Use of ALKBH5 protein inhibitor in preparation of medicine for reversing drug resistance of MTX-resistant tumor cells

Abstract

This invention discloses the application of an ALKBH5 protein inhibitor in the preparation of drugs to reverse drug resistance in MTX-resistant tumor cells. This invention targets MTX-resistant malignant tumor cells with highly amplified DHFR gene, reducing the degree of DHFR gene amplification and decreasing the number of DMs within the cells. Interference with ALKBH5 mainly works by inhibiting (m... 6 A) R-loop demethylation regulates R-loop stability, thereby inhibiting damage response signals to DNA double-strand breaks, reducing the repair effect of HR factors on DSBs, and thus reversing tumor drug resistance and improving the efficiency of tumor treatment. This invention provides a new targeted treatment strategy for the biotherapy of malignant tumors that are prone to developing resistance, where MTX is the main therapeutic drug. It also has very positive significance for a deeper understanding of the nature of chemotherapy resistance and for finding drug resistance targets for personalized treatment.

Description

Technical Field

[0001] This invention relates to the use of ALKBH5 protein inhibitors in the preparation of drugs that reverse tumor cell resistance to MTX, and particularly to the use of ALKBH5 protein inhibitors in the preparation of drugs that inhibit (m 6 A) The application of drugs that reduce the HR repair capacity of cells to DSBs by demethylation modification of R-loop, downregulate DHFR gene amplification, and thereby reverse the resistance of tumor cells to MTX, belongs to the field of pharmaceutical technology. Background Technology

[0002] Methotrexate (MTX), an antimetabolite antitumor drug, is widely used to treat diseases such as acute lymphoblastic leukemia, lymphoma, choriocarcinoma, breast cancer, head and neck cancer, bladder cancer, and lung cancer. MTX competitively inhibits the activity of dihydrofolate reductase (DHFR), impairing the synthesis of tetrahydrofolate from dihydrofolate, leading to inhibited DNA synthesis and ultimately cell death. However, some patients develop acquired resistance during MTX treatment, resulting in chemotherapy failure. Increased activity of DHFR, the main target enzyme of MTX, and amplification of its gene are among the main mechanisms by which tumors develop resistance to MTX.

[0003] Gene amplification, an increase in the copy number of chromosomal restriction regions, is prevalent in various tumors. Cytogenetic analysis indicates that gene amplification mainly exists in two forms: homogeneously staining regions (HSRs) on chromosomes and extrachromosomal double-minute chromosomes (DMs). The oncogenes or drug resistance genes carried by DMs drive tumor progression and drug resistance. The formation mechanisms of DMs mainly include the replication fork break model, the chromosome looping model, the "break-fusion-bridge" model, and the "translocation-break-amplification" model. Although the formation mechanism of DMs is still highly controversial, a prerequisite is DNA double-strand breakage, and repair after double-strand breakage promotes DM formation. DNA double-strand breaks (DSBs) are the most severe form of DNA damage. To maintain the integrity and stability of the genome, organisms have evolved sophisticated DNA damage responses (DDRs) to repair DNA double-strand damage. This process is a specific, complex, and interconnected signaling cascade network triggered by the affected cells, including sensing and transmitting damage signals to effector proteins, arresting the cell cycle, activating DNA repair pathways, and cell death.

[0004] There are three main repair pathways for DSBs in cells: homologous recombination (HR), non-homologous end joining (NHEJ), and alternative non-homologous end joining (A-EJ). Previous studies have shown that multiple repair pathways, including HR, NHEJ, and A-EJ, can promote the formation of DMs.

[0005] RNA epigenetics is a new research direction in epigenetics that emerged after DNA epigenetics. In mammals, RNA epigenetic modifications can regulate RNA processing and metabolism, and modulate numerous biological processes. RNA methylation accounts for more than 60% of all RNA modifications, with N6-methyladenosine (M...) being a particularly significant component. 6 A) is the most common modification on mRNA and lncRNAs in higher organisms. 6 A-modification primarily occurs on adenine in RRACH sequences, and studies have shown that its function is determined by an "encoder (writer)," an "eraser (eraser)," and a "reader." The "encoder (writer)" is a methyltransferase, and the known components of this complex include METTL3, METTL14, WTAP, and KIAA1429; while ALKBH5 and FTO act as demethylases (erasers) that can reverse methylation; m 6 A is from m 6 A-binding protein recognition, currently found m 6 A-binding proteins (code readers) include YTH domain proteins (including YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2) and the nuclear heterogeneous protein HNRNP family (HNRNPA2B1 and HNRNPC). M-binding proteins occur on microRNA, circRNA, rRNA, tRNA, and snoRNA. 6 A modification, however, it has been found that METTL3 can catalyze the R-loop m in genomic DNA. 6 A methylation modification, and through the corresponding m 6 A reading protein performs biological functions.

[0006] R-loops are triple-stranded structures composed of a single ssDNA strand and a DNA-RNA hybrid. The presence of R-loops disrupts the stable double-stranded DNA structure, thus affecting genome stability. R-loop structures are widely distributed throughout the genome. R-loops located in gene promoters, terminators, and DSBs can promote gene initiation and termination, and inhibit damage repair. Studies have shown that R-loops also exist in intergenic regions, located in enhancer or superenhancer regions, and can promote the transcription of multiple genes. R-loops primarily function in the genome through their own stability, which depends on the proteins they bind to, including DNA-RNA helicases, nucleases, and m... 6 Proteins involved in DNA modification and DNA damage repair regulate R-loop stability by degrading or stabilizing it. As their names suggest, helicases and nucleases primarily regulate R-loop stability through enzymatic activity. Furthermore, literature reports indicate that promoters, terminators, and intergenic regions of genomic DNA possess m... 6 A modification site, while theoretically this type of DNA should not have m 6 A modification site; therefore, the R-loop, which is widespread in genomic DNA, may be m 6 The target site for A modification, and ultimately through the m of the R-loop. 6 A modification modifies the stability of the R-loop. Because m 6 The demethylation modifier ALKBH5 is also an R-loop binding protein. Therefore, ALKBH5 may regulate R-loop stability by catalyzing R-loop demethylation modification, thereby playing a role in gene transcription regulation and DNA damage repair.

[0007] This invention discovers that ALKBH5 can catalyze (m 6 A) R-loop demethylation modification, therefore with m 6 After interfering with the demethylase ALKBH5 as the target, it was found that ALKBH5 catalyzes (m 6 A) R-loop demethylation regulates R-loop stability, further promoting the HR damage repair response and the formation of DMs, leading to increased cellular resistance to MTX. Therefore, ALKBH5 acts on (m 6 A) R-loop shows promise as a new target for treating MTX-resistant tumors and other tumors containing gene amplification. Summary of the Invention

[0008] The purpose of this invention is to address the common problem of chemotherapy failure due to drug resistance to the tumor chemotherapy drug MTX during treatment, by providing a (m) catalyst catalyzed by ALKBH5.6 A) A method to reverse MTX-resistant tumor cell resistance by regulating HR repair function using R-loop.

[0009] To achieve the above objectives, the inventors of this invention previously constructed a human colon cancer cell line HT29-4, which exhibited a tolerance level of 10 to MTX. -4 In human colon cancer cells with a concentration of mol / L, both DMs and HSRs amplify simultaneously, with DMs being the predominant amplification form.

[0010] This invention utilizes Western blotting to detect the expression levels of ALKBH5 in DM-free HT29 parental cells and DM-containing HT29-4 cells. Then, shALKBH5 is stably transfected into DM-containing HT29-4 cells using a lentiviral vector to establish the HT29-4 shALKBH5 cell line. Subsequent CHIP-seeker analysis using Merip-seq data revealed the widespread presence of ALKBH5 catalyzing (m...) in the genomic DNA. 6 A) Sites for R-loop demethylation modification; utilizing m 6 A quantitative technique was used to detect the presence of ALKBH5-catalyzed (m) molecule in genomic DNA before and after ALKBH5 interference. 6 A) R-loop demethylation modification sites. Immunofluorescence and Dotblot techniques were used to detect changes in R-loop accumulation in cells before and after ALKBH5 knockdown. Immunofluorescence was also used to detect changes in hnRNPC fluorescence expression before and after ALKBH5 knockdown in HT29-4 cells. S9.6 co-IP technology was used to detect the interaction between R-loop and hnRNPC in HT29-4 cells. Then, changes in HR, A-EJ, and NHEJ repair pathway proteins in HT29-4 cells before and after ALKBH5 knockdown were examined. Immunofluorescence co-localization was then used to detect the co-localization of R-loop with the damage signal γ-H2AX and the HR repair factor RAD51 in HT29-4 cells before and after ALKBH5 knockdown. Finally, changes in MTX resistance in HT29-4 cells before and after ALKBH5 knockdown were detected.

[0011] In MTX-resistant HT29-4 cells containing DMs, this invention found that interfering with ALKBH5 can increase the m of the R-loop. 6 A-modification and binding to the reading protein hnRNPC stabilizes the R-loop while inhibiting the repair of DSBs by HR and A-EJ, thereby reducing the number of intracellular DMs. This suggests that ALKBH5, through the m-loop of the R-loop, 6The ALKBH5 demethylation pathway may play an important role in the formation of tumor demyelinating cytokines (DMs). Interference with ALKBH5 increased the sensitivity of DM-containing, MTX-resistant HT29-4 cells to MTX, suggesting that ALKBH5 plays a crucial role in the progression of tumor MTX resistance. (The last sentence appears to be incomplete and possibly refers to a different topic.) 6 A) R-loop provides a scientific basis for targeting and reversing tumor cell resistance to MTX.

[0012] Specifically, the present invention employs the following technical means:

[0013] 1.HT2910 -4 Establishment of a DM-containing cell model with drug resistance concentration of mol / L

[0014] In the early stages of this invention, human colon cancer HT29 cells were continuously cultured with gradually increasing concentrations of MTX until the MTX-resistant concentration of HT29 cells reached 10. -4 The concentration of mol / L was determined by karyotype and FISH experiments, and the cells were identified as containing DMs and named HT29-4 cells.

[0015] 2. Determination of ALKBH5 protein expression levels in HT29 parental cells without DMs and HT29-4 drug-resistant cells containing DMs.

[0016] Proteins were extracted from HT29 parental cells and HT29-4 drug-resistant cells, and the expression level of ALKBH5 protein was detected by Western blotting. The results showed that the expression of ALKBH5 protein in HT29-4 cells was significantly higher than that in HT29 parental cells.

[0017] 3. Construction of a stable ALKBH5 (two sequences) interference cell line in HT29-4 and determination of interference effect.

[0018] The sensitivity of DM-containing HT29-4 resistant cells to puromycin was determined, and an appropriate puromycin concentration was selected as the drug selection concentration for stable transfection. DM-containing HT29-4 resistant cells in logarithmic growth phase were seeded in 24-well culture plates. When the cells reached 80% confluency, stable transfection was performed. Control lentivirus Control shRNA and two interference lentiviruses ALKBH5 shRNA were transfected, respectively. After 72 hours of culture, the appropriate concentration of puromycin was added to each well for stable selection, and the medium was continuously changed. The control clone of DM-containing HT29-4 cells was named shControl, and the interference clones of HT29-4 cells were named shALKBH5-1 and shALKBH5-2.

[0019] Western blotting was used to detect the expression of ALKBH5 in DM-containing HT29-4 drug-resistant cells before and after ALKBH5 interference. The expression level of ALKBH5 in the interference group was significantly lower than that in the control group, indicating that we have successfully established a stable ALKBH5 knockdown cell line in DM-containing HT29-4 drug-resistant cells.

[0020] 4. ALKBH5 catalyzes the genome (m 6 A) Analysis of R-loop demethylation modification

[0021] (1) In the NCBI database, Merip-seq data of head and neck squamous cell carcinoma (HNSCC) were analyzed to determine the differences in ALKBH5 levels before and after interference. 6 A modification revealed ALKBH5-related differences in regions such as the 5'UTR, 3'UTR, and exons that can transcribe RNA. 6 With A modification, we found that promoters, introns, and intergenic regions of genes that cannot transcribe RNA contained ALKBH5-related differential m. 6 A modifies the difference m here. 6 The A modification is most likely catalyzed by ALKBH5 (m 6 A) R-loop.

[0022] (2) Extract genomic DNA from cells before and after HT29-4 interference with ALKBH5, and utilize m 6 A quantitative reagent kit technique was used to detect (m) in genomic DNA after ALKBH5 interference. 6 A) A significant increase in R-loop.

[0023] 5. ALKBH5 via (m 6 A) Detection of R-loop demethylation modification and regulation of R-loop stability by the hnRNPC pathway of the reading protein.

[0024] (1) Immunofluorescence was used to detect the changes in R-loop accumulation before and after HT29-4 interference with ALKBH5. The results showed that the focal signal of S9.6 increased significantly after ALKBH5 interference, and the R-loop accumulation increased.

[0025] (2) Extracting HT29-4 before and after ALKBH5 interference, and performing Dotblot detection, it was found that the foci signal of S9.6 increased significantly after ALKBH5 interference, and the R-loop accumulation increased.

[0026] (3) According to literature reports, hnRNPC is both an R-loop binding protein and an m 6This protein is a reading protein and also functions to stabilize the R-loop. We used S9.6co-IP technology to detect and demonstrate the interaction between hnRNPC and the R-loop in HT29-4 cells.

[0027] (4) Immunofluorescence technique was used to detect the changes in hnRNPC protein expression before and after HT29-4 interfered with ALKBH5. The results showed that the hnRNPC focal signal increased and the hnRNPC expression increased after ALKBH5 interference.

[0028] 6. Detection of HR repair proteins promoting HR repair through R-loop stability

[0029] (1) Nuclear proteins were extracted before and after ALKBH5 interference, and the expression of key DDR proteins was detected by Western blotting. The results showed that the expression of BRCA1, NBS1, and Mre11 proteins in the HR repair pathway was significantly reduced, the expression of PARP1 protein in the A-EJ repair pathway was significantly reduced, and the expression of KU70 protein in the NHEJ repair pathway did not change significantly.

[0030] (2) The changes in R-loop accumulation in HT29-4 cells before and after BRCA1 interference were detected using Dotblot technology. The results showed that after BRCA1 interference, the signal at the S9.6 foci increased significantly, and R-loop accumulation increased. This indicates that HR repair protein can degrade R-loop.

[0031] (3) Immunofluorescence colocalization technology was used to compare the colocalization signals of R-loop damage signals γ-H2AX and RAD51 with the R-loop in HT29MTX-resistant cells before and after ALKBH5 interference. The results showed that the colocalization signal of γ-H2AX with S9.6 was significantly increased after ALKBH5 interference, while the colocalization signal of RAD51 with S9.6 was significantly decreased. ALKBH5 promotes the interaction between HR repair factors and the R-loop, promoting HR repair by degrading the R-loop; furthermore, the increased colocalization of DSBs with the R-loop prevents the damage from being repaired.

[0032] 7. Changes in DMs after stable ALKBH5 interference in HT29-4 cells

[0033] (1) The metaphase chromosome karyotypes of shControl, shALKBH5-1, and shALKBH5-2 cells were analyzed using karyotype preparation technology. It was found that after interfering with ALKBH5, the number of DMs in HT29-4 drug-resistant cells containing DMs decreased significantly.

[0034] (2) Real-time PCR was used to detect the changes in the copy number of the DHFR gene amplified on DMs before and after ALKBH5 interference. Compared with the control group, the copy number of the DHFR gene was significantly reduced in cells with stable ALKBH5 interference.

[0035] 8. Stable interference in HT29MTX-resistant cells enhances the sensitivity of ALKBH5 cells to MTX and reduces the growth capacity of tumor cells.

[0036] We used the CCK8 assay to compare the IC50 of MTX on cells containing DMs before and after ALKBH5 interference. 50 The value (median lethal concentration) was shown in HT29MTX-resistant cells, where the IC50 value of the control clone was significantly lower. 50 The values ​​were 2.58 and 2.63 times higher than those in the ALKBH5 interference group, indicating that cell sensitivity to MTX increased after ALKBH5 inhibition. This suggests that ALKBH5 may be a novel target for reversing MTX-resistant tumor cells.

[0037] Based on the above research, this invention proposes the application of ALKBH5 protein inhibitors in the preparation of drugs that reverse MTX-resistant tumor cell resistance and reduce malignant growth of tumor cells.

[0038] Preferably, the ALKBH5 protein inhibitor is a drug that inhibits the expression of ALKBH5 protein at the transcriptional level.

[0039] Preferably, the drug is an interference vector containing ALKBH5shRNA.

[0040] Preferably, the interference vector containing ALKBH5shRNA is a lentiviral vector containing ALKBH5shRNA.

[0041] Preferably, the sequence of the ALKBH5shRNA is shown in SEQ ID NO.1 or 2.

[0042] Preferably, the tumor cells are MTX-resistant cells containing DMs and with highly amplified DHFR gene.

[0043] Preferably, the tumor cells are human colon cancer HT29MTX resistant cells containing DMs and with highly amplified DHFR gene.

[0044] Preferably, the tumor cells have a tolerance level of 10 to MTX. -4 mol / L.

[0045] Preferably, the drug reduces the amplification of the DHFR gene, decreases intracellular DMs, and simultaneously inhibits the repair effect of HR and A-EJ on DSBs, thereby reversing tumor drug resistance and improving the efficiency of tumor treatment.

[0046] Compared with the prior art, the beneficial effects of the present invention are:

[0047] This invention provides a novel targeted therapy for malignant tumors where MTX is the primary treatment drug and resistance is easily developed, offering a scientific basis for effectively combating MTX resistance. This invention targets MTX-resistant malignant tumor cells with highly amplified DHFR genes, using ALKBH5 and its catalyzed (m... 6 A) Targeting the R-loop reduces gene amplification, eliminates intracellular DMs, and inhibits cellular repair of DSBs, thereby reversing tumor drug resistance and improving the efficiency of tumor treatment. Furthermore, it comprehensively elucidates the mechanism by which ALKBH5 participates in DM formation and tumor drug resistance in drug-resistant tumor cells, contributing to a more comprehensive understanding of the process of tumor development and drug resistance caused by DSBs, and providing new strategies for reversing tumor drug resistance. Attached Figure Description

[0048] Figure 1 The protein expression of ALKBH5 in HT29 cells containing and without DMs;

[0049] Figure 2 To establish stable cell lines in HT29-4 cells before and after ALKBH5 interference, Western blotting was used to verify ALKBH5 expression.

[0050] Figure 3 In Merip-seq data, ALKBH5 catalyzes the DNA of gene promoters, terminators, intergenic regions, etc., which are also known as (m 6 A) m of R-loop 6 Figure A shows the results of demethylation modification; and m 6 A quantitative detection of HT29-4 cells before and after ALKBH5 interference catalysis of genomic DNA, i.e. (m 6 A) Graph showing the results of R-loop demethylation modification (B);

[0051] Figure 4Figure (A) shows the immunofluorescence results of R-loop accumulation in HT29-4 cells before and after ALKBH5 interference. RNaseH (enzyme) specifically degrades R-loop and was used as a negative control. The right figure shows the statistical analysis results. Figure (B) shows the Dotblot detection results of R-loop accumulation in HT29-4 cells before and after ALKBH5 interference. Figure (C) shows the immunofluorescence detection results of hnRNPC protein expression in HT29-4 cells before and after ALKBH5 interference. The right figure shows the statistical analysis results. Figure (D) shows the interaction results of R-loop and hnRNPC detected by S9.6co-IP in HT29-4 cells.

[0052] Figures 5A-F as well as Figure 5G-H Figure (A) shows the results of Western blotting analysis of changes in key DDR proteins in HT29-4 cells before and after ALKBH5 interference. Figure 3 The following are the statistical analysis results: Figure B shows the interaction between the R-loop and the key HR repair proteins RAD50 and RAD51 in HT29-4 cells detected by S9.6co-IP; Cyclin B1 protein is not expressed in G1 phase, and the expression of Cyclin B1 in HT29-4 cells was detected by Western blotting within a time gradient (0-6 hours), showing the optimal time from G2 / M to the next G1 phase; Figure D shows the changes in R-loop accumulation in HT29-4 cells before and after BRCA1 interference in G2 phase using Dotblot detection, with DNA-PKcs and PARP1 interference in G2 phase serving as negative controls; Figures E and F show the co-localization of γ-H2AX and RAD51 with the R-loop before and after ALKBH5 interference in HT29-4 cells, and the statistical analysis results are shown in Figures G and H.

[0053] Figure 6 Figure (A) shows the results of Real-time PCR detection of DHFR copy number changes before and after ALKBH5 interference in HT29-4 resistant cells; Figure (B) shows the results of karyotype analysis of DMs number changes before and after ALKBH5 interference in HT29-4 resistant cells; and Figure (C) shows the results of statistical analysis. Detailed Implementation

[0054] The present invention will be further described below with reference to specific embodiments to more clearly illustrate its advantages and features. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0055] Example 1

[0056] 1. Cell Culture

[0057] Human colon cancer cell line HT29 was continuously cultured with MTX at progressively increasing concentrations to screen for MTX-resistant cells at a concentration of 10. -4 Cells were cultured at mol / L for another 5 months until they developed stable drug resistance, and then further experiments were conducted. These cells were named HT29-4 cells.

[0058] HT29 parental cells and drug-resistant cells were cultured in DMEM high-glucose medium containing 15% fetal bovine serum. The drug-resistant cell line medium was supplemented with the corresponding concentration of MTX. All cells were cultured in a 5% CO2, 37°C incubator.

[0059] (1) Western blot detection of cellular protein expression levels

[0060] Collect HT29-4 cells in the logarithmic growth phase, discard the culture medium, and wash the cells three times with pre-chilled PBS at 4°C, removing any residual PBS. Place the cell culture dish on ice and prepare a cell lysis buffer at a ratio of RIPA buffer:protease inhibitor:phosphatase inhibitor of 100:10:1 (volume ratio). Add an appropriate amount to the cells and incubate for 3 minutes. Scrape off the cells and collect the cell suspension into a 1.5 ml Eppendorf tube. Vortex for 15 seconds, then place on ice and sonicate for vortex lysis. Let stand for 10 minutes, vortexing every 5 minutes during this period. Centrifuge at 12000 rpm at 4°C for 20 minutes. Collect the supernatant into a new Eppendorf tube, determine the protein concentration using the BCA method, and adjust the sample to a uniform concentration using RIPA buffer. Add 1 / 4 volume of 5× loading buffer to the sample, denature in boiling water for 5 minutes, and store at -20°C.

[0061] (2) SDS-PAGE gel electrophoresis

[0062] Prepare a 30% gel stock solution using a ratio of methylenebisacrylamide to acrylamide of 1:29, and use this stock solution to prepare SDS-PAGE gels. Prepare separating gels of different concentrations according to the size of the target protein. Mix the protein samples thoroughly, centrifuge briefly, and then load the samples. Prepare electrophoresis buffer (100 ml of 10× electrophoresis buffer + 10 ml of 10% SDS + deionized water to a final volume of 1000 ml). Perform electrophoresis at a constant voltage of 80 volts. Once the protein marker enters the separating gel, adjust the voltage to 120 volts and continue electrophoresis at this constant voltage until the marker band with a molecular weight close to that of the target protein reaches one-third of the way up the separating gel. Stop electrophoresis at this point.

[0063] (3) Wet transfer method

[0064] Prepare a transfer buffer by mixing 100 ml of 10× electrophoresis buffer and 200 ml of methanol with deionized water to a final volume of 1000 ml. Remove the gel and place it in the transfer buffer. Cut a PVDF membrane according to the gel size and activate it by soaking it in methanol for 30 seconds. Assemble the transfer apparatus in the following order: cathode, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, anode. Transfer the apparatus to a transfer tank containing the transfer buffer. Place an ice pack in the transfer tank to cool the tank and perform a constant current transfer at 300 mA.

[0065] (4) Immune response

[0066] After transfer, place the PVDF membrane in a hybridization chamber, add TBS-T, and wash the membrane three times on a shaker for 5-10 minutes each time. Discard the TBS-T. Prepare a 5% skim milk powder solution using TBS-T as the blocking buffer. Add the blocking buffer to the hybridization chamber, ensuring the liquid surface completely covers the PVDF membrane. Block on a shaker at room temperature for 1-2 hours. Discard the blocking buffer, add diluted primary antibody, and hybridize overnight on a shaker at 4°C. On the second day, recover the primary antibody, wash the membrane three times with TBS-T for 10 minutes each time, then add diluted secondary antibody and hybridize at room temperature in the dark for 1 hour. Recover the secondary antibody and wash the membrane three times with TBS-T. Finally, scan the membrane using the Odyssey Infrared Imaging System to obtain the results.

[0067] The results showed that the protein expression level of ALKBH5 was significantly increased in HT29-4 drug-resistant cells. This suggests that ALKBH5 may be associated with drug resistance and gene amplification. Figure 1 ).

[0068] 2. Establishment of HT29-4 resistant cell line stabilizing ALKBH5 cell line

[0069] (1) Determine the sensitivity of HT29 MTX-resistant cells to puromycin.

[0070] Count the HT29 cells and divide them into groups of 10 per well. 5 Cells were seeded at a density of 1000 mcg / mL in 12-well plates. After cell attachment, the culture medium was changed, and puromycin was added at final concentrations of 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1.0 μg / mL, 1.1 μg / mL, and 1.2 μg / mL, respectively. It was observed that HT29-DMs cells containing DMs died completely on day 7 at a puromycin concentration of 0.8 μg / mL. Therefore, a final puromycin concentration of 0.8 μg / mL was selected for treatment of DMs-transfected cells.

[0071] (2) Stable transfection

[0072] The virus provided by Jikai Biotechnology Co., Ltd. was used for transfection. The ALKBH5 shRNA lentiviral vectors were named sh ALKBH5-1 and shALKBH5-2, with sequences of 5'-GCUGCAAGUUCCAGUUCAATT-3' (SEQ ID NO.1) and 5'-UGGAUAUGCUGCUGAUGAAT-3' (SEQ ID NO.2). This virus, after infecting host cells, does not infect other cells or utilize host cells to produce new viral particles. The following procedures were followed for virus transfection experiments: A biosafety cabinet was used for virus transfection. Before handling the virus, wear a lab coat, mask, and gloves. Handle the virus with extreme care to avoid generating aerosols or splashes. Prepare 1% SDS solution; if the work surface becomes contaminated with the virus during operation, immediately wipe it clean with 1% SDS solution. Pipe tips, centrifuge tubes, culture plates, and other items that have come into contact with the virus were soaked in diluted 84 disinfectant. After the procedure, add 84 disinfectant (approximately 1:8) to the discarded virus-containing culture medium, soak for one day, and then discard. After completing the procedure, remove gloves and wash hands with soap and water.

[0073] One day before viral transfection, HT29 MTX-resistant cells containing homogeneous stained regions and DMs were harvested and trypsin-digested, then suspended in culture medium to form single-cell suspensions. Cells were seeded into 24-well plates at approximately 30%–50% confluence and cultured overnight. The next day, the old culture medium was discarded, and 485 μl of fresh culture medium was added. Negative control lentivirus and ALKBH5 knockdown lentivirus were thawed on ice. Based on the MOI (MOI = 20) of viral transfection into HT29 cells, 15 μl of lentivirus was added per well for transfection. After mixing, the cells were incubated for 16 hours. The medium was changed, and fresh culture medium was added for further incubation until 72 hours. Transfection efficiency was observed under a fluorescence microscope, and puromycin was added for screening to estimate lentivirus infection efficiency. Final concentrations of 0.5 μg / mL and 0.8 μg / mL were selected as suitable drug screening concentrations for stable transfection of cells containing homogeneous stained regions and DMs, respectively.

[0074] The control group clone containing DMs-resistant HT29-4 cells was named shControl, and the interference group clones were named shALKBH5-1 and shALKBH5-2.

[0075] 3. ALKBH5 catalytic genome (m 6 A) Analysis of R-loop demethylation modification

[0076] (1) Merip-seq data of head and neck squamous cell carcinoma (HNSCC) and hepatocellular carcinoma (HCC) were downloaded from the NCBI database. The database was analyzed before and after ALKBH5 interference using the GALAXY TEST server. CHIP-seeker annotation was performed, and the annotation peak scope included exons, introns, 5'UTR, 3'UTR, promoters, gene terminus extensions to the intergenic region, and distinct intergenics. Results are as follows: Figure 3 A shows that, in addition to ALKBH5-related differences in regions such as the 5'UTR, 3'UTR, and exons that can transcribe RNA, m 6 A-modified gene promoters, introns, and intergenic regions that cannot transcribe RNA contain ALKBH5-related differential m. 6 A modifies the difference m here. 6 The A modification is most likely catalyzed by ALKBH5 (m 6 A) R-loop. Hint: The m6A demethylation modification of ALKBH5 may act not only on mRNA, but also on the R-loop structure formed by lncRNA and genomic double-stranded DNA.

[0077] (2) Genomic DNA was extracted from HT29-4 cells before and after ALKBH5 gene knockdown, i.e. (m 6 A) Changes in the total amount of R-loop.

[0078] a. Extracting genomic DNA

[0079] DNA extraction was performed using the QIAmp DNA mini kit. Cell pellet was collected (not exceeding 5 × 10⁻⁵). 6 Add 200 μl PBS, 20 μl proteinase K, and 200 μl Buffer AL to the collection tube. Vortex and heat at 56°C for 10 min. Add 200 μl anhydrous ethanol, mix well, and transfer to a QIAmp mini centrifuge column. Centrifuge at 8000 rpm for 1 min. Discard the waste liquid in the collection tube and replace with a new collection tube. Add 500 μl Buffer AW1 to the centrifuge column and centrifuge at 8000 rpm for 1 min. Add 500 μl Buffer AW2 to the centrifuge column and centrifuge at 14000 rpm for 3 min. Transfer the column to a new EP tube, add 150 μl Buffer AE, let stand at room temperature for 5 min, and centrifuge at 8000 rpm for 1 min. The liquid in the EP tube is the DNA sample. Use NanoDrop to determine the DNA concentration and perform Real-time PCR directly or store at -20°C.

[0080] bm 6 A quantitative detection of genome (m 6 A) R-loop quantity:

[0081] Genomic DNA was extracted, as above; and EpiQuik m was used. 6 An RNA Methylation Quantification Kit (Catalog No.: P-9005) is used. First, prepare the solutions and buffers: 1×Wash Buffer + 26 mL WB (10×Wash Buffer) + 234 mL RNase-free distilled water; CA dilution (capture antibody): Dilute the CA solution 1:1000 with the diluted WB; DA dilution (detection antibody): Dilute the DA solution 1:2000 with the diluted WB; ES dilution (enhancement solution): Dilute the ES solution 1:5000 with the diluted WB; Diluted positive control (PC): (Single-point control method) Dilute PC (positive control) with 1×TE to 0.5 ng / ul (1ul PC + 3ul TE). Note: All diluted solutions (except diluted WB) should be incubated on ice until use. Any diluted solution, except for diluted WB, must be used on the same day and discarded the following day.

[0082] RNA binding: Calculate the required number of 8-tube strips beforehand. Carefully remove any unnecessary tubes from the plate rack and return them to the bag. Seal the bag tightly and store at 4°C. Add 80 μL of BS binding buffer to each well. Add 2 μL of NC, 2 μL of diluted PC, and 200 ng of DNA sample to each well. Cover the plate with sealing film or sealing strip and incubate at 37°C for 90 minutes. Remove the BS solution from each well, then wash each well with 150 μL of diluted WB solution. Repeat the washing process three times, removing the diluted WB solution each time.

[0083] m6A RNA capture: Add 50 μL of diluted CA solution to each well, then cover and incubate at room temperature for 60 minutes. Remove the diluted CA solution from each well using a pipette. Add 150 μL of diluted WB solution to each well and wash three times. Add 50 μL of diluted DA solution to each well, then cover and incubate at room temperature for 30 minutes. Remove the diluted DA solution from each well using a pipette. Add 150 μL of diluted WB solution to each well and wash four times. Add 50 μL of diluted ES solution to each well, then cover and incubate at room temperature for 30 minutes. Remove the diluted ES solution from each well using a pipette. Add 150 μL of diluted WB solution to each well and wash five times.

[0084] Signal detection: Add 100 μL of DS to each well and incubate at a warm temperature in the dark for 1-10 minutes. Begin detecting color changes in the sample and positive control wells. In a sufficiently moist environment... 6 Under condition A, the DS solution turns blue. When the color of the positive control well turns moderately blue, add 100 μL of SS to each well to inhibit the enzyme reaction. After adding SS, the color will turn yellow; and read the absorbance at 450 nm using a microplate reader within 2–15 minutes.

[0085] m 6 A. Calculation: Since a relative quantitative method was used in this experiment, the relative (m) values ​​in the genomic DNA of two different samples were analyzed. 6 A) R-loop methylation status, calculated using the following formula (m 6 A) R-loop percentage: (S is the amount of sample DNA input, P is the amount of positive control input, both in ng.)

[0086]

[0087] The results are as follows Figure 3 As shown in B: (m) 6 A) R-loop structures account for approximately 0.1%-0.25% of genomic DNA. After knocking down ALKBH5 (m 6 A) Increased R-loop content, with R-loop sites affected by ALKBH5 accounting for approximately 0.15% (0.25%-0.1%) of genomic DNA.

[0088] 4. Detection of R-loop stability regulated by ALKBH5

[0089] (1) Immunofluorescence assay was used to detect the changes in R-loop accumulation before and after ALKBH5 knockdown in HT29-4 cells. Since RNase H can specifically degrade R-loop, we used RNase H as a negative control.

[0090] a. Immunofluorescence analysis

[0091] Single staining analysis: For adherent cells, coverslips were sterilized the night before and placed in six-well plates. 100,000 cells were seeded and 1 ml of culture medium was added overnight. The cells were washed twice with PBS pre-cooled at 4°C, 1 ml of 4% paraformaldehyde was added, and the cells were fixed at room temperature for 15 minutes. The cells were then washed with PBS-Tw for 3 × 5 minutes on a shaker, 1 ml of PBS-Tr was added, and the cells were permeated at room temperature for 10 minutes. The cells were washed with PBS-Tw on a shaker, 1 ml of PBS-T was added to the six-well plates, and the cells were blocked at room temperature for 1 hour. The cells were then incubated overnight with antibody diluted with PBS-T. The next day, the cells were incubated with secondary antibody at room temperature for 2 hours. After mounting with DAPI containing mounting medium, the cells were observed and photographed under a fluorescence microscope and analyzed using ImageJ.

[0092] The results are as follows Figure 4 A shows that after knocking down ALKBH5, the S9.6 signal increased, indicating an increase in R-loop accumulation. After adding RNaseH, the S9.6 signal decreased significantly, indicating that the R-loop was degraded. Figure 4 The right side of A shows the statistical analysis of the results, with ***P<0.005 (n=100).

[0093] (2) The accumulation of R-loop in the genomic dsDNA of HT29-4 cells before and after ALKBH5 knockdown was detected by Dotblot experiment (sample loading amount: 500ng, 1000ng, 2000ng).

[0094] Dotblot detection of R-loop accumulation:

[0095] Prepare an appropriate amount of tumor cell samples in advance. The Dotblot samples used in this experiment are all genomic DNA from cells, and DNA extraction is performed as described above. Clean the Dotblot sample loading instrument with an alcohol swab. Prepare the loading buffer (for a 16-well sample, 500 μl of 10×MOPS + 915 μl of formaldehyde + 4.5 ml of deionized formamide). Clamp the nylon membrane to the loading wells, seal the remaining wells with plastic film, and finally connect the negative pressure aspirator. Add 100 μl of the loading buffer to the wells, turn on the negative pressure aspirator until all the liquid is aspirated, add the calculated DNA sample (e.g., 1000 μg, 1500 μg, 2000 ng DNA), mix with the loading buffer into the wells (100 μl), turn on the negative pressure aspirator until the liquid is aspirated, and then wash the wells twice with 100 μl of the loading buffer. Remove the nylon membrane and place it in a ventilated area to air dry. Perform UV cross-linking for 20 minutes, rinse with deionized water, and block with 5% skim milk powder on a shaker at room temperature for 1-2 hours. Add m6A antibody and S9.6 antibody respectively and incubate at 4°C overnight. The subsequent immune response is the same as that of Western blotting.

[0096] The results are as follows Figure 4 As shown in B: After knocking down ALKBH5, (m 6 A) Increased R-loop activity: At a total DNA loading of 500-1000 ng, the S9.6 signal increased, indicating increased R-loop accumulation. Our results suggest that ALKBH5 can promote R-loop degradation.

[0097] (3) Because ALKBH5 can catalyze the formation of m in the genome 6 A) R-loop, and also to explore the reason why ALKBH5 promotes R-loop degradation, therefore, we will next look for the relationship with (m 6A) R-loop-binding reading protein. Due to the differential upregulation of m before and after ALKBH5 knockdown in HEK293T cells. 6 The A-modified reading proteins include hnRNPC, and hnRNPC is an R-loop binding protein. Immunofluorescence was used to detect the expression of hnRNPC in HT29-4 cells before and after ALKBH5 knockdown. Results are as follows... Figure 4 As shown in Figure C: After knocking down ALKBH5, the hnRNPC signal increased significantly. Figure 4 C. The right side shows the statistical analysis of the results, ****P<0.001 (n=100). Simultaneously, we detected (m) using the co-IP experiment of S9.6. 6 A) Does the R-loop and hnRNPC interact?

[0098] a.S9.6co-IP technology

[0099] Chromatin Extraction: (Using the Epigentek Chromatin Extraction Kit) Culture HT29-4 (adherent cells) at 80%-90% confluence in 100 mm plates, digest with trypsin and collect in a 15 ml centrifuge tube. Count cells using a hemocytometer, taking 10⁵-10⁶ cells per tube. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Wash cells once with 10 mL PBS, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 9 mL of fresh cell culture medium containing 1% formaldehyde (i.e., add 270 μl of 37% formaldehyde to 10 mL of cell culture medium). Incubate at room temperature (20-25°C) on a rocking platform (50-100 rpm) for 10 minutes. Add 1 mL of 1.25 M glycine to 9 mL of crosslinked solution, mix well, and centrifuge at 1000 rpm for 5 minutes. Remove the culture medium and wash the cells once with 10 mL of ice-cold PBS. Centrifuge at 1000 rpm for 5 minutes. For adherent cells, add 200 μl of Working Lysis Buffer (1 mL 10×Lysis Buffer + 6 μl Protease Inhibitor Cocktail + 9 mL distilled water) to resuspend the cell particles (100 μl / 1×10⁶ cells). Transfer the cell suspension to a 1.5 mL EP tube and incubate on ice for 10 minutes. Vortex vigorously for 10 seconds, centrifuge at 5000 rpm for 5 minutes. Carefully remove the supernatant. For each ×10⁶ cell, add 50 μl (maximum 500 μl per EP tube) of Working Extraction Buffer (1 μl Protease Inhibitor Cocktail + 1 mL Extraction Buffer) to resuspend the chromatin. Incubate the sample on ice for 10 minutes, vortexing occasionally. The sample was resuspended and sonicated for 2 x 20 seconds to increase chromatin extraction efficiency. The sample was then cooled on ice for 30 seconds between sonications. It was then centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new EP tube and Chromatin Buffer was added at a 1:1 ratio.

[0100] S9.6 Immunoprecipitation: (Using the Active Motif co-IP kit) First, prepare 2.5 mL of immunoprecipitation buffer (500 μl for immunoprecipitation, 2 mL for washing): 2.325 mL co-IP / Wash Buffer + 25 μl Phosphatase Inhibitors + 5 μl Deacetylase Inhibitor + 2.5 μl Protease Inhibitor Cocktail + 2.5 μl 100 mM PMSF, and store at 4°C. Add 500 μl co-IP / Wash Buffer and 5 μl S9.6 antibody to the extracted chromatin sample to prepare sample-antibody buffer, and incubate overnight at 4°C in a centrifuge. The next day, centrifuge at 4000 rpm for 1 min after pre-cooling at 4°C, and do not perform any further processing after rapid centrifugation. Add 25 μl of protein G magnetic beads to the sample-antibody buffer, incubate at 4°C for 4 hours in a vortex mixer, then centrifuge at 4000 rpm for 1 minute after pre-cooling at 4°C. Do not perform any further processing after centrifugation. Place the sample tube on a magnetic rack to allow the magnetic beads to settle on the side of the tube. Carefully remove and discard the supernatant, being careful not to let the beads stick to the pipette tip. The tube may need to be removed from the magnet before resuspending. Add 500 μl of immunoprecipitation buffer and completely resuspend the precipitate using a pipette. Carefully remove and discard the supernatant. Add another 500 μl of immunoprecipitation buffer and repeat this washing step 4 times. After the final wash, remove as much supernatant as possible with a 200 μl pipette. Resuspend the magnetic beads in 20 μl of 2×loading buffer, vortex to mix thoroughly, and store at -20°C or proceed directly to Western blotting.

[0101] S9.6co-IP technology was used to detect the interaction between R-loop and hnRNPC in HT29-4 cells. Figure 4 D), therefore, ALKBH5 inhibits the expression of the reading protein hnRNPC while simultaneously catalyzing (m 6 A) R-loop demethylation modification thereby inhibits the interaction between hnRNPC and (m 6 A) R-loop binding promotes R-loop degradation and regulates R-loop stability.

[0102] 5. ALKBH5 regulates the HR repair pathway through R-loop stability.

[0103] (1) To investigate whether ALKBH5 can affect the stability of the R-loop through three repair pathways, we used Western blotting to detect the expression of proteins in the HR (RAD51, BRCA1, MRN complex), α-NHEJ (PARP1), and c-NHEJ (KU70) pathways before and after ALKBH5 knockdown in HT29-4 cells, and performed grayscale analysis using ImageJ. The results are shown in Figure 5A. ALKBH5 expression promoted the expression of proteins such as the MRN complex and BRCA1, and PARP1 protein expression was also significantly increased, while KU70 showed no significant change. The right figure shows the statistical analysis results, which showed significant differences (*P<0.05, **P<0.01). These results indicate that ALKBH5 promotes the expression of HR and α-NHEJ repair proteins.

[0104] (2) In order to verify whether there is an interaction between R-loop and repair protein, we performed co-IP of S9.6 and repair protein in HT29-4 cells. The results are shown in Figure 5B: the key HR repair proteins RAD51 and RAD50 are bound to R-loop.

[0105] (3) To investigate whether HR repair pathway proteins exert their damage repair function by degrading R-loops, we further examined the accumulation of R-loops before and after knocking down the key gene BRCA1 in the HR repair pathway. At the same time, to more accurately detect the role of HR repair pathway proteins (which can exert their repair function in G2 phase) in R-loop accumulation during G2 phase.

[0106] a. Dotblot detection of G2 phase R-loop accumulation:

[0107] A suitable amount of cells were placed in fresh culture medium and incubated statically for 12 hours until they adhered to the culture wall. G2 / M cell cycle arrestor RO-3306 was added to the medium to a final concentration of 10 μM. The cells were then incubated statically at 37°C for 24 hours. The culture medium was discarded, and the cells were washed three times with PBS. Fresh culture medium was then added, and the cells were cultured for 0, 2, 3, 4, and 6 hours respectively. Cells were collected for DNA extraction, following the same procedure as above. The Dotblot sample loading instrument was first cleaned with an alcohol swab. A loading buffer was prepared (for a 16-well sample, 500 μl 10× MOPS + 915 μl formaldehyde + 4.5 mL deionized formamide). A nylon membrane was clamped at the loading well, and the remaining wells were sealed with plastic film. Finally, a negative pressure suction device was connected. Add 100 μl of loading buffer to each well. Turn on the negative pressure aspirator until all liquid is aspirated. Add the calculated DNA sample (e.g., 1000 μg, 1500 μg, 2000 ng DNA), mix with the loading buffer, and add 100 μl to each well. Turn on the negative pressure aspirator until all liquid is aspirated. Repeat the washing process twice with 100 μl of loading buffer. Remove the nylon membrane and place it in a ventilated area to air dry. Perform UV crosslinking for 20 minutes, rinse with deionized water, and block with 5% skim milk powder on a shaker at room temperature for 1-2 hours. Add m... 6 Antibody A and S9.6 were incubated overnight at 4°C, followed by the same immune response as Western blot (WB).

[0108] Cells were arrested in the G2 phase using RO-3306, and Western blot analysis is shown in Figure 5C: from 6 hours after release from G2 phase to before G1 phase (G2 / M to before G1 phase of the next cycle). Dotblot analysis was then used to detect R-loop accumulation in the G2 / M phase, and the results are shown in Figure 5D: BRCA1, compared to DNA-PKcs and PARP1, exhibited a degrading effect on R-loops in the G2 phase (dsDNA loading amounts: 500 ng, 1000 ng, 1500 ng). Our results suggest that HR repair factors can degrade R-loops in the G2 phase.

[0109] (4) In order to investigate the effect of inhibiting ALKBH5 (m 6 A) Changes in the binding ability of R-loop with repair factors. We used immunofluorescence to detect the co-localization of the cell damage signal γ-H2AX and the repair factor RAD51 with R-loop before and after ALKBH5 knockdown in HT29-4 cells, and performed statistical analysis on the results.

[0110] a. Immunofluorescence colocalization analysis:

[0111] Before blocking, the process was the same as for single staining. For double staining, the two diluted antibodies were mixed and added to a six-well plate simultaneously. The plate was incubated overnight at 4°C, and the slide was covered with a small square cut from a PE glove to prevent the diluted antibodies from evaporating. The next day, the plate was incubated with fluorescent secondary antibodies for 2 hours each. Different species of antibodies correspond to different detection wavelengths of fluorescent secondary antibodies. In this experiment, a 568nm fluorescent rabbit secondary antibody and a 647nm fluorescent mouse secondary antibody were used for localization, which were respectively linked to the rabbit primary antibody of the repair factor and the mouse primary antibody of S9.6. Fluorescence microscopy was used to capture and photograph the fluorescence signals of the cy3 and cy5 channels, and ImageJ was used for colocalization analysis.

[0112] Before and after ALKBH5 knockdown in HT29-4 cells, the co-localization of the cell damage signal γ-H2AX and the repair factor RAD51 with the R-loop was analyzed (Figures 5E and 5F). The nuclear co-localization before and after ALKBH5 knockdown was statistically analyzed (Figures 5G and 5H, n=100), showing a significant difference (***P<0.005). These results indicate that HR repair proteins can degrade the R-loop, and ALKBH5 promotes the binding of the R-loop to HR repair proteins. Therefore, we believe that ALKBH5 promotes HR repair of DSBs by facilitating the degradation of the R-loop by HR repair proteins.

[0113] 6. Changes in DMs after stable interference with ALKBH5 in HT29-4 resistant cells

[0114] (1) To clarify whether ALKBH5 is associated with the formation of DMs, we used Real-time PCR to detect the changes in DHFR gene copy number before and after ALKBH5 interference in HT29-4 drug-resistant cells containing DMs. Genomic DNA was extracted as above.

[0115] Real-time PCR

[0116] 20 μl PCR reaction system:

[0117]

[0118] PCR reaction conditions:

[0119] The reaction was carried out in 45 cycles: 95℃ for 6 minutes, 95℃ for 20 seconds, Tm for 20 seconds, and 72℃ for 20 seconds; the melting curve was 95℃ for 5 seconds, 65℃ for 1 minute, and 97℃.

[0120] Three independent replicate experiments were conducted, and 2 were applied. -ΔΔtThe experimental results were analyzed using a statistical method, and the results of the target gene were normalized using the internal reference gene ACTIN. Finally, a t-test was used for statistical analysis. The changes in gene amplification of the target gene in the shControl, shALKBH5-1, and shALKBH5-2 groups were compared, and the differences were expressed as fold changes. P-values ​​were calculated; a p-value < 0.05 was considered statistically significant between the two groups.

[0121] Metaphase chromosome karyotype preparation

[0122] Culture cells to the active mitotic phase, then add colchicine to the cells to a final concentration of 0.20 μg / mL and incubate for 1 hour. Collect the supernatant into a centrifuge tube, wash the cells with PBS, and digest them with trypsin. Centrifuge the supernatant and digested cells in the centrifuge tube at 1000 rpm for 5 minutes. After the cell pellet is separated, add 8 mL of preheated hypotonic buffer (0.075 mol / L KCl solution) at 37°C and incubate in a 37°C water bath for 13 minutes. Then add 1 mL of fixative (methanol: glacial acetic acid = 3:1) to the centrifuge tube and centrifuge at 1500 rpm for 6 minutes. Discard the supernatant, separate the pellet, add 10 mL of fixative, fix at room temperature for 1 hour, and then centrifuge again under the same conditions. Repeat the previous step, then discard the supernatant again, break up the cell pellet and add an appropriate amount of fixative. Drop the cell suspension vertically onto a pre-cooled glass slide, stain it with crystal violet, acquire images using a Leica camera and analyze them using ImageJ.

[0123] The results are as follows Figure 6 As shown in Figure A, the DHFR gene copy number was significantly reduced after stable interference with ALKBH5. Further investigation was conducted in HT29-4 drug-resistant cells containing DMs to determine whether interference with ALKBH5 affected the number of DMs. Figure 6 B). Subsequently, we randomly selected 100 karyotypes from the three clone karyotypes and statistically analyzed that the number of DMs after interfering with ALKBH5 decreased significantly ( Figure 6 C).

[0124] 7. Stable interference with MTX in HT29-4 resistant cells enhances the sensitivity of ALKBH5 cells to MTX.

[0125] DHFR gene amplification is a major cause of cellular resistance to MTX. Stable interference with ALKBH5 in DM-containing HT29-4 resistant cells leads to a decrease in DHFR copy number and protein expression levels. To clarify whether ALKBH5 knockdown alters cellular sensitivity to MTX, we used the CCK8 assay to compare the IC50 of MTX before and after ALKBH5 knockdown. 50The median lethal concentration (LD50) was calculated, and the results are shown in Table 1. In MTX-resistant cells containing DMs, knocking down ALKBH5 increased cell sensitivity to MTX by 2.58 and 2.63 times. This indicates that ALKBH5 holds promise as a novel target for reversing MTX-resistant tumor cells.

[0126] The CCK8 assay method for assessing the 50% lethal concentration of cells is as follows:

[0127] Cells in logarithmic growth phase were digested with trypsin, resuspended in culture medium, and counted. Cells were seeded at a density of 5000 cells per well in 96-well plates. Continuous MTX-containing medium was prepared using a serial dilution method, and medium at the corresponding drug concentration was added to each well of the 96-well plate, with 6 auxiliary wells for each drug concentration. After 72 hours of continuous cell culture, the medium was aspirated from the 96-well plates, and 100 μl of medium and 20 μl of MTX mixture were added to each well. The plates were incubated at 37°C for 2–4 hours, and the absorbance at OD492 nm was measured using a microplate reader. Based on the absorbance values ​​of the control wells (without MTX) and the drug-treated wells (with MTX), the cell growth inhibition rate of different MTX concentrations was calculated. Finally, the median lethal concentration (IC50) of MTX was calculated based on the inhibition rate. 50 The results are shown in Table 1.

[0128] Table 1. Statistics on the increase in MTX sensitivity in HT29-4 resistant cells due to ALKBH5 interference

[0129]

Claims

1. The application of an ALKBH5 protein inhibitor in the preparation of a drug that reverses MTX-resistant tumor cell resistance and reduces malignant growth of tumor cells, wherein the ALKBH5 protein inhibitor is an interfering vector containing ALKBH5 shRNA, and the sequence of the ALKBH5 shRNA is shown in SEQ ID NO. 1 or 2; the tumor cells are human colon cancer HT29 MTX-resistant cells containing two microchromosomes and with highly amplified DHFR gene.

2. The application as described in claim 1, characterized in that, The interference vector containing ALKBH5 shRNA is a lentiviral vector containing ALKBH5 shRNA.

3. The application as described in claim 1, characterized in that, The tumor cells described above have a tolerance level of 10 to MTX. -4 mol / L.

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