Application of IGF2BP3 as target spot in preparation of colorectal cancer drugs

Abstract

The invention relates to the technical field of cancer drugs, in particular to application of IGF2BP3 serving as a target spot in preparation of colorectal cancer drugs. The invention provides application of IGF2BP3 serving as a target spot in preparation of medicines for treating colorectal cancer and reagents for diagnosing and / or evaluating colorectal cancer. The invention creatively finds that IGF2BP3 has an important effect in treatment of colorectal cancer as a target spot, and provides a new drug resistance mechanism for a treatment strategy of colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer drugs, and in particular to use of IGF2BP3 as a target in the preparation of colorectal cancer drugs. Background Art

[0002] Colorectal cancer (CRC) is a malignant tumor that occurs in the colon or rectum. Current treatment options for colorectal cancer include surgical resection of the tumor, chemotherapy, radiotherapy, targeted therapy, or immunotherapy. Among them, surgery combined with chemotherapy drugs is a more common treatment method. Fluorouracil (5-Fu) is a basic drug in colorectal cancer chemotherapy. The mechanism of action of this drug is to inhibit the biological activity of thymine synthase (TS), interfere with the synthesis of DNA precursor thymine, hinder DNA synthesis in CRC cells, and inhibit tumor growth and metastasis. However, a large number of clinical trials have found that the failure of chemotherapy based on 5-Fu is mostly attributed to the patient's resistance to 5-Fu during long-term medication. Especially for recurrent patients, their resistance to 5-Fu is stronger. Therefore, overcoming 5-Fu resistance has become a major challenge to improve the efficacy of CRC treatment. Summary of the invention

[0003] Therefore, the technical problem to be solved by the present invention is to overcome the defect of strong drug resistance in the use of drugs in the treatment of colorectal cancer in the prior art, thereby providing a use of IGF2BP3 as a target in the preparation of colorectal cancer drugs.

[0004] Another technical problem to be solved by the present invention is to overcome the defect of the prior art that it is unable to accurately diagnose colorectal cancer or evaluate the prognosis of colorectal cancer patients, thereby providing a use of IGF2BP3 as a target in an agent for diagnosing and / or evaluating colorectal cancer.

[0005] To this end, the present invention provides a use of IGF2BP3 as a target in the preparation of drugs for treating colorectal cancer, and reagents for diagnosing and / or evaluating colorectal cancer.

[0006] In some of these embodiments, the effect of IGF2BP3 as a target includes knocking down IGF2BP3 to reduce or reverse the drug resistance of colorectal cancer cells.

[0007] In some of these embodiments, knockdown of IGF2BP3 promotes apoptosis in colorectal cancer cells.

[0008] In some of these embodiments, knocking down IGF2BP3 increases the sensitivity of colorectal cancer cells to drugs.

[0009] Preferably, the increase in the m6A modification level of IGF2BP3 causes IGF2BP3 knockdown.

[0010] Preferably, overexpression of METTL16 causes an increase in the m6A modification level of IGF2BP3.

[0011] Preferably, reduction in the level of METTL16 S-glutathione modification results in IGF2BP3 knockdown.

[0012] Knocking down IGF2BP3 includes inhibiting the expression of IGF2BP3 gene and / or downregulating the level of IGF2BP3 protein.

[0013] On the other hand, the present invention also provides a product for inhibiting the expression of IGF2BP3 gene or down-regulating the expression of IGF2BP3 protein for use in preparing a drug for adjuvant therapy or treatment of colorectal cancer.

[0014] In some embodiments, the drug for adjuvant therapy or treatment of colorectal cancer has any of the following applications:

[0015] (I) Improving the sensitivity of drug-resistant colorectal cancer cells to fluorouracil;

[0016] (ii) reduce or reverse the resistance of colorectal cancer cells to fluorouracil;

[0017] (iii) promoting apoptosis of drug-resistant colorectal cancer cells;

[0018] The product includes: at least one of: IGF2BP3 knockdown virus particles, IGF2BP3 inhibitors of small molecule compounds, m6A modification inducers, METTL16 inducers or METTL16 S-glutathione modification inhibitors.

[0019] At the same time, the present invention also provides a pharmaceutical composition for treating colorectal cancer, which comprises fluorouracil and an inhibitor, wherein the inhibitor comprises a substance that inhibits the expression of the IGF2BP3 gene or downregulates the expression of the IGF2BP3 protein.

[0020] In some embodiments, the inhibitor is an IGF2BP3 knockdown virus particle, an IGF2BP3 inhibitor of a small molecule compound, 6 At least one of an A modification inducer, a METTL16 inducer or a METTL16 S-glutathione modification inhibitor.

[0021] In some embodiments, the m 6 A modification inducer is a substance that induces IGF2BP3 mRNA to undergo methyl group modification on the 6th nitrogen atom of the adenosine residue.

[0022] In some embodiments, in the drug for treating colorectal cancer, the molar ratio of fluorouracil to the inhibitor is (18-21):(6-8),

[0023] Preferably, the inhibitor is isoliquiritigenin.

[0024] The present invention also provides a reagent for diagnosing and / or evaluating colorectal cancer, wherein the primer sequences in the reagent are shown in SEQ ID NO.11-SEQ ID NO.12.

[0025] The technical solution of the present invention has the following advantages:

[0026] The present invention provides a use of IGF2BP3 as a target in the preparation of a drug for treating colorectal cancer, or a reagent for diagnosing and / or evaluating colorectal cancer. The present invention creatively discovers that IGF2BP3 as a target plays an important role in the treatment of colorectal cancer, and proposes a new drug resistance mechanism for the treatment strategy of colorectal cancer.

[0027] In the use provided by the present invention, the effects of IGF2BP3 as a target include: knocking down IGF2BP3 to reduce or reverse the drug resistance of colorectal cancer cells; knocking down IGF2BP3 to promote the apoptosis of colorectal cancer cells; knocking down IGF2BP3 to increase the sensitivity of colorectal cancer cells to drugs.

[0028] At the same time, in the use provided by the present invention, the overexpression of METTL16 causes the m 6 A modification level is increased, and then the m 6 The increase of S-glutathione modification level of METTL16 leads to the knockdown of IGF2BP3. The present invention found in the research process that the decrease of S-glutathione modification level of METTL16 leads to the increase of METTL16 protein expression, thereby improving the function of METTL16 in m 6 A modified the writing function, thereby increasing the m 6 A modification level, weakening the intracellular transport and shearing efficiency of IGF2BP3 mRNA, causing a decrease in the level of IGF2BP3 protein, thereby achieving the purpose of significantly enhancing the sensitivity of colorectal cancer cells to drugs, promoting the apoptosis of colorectal cancer cells, and reducing or reversing the drug resistance of colorectal cancer cells.

[0029] The present invention provides a drug for treating colorectal cancer, including fluorouracil and an inhibitor, wherein the inhibitor includes a substance that inhibits the expression of the IGF2BP3 gene or downregulates the expression of the IGF2BP3 protein. The present invention provides a compound of the inhibitor and fluorouracil, which can overcome the drug resistance of fluorouracil during use, inhibit the proliferation of tumor cells, increase the sensitivity of colorectal cancer cells to drugs, promote the apoptosis of colorectal cancer cells, and significantly improve the treatment effect of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The results of the cytotoxicity test of 5-Fu in non-drug-resistant colon cancer cells and drug-resistant colon cancer cells in Example 1 of the present invention are as follows;

[0032] Figure 2 is m in the non-drug-resistant colon cancer cells and the drug-resistant colon cancer cells in Example 2 of the present invention. 6 A methylation modification level, Figure 2 a in the figure indicates the total protein of non-resistant colorectal cancer cells HCT8 and resistant colorectal cancer cells HCT8 / 5Fu m 6 A modification degree, Figure 2 b in the figure indicates the total RNA amount of non-resistant colon cancer cell HCT8 and resistant colon cancer cell HCT8 / 5Fu. Figure 2 The c in the figure indicates the total protein of non-resistant colorectal cancer cells HCT15 and resistant colorectal cancer cells HCT15 / 5Fu. 6 A modification degree, Figure 2 The d in the figure indicates the total RNA amount of non-resistant colon cancer cell HCT15 and resistant colon cancer cell HCT15 / 5Fu;

[0033] Figure 3 is the mRNA of the total mRNA in the non-drug-resistant colon cancer cells and the drug-resistant colon cancer cells in Example 2 of the present invention. 6 A / A ratio comparison results;

[0034] Figure 4 is the RNA sequencing test result of drug-resistant colon cancer cells and non-drug-resistant colon cancer cells in Example 3 of the present invention, wherein the horizontal axis is the Log of drug-resistant colon cancer cells HCT8 / 5Fu and non-drug-resistant colon cancer cells HCT8 2 (Difference multiple), the vertical axis is the Log of resistant colon cancer cells HCT15 / 5Fu and non-resistant colon cancer cells HCT15 2 (Difference multiple);

[0035] Figure 5 is m in Example 3 of the present invention 6 A sequencing detected m 6 A peak density distribution in the mRNA transcriptome;

[0036] Figure 6 is m in the non-drug-resistant colon cancer cells and the drug-resistant colon cancer cells in Example 3 of the present invention. 6 A. Expression of related enzymes;

[0037] Figure 7 is the expression of S-glutathionylated METTL16 in non-drug-resistant colorectal cancer cells and drug-resistant colorectal cancer cells in Example 3 of the present invention;

[0038] Figure 8 is a Venn diagram of the gene intersection of each sample in Example 3 of the present invention;

[0039] Fig. 9 is the m of IGF2BP3 in the non-drug-resistant colon cancer cells and the drug-resistant colon cancer cells in Example 4 of the present invention. 6 A distribution IGV visualization;

[0040] Fig.10 is the mRNA of IGF2BP3 mRNA of drug-resistant colon cancer cell HCT8 / 5Fu and non-drug-resistant colon cancer HCT8 in Example 4 of the present invention. 6 A modification relative abundance;

[0041] Fig.11 is the mRNA of IGF2BP3 mRNA of drug-resistant colon cancer cell HCT15 / 5Fu and non-drug-resistant colon cancer HCT15 in Example 4 of the present invention. 6 A modification relative abundance;

[0042] Fig.12 is the intervention m in the non-drug resistant intestinal cancer cells in Example 4 of the present invention 6 A. The mRNA level of IGF2BP3 after the inhibitor 3-deazaadenosine;

[0043] Fig.13 is the intervention m in the non-drug resistant intestinal cancer cells in Example 4 of the present invention 6 A protein expression level of IGF2BP3 after the inhibitor 3-deazaadenosine;

[0044] Fig.14 is the mRNA level of IGF2BP3 in the non-drug-resistant colorectal cancer cells and the drug-resistant colorectal cancer cells in Example 5 of the present invention;

[0045] Fig.15 is the protein expression level of IGF2BP3 in the non-drug-resistant colorectal cancer cells and the drug-resistant colorectal cancer cells in Example 5 of the present invention;

[0046] Fig.16 is the IGF2BP3 mRNA level after overexpression of the METTL16 plasmid in the drug-resistant intestinal cancer cells in Example 5 of the present invention;

[0047] Fig.17 is the expression level of IGF2BP3 protein after overexpression of METTL16 plasmid in drug-resistant intestinal cancer cells in Example 5 of the present invention;

[0048] Fig.18 is a graph showing the proportion of IGF2BP3 mRNA in the cytoplasm and nucleus of non-drug-resistant colorectal cancer cells and drug-resistant colorectal cancer cells in Example 5 of the present invention;

[0049] Fig.19 is the relative expression of precursor RNA and mature RNA of IGF2BP3 in the cytoplasm and nucleus of non-drug-resistant colorectal cancer cells and drug-resistant colorectal cancer cells in Example 5 of the present invention;

[0050] Fig. 20 This is a diagram showing the RNA level verification result of knocking down the IGF2BP3 gene in Example 6 of the present invention;

[0051] Fig.21 This is a graph showing the protein level verification result of knocking down the IGF2BP3 gene in Example 6 of the present invention;

[0052] Fig. 22 This is a graph showing the cell proliferation results of the control group of drug-resistant HCT8 / 5Fu cells and the IGF2BP3 knockdown group under treatment with different concentrations of 5-Fu in Example 7 of the present invention;

[0053] Fig.23 This is a graph showing the cell proliferation results of the control group of drug-resistant HCT15 / 5Fu cells and the IGF2BP3 knockdown group under treatment with different concentrations of 5-Fu in Example 7 of the present invention;

[0054] Fig.24 is a histogram of the relative amount of clone formation of the control group of drug-resistant HCT8 / 5Fu cells and the IGF2BP3 knockdown group under different concentrations of 5-Fu treatment in Example 7 of the present invention;

[0055] Fig.25 is a histogram of the relative amount of clone formation of the control group of drug-resistant cells HCT15 / 5Fu and the group with IGF2BP3 knockdown under different concentrations of 5-Fu treatment in Example 7 of the present invention;

[0056] Fig.26 This is a graph showing the results of cell apoptosis in the control group of drug-resistant HCT8 / 5Fu cells and the IGF2BP3 knockdown group under treatment with different concentrations of 5-Fu in Example 7 of the present invention;

[0057] Fig. 27 is a histogram of cell apoptosis rates of a control group of drug-resistant HCT8 / 5Fu cells and a group with IGF2BP3 knockdown under treatment with different concentrations of 5-Fu in Example 7 of the present invention;

[0058] Fig.28 This is a graph showing the results of cell apoptosis in the control group of drug-resistant HCT15 / 5Fu cells and the IGF2BP3 knockdown group under treatment with different concentrations of 5-Fu in Example 7 of the present invention;

[0059] Fig.29 is a histogram of cell apoptosis rates of the control group of drug-resistant HCT15 / 5Fu cells and the IGF2BP3 knockdown group under different concentrations of 5-Fu treatment in Example 7 of the present invention;

[0060] Fig.30 This is a tumor image of the control group of nude mice subcutaneously xenografted with drug-resistant HCT8 / 5Fu cells and the group with stable knockdown of IGF2BP3 intervened with 5-Fu or normal saline in Example 7 of the present invention;

[0061] Fig.31 It is a tumor volume curve of the control group of nude mice subcutaneously transplanted with drug-resistant cells HCT8 / 5Fu and the group with stable knockdown of IGF2BP3 intervened with 5-Fu or normal saline in Example 7 of the present invention, wherein, Fig.31 A represents the stable knockdown IGF2BP3 group of drug-resistant HCT8 / 5Fu cells. Fig.31 B in the figure indicates the control group;

[0062] Fig.32 This is a tumor image of the control group of nude mice subcutaneously xenografted with drug-resistant cells HCT15 / 5Fu and the group with stable knockdown of IGF2BP3 intervened with 5-Fu or normal saline in Example 7 of the present invention;

[0063] Fig.33 This is a tumor volume curve of the control group of nude mice subcutaneously transplanted with drug-resistant cells HCT15 / 5Fu and the group with stable knockdown of IGF2BP3 intervened with 5-Fu or saline in Example 7 of the present invention. In the figure, ctrl represents the saline treatment group, 5-Fu represents the 5-Fu treatment group, Fig.33 C represents the stable knockdown IGF2BP3 group of drug-resistant cells HCT15 / 5Fu. Fig.33 D in the figure indicates the control group;

[0064] Fig.34 is a forest plot of multi-factor regression analysis of intestinal cancer microarray in Example 8 of the present invention;

[0065] Fig.35 The results of IGF2BP3 immunohistochemistry of intestinal cancer microarray in Example 8 of the present invention are Figure 1 , where T represents tumor tissue and N represents adjacent tissue;

[0066] Fig.36 The results of IGF2BP3 immunohistochemistry of intestinal cancer microarray in Example 8 of the present invention are Figure 2 , where T represents tumor tissue and N represents adjacent tissue;

[0067] Fig.37 is a comparison of the H-score quantification results of paired cancer and adjacent paracancerous immunohistochemistry results in Example 8 of the present invention;

[0068] Fig.38 It is a survival analysis result and cumulative risk result analysis diagram of the difference between high and low expression of IGF2BP3 in Example 8 of the present invention;

[0069] Fig.39 This is a survival analysis result analysis diagram of the difference between high and low expression of IFG2BP3 in the intestinal cancer microarray group treated with fluorouracil in Example 8 of the present invention;

[0070] Fig.40 This is a comparison chart of clinical remission rates of high and low expression of IFG2BP3 in the intestinal cancer microarray of Example 8 of the present invention for the population receiving fluorouracil treatment;

[0071] Fig.41 This is a comparison chart of IGF2BP3 mRNA levels between the 5-Fu resistant group and the non-resistant group in the GSE40967 cohort in the GSE database in Example 8 of the present invention;

[0072] Fig.42 is the Western blotting result of different drug monomers intervening in colorectal cancer resistant colorectal cancer cells in Example 9 of the present invention, wherein TET is tetrahydropalmatine, QUE is quercetin, ISO is isoliquiritigenin, AF is paeoniflorin, HSP is hesperidin, LIQ is liquiritigenin, BBR is berberine, and CUR is curcumin;

[0073] Fig.43 This is the Western blotting result of different concentrations of isoliquiritigenin intervening in drug-resistant intestinal cancer cells in Example 9 of the present invention;

[0074] Fig.44 is a molecular docking visualization diagram of the binding of isoliquiritigenin to the IGF2BP3 protein in Example 9 of the present invention, in which Acidic represents acidity, Basic represents alkalinity, and lonizability represents ionizability;

[0075] Fig.45 This is a schematic diagram of the prediction of the combined medication index CI in Example 9 of the present invention;

[0076] Fig.46 is the cell proliferation capacity of the control group, the isoliquiritigenin group, the 5-Fu group, and the combined medication group in Example 9 of the present invention;

[0077] Fig.47This is a picture of the tumor body after the intervention of the nude mouse xenograft tumor model by the control group, the isoliquiritigenin group, the 5-Fu group, and the combined drug group in Example 9 of the present invention;

[0078] Fig.48 This is a graph showing the changes in tumor volume after intervention in the nude mouse xenograft tumor model by the control group, the isoliquiritigenin group, the 5-Fu group, and the combined drug group in Example 9 of the present invention;

[0079] Fig.49 It is the mass spectrum of the S-glutathionylated peptide fragment containing Cys548 in Example 11 of the present invention. DETAILED DESCRIPTION

[0080] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0081] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0082] The experimental methods in the following examples of the present invention without specifying specific conditions are generally conventional methods in the art and are carried out according to the conditions recommended by the reagent manufacturers.

[0083] The data in the following embodiments and experimental examples of the present invention are expressed as the mean ± standard deviation of three independent experiments. In each figure, P < 0.05, *; P < 0.01, **; P < 0.001, ***. The concentration units in the embodiments are written in the usual way in the field: M, mM or μM represent mol / L, mmol / L or μmol / L respectively.

[0084] In the following embodiments of the present invention, the experimental supplies involved mainly include the following categories.

[0085] 1. Material Description

[0086] Fluorouracil (5-Fu) was purchased from Sigma; fetal bovine serum (FBS), DMEM culture medium, and trypsin were purchased from Gibco; antibodies (primary antibody, secondary antibody) were purchased from Proteintech; DMEM complete culture medium is DMEM culture medium containing 10% FBS, 100U / mL penicillin and 100μg / mL streptomycin; control knockdown lentiviral particles and IGF2BP3 knockdown lentiviral particles were purchased from Santa Cruz; TRIzol reagent was purchased from Invitrogen, and m6A RNA methylation quantitative detection kit (colorimetric method) was purchased from Epigentek; RNA rapid purification kit, all-in-one RT kit, and 2x Super SYBRGreen qPCR Master Mix kit were purchased from Shanghai Yishan Biotechnology Co., Ltd.; RNA subcellular fractionation kit was purchased from Active Motif.

[0087] Human colorectal cancer cell lines HCT8 and HCT15 were purchased from ATCC cell bank; control overexpression particles and F1ag-METTL16 plasmids were constructed by Vigene Bioscience.

[0088] BALB / c nude mice, 6 weeks old, female, were purchased from Shanghai Slake Laboratory Animal Co., Ltd., with the experimental animal license number of SCXK (Shanghai) 2017-0005. All experimental animals were kept in SPF animal rooms.

[0089] In the following embodiments of the present invention, the construction methods of drug-resistant cell lines HCT8 / 5Fu and HCT15 / 5Fu are as follows:

[0090] HCT8 cells were subcultured into 24-well plates and 2 × 10 5 cells, placed in 5% CO 2 Incubator, culture in DMEM complete culture medium for 24 hours at 37°C; then, add 20 μM 5-Fu to the DMEM complete culture medium and continue to culture for 72 hours; then, change to fresh DMEM complete culture medium and continue to culture for 24 hours, then add 30 μM 5-Fu to the DMEM complete culture medium and continue to culture for 72 hours; continue to adjust the 5-Fu concentration in the DMEM complete culture medium (increase in the following order 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, final concentration 100 μM), repeat the above culture process to obtain 5-Fu-resistant HCT8 / 5Fu cell line.

[0091] HCT15 cells were subcultured into 24-well plates and 2 × 10 5cells were placed in a 5% CO2 incubator at 37°C and cultured in DMEM complete culture medium for 24 h; 2 μM 5-Fu was then added to the DMEM complete culture medium and the culture was continued for 72 h; then, fresh DMEM complete culture medium was used for the culture to continue for 24 h, and 3 μM 5-Fu was then added to the DMEM complete culture medium and the culture was continued for 72 h; the 5-Fu concentration in the DMEM complete culture medium was adjusted (increasing in the following order: 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and the final concentration was 10 μM), and the above culture process was repeated to obtain a 5-Fu-resistant HCT15 / 5Fu cell line.

[0092] The HCT8 / 5Fu and HCT15 / 5Fu resistant cell lines were routinely cultured with DMEM complete culture medium and maintained with 100 μM and 10 μM 5-Fu, respectively, to ensure the stability of their drug resistance.

[0093] 2. Method Description

[0094] 2.1. Cell survival and growth assay (MTT)

[0095] Cells in the logarithmic growth phase were digested with 0.25% trypsin and pipetted to obtain a cell suspension. The cells were inoculated into a 96-well cell culture plate with a final volume of 200 μL per well (5,000 cells per well). The 96-well plate was moved to a 37°C, 5% CO 2 After 24 hours of culture in a saturated humidity incubator, the DMEM complete culture medium was discarded and replaced with DMEM complete culture medium containing different concentrations of drugs for culture. Four replicate wells were set for each concentration and placed at 37°C, 5% CO 2 The cells were incubated in a saturated humidity incubator for 48 h. MTT solution was added to each well and incubated at 37 °C for 4 h. The absorbance was detected, the cell survival rate was calculated, and a heat map was drawn.

[0096] 2.2 RNA dot blot method

[0097] The total RNA of non-resistant colorectal cancer HCT8, HCT15 cells and resistant colorectal cancer HCT8 / 5Fu, HCT15 / 5Fu cells was extracted using RNA rapid purification kit, and the concentration and purity of RNA were detected by Nanodrop 2000. Then RNA was denatured at 65°C for 10 min, immediately cooled on ice, and dropped into nitrocellulose membrane. After UV cross-linking, the membrane was washed twice with TBST, blocked with 5% fetal bovine serum for 2 h, and m 6 A. Incubate with primary antibody at 4°C overnight; incubate with secondary antibody at room temperature for 1 hour before detection, and methylene blue staining shows the overall RNA loading amount.

[0098] 2.3. Colorimetric quantitative detection of m 6A Method

[0099] 200 ng RNA isolated from non-resistant colon cancer HCT8, HCT15 cells and resistant colon cancer HCT8 / 5Fu, HCT15 / 5Fu cells were added to the detection wells, and then m 6 A RNA methylation quantification kit detects m 6 The absolute quantitative calculation method is as follows: draw a standard curve and use linear regression to determine the slope (in OD / ng). Use formula (Ⅰ) to calculate the m 6 The amount and percentage of A.

[0100]

[0101] In formula (I), S is the loading amount of sample RNA (ng), and NC represents the OD value of the negative control (the negative control is an RNA sample without m6A modification, which is the sample provided by the kit).

[0102] 2.4 RNA Sequencing Methods

[0103] Total RNA was extracted using TRIzol according to the instructions to separate and purify the total RNA of non-resistant colorectal cancer HCT8 and HCT15 cells and resistant colorectal cancer HCT8 / 5Fu and HCT15 / 5Fu cells. After the concentration was determined, the RNA was denatured.

[0104] mRNA was enriched using oligo(dT) magnetic beads (Dynabeads Oligo(dT), Thermo Fisher) and then fragmented using a magnesium ion fragmentation kit ( Magnesium RNA Fragmentation Module) for fragmentation at 86°C for 7 minutes; set up a first-chain reaction system to synthesize a first-chain cDNA; set up a second-chain reaction system to synthesize a second-chain cDNA.

[0105] End repair, adding "A" and linker connection, set the reaction program, add A base at the 3' end and repair the double-stranded cDNA end, so that the linker and cDNA can be connected; then PCR is performed to form a sequencing library. The program is: pre-denaturation at 95℃ for 3 minutes, denaturation at 98℃ for a total of 8 cycles of 15 seconds each, annealing to 60℃ for 15 seconds, extension at 72℃ for 30 seconds, and finally extension at 72℃ for 5 minutes.

[0106] Finally, Illumina Novaseq TMDouble-end sequencing was performed using LC Bio Technology Co., Ltd. Hangzhou, China (LC Bio Technology Co., Ltd. Hangzhou, China) according to standard procedures, and the sequencing mode was PE150.

[0107] 2.5, m 6 Sequencing method of A

[0108] According to the operating instructions, TRIzol was used to separate and purify the total RNA of non-resistant colorectal cancer HCT8, HCT15 cells and resistant colorectal cancer HCT8 / 5Fu, HCT15 / 5Fu cells. NanoDrop 2000 was used to measure RNA concentration and purity for quality control, and Bioanalyzer 2100 was used for integrity detection; concentration>50ng / μL, RIN value>7.0, OD260 / 280>1.8, totalRNA>50μg met the requirements for downstream experiments.

[0109] After mRNA was enriched using oligo(dT) magnetic beads (Dynabeads Oligo(dT, Thermo Fisher), the mRNA was fragmented using a magnesium ion fragmentation kit ( The fragmented RNA was fragmented at 86°C for 7 minutes. The fragmented RNA was mixed with immunomagnetic beads Dynabeads Antibody Coupling Kit (ThermoFisher) and m 6 Antibody A (Synaptic Systems) was premixed in an immunoprecipitation buffer (IP buffer, 50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630) and immunoprecipitated (IP). The IP product was reverse transcriptase (Invitrogen SuperScript TMII Reverse Transcriptase, USA) to synthesize cDNA, and then use E. coli DNA polymerase I (NEB) and RNase H (NEB) for double-strand synthesis, convert these DNA and RNA composite double strands into DNA double strands, and incorporate dUTP (Thermo Fisher) into the double strands. The ends of the double-stranded DNA were filled, and then an A base was added to each end so that it could be connected to the adapter with a T base at the end. The fragment size was screened and purified using magnetic beads (insert size: 180-220bp). The second strand was digested with UDG enzyme (NEB, catalog number m0280, MA, US), and then PCR was performed to form a sequencing library. The program was pre-denaturation at 95°C for 3 minutes, denaturation at 98°C for a total of 8 cycles of 15 seconds each, annealing to 60°C for 15 seconds, extension at 72°C for 30 seconds, and finally extension at 72°C for 5 minutes. Finally, the illumina Novaseq TM 6000, double-end sequencing was performed according to standard procedures, and the sequencing mode was PE150.

[0110] 2.6. Western Blotting Method

[0111] After adding cell lysis buffer RIPA, cells of each component treated with drugs were lysed in an ice bath for 30 minutes, centrifuged at 16000g for 15 minutes at 4°C, and the supernatant was collected. After BCA determination of protein content, equal amounts of protein were used for different samples to separate proteins by SDS-PAGE. After electrophoresis, the proteins were transferred to PVDF membranes, blocked with 5% skim milk powder, and the primary antibody (m 6 A-related protein, IGF2BP3 protein, GAPDH protein) were blocked overnight, and then blocked with horseradish peroxidase-labeled secondary antibody blocking solution for 2 h, and the color was developed with diaminobenzidine (DAB) solution, and then scanned for analysis.

[0112] 2.7. Glutathione (GSH) modification detection

[0113] Biotin-coupled GSH (BioGEE) was added to 10 cm dishes seeded with non-resistant colorectal cancer HCT8, HCT15 cells and resistant colorectal cancer HCT8 / 5Fu, HCT15 / 5Fu cells the day before, and the cells were incubated at 37°C for 2 hours. The cells were lysed with IP lysis buffer, and co-immunoprecipitation (Co-IP) was performed using magnetic beads labeled with streptavidin. The enriched protein samples were subjected to protein immunoblotting in non-reduced (no reducing agent was added to the loading buffer) SDS-PAGE.

[0114] 2.8. Methylated RNA Immunoprecipitation (MeRIP)

[0115] The total RNA of non-resistant colorectal cancer HCT8, HCT15 cells and resistant colorectal cancer HCT8 / 5Fu, HCT15 / 5Fu cells was separated and purified respectively, and the RNA concentration was measured and adjusted to 1μg / μl. 10×Fragmentation buffer was added to the RNA, and EDTA was immediately added after incubation at 95°C for 5 minutes. The mixture was vortexed and placed on ice until all RNA was fragmented. 1 / 10 of the volume of 3 mol / L sodium acetate, 2.5 times the volume of 100% ethanol, and glycogen (final concentration 100μg / ml) were added to the fragmented RNA solution, mixed, and incubated at -80°C overnight. 10% of the RNA was taken in a new EP tube, marked as input, and stored at -80C for subsequent use as a control. The remaining RNA was added to Protein A / G magnetic beads and mRNA antibodies (m 6 A or negative control) premix, incubate at 4℃ for 2h; elute the enriched RNA; purify the eluted RNA using RNeasy mini kit to obtain the RNA after IP. Reverse transcribe the input RNA and IP RNA to obtain cDNA. 6 Five pairs of specific primers for MeRIP-qPCR were designed for the methylation modification region A. The primer sequences are shown in Table 1. The cDNA of the reverse transcription product was amplified by RT-PCR, and the relative abundance was calculated as shown in formula (II).

[0116]

[0117] ΔCT nomalized RIP / negetive control =ΔCT normalized RIP -ΔCT negative control

[0118]

[0119] In formula (II), ΔCT normalized RIP is the normalized CT change, Average CT RIP Average CT is the average CT value obtained from three technical replicates of the m6A-IP product. Input is the average CT value obtained from three technical replicates of the Input RNA product, the Input dilution factor is the ratio of the input RNA to the m6A-IP RNA, and ΔCT negative control is the normalized CT change of the negative control sample, and Fold Enrichment is the methylation enrichment fold.

[0120] Table 1 IGF2BP3 specificity m 6 A modified primer sequence

[0121]

[0122]

[0123] 2.9. Real-time quantitative PCR determination of IGF2BP3 mRNA expression level

[0124] The total RNA of non-resistant colorectal cancer HCT8, HCT15 cells and resistant colorectal cancer HCT8 / 5Fu, HCT15 / 5Fu cells was extracted using RNA rapid purification kit. cDNA was synthesized by reverse transcription using All-In-One RT kit, and the reverse transcription conditions were 42°C for 15 minutes. Real-time quantitative PCR was performed using 2x Super SYBR Green qPCR Master Mix kit and ABI7300PCR instrument, and the PCR program is shown in Table 2. The PCR primers for IGF2BP3 are shown in Table 3.

[0125] Table 2 PCR program

[0126]

[0127] Table 3 PCR primer sequences of IGF2BP3

[0128] name sequence Sequence number Forward Primer F 5'-TCCTGGTGAAGACTGGCTACG-3' SEQ ID NO.11 Reverse primer R 5'-GACCGAGTGCTCAACTTCTATGG-3' SEQ ID NO.12

[0129] The parental cells HCT8 and HCT15 were intervened with different concentrations of 0μM, 20μM, and 40μM of the methylation inhibitor 3-DAA, and total RNA was extracted using an RNA rapid purification kit. cDNA was reverse transcribed by an all-in-one RT kit, and the reverse transcription conditions were: 42°C, 15 minutes. Real-time quantitative PCR was performed using a 2x Super SYBR Green qPCR Master Mix kit and an ABI 7300PCR instrument. The PCR program is shown in the table. The PCR primers for IGF2BP3 are shown in Table 3.

[0130] 2.10. IGF2BP3 mRNA nuclear-cytoplasmic ratio and splicing efficiency

[0131] The RNA subcellular fractionation kit was used to extract the cytoplasmic and nuclear RNA of drug-resistant and non-resistant colorectal cancer cells, and the mRNA expression level of IGF2BP3 was determined by real-time quantitative PCR. IGF2BP3-specific precursor RNA (pre-IGF2BP3) primers and mature RNA (mat-IGF2BP3) primers were designed, and the real-time quantitative PCR was performed on the isolated and purified cell RNA of each group to analyze the IGF2BP3 mRNA shearing efficiency.

[0132] 2.11. METTL16 liposome transfection method

[0133] Cells in the logarithmic growth phase were taken and digested into a single cell suspension. 5 Cells / well were seeded in a six-well cell culture plate and incubated at 37°C and 5% CO 2 Culture in an incubator with saturated humidity for 24 hours to adhere to the wall. The next day, first prepare the transfection complex and the overexpression complex. To prepare the transfection complex, add 4μL Lipo3000 and 125μL Opti-MEM culture medium. To prepare the overexpression complex, add 2μg METTL16 plasmid or control plasmid and 4μL P3000 and 125μL Opti-MEM culture medium. After the preparation is completed, mix them separately and let them stand at room temperature for 5 minutes, then mix the two mixtures together and let them stand at room temperature for 10 minutes. Finally, add the mixed mixture to the corresponding culture wells, and replace it with DMEM complete culture medium after standing in the incubator for 6 hours.

[0134] 2.12. Construction of IGF2BP3 knockdown cells by lentiviral particle transfection

[0135] Cells in the logarithmic growth phase were taken and digested into a single cell suspension. 5 Cells / well were seeded in 12-well cell culture plates and incubated at 37°C and 5% CO 2 The cells were cultured in a saturated humidity incubator for 24 hours to adhere to the wall. The next day, the cells were cultured in DMEM complete culture medium containing 5 μg / mL Polybrene, and 5 μL of lentivirus (IGF2BP3 knockdown lentiviral particles, control knockdown lentiviral particles) was added to each well of cells. Fresh DMEM complete culture medium was replaced on the third day, and the cells were subcultured and the cells successfully transfected with lentivirus were selected with puromycin on the fourth day to construct: IGF2BP3 knockdown cells HCT8 / 5Fu-shIGF2BP3 and HCT15 / 5Fu-sh IGF2BP3, and the corresponding vector cells HCT8 / 5Fu-Scramble and HCT15 / 5Fu-Scramble.

[0136] 2.13 Plate cloning experiment

[0137] Non-drug-resistant colorectal cancer HCT8 and HCT15 cells in logarithmic growth phase and drug-resistant colorectal cancer HCT8 / 5Fu and HCT15 / 5Fu cells were inoculated in 6-well plates at a density of 3000 cells / well and placed in a 5% CO 2, 37℃ incubator for 24h, discard the supernatant, wash once with 1×PBS, and add DMEM medium containing different concentrations of 5-Fu to each well. Two weeks later, discard the supernatant, wash with 1×PBS, add 1mL pre-cooled methanol to fix for 15min, wash with ultrapure water, add 1mL of 0.1% crystal violet staining solution to stain for 15min, rinse the 6-well plate gently, dry it, and then take pictures and analyze it.

[0138] 2.14 Flow cytometry for apoptosis

[0139] Cells in the logarithmic growth phase were taken and digested into a single cell suspension. 5 Cells / well were seeded in 6-well cell culture plates and incubated at 37°C and 5% CO 2 After culturing in a saturated humidity incubator for 24 h, the cells were grouped and treated: DMEM complete culture medium containing different group drugs was added to each cell and incubated at 37°C and 5% CO 2 The cells were cultured in a saturated humidity incubator for 48 h, and then the cells were collected and flow cytometry was used to detect cell apoptosis using the Annexin V FITC / PI kit from BD.

[0140] 2.15 RTCA real-time cell analysis technology to detect cell proliferation

[0141] 50 μM DMEM complete medium was added to each well of the E-plate 16 plate, and a control group, a single drug group, and a combined drug group were set up. The RTCA system baseline was detected on the RTCA Station instrument. Then 100 μL of a 7.5×10 4 The logarithmic growth phase colorectal cancer resistant cells were plated on E-plate 16 plates and treated with different concentrations of drugs according to the group. They were kept at room temperature for 30 minutes and then incubated at 37°C and 5% CO. 2 The RTCA Station instrument in the incubator performs real-time dynamic scanning of cell proliferation, and the program is set to collect data once every 30 minutes. The cell proliferation is observed within 100 hours to obtain a complete cell proliferation curve.

[0142] 2.16 Immunohistochemistry

[0143] The colorectal cancer microarray chip was baked in an oven at 60°C for 90 min, and routinely dewaxed, hydrated, antigen repaired, blocked endogenous peroxidase, blocked, and incubated with primary antibody. After overnight at 4°C, it was rewarmed at 37°C for 45 min, incubated with secondary antibody (1:100), incubated at 37°C for 30 min, and streptavidin-POD (1:100) was added for 30 min at 37°C. Diaminobenzidine (DAB) staining was performed and the color development time was controlled under a microscope; the chip was washed with tap water; lightly counterstained with hematoxylin, washed with tap water; routinely dehydrated, transparent, and sealed with neutral gum, observed under a microscope, and photographed.

[0144] 2.17 Immunoprecipitation detection of S-glutathionylation on Flag-IGF2BP3 protein and its mass spectrometry analysis

[0145] The cells were collected and lysed at 4°C for 1 h using IP buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 (V / V), 2 mM EDTA) containing protease inhibitors. After centrifugation of the cell lysate at 16,000 × g for 10 min, the supernatant was collected and the protein concentration was determined. 800 μg of cell lysate and 2 μg of Flag antibody were incubated overnight at 4°C. The protein S-glutathionylation level was analyzed by immunoblotting using anti-glutathione antibody. Flag-IGF2BP3 protein was captured from the cell lysate using protein A / G agarose and digested with trypsin at 37°C overnight. The S-glutathionylation sites were analyzed by liquid chromatography-mass spectrometry, and the LC-MS / MS data were analyzed by Proteome Discoverer 1.4 software (Thermo Fisher).

[0146] Example 1

[0147] This embodiment provides a method for determining the in vitro cytotoxicity of 5-Fu on various cell lines. The specific steps and parameters refer to Method Design 2.1 for detecting cell survival and growth. MTT experiments are performed on non-resistant colorectal cancer HCT8, HCT15 cells and resistant colorectal cancer HCT8 / 5Fu, HCT15 / 5Fu cells, wherein the concentration gradient of 5-Fu in the DMEM complete culture medium containing 5-Fu is set to 0 μM, 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, and 1000 μM.

[0148] Results Figure 1 It can be seen that under different concentration treatments, the resistant cells showed better cell activity than the corresponding parental cells, proving that colorectal cancer cells are resistant to 5-Fu.

[0149] Example 2

[0150] This embodiment provides a method for providing an mRNA mRNA in intestinal cancer cells. 6 A. Method for detecting methylation modification level. For specific steps and parameters, refer to 2.2 RNA dot blot method and 2.3 colorimetric quantitative detection of m 6 A method.

[0151] Results Figure 2-Figure 3 , it can be seen that according to Figure 2 The results of mRNA dot blot showed that m 6 A modification level is lower than that of non-resistant colorectal cancer cells. Figure 3The results show that the colorimetric method was used to quantitatively determine the mRNA mRNA isolated and purified from non-resistant colorectal cancer HCT8, HCT15 cells and resistant colorectal cancer HCT8 / 5Fu, HCT15 / 5Fu cells. 6 The A / A contents were 0.24±0.0132%, 0.20±0.0086%, 0.18±0.0124% and 0.17±0.0027% respectively. 6 The A / A ratio can indicate the intracellular m 6 A modification level, it can be seen that this result is consistent with the dot blot result, both indicating that m 6 A modification level decreased, among which m 6 The A in A / A represents the number of adenine A in the RNA.

[0152] Example 3

[0153] This embodiment provides a method based on m 6 A sequencing results to find the key target gene mediating drug resistance, the specific steps and methods use 2.4 RNA sequencing method and 2.5m 6 A sequencing method was used to sequence non-resistant cell lines and resistant cell lines. The sequencing results are shown in Figure 4 ,according to Figure 4 It can be seen that there are 199 genes upregulated in both HCT8 / 5Fu and HCT15 / 5Fu resistant cells (log2 FC>1.4or log2 FC<-1.4, p<0.05).

[0154] according to Figure 5 It can be seen that m 6 A sequencing results showed that the mRNA mRNA of non-resistant colorectal cancer cells and resistant colorectal cancer cells 6 A methylation modification mainly revolves around the 3'UTR region, among which, 3'UTR modification accounts for 56.33% in HCT8, while 3'UTR modification accounts for 53.86% in HCT8 / 5Fu; 3'UTR modification accounts for 54.13% in HCT15, while 3'UTR modification accounts for 50.02% in HCT8 / 5Fu (wherein, the percentage of each region is determined by dividing the 5' untranslated region (5'UTR), coding region (CDS) and 3' untranslated region (3'UTR) into 100 segments, and then determining the m 6 A peak percentage).

[0155] m 6 A variety of proteins play an important role in the process of A modification, among which METTL3, METTL14, and METTL16 are written as 6 A modified methyltransferase; ALKBH5 and FTO are for erasing m 6A modified demethylase. Figure 6 Western blotting results showed that the protein expression of METTL16 decreased significantly in drug-resistant colorectal cancer cells, while other proteins showed no significant changes. The inventors have confirmed in previous studies that glutathione modification increases in drug-resistant cells. Therefore, BioGEE was used for immunoprecipitation to extract proteins with glutathione modification. The results of Western blotting are shown in Figure 2. Figure 7 As shown, compared with non-resistant colorectal cancer cells, the glutathione of METTL16 in resistant colorectal cancer cells increased, while the protein level of METTL16 decreased, suggesting that METTL16 may enhance the induction of protein level decrease through glutathione modification.

[0156] The decreased protein level of METTL16 in drug-resistant colorectal cancer cells must have caused m 6 A modification change was observed, and 7164 RNAs that could bind to Mettl16 were obtained from the online database RIPseq (http: / / rm2target.canceromics.org). Based on the above results, 199 genes with significantly upregulated mRNA in RNA-seq and 1532 genes with significantly upregulated mRNA in HCT8 / 5Fu were selected. 6 A-seq has significant m 6 A peak genes, 1563 in HCT15 / 5Fu m 6 A-seq has significant m 6 The genes in peak A and 7164 RNAs that bind to Mettl16 were analyzed by Venn diagram. Figure 8 As shown in the figure, the intersection revealed a gene, IGF2BP3. This proved that while the transcription level of IGF2BP3 increased in drug-resistant cells, there was also m 6 The A modification level increased and it could bind to METTL16, indicating that IGF2BP3 may play a key role in mediating the occurrence of colorectal cancer resistance.

[0157] Example 4

[0158] This embodiment provides an IGF2BP3 mRNA m 6 A modification level verification method, according to m 6 A The sequencing results were visualized and analyzed using IGV software for the IGF2BP3 gene. Fig. 9 As shown, it can be seen that the modification ratio of IGF2BP3 at the 3'UTR end in drug-resistant colorectal cancer cells with equal transcription levels is lower than that in parental cells.

[0159] In order to verify the sequencing conclusion, 5 mRNAs were designed based on the sequencing results of IGF2BP3 and the modification sites predicted by SRAMP (http: / / www.cuilab.cn / sramp).6 Primer A, referring to the methylated RNA immunoprecipitation method in 2.8, experimentally verified the modification differences of IGF2BP3 in parental cells and drug-resistant cells. The results are as follows Fig.10 and Fig.11 As shown, the difference in primer pair 3 was the most significant.

[0160] 3-Deazaadenosine (3-DAA) is an effective methylation inhibitor. A certain amount of 3-DAA was injected into non-resistant colorectal cancer cells to verify whether the target gene was affected by m 6 A regulation. The results are as follows Fig.12 and Fig.13 As shown in Figure 2, in non-resistant cells, 3-DAA at final concentrations of 0 μM, 20 μM, and 40 μM was used to intervene, and it was found that the transcription and translation levels of IGF2BP3 increased. This indicates that IGF2BP3 is regulated by m 6 A methylation modification regulation.

[0161] Example 5

[0162] This embodiment provides a 6 The specific steps and parameters refer to 2.9 real-time quantitative PCR to determine the mRNA expression level of IGF2BP3 and 2.6 protein immunoblotting method to verify the background transcription and translation levels of IGF2BP3 in non-resistant colorectal cancer cells and resistant colorectal cancer cells. The results are shown in Fig.14 and Fig.15 , it can be seen that compared with high m 6 A modified level of non-resistant colorectal cancer cells, low levels of m 6 The transcription level of IGF2BP3 was upregulated and the protein expression level was increased in drug-resistant colorectal cancer cells with A modification level.

[0163] Combined with the METTL16 liposome transfection method provided in 2.11 to verify m 6 Does the effect of A modification level on the transcription and translation of IGF2BP3 have any relationship with m 6 A methyl transfer protein METTL16 is downregulated in drug-resistant colorectal cancer cells. Fig.16 and Fig.17 It can be seen that the increased expression of METTL16 downregulated the mRNA level of IGF2BP3, and the protein level decreased to a certain extent. This shows that the overexpression of METTL16 in drug-resistant colorectal cancer cells causes m 6 Increased A modification can downregulate IGF2BP3 mRNA and protein levels.

[0164] The cytoplasmic and nuclear RNA obtained by the METTL16 liposome transfection method provided in 2.11 was subjected to real-time quantitative PCR experiments. The specific steps and parameters refer to 2.9. The results are shown in Fig.18 It can be seen that the proportion of intracellular IGF2BP3 in drug-resistant colorectal cancer cells is higher than that in non-resistant colorectal cancer cells. Then, the inventors designed Precursor mRNA primers containing IGF2BP3 introns and Mature-mRNA primers excluding introns, and detected and analyzed the proportion of Pre-IGF2BP3 (precursor RNA) and mat-IGF2BP3 (mature RNA) in RNA of non-resistant cells and drug-resistant cells by qPCR. The results are as follows Fig.19 As shown in the figure, the difference between precursor RNA and mature RNA in non-resistant and resistant cells is significantly lower than that in non-resistant and resistant cells. 6 A modification level decreased, and the transcription and translation levels of IGF2BP3 were upregulated. 6 A modification affects the splicing, maturation and transport process of IGF2BP3 mRNA.

[0165] Example 6

[0166] This example provides a verification of the mRNA level and protein level of IGF2BP3 knockdown cells. The mRNA level and protein level of IGF2BP3 knockdown cells and vector cells constructed in 2.12 are detected using the methods of 2.6 and 2.9. Fig. 20 and Fig.21 It can be seen that compared with the control group formed by vector cells HCT8 / 5Fu-Scramb1e and HCT15 / 5Fu-Scramble, the mRNA level and protein level of IGF2BP3 in the IGF2BP3 knockdown group, i.e., HCT8 / 5Fu-shIGF2BP3 and HCT15 / 5Fu-sh IGF2BP3 cells, were significantly reduced.

[0167] Example 7

[0168] This example provides a drug resistance phenotype experiment of IGF2BP3 knockdown cells, and the specific steps and parameters are as follows:

[0169] 7.1 Cell experiments

[0170] The IGF2BP3 knockdown cells and vector cells constructed in 2.12 were subjected to cloning plate experiments using the method in 2.13, where the concentrations of 5-Fu were set to 0 μM, 100 μM, and 200 μM, respectively. Figure 22-Figure 25It can be seen that under the treatment of different concentrations of 5-Fu, compared with the control group formed by vectors HCT8 / 5Fu-Scramble and HCT15 / 5Fu-Scramble, the IGF2BP3 knockdown groups formed by HCT8 / 5Fu-shIGF2BP3 and HCT15 / 5Fu-shIGF2BP3 showed lower clone formation ability and decreased proliferation ability, indicating that IGF2BP3 knockdown cells have lower resistance to 5-Fu.

[0171] Cell apoptosis experiments were performed on vector cells HCT8 / 5Fu-Scramble and HCT15 / 5Fu-Scramble, knockdown cells HCT8 / 5Fu-shIGF2BP3 and HCT15 / 5Fu-shIGF2BP3 with intervention of different concentration gradients of 5-Fu according to method 2.14. The concentration gradient of 5-Fu was set to 0, 25μM, 50μM, 100μM, 200μM, and 400μM. The cell apoptosis results of HCT8 / 5Fu-Scramble and HCT8 / 5Fu-shIGF2BP3 and their quantitative histograms are shown in Figure 2. Fig.26 and Fig. 27 The apoptosis results and quantification histograms of HCT15 / 5Fu-Scramble and HCT15 / 5Fu-shIGF2BP3 are shown in Fig.28 and Fig.29 The results showed that under treatment with different concentrations of 5-Fu, compared with the control group formed by vectors HCT8 / 5Fu-Scramble and HCT15 / 5Fu-Scramble, the IGF2BP3 knockdown group formed by HCT8 / 5Fu-shIGF2BP3 and HCT15 / 5Fu-shIGF2BP3 had higher apoptosis caused by 5-Fu, indicating that IGF2BP3 knockdown cells were more sensitive to 5-Fu.

[0172] 7.2 Animal experiments

[0173] Xenograft tumor models of vector cells HCT8 / 5Fu-Scramble and HCT15 / 5Fu-Scramble, and knockdown cells HCT8 / 5Fu-shIGF2BP3 and HCT15 / 5Fu-shIGF2BP3 were constructed in BALB / c nude mice to evaluate the therapeutic effect of 5-Fu on xenograft tumors. Specifically, the method for constructing vector cells and knockdown cells in nude mice is as follows:

[0174] The mice were randomly divided into 8 groups, with 6 mice in each group.

[0175] The vector cells HCT8 / 5Fu-Scramble and HCT15 / 5Fu-Scramble and the knockdown cells HCT8 / 5Fu-shIGF2BP3 and HCT15 / 5Fu-shIGF2BP3 were injected into nude mice (5×10 6 The xenograft tumor models constructed by vector cells HCT8 / 5Fu-Scramble and HCT15 / 5Fu-Scramble were control group 1 and control group 2, and the models constructed by knockdown cells HCT8 / 5Fu-shIGF2BP3 and HCT15 / 5Fu-shIGF2BP3 were IGF2BP3 knockdown group 1 and IGF2BP3 knockdown group 2. When the tumor grew to 100 mm 3 At about 1:10 pm, each group was given the following medication: the saline group was given 0.95% saline solution; the 5-Fu group was given 25 mg / kg (5-Fu weight: mouse body weight), and the frequency of medication for each group of mice was once every two days. Specific grouping and medication information are shown in Table 4.

[0176] After administration, the tumor volume and weight of mice in each group were recorded every 3 days (the size of subcutaneous transplanted tumors in living mice was measured with a vernier caliper), and the mice were killed on the 20th day of intervention, and the tumor was taken. The calculation formula of tumor volume (V) was: V = 1 / 2 × a × b 2 , a is the long diameter of the subcutaneous tumor, b is the short diameter of the subcutaneous tumor.

[0177] Table 4 Experimental groups and medication information

[0178]

[0179] Results Figure 30-Figure 33 It can be seen that compared with the saline group, after 5-Fu treatment, there was no significant difference in the tumor volume of the control group 1 and control group 2 formed by the vector cells HCT8 / 5Fu-Scramble and HCT15 / 5Fu-Scramble, while the tumor volume of the model cells of IGF2BP3 knockdown group 1 and IGF2BP3 knockdown group 2 was significantly reduced after 5-Fu treatment. This proves that after knocking down IGF2BP3, the resistance of colorectal cancer cells to 5-Fu decreases.

[0180] In summary, knockdown of IGF2BP3 significantly reduced the resistance of HCT8 / 5Fu and HCT15 / 5Fu cells to 5-Fu. In other words, stable knockdown of IGF2BP3 can reverse the resistance of resistant cells to 5-Fu.

[0181] Example 8

[0182] This example provides a method for detecting the expression level of IGF2BP3 protein in tumor tissue of clinical colorectal cancer patients. The specific steps and parameters are as follows:

[0183] Tumor tissues from 112 colorectal cancer cases were used to form a tumor tissue chip team. The 2.15 method was used to perform immunohistochemical staining on the tumor tissue chip team, and the immunohistochemical score was calculated.

[0184] Results Fig.34 It can be seen that age and IGF2BP3 expression are both related to prognosis ( Fig.34 In the table, T (Tumor) represents the size and invasion range of the primary tumor. N (Node) represents the involvement of regional lymph nodes, which assesses whether the cancer has spread to surrounding lymph nodes. M (Metastasis) refers to distant metastasis, that is, whether the cancer has spread to other parts of the body. Figure 35-Figure 37 It can be seen that the expression level of IGF2BP3 in colorectal cancer tissue is significantly higher than that in adjacent tissues (p<0.0001), proving that IGF2BP3 is associated with the occurrence of colon cancer. Fig.38 It can be seen that patients with colon cancer with higher levels of IGF2BP3 showed shorter overall disease survival time (p = 0.0064), proving that high expression of IGF2BP3 is associated with poor prognosis of colon cancer. Then, 43 cases of these 112 cases who received fluorouracil treatment were screened out, and survival analysis was performed on these 43 cases. The results are shown in Fig.39 , found that in the population treated with 5-Fu, the overall survival time of colon cancer patients with higher IGF2BP3 levels was significantly shortened (p = 0.00076). Then the clinical benefit rate in the high and low expression groups of IGF2BP3 was analyzed, and the results were as follows Fig.40 As shown in the figure, the clinical benefit rate of the IGF2BP3 high expression group was significantly lower than that of the IGF2BP3 low expression group. The above results show that in the colorectal cancer tissue chip cohort, high expression of IGF2BP3 is associated with poor prognosis of colorectal cancer and is related to the degree of clinical benefit after 5-Fu treatment.

[0185] Based on the gene information and clinical data in the GSE4096 dataset, the transcription level of IGF2BP3 in tumor tissues of 5-Fu sensitive and 5-Fu resistant patients was compared. Fig.41 As shown, higher IGF2BP3 mRNA levels were found in 5-Fu-resistant patients, suggesting a correlation between IGF2BP3 and 5-Fu resistance.

[0186] In conclusion, high expression of IGF2BP3 is associated with poor prognosis of colon cancer.

[0187] Example 9

[0188] This example provides a pharmaceutical composition and verifies the therapeutic effect of the pharmaceutical composition on colorectal cancer. The specific steps and parameters are as follows:

[0189] 9.1 Drug Screening

[0190] Eight small molecule drugs that may target IGF2BP3 protein were screened out through online database and literature search, as shown in Table 5. The minimum binding energy between small molecule drugs and IGF2BP3 was predicted by CB-Dock2 software. When the binding energy was less than -4.25 kcal·mol -1 When the binding energy is less than -5.0 kcal·mol -1 The binding activity is better when the binding energy is less than -7.0 kcal·mol -1 The binding activity is stronger when the ligands in the table have a certain binding activity with IGF2BP3.

[0191] Table 5 Prediction of minimum binding energy of IGF2BP3 protein

[0192]

[0193] The small molecule drug was used to intervene in the drug-resistant cells HCT15 / 5Fu at a working concentration of 100 μM, and the total cell protein was extracted. The results of Western blotting are as follows Fig.42 As shown in Figure 2, it was found that isoliquiritigenin can significantly downregulate the protein level of IGF2BP3. The drug-resistant cells HCT15 / 5Fu were intervened with isoliquiritigenin at a concentration gradient of 0, 50μM, 100μM, and 200μM. The results are shown in Figure 2 Fig.43 As shown in Figure 2, it was found that as the concentration of isoliquiritigenin increased, the level of IGF2BP3 protein decreased, proving that isoliquiritigenin reduced the protein level of IGF2BP3 in a concentration-dependent manner. Further, CD-Dock2 was used to simulate molecular docking and perform visual analysis. The results are shown in Figure 2. Fig.44 As shown, it indicates the possibility of small molecule drugs binding to IGF2BP3 protein.

[0194] At the same time, in order to explore whether isoliquiritigenin can enhance the cytotoxicity of 5-FU, the combination index (CI) was analyzed using Compusyn software developed by Dr. Dorothy Chou. The results are as follows Fig.45 As shown, 1 / CI>1 indicates that the two drugs have a synergistic effect. The results show that the combined therapeutic effect is best when the 5-FU concentration range is 180-210μM and the isoliquiritigenin concentration range is 60-80μM.

[0195] 9.2 Cell experiments

[0196] The 2.15 method was used to intervene in the drug-resistant cells HCT15 / 5Fu, wherein the control group was DMEM complete culture medium without drug, the isoliquiritigenin group was DMEM complete culture medium containing 80 μM isoliquiritigenin, the 5-Fu group was DMEM complete culture medium containing 200 μM 5-Fu, and the combination group was DMEM complete culture medium containing 80 μM isoliquiritigenin and 200 μM 5-Fu.

[0197] Results Fig.46 It can be seen that compared with the control group, the proliferation efficiency of HCT15 / 5Fu cells in the isoliquiritigenin group and the 5-Fu group was reduced to a certain extent, while in the 5-Fu and isoliquiritigenin combined group, the proliferation efficiency of HCT15 / 5Fu cells was significantly inhibited. This means that isoliquiritigenin can effectively cooperate with 5-Fu to enhance cytotoxicity.

[0198] 9.3 Animal experiments

[0199] A xenograft tumor model of vector-resistant HCT15 / 5Fu cells was constructed in BALB / c nude mice to evaluate the sensitization effect of isoliquiritigenin on 5-Fu treatment of xenograft tumors. The specific method is as follows: Intestinal cancer cells were injected into nude mice (5×10 6 cells / cell), and wait until the tumor grows to 100 mm 3 The tumor-bearing mice were randomly divided into groups of 6 mice each and given drugs daily according to the following groups:

[0200] The drug of the control group was 0.95% saline solution, the drug of the isoliquiritigenin group was isoliquiritigenin at 50 mg / kg (mass of isoliquiritigenin: weight of mouse), the drug of the 5-Fu group was 5-Fu at 25 mg / kg (mass of 5-Fu: weight of mouse), and the drug of the combined drug group was isoliquiritigenin at 50 mg / kg (mass of isoliquiritigenin: weight of mouse) and 5-Fu at 25 mg / kg (mass of 5-Fu: weight of mouse).

[0201] The tumor volume of mice was measured once every three days, and the weight of mice was recorded. The calculation formula of tumor volume (V) is: V = 1 / 2 × a × b 2 , a is the long diameter of the subcutaneous tumor, b is the short diameter of the subcutaneous tumor.

[0202] Results Fig.47 and Fig.48It can be seen that compared with the control group, there was no significant difference in the tumor volume of the 5-Fu group, while the volume of the isoliquiritigenin monotherapy group was slightly reduced, and the tumor volume of the combined drug treatment group decreased significantly. In summary, isoliquiritigenin combined with 5-Fu treatment has a good ability to inhibit the growth of tumor-resistant cells in mice. Isoliquiritigenin can enhance the cytotoxicity of 5-Fu in vivo and in vitro, and play a role in reversing 5-Fu resistance.

[0203] Example 10

[0204] Take non-drug-resistant colon cancer cells HCT15 and drug-resistant colon cancer cells HCT15 / 5Fu, use METTL16 antibody to pull the bound protein in the cell lysate according to the method in 2.17, and display the LC-MS / MS results of the captured METTL16 protein. Fig.49 : In the non-resistant colorectal cancer cell HCT15 sample, no S-glutathionylation of Cys548 was observed; in the resistant colorectal cancer cell HCT15 / 5Fu sample, only one S-glutathionylation peptide containing Cys548 was detected, and its mass spectrum is shown in Fig.49 Specifically, at m / z 558.91414[(m+3H) 3+ The parent ion of the peptide DLMNQLCTYIR containing S-glutathionylated Cys548 was detected at 40.92 min, and a series of fragment ions were also observed (see Table 6), at m / z 960.39166 [(m+H) + ] containing a molecule of glutathione (305.06816Da) was detected 5 ions (ICLILLDVNPK) further confirmed the S-glutathionylation of Cys548. That is, C548 is the main site of S-glutathionylation modification on METTL16.

[0205] Table 6 Peptide ion information

[0206]

[0207] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of IGF2BP3 as a target in the preparation of drugs for treating colorectal cancer, and reagents for diagnosing and / or evaluating colorectal cancer.

2. The use according to claim 1, characterized in that The effect of IGF2BP3 as a target includes knocking down IGF2BP3 to reduce or reverse the drug resistance of colorectal cancer cells; and / or, Knockdown of IGF2BP3 promotes apoptosis of colorectal cancer cells; and / or, Knockdown of IGF2BP3 increases the sensitivity of colorectal cancer cells to drugs.

3. The use according to claim 2, characterized in that: The m 6 Increased levels of A modification resulted in IGF2BP3 knockdown.

4. The use according to claim 3, characterized in that: Overexpression of METTL16 induces the expression of IGF2BP3 6 An increase in the level of A modification; and / or, Reduction of METTL16 S-glutathione modification levels causes IGF2BP3 knockdown; and / or Knocking down IGF2BP3 includes inhibiting the expression of IGF2BP3 gene and / or downregulating the level of IGF2BP3 protein.

5. Use of a product that inhibits IGF2BP3 gene expression or downregulates IGF2BP3 protein expression in the preparation of drugs for adjuvant therapy or treatment of colorectal cancer.

6. The use according to claim 5, characterized in that: The drug for adjuvant therapy or treatment of colorectal cancer has any of the following applications: (I) Improving the sensitivity of drug-resistant colorectal cancer cells to fluorouracil; (ii) reduce or reverse the resistance of colorectal cancer cells to fluorouracil; (iii) promoting apoptosis of drug-resistant colorectal cancer cells; The products include: IGF2BP3 knockdown virus particles, small molecule IGF2BP3 inhibitors, m 6 At least one of an A modification inducer, a METTL16 inducer or a METTL16 S-glutathione modification inhibitor.

7. A pharmaceutical composition for treating colorectal cancer, characterized in that: The pharmaceutical composition for treating colorectal cancer comprises fluorouracil and an inhibitor, wherein the inhibitor comprises a substance that inhibits the expression of the IGF2BP3 gene or downregulates the expression of the IGF2BP3 protein.

8. The pharmaceutical composition for treating colorectal cancer according to claim 7, characterized in that: The inhibitor is an IGF2BP3 knockdown virus particle, an IGF2BP3 inhibitor of a small molecule compound, 6 At least one of an A modification inducer, a METTL16 inducer or a METTL16 S-glutathione modification inhibitor.

9. The pharmaceutical composition for treating colorectal cancer according to claim 8, characterized in that: The m 6 A modification inducer is a substance that induces IGF2BP3 mRNA to undergo methyl group modification on the 6th nitrogen atom of the adenosine residue; and / or, In the drug for treating colorectal cancer, the molar ratio of fluorouracil to the inhibitor is (18-21):(6-8), and the inhibitor is isoliquiritigenin.

10. A reagent for diagnosing and / or evaluating colorectal cancer, characterized in that The primer sequences in the reagent are shown as SEQ ID NO.11-SEQ ID NO.12.

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