Application of METTL2 in radiotherapy sensitization of digestive system tumor

Abstract

The present application relates to the application of METTL2 in radiotherapy sensitization of cancer. Through a large number of research and screening, it is found that METTL2 is a gene closely related to radiotherapy tolerance of liver cancer, and the higher expression level of METTL2 is positively correlated with radiotherapy tolerance, which can be used to predict the radiotherapy tolerance and sensitivity degree of liver cancer, and provide early evaluation for the connection of preoperative treatment of patients. By revealing the correlation between METTL2 gene and radiotherapy tolerance of liver cancer, the present application has important practical significance for solving the problems of clinical efficacy difference among individuals and blank of retreatment effect / prognosis evaluation, and better realizing precise treatment. A new drug treatment target for human to conquer liver cancer is provided.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of METTL2 in radiosensitization of digestive system tumors. Background Technology

[0002] Hepatocellular carcinoma (HCC) is currently the fourth most common malignant tumor and the second leading cause of cancer death in my country, seriously threatening people's lives and health. Treatment for HCC mainly includes surgery, local ablation therapy, radiotherapy, transarterial chemoembolization (TACE), and systemic drug therapy. Among these, new radiotherapy techniques, such as three-dimensional or intensity-modulated radiotherapy (3D-CRT or IMRT) and stereotactic body radiotherapy (SBRT), are gradually becoming an important part of HCC treatment. In recent years, scholars at home and abroad have reported the unique advantages of radiotherapy in terms of efficacy: it can improve local control rates and significantly prolong survival time (the 3-year survival rate can reach about 30%); it can also alleviate symptoms such as pain, obstruction, or bleeding caused by metastases to lymph nodes, lungs, bones, brain, or adrenal glands; and some tumors that cannot be operated on shrink or downstage after radiotherapy, allowing them to be converted to surgical resection.

[0003] Both the NCCN and CSCO guidelines recommend radiotherapy (radical or palliative) as the standard treatment for liver cancer. However, radiotherapy for liver cancer has several limitations, including low overall efficacy, short duration of effect, and a high recurrence rate, which severely restricts its widespread application in the field. It is generally believed that the high heterogeneity of liver cancer cells, with some cells being insensitive to radiation or exhibiting resistance, is the main reason for the poor efficacy of radiotherapy. Therefore, screening and identifying key targets mediating radiotherapy resistance in liver cancer, and developing corresponding intervention strategies based on these mechanisms of action to improve the clinical efficacy of radiotherapy for liver cancer, is of great practical significance. Summary of the Invention

[0004] The purpose of this invention is to address the problem of unclear radiotherapy tolerance mechanisms in the prior art for liver cancer. By starting from the essence (physical basis) of radiation and expanding on the biological effects of DNA damage, this invention expands on other biological effects caused by radiation and clarifies that METTL2 is a target related to radiotherapy resistance in liver cancer, thus providing a more comprehensive scientific basis and solution for radiosensitization of liver cancer.

[0005] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0006] The first aspect of this invention provides the use of METTL2 inhibitors in the preparation of drugs that improve 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 sgRNAs 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] A second aspect of the present invention provides the use of a reagent for detecting METTL2 expression levels in the preparation of products for detecting radiosensitivity and / or predicting the efficacy of radiotherapy for digestive system tumors.

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

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

[0013] Primer pair 1: The upstream sequence is shown in SEQ ID NO: 1 (5'-GCAGTCCTCGCCGATAAGAG-3'), and the downstream sequence is 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'-TCCTCAGCCACCTACCGAATA-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'-GCGAGCTCATGGCCGGCTCCTACCCT-3'), and the downstream sequence is 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] A third aspect of this invention provides the use of METTL2 inhibitors in the preparation of products that promote ferroptosis in 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 radiosensitivity and / or efficacy of digestive system tumors, comprising primers for detecting the expression level of the METTL2 gene and / or reagents for detecting the content of METTL2 protein; and one or more of PCR enzyme, PCR buffer, dNTPs, and fluorescent substrate.

[0025] Preferably, the primers for detecting the METTL2 gene expression level 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 in SEQ ID NO: 7, and the downstream sequence is 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 fluorescently labeled probes.

[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 fillers, binders, lubricants, disintegrants, flavoring agents, colorants, antioxidants, antibacterial agents, chelating agents, surfactants, and solvents.

[0039] The sixth aspect of the present invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for improving 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 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 this invention, the primers and / or primer pairs refer to PCR primers used to synthesize the METTL2 gene cDNA strand in PCR, thereby detecting the expression level of the METTL2 gene mRNA. Besides the primers and / or primers listed in this invention, those skilled in the art are fully capable of designing corresponding primers and / or primer pairs based on the METTL2 gene sequence using conventional methods and techniques in the art, including but not limited to molecular biology, and screening the designed primers and / or primer pairs using conventional experimental methods, as long as they can specifically detect the METTL2 expression level. The METTL2 inhibitor refers to a substance that can specifically downregulate the expression level of METTL2 and / or the transcriptional level of its mature mRNA and / or the expression level or activity of the METTL2 protein. For example, it can be downregulated by methods such as antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA erasers, target masking and / or multiple targets. Any method that can reduce the level and / or activity of METTL2 is acceptable.

[0044] X-rays (or gamma rays) used in radiotherapy are high-energy rays with wave-particle duality, possessing photon properties. When X-rays (or gamma rays) collide with molecules, energy transfer occurs, causing ionizing radiation (IR) and 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, reactive oxygen species (ROS) generated by the ionization of water molecules (H₂O) by X-rays (or gamma rays) can not only attack nucleic acid linker sites, causing DNA breaks, but can also accept ferrous iron (Fe²⁺). 2+ Electron transfer from oxygen into peroxide free radicals provides the raw materials and a highly reactive oxygen species (ROS) environment for the peroxidation of biomolecules such as nucleic acids, lipids, and proteins. Notably, lipids, catalyzed by lipoxygenases, form lipid peroxides, leading to oxidative damage to the membrane and inducing programmed cell death, specifically ferroptosis. Therefore, it can be concluded that radiotherapy creates conditions for ferroptosis in tumor cells.

[0045] To clarify the relationship between radiotherapy and ferroptosis in liver cancer, it is first necessary to determine whether ferroptosis contributes to the killing effect of radiotherapy on liver cancer cells. To this end, this invention uses the iron chelating agent DFO to block Fe... 2+ The study aimed to inhibit ferroptosis at its source and then observe its effect on radioresistance. Results showed that this inhibitor slightly promoted radioresistance in liver cancer cells (SKHep1 / Huh7 / SNU449). Furthermore, the use of the acetyl-CoA carboxylase (ACCA) allosteric inhibitor TOFA to block fatty acid synthesis yielded similar results to DFO; TOFA also only slightly promoted radioresistance. These results indicate that blocking the underlying mechanisms of ferroptosis at its source has little contribution to radioresistance. This may be due to the presence of cellular factors mediating ferroptosis escape, resulting in a relatively small contribution of ferroptosis to the radiotoxic effect.

[0046] Lipid peroxidation is a key marker of ferroptosis. Therefore, this study aimed to assess the accumulation of lipid peroxides (PUFA-PLs-OOH) in hepatocellular carcinoma cells after radiotherapy to determine whether ferroptosis escape is mediated by PUFA-PLs-OOH downregulation. First, various radiotherapy modalities were applied to hepatocellular carcinoma cells. Results showed that PUFA-PLs-OOH accumulation was only observed in hepatocellular carcinoma cells with a single irradiation dose of 12 Gy or higher, or with multiple consecutive moderate-dose irradiations (6 Gy / day for 3 or 5 consecutive days), and even then, the accumulation was at a low level. Combined in vitro and in vivo results indicate that insufficient accumulation of lipid peroxides is the main reason why ferroptosis is difficult to trigger after radiotherapy. Analysis of the PUFA-PLs-OOH generation stage revealed that radiotherapy created favorable conditions for PUFA-PLs-OOH generation, promoting its formation. Meanwhile, research on the PUFA-PLs-OOH clearance (ferroptosis defense) showed that after radiotherapy, liver cancer cells were protected by a combined SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ10 reduction system, rapidly clearing lipid peroxide-mediated ferroptosis escape.

[0047] Screening using a whole-genome sgRNA library and subsequent validation revealed 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 dual defense system, mediating ferroptosis escape and thus enhancing radiotherapy resistance in hepatocellular carcinoma (HCC) in vitro and in vivo. In vitro experiments showed that METTL2 knockdown significantly increased the radiosensitivity of HCC cells, while reintroducing METTL2 into METTL2 knockout cells restored the original pro-resistance level. Simultaneously, in vivo experiments confirmed that METTL2 knockdown enhances the radiosensitivity of HCC cells. Therefore, METTL2 is identified as a radiotherapy tolerance factor for HCC, and knocking out or inhibiting METTL2 expression can effectively promote radiosensitivity and prevent radiotherapy tolerance. Mechanistic studies show that METTL2 can rapidly sense radiotherapy signals, catalyze m3C modification of certain tRNAs, and 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] The present invention has the following technical advantages over the prior art:

[0049] (1) Through extensive research and screening, this invention has found that METTL2 is a gene that is highly associated with radiotherapy tolerance in liver cancer. The high expression level of METTL2 is significantly positively correlated with radiotherapy resistance in liver cancer, and can be used to predict radiotherapy tolerance and sensitivity in liver cancer, providing an early assessment for patients to receive preoperative treatment.

[0050] (2) This invention reveals the association between the METTL2 gene and radiotherapy tolerance in liver cancer, which is of great practical significance for solving the problem of inter-individual differences in clinical efficacy and the gap in regression / prognostic assessment, and for better achieving precision treatment. It provides a new drug target for conquering liver cancer, thus providing a new direction for subsequent drug development and clinical treatment, and has extremely high social value and market application prospects. Attached Figure Description

[0051] Figure 1 A schematic diagram showing the contribution of ferroptosis inhibitors DFO and TOFA to the radiotherapy resistance of liver cancer cells.

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

[0053] Figure 3 This is a schematic diagram showing the effects of Ferrostatin-1 on intracellular ROS levels and radioresistance after radiotherapy.

[0054] Figure 4 This is a schematic diagram showing the effect of DFO on lipid peroxidation after radiotherapy.

[0055] Figure 5 This is a schematic diagram showing the effect of combined radiotherapy and ferroptosis inducers on lipid peroxidation in liver cancer cells.

[0056] Figure 6 This is a schematic diagram showing the effect of ferroptosis inducers on the radiosensitivity of liver cancer cells.

[0057] Figure 7 GSH and CoQ in liver cancer cells before and after radiotherapy 10 Schematic diagram of H2 content determination results.

[0058] Figure 8 This is a schematic diagram illustrating the screening process and screening criteria for whole-genome sgRNA libraries.

[0059] Figure 9 This is a schematic diagram showing the effects of radiotherapy and its combination with ferroptosis inducers on lipid peroxidation in METTL2 knockout cells.

[0060] Figure 10 This is a schematic diagram showing the effect of METTL2 knockout on the radiosensitivity of liver cancer cells.

[0061] Figure 11 A schematic diagram showing the effect of METTL2 reinstatement on radiosensitivity in METTL2 knockout hepatocellular carcinoma cells.

[0062] Figure 12 This is a schematic diagram illustrating the effect of METTL2 knockout on tumor growth rate in tumor-bearing mice.

[0063] Figure 13 This is a schematic diagram showing the effect of METTL2 on the translation efficiency of ferroptosis and defense-related factors after radiotherapy.

[0064] Figure 14 This diagram illustrates the impact of METTL2 knockout followed by METTL2 reinstatement on the translation efficiency of GPX4 and FSP1. Detailed Implementation

[0065] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0066] Unless otherwise specified, the cell lines listed in this invention, including Huh7, SNU449, and MHCC97H, were cultured according to existing techniques. All cell lines were identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan) and their presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci). The cells were then cryopreserved in liquid nitrogen for subsequent experiments. All reagents used in this invention were commercially available. Informed consent was obtained from patients for all clinical specimens used, and the relevant procedures and methods complied with medical ethics requirements and Good Clinical Practice (GCP) guidelines. The experimental methods used in this invention, such as DNA extraction, whole-genome sequencing, primer design, immunohistochemistry, Western blot, cell experiments, and animal experiments, are all conventional methods and techniques in the field. For intracellular / in vivo METTL2 expression levels, conventional methods in the art (such as PCR, Western blot, etc.) can be used for detection. This invention has verified the specificity of the primer pairs (primer pairs 1-4) and antibody ab101510 described in this invention for detecting METTL2 expression levels through specific experiments. However, given that the detection of specific gene / protein expression levels is a conventional method in the art and not the main focus of this invention, the relevant detection results are not specifically presented in this invention. Those skilled in the art can perform detection and verification according to the experimental methods described in this invention or other conventional methods in the prior art as needed. Furthermore, in addition to the primer pairs and antibodies listed in this invention, those skilled in the art can also design relevant primer pairs / antibodies based on the METTL2 gene sequence and / or protein structure, or obtain commercially available reagents to complete the detection of METTL2 expression levels. Therefore, the specific information such as primer pairs and antibodies listed in the context of this invention does not constitute a limitation on the actual scope of protection of this invention.

[0067] Representative results from biological experiments were selected from replicates and presented in the contextual figures. Data were displayed 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. Standard medical statistical methods such as t-tests, chi-square tests, and ANOVA were used to compare differences in means between two or more groups. p < 0.05 was considered statistically significant.

[0068] Example 1

[0069] To investigate the relationship between radiotherapy and ferroptosis in liver cancer, it is first necessary to determine whether ferroptosis contributes definitively to the killing effect of radiotherapy on liver cancer cells. To this end, the iron chelator DFO was used to block Fe...2+ The supply of [the substance] inhibits ferroptosis at its source, and then its effect on radiotherapy resistance is observed. The specific steps are as follows:

[0070] (1) Log-phase liver cancer cells (SKHep1 / Huh7 / SNU449) were seeded at 1000 per well in 6-well plates.

[0071] (2) After 24 hours, the cells were subjected to 6 Gy of X-ray radiation and treated with the drug DFO (10 μM).

[0072] (3) After culturing 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, wash off the residual methanol with water, and cell clones can be observed.

[0073] Under a microscope, a cell count greater than 50 is considered a valid clone. The total number of clones in each group is counted. The calculation is as follows:

[0074]

[0075]

[0076] The results are as follows Figure 1 As shown, the results indicated that the ferroptosis inhibitor DFO slightly promoted radioresistance in liver cancer cells (SKHep1 / Huh7 / SNU449); subsequently, the acetyl-CoA carboxylase (ACCA) allosteric inhibitor TOFA was used to block lipase synthesis to observe the effect of ferroptosis on radioresistance. The results are as follows... Figure 1 As shown, the results are similar to those of DFO, indicating that TOPA only slightly promotes radiotherapy resistance. These results collectively suggest that blocking the underlying material basis for ferroptosis at its source has little contribution to radiotherapy resistance. This may be due to the presence of factors in cells that mediate ferroptosis escape, resulting in a smaller contribution from the radiotoxic effects leading to ferroptosis.

[0077] Subsequently, the accumulation of lipid peroxides (PUFA-PLs-OOH) in liver cancer cells after radiotherapy was evaluated to determine whether ferroptosis escape was mediated by the downregulation of PUFA-PLs-OOH. First, Huh7 and SNU449 cells were treated with different radiotherapy modalities, and then stained with lipid peroxide dye (C11-BODIPY staining). The results were then analyzed by flow cytometry. Figure 2 As shown in the figure. The results showed that only when the single irradiation dose was as high as 12 Gy or above, or when multiple moderate doses were continuously irradiated (6 Gy / day, for 3 or 5 consecutive days), PUFA-PLs-OOH accumulation in liver cancer cells could be observed, and the degree was low.

[0078] Furthermore, when the antioxidant Ferrostatin-1, which scavenges reactive oxygen species (ROS) free radicals, was added during radiotherapy for liver cancer cells, it was found that it did not significantly lower the radiotherapy-induced ROS levels, and Ferrostatin-1 had no effect on radiosensitivity (see [link to study]). Figure 3 This indicates that the high ROS environment after radiotherapy is not easily reversed. Furthermore, DFO was used to treat liver cancer cells simultaneously with radiotherapy for Fe... 2+ The blockade resulted in a significant downregulation of intracellular PUFA-PLs-OOH accumulation (see [link to article]). Figure 4 In summary, radiotherapy creates favorable conditions for the formation of PUFA-PLs-OOH, thus promoting its formation.

[0079] To further investigate the effects of radiotherapy on PUFA-PLs-OOH clearance (ferroptosis defense), liver cancer cells (Huh7) were treated with either the glutathione peroxidase 4 (GPX4) inhibitor RSL3, the ferroptosis inhibitor protein 1 (FSP1) inhibitor Ifsp1, or the GTP cyclase 1 (GCH1) inhibitor DAHP. The results are as follows: Figure 5 As shown in the figure. The results showed that neither the GPX4-dependent reduction pathway (RSL3) nor the GPX4-independent reduction pathway (FSP1, DAHP) could promote the accumulation of PUFA-PLs-OOH after radiotherapy, nor could they effectively improve radiosensitivity. This means that the three reduction pathways that inhibit ferroptosis cannot break through ferroptosis defense. Based on this, it is speculated that ferroptosis defense after radiotherapy may be constructed by multiple reduction pathways. In this regard, the three ferroptosis inducers were combined in pairs. It was found that when RSL3 was combined with iFSP1, it could significantly promote the accumulation of PUFA-PLs-OOH after radiotherapy and greatly improve radiosensitivity; no improvement effect was observed when RSL3 or iFSP1 was combined with DAHP (see Figure 6 GSH and CoQ in liver cancer cells before and after radiotherapy were detected by high performance liquid chromatography-mass spectrometry (LC-MS). 10 The levels of H2 were found to be related to the aforementioned core mechanisms, including intracellular GSH and CoQ levels after radiotherapy. 10 This was validated by the results of a surge in H2 levels (see [link]). Figure 7 However, without radiation treatment, even the simultaneous use of three classes of ferroptosis inducers did 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 is clear that after radiotherapy, liver cancer cells are affected by SLC7A11-GSH-GPX4 and NAD(P)H-FSP1-CoQ... 10The reducing system together constructs a defense against ferroptosis, rapidly clearing lipid peroxides-mediated ferroptosis escape.

[0080] Example 2

[0081] To clarify the regulatory factors governing the construction of the ferroptosis defense system in hepatocellular carcinoma cells after radiotherapy, a human whole-genome sgRNA library was used to screen for lipid peroxidation in radiotherapy-treated hepatocellular carcinoma cells. The screening process is illustrated in the diagram below. Figure 8 As shown, the specific steps are as follows:

[0082] (1) In liver cancer cells, a stable cell line containing a whole genome sgRNA library was constructed (a hybrid cell line, ensuring that each cell contains one sgRNA).

[0083] (2) Cells were distinguished by lipid peroxide dyes after being irradiated; then, the genes corresponding to the decrease in sgRNA copy number in the library cells with lipid peroxidation accumulation were analyzed.

[0084] (3) The following screening criteria were set: |Log2(Fold Change)|>4, the number of captured sgRNAs>3, and the criteria were met in all three liver cancer cells (SKHep1 / Huh7 / SNU449). The results showed that METTL2 may have the potential to downregulate lipid peroxide levels after radiotherapy.

[0085] Since lipid peroxidation is an important marker of ferroptosis, this study first investigated the effect of METTL2 on lipid peroxidation after radiotherapy. Peroxidized lipids (PUFA-PLs-OOH) on the membrane of Hu7 cells were stained with BODIPY 581 / 591 C11 dye, and then detected by flow cytometry. The results are as follows: Figure 9 As shown in the figure. The results showed that METTL2 knockout significantly increased the level of lipid peroxidation in liver cancer cells after radiotherapy, and METTL2 knockout led to the inactivation of the promoting effect of GPX4 inhibitor combined with FSP1 inhibitor on lipid peroxidation after radiotherapy. In addition, it was found that cell survival rate was significantly reduced in METTL2 knockout cells, while the addition of DFO to block Fe 2+ The supply of this substance can significantly enhance the radiotherapy resistance of cells (not shown in the figure), indicating that the ferroptosis defense in METTL2 knockout cells is broken.

[0086] Furthermore, the effect of METTL2 on radiosensitivity was verified through in vitro and in vivo experiments. First, a cell colony formation experiment was conducted, and the specific steps are as follows:

[0087] (1) METTL2 knockout stable cell lines were constructed in Huh7 and SUN449 cells using CRISPR-cas9-sgRNAs (Sg1, sequence as shown in SEQ ID NO: 9, 5'-GCTGCTCCACAGGATGCAGA-3').

[0088] (2) The cells in the logarithmic growth phase were seeded at 1000 per well in a 6-well plate.

[0089] (3) 0-4 Gy radiotherapy intervention was performed 24 hours later; culture continued for 10-14 days.

[0090] (4) 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, wash off the residual methanol with water, and cell clones can be observed; observe under a microscope, only cells with more than 50 cells are counted as one effective clone, and count the total number of clones in each group.

[0091] .

[0092] The results are as follows Figure 10 As shown in the figure. The results showed that knocking out METTL2 significantly improved the sensitivity of hepatocellular carcinoma cells to radiotherapy, meaning that inhibiting intracellular METTL2 expression effectively improved the therapeutic effect of radiotherapy and inhibited the development of radiotherapy tolerance (**p<0.01, ***p<0.001). This demonstrates that the presence and expression level of METTL2 are positively correlated with the degree of radiotherapy resistance in hepatocellular carcinoma.

[0093] Subsequently, after introducing wild-type METTL2 (WT) into METTL2-KO Huh7 and SUN449 cells, radiosensitivity (using a single radiation dose of 2 Gy) was examined. Specifically, the colony formation experiment described above was used. The results showed that METTL2 complementation significantly improved cell viability after radiotherapy, restoring them to their original pro-resistance level (***p < 0.001, ****p < 0.0001) (see [link to study]). Figure 11 ).

[0094] Furthermore, the effect of METTL2 on radiosensitivity in vivo was verified through tumor-bearing mouse experiments. The specific steps are as follows:

[0095] (1) Five-week-old female BALB / c-nu / nu mice were selected and divided into two groups. One group was injected subcutaneously with normal Huh7 (METTL2-WT) cells, and the other group was injected subcutaneously with METTL2 stably knocked-out MHCC97H (METTL2-KO) cells constructed from sgMETTL2 (Sg1, sequence as shown in SEQ ID NO: 9, 5'-GCTGCTCCACAGGATGCAGA-3') lentivirus.

[0096] (2) After tumor formation, the mice in the METTL2-WT group were randomly divided into two groups of 10 each, namely group 1 and group 2; the mice in the METTL2-KO group were also randomly divided into two groups of 10 each, namely group 3 and group 4.

[0097] (3) No treatment was given to mice in groups 1 and 3. Mice in groups 2 and 4 were treated with tumor irradiation (RS2000 X-ray irradiator, source-to-skin distance of 100cm, medium-to-long-range daily equal dose irradiation; non-tumor areas were shielded with a mouse-specific lead container) at 2 Gy / day, once every other day for 5 consecutive treatments. Tumor volume was measured regularly during the treatment period. Seven weeks after treatment, the mice were sacrificed, the tumors were removed, and the tumor inhibition rate was calculated by weighing.

[0098] The results are as follows Figure 12 As shown in the figure. The results showed that, for mice that had not undergone radiotherapy, there was no significant difference in tumor growth rate and tumor volume between METTL2 knockout tumor-bearing mice and METTL2 wild-type tumor-bearing mice, and the difference was not statistically significant. However, 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 was statistically significant (p < 0.0001).

[0099] METTL2 knockout stable cell lines were constructed in Huh7 and SUN449 cells using sg2 (sequence shown in SEQ ID NO: 10, 5'-CGCAGAAGCATCATCCCGCC-3') or sg3 (sequence shown in SEQ ID NO: 11, 5'-GTGTCTCCACAGGATGCAGA-3') instead of sg1, and the cell colony formation experiment and in vivo radiosensitivity experiment were repeated. The results were similar to those of sg1, so they will not be repeated here.

[0100] As shown above, simply inhibiting the expression level of METTL2 in liver cancer cells does not significantly inhibit tumor cell growth. However, during radiotherapy for liver cancer cells, METTL2 knockout significantly increases the sensitivity of tumor cells to radiotherapy, reduces the growth rate and size of tumor cells, and significantly improves the tumor-suppressing effect, effectively preventing the development of radiotherapy tolerance. In summary, these results clearly indicate that METTL2 is a radiotherapy tolerance factor in liver cancer, and knocking out or inhibiting METTL2 expression can effectively promote sensitivity to radiotherapy and prevent the development of radiotherapy tolerance.

[0101] Example 3

[0102] To investigate the mechanism of action of METTL2 in radioresistance formation, we first used ribosome-new chain complex mRNA content assay (RNC-qPCR) to examine the types of proteins translated by METTL2 after radiotherapy. This method is based on the capture of full-length mRNA that is being translated and bound to ribosomes, and can be used to determine the translation efficiency of proteins, as detailed below:

[0103] (1) 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). Recovery experiment: METTL2 overexpression plasmid was added or not added after knockdown in the above two groups.

[0104] (2) After the cell samples were treated with cycloheximine for 15 minutes, they were lysed with lysis buffer at 4°C for 30 minutes. The cell lysis buffer was centrifuged at 16000g at 4°C for 10 minutes.

[0105] (3) RNA was extracted from the supernatant and sequenced and qRT-PCR was performed for each gene. The remaining supernatant was added to 30% sucrose solution and centrifuged at 185,000 g and 4 °C for 5 hours. RNA was extracted from the sediment and sequenced and qRT-PCR was performed using conventional methods in the field. 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).

[0106] The results showed that after radiotherapy, METTL2 knockout led to a significant decrease in the translational efficiency (TE) of glutathione peroxidase 4 (GPX4) and ferroptosis inhibitor 1 (FSP1) (see [link to relevant documentation]). Figure 13 To further validate these results, reintroducing METTL2 into knockout cell lines significantly improved the translation efficiency of GPX4 and FSP1 (see [link to relevant documentation]). Figure 14 ).

[0107] Furthermore, tRNA microarrays (ArrayStar) were employed. TM tRNA PCR chip analysis revealed that after radiotherapy, the levels of nine types of tRNAs in METTL2 knockout liver cancer cells were significantly lower than 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). Analysis of the frequency of anticodons of these nine tRNAs in the mRNAs of ferroptosis-related genes revealed that the frequency was significantly higher in GPX4 and FSP1 mRNAs than in other genes. Based on these results, the decisive factor in the specific regulation of GPX4 and FSP1 translation by METTL2 may be the mediator of the anticodons of these nine tRNAs. 3 C-methylation modification.

[0108] Table 1. Changes in tRNA copy number after radiotherapy in the METTL2 knockout group compared to the control group.

[0109]

[0110] Following radiotherapy, hepatocellular carcinoma cells exhibit rapid clearance of lipid peroxidation, mediating ferroptosis escape and thus contributing less to the radiotherapy-induced killing effect. To investigate the regulatory mechanism of ferroptosis defense construction, this invention screened a genome-wide sgRNA library and subsequently 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 dual defense system, mediating ferroptosis escape and thereby promoting radiotherapy resistance in hepatocellular carcinoma in vitro and in vivo. This confirms that METTL2 is a radiotherapy tolerance factor for hepatocellular carcinoma, and knocking out or inhibiting METTL2 expression can effectively promote radiosensitivity and prevent the development of radiotherapy tolerance. Mechanistic studies show that METTL2 can rapidly sense radiotherapy signals, catalyze m3C modifications of certain tRNAs, and specifically enhance the translation efficiency of GPX4 and FSP1, increasing their protein levels and promoting the continuous activation of their respective defense systems, thus accelerating the clearance of lipid peroxides. It is clear from the above that METTL2 is a gene highly associated with radiotherapy tolerance in liver cancer. A high expression level of METTL2 is significantly positively correlated with radiotherapy tolerance in liver cancer, and it can be used to predict radiotherapy tolerance and sensitivity in liver cancer, providing an early assessment for patients' preoperative treatment. Furthermore, this invention, by revealing the association between the METTL2 gene and radiotherapy tolerance in liver cancer, has significant practical implications for addressing the challenges of inter-individual differences in clinical efficacy and the gaps in regression / prognostic assessment, thus better achieving precision medicine. It provides a new drug therapeutic target for conquering liver cancer, offering a new direction for subsequent drug development and clinical treatment, and possesses extremely high social value and market application prospects.

[0111] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. The application of METTL2 inhibitors in the preparation of drugs to improve the radiosensitivity of digestive system tumors, characterized in that, The METTL2 inhibitor is selected from sgRNA designed based on METTL2; 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; the digestive system tumor is liver cancer.

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