACCCT-3’), and the downstream sequence is as shown in SEQ ID NO: 8 (5’-GCGCGGCCGCTCAGCTGGTGCTGGACAG-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 application of METTL2 inhibitor in the preparation of products for promoting ferroptosis of tumor cells.

[0021] Preferably, the METTL2 inhibitor is selected from one or more of shRNA, sgRNA, 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, 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 radiotherapy sensitivity and / or efficacy of digestive system tumors, comprising primers for detecting the expression level of METTL2 gene and / or reagents for detecting the content of METTL2 protein; and one or more of PCR enzymes, PCR buffers, dNTPs, and fluorescent substrates.

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

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

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

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

[0029] Primer pair 4: The upstream sequence is as shown in SEQ ID NO: 7, and the downstream sequence is as shown in 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] The fifth aspect of the present invention provides a pharmaceutical composition for enhancing 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 fillers, binders, lubricants, disintegrants, flavoring agents, coloring agents, antioxidants, bacteriostatic agents, chelating agents, surfactants, and solvents.

[0039] The sixth aspect of the present invention provides the use of the above pharmaceutical composition in the preparation of a drug for enhancing the radiosensitivity 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 sgRNAs designed based on METTL2; most preferably, the sgRNA sequences designed based on METTL2 are 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 primer and / or primer pair refers to a PCR primer used to synthesize the cDNA strand of the METTL2 gene in PCR, so as to detect the expression level of 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 according to the gene sequence of METTL2 by using conventional methods and means in the art, including but not limited to molecular biology, and screening the designed primers and / or primer pairs through conventional experimental means, as long as the specific detection of the METTL2 expression level can be achieved. The METTL2 inhibitor refers to a substance that can specifically down-regulate 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, methods such as antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA Erasers, Target Masking, and / or multi-targets are used to down-regulate the expression level and / or activity of METTL2, as long as the reduction of the level and / or activity of METTL2 can be achieved.

[0044] The X (or γ) rays used in radiotherapy are high-energy rays with wave-particle duality and have the properties of photons. After the X (or γ) rays collide with molecules, energy transfer and exchange occur, prompting molecules or atoms to undergo ionizing radiation (IR) and generating a large number of free radicals. Generally, it is believed that the biological effects of IR are mainly caused by free radical-mediated DNA damage. However, the reactive oxygen free radicals generated by the ionization of water molecules (H 2 O) by X (or γ) rays can not only attack the nucleic acid linkage sites to cause DNA breaks, but also accept divalent iron (Fe 2+) The electron transfer becomes a peroxyl radical, which provides the raw materials and a reactive oxygen species (ROS) reaction environment 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 further causes membrane oxidative damage and induces cells to enter the programmed cell death process, that is, ferroptosis occurs. In summary, it can be judged that radiotherapy creates conditions for tumor cells to undergo ferroptosis.

[0045] 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. In this regard, the present invention uses the iron chelator DFO to inhibit the occurrence of ferroptosis at the source by blocking the supply of Fe 2+ , and then observes its effect on the radiotherapy resistance function. The results show that this inhibitor can slightly promote the radiotherapy resistance of liver cancer cells (SKHep1 / Huh7 / SNU449); at the same time, the allosteric inhibitor of acetyl-CoA carboxylase (ACCA)-TOFA is further used to block the synthesis of fatty acids, and the result is similar to DFO, and TOFA can also only slightly promote radiotherapy resistance. Based on the above results, blocking the material basis for triggering ferroptosis at the source contributes little to the radiotherapy resistance effect, which may be due to the existence of elements mediating ferroptosis escape in cells, resulting in less contribution of ferroptosis to the radiotherapy killing effect.

[0046] Lipid peroxidation is an important marker for cells to undergo ferroptosis. Therefore, by evaluating the accumulation of lipid peroxides (PUFA-PLs-OOH) in liver cancer cells after radiotherapy, it is used to judge whether the ferroptosis escape process is mediated by "downregulation of PUFA-PLs-OOH". First, various radiotherapy methods are set in liver cancer cells, and the results show that: only when the single irradiation dose is as high as more than 12 Gy or multiple medium doses are continuously irradiated (6 Gy / day, continuously for 3 days or 5 days), can the accumulation of PUFA-PLs-OOH in liver cancer cells be observed, and the degree is relatively low. Based on the in vivo and in vitro results, 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 is found that radiotherapy creates favorable conditions for the generation of PUFA-PLs-OOH and promotes its generation; while the research on the clearance (ferroptosis defense) link of PUFA-PLs-OOH shows that after radiotherapy of liver cancer cells, the ferroptosis defense is jointly constructed by the SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ10 reduction systems, and lipid peroxides are rapidly cleared to mediate ferroptosis escape.

[0047] Screening was carried out through a whole-genome sgRNA library, and it was verified that the tRNA methyltransferase METTL2 promoted the clearance of lipid peroxides by positively regulating the construction of the SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ10 double defense systems, mediated ferroptosis escape, and thus promoted radiotherapy resistance of liver cancer in vitro and in vivo. In vitro experiments showed that knockdown of METTL2 could significantly enhance the radiosensitivity of liver cancer cells, while re-supplementation of METTL2 in METTL2 knockout cells could restore the original degree of promoting resistance; at the same time, in vivo experiments also confirmed the function of METTL2 knockout in sensitizing liver cancer cells to radiotherapy. Thus, it was clarified 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 studies showed that METTL2 could rapidly 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 the respective leading defense systems, and accelerate the clearance of lipid peroxides.

[0048] The present invention has the following technical effects compared with the prior art:

[0049] (1) Through a large number of studies and screenings, the present invention found that METTL2 is a gene extremely related to radiotherapy tolerance of liver cancer. There is an obvious positive correlation between the relatively high expression level of METTL2 and radiotherapy resistance of liver cancer, which can be used to predict radiotherapy tolerance and sensitivity of liver cancer, and provide an early assessment for the connection of patients to receive preoperative treatment.

[0050] (2) By revealing the correlation between the METTL2 gene and radiotherapy tolerance of liver cancer, the present invention has important practical significance for solving the problems of individual differences in clinical efficacy and the blank of regression effect / prognosis evaluation, and better realizing precise treatment. It provides a new drug treatment target for humans to overcome liver cancer, thus providing a new direction for subsequent drug research and 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 results of the ferroptosis inhibitors DFO and TOFA to radiotherapy resistance of liver cancer cells.

[0052] Figure 2 Schematic diagram of the results of the situation of lipid peroxidation in liver cancer cells after radiotherapy.

[0053] Figure 3 Schematic diagram of the results of the influence of Ferrostatin-1 on the intracellular ROS level and radiotherapy resistance after radiotherapy.

[0054] Figure 4 Schematic diagram of the effect of DFO on lipid peroxidation after radiotherapy.

[0055] Figure 5 Schematic diagram of the effect of the combination of radiotherapy and ferroptosis inducer on lipid peroxidation in hepatocellular carcinoma cells.

[0056] Figure 6 Schematic diagram of the effect of ferroptosis inducer on the radiosensitivity of hepatocellular carcinoma cells.

[0057] Figure 7 For the content determination results of GSH and CoQ 10 H 2 in hepatocellular carcinoma cells before and after radiotherapy.

[0058] Figure 8 Schematic diagram of the screening process and screening conditions of the whole-genome sgRNAs library.

[0059] Figure 9 Schematic diagram of the effect of radiotherapy and the combination with ferroptosis inducer on lipid peroxidation in METTL2 knockout cells.

[0060] Figure 10 Schematic diagram of the effect of METTL2 knockout on the radiosensitivity of hepatocellular carcinoma cells.

[0061] Figure 11 Schematic diagram of the effect of re-supplementing METTL2 on radiosensitivity in METTL2-knockout hepatocellular carcinoma cells.

[0062] Figure 12 Schematic diagram of the effect of METTL2 knockout on the tumor growth rate in tumor-bearing mice.

[0063] Figure 13 Schematic diagram of the effect of METTL2 on the translation efficiency of ferroptosis occurrence and defense-related factors after radiotherapy.

[0064] Figure 14 Schematic diagram of the effect of re-supplementing METTL2 on the translation efficiency of GPX4 and FSP1 after knocking out METTL2. Detailed implementation manners

[0065] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to the 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.

[0066] Unless otherwise specified, cell lines such as Huh7, SNU 449, and MHCC97H listed in the context of the present invention are cultured according to the prior art. All cell lines are identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan) and verified for mycoplasma contamination using a PCR detection kit (Shanghai Biothrive Sci). They are also cryopreserved in liquid nitrogen and used for subsequent experiments. All reagents used in the present invention are commercially available. For the use of clinical specimens, informed consent forms have been signed with the patients, and the relevant procedures and methods comply with the requirements of medical ethics and Good Clinical Practice. 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 art. 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 has verified through specific experiments the detection specificity of the primer pairs (primer pairs 1 - primer pair 4) and antibody ab101510, etc., described in the present invention for the METTL2 expression level. Given that the detection of the expression level of a specific gene / protein is a conventional means in the art and not the main improvement direction of the present invention, the relevant detection results are not specifically presented in the present invention. 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 based on the METTL2 gene sequence and / or protein structure by themselves, or obtain commercial reagents through commercial channels to complete the detection of the METTL2 expression level. Therefore, the specific information such as primer pairs and antibodies listed in the context of the present invention does not constitute a limitation to the actual protection scope of the present invention.

[0067] Representative results from biological experiment replicates are presented in the context figures, and data are shown as mean ± SD and mean ± SEM as specified in the figures. 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 SPSS 22.0 software. Conventional medical statistical methods such as t-tests, chi-square tests, and analysis of variance were used to compare the mean differences between two or more groups. p < 0.05 was considered a significant difference.

[0068] Example 1

[0069] To explore the relationship between radiotherapy and ferroptosis in liver cancer, it is first necessary to clarify whether ferroptosis makes an exact contribution to the killing effect of radiotherapy on liver cancer cells. For this purpose, the iron chelator DFO was used to block Fe2+ The supply inhibits the occurrence of ferroptosis at the source, and then observes its effect on the radioresistance function. The specific steps are as follows:

[0070] (1) Inoculate hepatoma cells (SKHep1 / Huh7 / SNU449) in the logarithmic growth phase into 6-well plates at 1000 cells / well.

[0071] (2) After 24 hours, irradiate the cells with 6 Gy of X-rays and treat them with the drug DFO (10 μM).

[0072] (3) After continuing to culture for 10 - 14 days, discard the culture medium, add 1 mL of methanol containing 0.5% crystal violet to each well, and stain for 30 min; discard the methanol and wash the remaining methanol with water, then cell clones can be observed.

[0073] Observed under a microscope, only when the number of cells > 50 is counted as an effective clone, and the total number of clones in each group is counted. The calculation is as follows:

[0074]

[0075] The results are as Figure 1 shown. The results show that the ferroptosis inhibitor DFO can slightly promote the radioresistance of hepatoma cells (SKHep1 / Huh7 / SNU449); subsequently, the allosteric inhibitor TOFA of acetyl-CoA carboxylase (ACCA) is used to block the synthesis of lipase to observe the effect of ferroptosis on radioresistance. The results are as Figure 1 shown, and its results are similar to those of DFO, that is, TOPA can also only slightly promote radioresistance. The above results together indicate that blocking the material basis of ferroptosis at the source contributes little to radioresistance, which may be due to the existence of elements mediating ferroptosis escape in cells, resulting in less contribution of ferroptosis to the radiotherapy killing effect.

[0076] Subsequently, by evaluating the accumulation of lipid peroxides (PUFA-PLs-OOH) in hepatoma cells after radiotherapy, it is judged whether the ferroptosis escape process is mediated by the down-regulation of PUFA-PLs-OOH. First, Huh7 and SNU449 cells are treated with different radiotherapy methods respectively, and then the cells are stained with lipid peroxide fuel (C11-BODIPY staining) and detected by flow cytometry. The results are as Figure 2 shown. The results show that only when the single irradiation dose is as high as more than 12 Gy or multiple medium-dose continuous irradiations (6 Gy / day, for 3 or 5 consecutive days), can the accumulation of PUFA-PLs-OOH in hepatoma cells be observed, and the degree is relatively low.

[0077] Furthermore, it was found that when the antioxidant Ferrostatin-1, which scavenges reactive oxygen species (ROS) free radicals, was added during the radiotherapy treatment of liver cancer cells, it could not significantly downregulate the ROS level induced by radiotherapy, and Ferrostatin-1 had no effect on the sensitivity of radiotherapy (see Figure 3 ), indicating that the high ROS environment after radiotherapy is not easily reversed. Moreover, when DFO was used to block Fe 2+ during the radiotherapy treatment of liver cancer cells, it was found that the intracellular accumulation of PUFA-PL s-OOH was significantly downregulated (see Figure 4 ). From the above, it can be seen that radiotherapy creates favorable conditions for the generation of PUFA-PLs-OOH and promotes its generation.

[0078] To further investigate the effect of radiotherapy on the clearance (ferroptosis defense) of PUFA-PLs-OOH, the liver cancer cells (Huh7) were treated with the inhibitor RSL3 of glutathione peroxidase reductase 4 (GPX4), the inhibitor Ifsp1 of ferroptosis suppressor protein 1 (FSP1), or the inhibitor DAHP of GTP cyclohydrolase 1 (GCH1) respectively, and the results were as Figure 5 shown. The results showed that neither in the GPX4-dependent reduction pathway (RSL3) nor in the GPX4-independent reduction pathways (FSP1, DAHP) could promote the accumulation of PUFA-PLs-OOH after radiotherapy, nor could it effectively improve the radiotherapy sensitivity, which means that inhibiting the three reduction pathways of ferroptosis respectively cannot break the ferroptosis defense. It is speculated that the ferroptosis defense after radiotherapy may be constructed by multiple reduction pathways. In this regard, when the above three ferroptosis inducers were combined in pairs, it was found that when RSL3 and iFSP1 were used in combination, it could significantly promote the accumulation of PUFA-PLs-OOH after radiotherapy and greatly improve the radiotherapy sensitivity; no improvement effect was seen when RSL3 or iFSP1 was combined with DAHP (see Figure 6 ). By detecting the levels of GSH and CoQ 10 H 2 in liver cancer cells before and after radiotherapy by high performance liquid chromatography-mass spectrometry (LC-MS), it was found that the above core mechanism was also verified in the results of the significant increase in the levels of GSH and CoQ 10 H 2 in cells after radiotherapy (see Figure 7 ). Without radiotherapy treatment, even when the three types of ferroptosis inducers were used in combination, it could not increase the level of PUFA-PLs-OOH to induce ferroptosis in liver cancer cells, indicating that radiotherapy is a prerequisite for ferroptosis. In summary, it can be clearly seen that after radiotherapy of liver cancer cells, SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ 10The reduction system jointly constructs an iron death defense and rapidly clears lipid peroxides to mediate iron death escape.

[0079] Example 2

[0080] To identify the regulatory factors in the construction of the iron death defense system in hepatocellular carcinoma cells after radiotherapy, the lipid peroxidation situation in hepatocellular carcinoma cells after radiotherapy was screened using a human whole-genome sgRNA library. The schematic diagram of this screening process is as Figure 8 shown, and the specific steps are as follows:

[0081] (1) In hepatocellular carcinoma cells, a stable cell line containing a whole-genome sgRNA library (mixed type, ensuring that each cell contains 1 sgRNA) was constructed.

[0082] (2) After the cells were irradiated, a lipid peroxide dye was used to distinguish the cells; subsequently, the genes corresponding to the decrease in the sgRNA copy number in the library cells with accumulated lipid peroxidation were analyzed.

[0083] (3) The following screening conditions were set: |Log2(Fold Change)| > 4, the number of captured sgRNAs > 3, and all were satisfied in three hepatocellular carcinoma cells (SKHep1 / Huh7 / SNU449). The results showed that METTL2 might have the potential to down-regulate the level of lipid peroxides after radiotherapy.

[0084] Since lipid peroxidation is an important marker of cell iron death, first, the effect of METTL2 on lipid peroxidation after radiotherapy was studied. The peroxidized lipids (PUFA-PLs-OOH) on the membrane of Hu7 cells were stained with BODIPY 581 / 591C11 dye, and then detected by flow cytometry. The results are as Figure 9 shown. The results showed that knocking out METTL2 indeed significantly increased the level of lipid peroxidation in hepatocellular carcinoma cells after radiotherapy, and the knockout of METTL2 led to the failure of the combined effect of GPX4 inhibitor and FSP1 inhibitor on promoting lipid peroxidation after radiotherapy. In addition, it was also found that in cells with METTL2 knocked out, the cell survival rate decreased significantly, and after adding DFO to block the supply of Fe 2+ it was able to significantly improve the radiotherapy resistance effect of the cells (not shown in the figure), indicating that the iron death defense in METTL2-knocked-out cells was disrupted.

[0085] Furthermore, in vitro and in vivo experiments were respectively carried out to verify the effect of METTL2 on radiotherapy sensitivity. First, a cell colony formation experiment was conducted, and the specific steps are as follows:

[0086] (1) The METTL2 knockout stable cell lines were constructed in Huh7 and SUN449 cells using CRISPR-cas9-sgRNAs (Sg1, the sequence is shown in SEQ ID NO: 9, which is 5’-GCTGCTCCACAGGATGCAGA-3’).

[0087] (2) Cells in the logarithmic growth phase were seeded at 1000 cells / well in 6-well plates.

[0088] (3) After 24 hours, radiotherapy intervention with 0 - 4 Gy was performed; the cells were further cultured for 10 - 14 days.

[0089] (4) The culture medium was discarded, 1 mL of methanol containing 0.5% crystal violet was added to each well, and the cells were stained for 30 min; the methanol was discarded, and the residual methanol was washed with water, then cell clones could be observed; under the microscope, only when the number of cells > 50 was counted as a valid clone, and the total number of clones in each group was counted.

[0090]

[0091] The results are as Figure 10 shown. The results showed that knocking out METTL2 could significantly improve the radiosensitivity of liver cancer cells and liver cancer cells to radiotherapy, that is, inhibiting the expression of METTL2 in cells could effectively improve the therapeutic effect of radiotherapy and inhibit the generation of radiotherapy tolerance (**p < 0.01, ***p < 0.001). It was proved that the presence of METTL2 and its expression level were positively correlated with the degree of radiotherapy resistance in liver cancer.

[0092] Subsequently, wild-type METTL2 (WT) was respectively introduced into METTL2-KO Huh7 and SUN449 cells, and then the radiosensitivity (selecting a single radiation dose of 2 Gy) was investigated. Specifically, the above colony formation experiment was still used. The results showed that the reconstitution of METTL2 could significantly improve the survival ability of cells after radiotherapy, making it return to the original resistance-promoting degree (***p < 0.001, ****p < 0.0001) (see Figure 11 ).

[0093] Furthermore, a tumor-bearing mouse experiment was carried out to verify the role of METTL2 in radiosensitivity in vivo. The specific steps are as follows:

[0094] (1) Five-week-old female BALB / c-nu / nu mice were selected and divided into two groups. One group was subcutaneously injected with normal Huh7 (METTL2-WT) cells, and the other group was subcutaneously injected with METTL2-stably knocked-out MHCC97H (METTL2-KO) cells constructed by sgMETTL2 (Sg1, the sequence is as shown in SEQ ID NO: 9, 5'-GCTGCTCCACAGGATGCA GA-3') lentivirus.

[0095] (2) After tumor formation, the mice in the METTL2-WT group were randomly divided into two groups, with 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, with 10 mice in each group, namely group 3 and group 4.

[0096] (3) Mice in group 1 and group 3 were not treated with any treatment. Mice in group 2 and group 4 were treated by tumor area irradiation (RS2000 X-ray irradiator, the source-skin distance was based on 100 cm, and medium-long-range daily equal-dose radiotherapy was used; the non-tumor area was blocked by a special lead container for mice), 2 Gy per day, treated once every other day for 5 consecutive times; the tumor volume was measured regularly during the treatment, and the mice were sacrificed 7 weeks after the treatment, the tumors were dissected, and the tumor inhibition rate was calculated by weighing.

[0097] The results are as Figure 12 shown. The results showed that for mice without radiotherapy, there was no significant difference in the tumor growth rate and tumor volume between METTL2-knockout tumor-bearing mice and METTL2-wild-type tumor-bearing mice, and the difference between the two was not statistically significant; while after radiotherapy, the tumor growth rate of METTL2-knockout tumor-bearing mice was significantly slower than that of METTL2-wild-type tumor-bearing mice, the tumor volume was significantly smaller, and the tumor inhibition rate was significantly increased, and the difference between the two was statistically significant (****p < 0.0001).

[0098] sg2 (the sequence is as shown in SEQ ID NO: 10, 5'-CGCAGAAGCATC ATCCCGCC-3') or sg3 (the sequence is as shown in SEQ ID NO: 11, 5'-GTGTC TCCACAGGATGCAGA-3') was used to replace sg1 to construct METTL2-knockout stable cell lines in Huh7 and SUN449 cells by CRISPR-cas9 technology, and the above cell colony formation experiment and in vivo radiotherapy sensitivity experiment were repeated. The results were similar to those of sg1 above, so they were not shown again.

[0099] As described above, simply inhibiting the expression level of METTL2 in liver cancer cells cannot significantly inhibit the growth of tumor cells. However, when radiotherapy is performed on liver cancer cells, the knockout of METTL2 can significantly improve the sensitivity of tumor cells to radiotherapy, reduce the growth rate and volume of tumor cells, significantly improve the antitumor effect, and effectively prevent the occurrence of radiotherapy tolerance. Based on the above results, it is clear that METTL2 is a radiotherapy tolerance factor in liver cancer, and knocking out or inhibiting the expression of METTL2 can effectively promote the sensitivity to radiotherapy and prevent the occurrence of radiotherapy tolerance.

[0100] Example 3

[0101] To study the mechanism of action of METTL2 in the formation of radiotherapy resistance, first, the method of measuring the mRNA content in ribosome-nascent chain complexes (RNC-qPCR) was used to examine the types of proteins translated by METTL2 after radiotherapy. This method is based on the capture of full-length mRNAs that are being translated and bound to ribosomes, and can be used to judge the translation efficiency of proteins, as follows:

[0102] (1) Cells were treated with sgRNA (sg1) knockout or METTL2 overexpression plasmid and divided into 4 groups: METTL2-WT (untreated group), METTL2-KO (METTL2 knockout group). For the rescue experiment, after knocking down in the above two groups, the overexpression METTL2 plasmid was added or not added.

[0103] (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 for 10 minutes at 4°C.

[0104] (3) The supernatant RNA was extracted, and sequencing and qRT-PCR were performed for each gene. 30% sucrose solution was added to the remaining supernatant, and centrifuged at 185000g for 5 hours at 4°C. The sediment RNA was extracted and sequenced and qRT-PCR were performed using conventional methods in the art. Enrichment analysis was performed through an open-source website (http: / / www.webgestalt.org). The FPKM method was used to normalize the gene expression level. The calculation formula for the translation ratio (TR) is as follows: TR = (FPKM in RNA-seq) / (FPKM in input RNA-seq).

[0105] The results showed that after radiotherapy, the translation efficiency (TE) of glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) decreased significantly due to the knockout of METTL2 (see Figure 13 ). To further verify the above results, the reconstitution of METTL2 in the knockout cell line would significantly increase the translation efficiency of GPX4 and FSP1 (seeFigure 14 )。

[0106] Furthermore, detection and analysis using a tRNA chip (ArraystarnStar TM tRNA PCR chip) found that after radiotherapy, the contents of nine types of tRNAs in hepatocellular carcinoma cells with METTL2 knockout were significantly lower than those 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). Analyzing the frequencies of the anticodons of these nine types of tRNAs in the mRNAs of ferroptosis-related genes found that their frequencies in GPX 4 and FSP1-mRNA were much higher than those in other genes. Based on the above results, it can be known that the decisive factor for METTL2 to specifically regulate the translation of GPX4 and FSP1 may be the mediation of m 3 C methylation modification of these nine types of tRNAs.

[0107] Table 1 Changes in the copy numbers of tRNAs after radiotherapy in the METTL2 knockout group compared with the control group

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[0110]

[0111] After radiotherapy, due to the rapid clearance of lipid peroxidation-mediated ferroptosis escape, ferroptosis contributes less to the killing effect caused by radiotherapy. Therefore, in order to explore the regulatory mechanism of ferroptosis defense construction, the present invention screened through a whole-genome sgRNA library, and then verified that the tRNA methyltransferase METTL2 promotes the clearance of lipid peroxides by positively regulating the construction of the SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ10 double defense systems, mediates ferroptosis escape and then promotes radiotherapy resistance of liver cancer in vitro and in vivo, and identified METTL2 as a liver cancer radiotherapy tolerance factor. By knocking out or inhibiting the expression of METTL2, the sensitivity to radiotherapy can be effectively promoted and the occurrence of radiotherapy tolerance can be prevented. 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 the defense systems they respectively lead, and accelerate the clearance of lipid peroxides. It can be clearly seen from the above that METTL2 is a gene extremely related to liver cancer radiotherapy tolerance. A higher expression level of METTL2 has an obvious positive correlation with liver cancer radiotherapy tolerance, which can be used to predict the radiotherapy tolerance and sensitivity of liver cancer and provide an early assessment for the connection of patients receiving preoperative treatment. At the same time, by revealing the correlation between the METTL2 gene and liver cancer radiotherapy tolerance, the present invention has important practical significance for solving the problems of individual differences in clinical efficacy and the blank of regression effect / prognosis evaluation, and better realizing precision treatment. It provides a new drug treatment target for humans to overcome liver cancer, thus providing a new direction for subsequent drug research and development, clinical treatment, etc., and has extremely high social value and market application prospects.

[0112] The above specific implementation part specifically introduces the analysis 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 idea of the present invention, rather than a limitation on the relevant content. Without departing from the principle of the present invention, those skilled in the art can also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications should also belong to the protection scope 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.

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