Hepatocellular carcinoma TKI drug resistance marker and application thereof

Abstract

The invention relates to the field of tumor detection, in particular to a hepatocellular carcinoma TKI drug resistance marker and application thereof. The invention reveals the expression level of methyltransferase SMYD3, the expression level of MELK, MELK trimethylation and direct association between IGF1R activity and the drug resistance of hepatocellular carcinoma for the first time, and through systematic experimental verification, it is confirmed that SMYD3 is remarkably and highly expressed in a drug-resistant hepatocellular carcinoma cell line of the revatinib and tissues of a drug-resistant patient of the hepatocellular carcinoma. According to the present invention, the recombinant plasmid is capable of regulating the expression and the trimethylation of MELK so as to activate the activity of IGF1R, such that the hepatocellular carcinoma lenvatinib drug resistance is finally caused, and the recombinant plasmid has significant potential as the lenvatinib drug resistance detection marker. According to the present invention, with the detection of the SMYD3 expression level, the MELK expression level, the MELK trimethylation and the IGF1R activity level, the drug resistance condition of the patient to the lenvatinib can be accurately reflected, the powerful tool is provided for the clinical diagnosis, and the kit can be used for guiding the timely adjustment of the treatment strategy, and has important social and economic benefits.

Description

Technical Field

[0001] The present invention relates to the field of tumor detection, and in particular to a TKI resistance marker for hepatocellular carcinoma and applications thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, with over 800,000 new cases and approximately 700,000 deaths each year. Treatment options for HCC include surgical resection, liver transplantation, localized radiation therapy or chemotherapy, systemic therapy, and combination therapy. However, due to the lack of obvious symptoms in the early stages of HCC and the lack of specific diagnostic markers, many patients are diagnosed at an advanced stage, at which point surgical treatment is no longer effective.

[0003] In the treatment of hepatocellular carcinoma (HCC), drugs such as lenvatinib, sorafenib, and regorafenib have demonstrated significant efficacy. However, monotherapy with these multi-targeted tyrosine kinase inhibitors (TKIs) only prolongs HCC patient survival by two to three months. Lenvatinib is an oral, multi-targeted receptor tyrosine kinase inhibitor that primarily targets the following pathways: 1) angiogenesis pathways: VEGFR1-3, FGFR1-4, and PDGFRα; 2) proliferation pathways: RET, KIT, and c-MET; and 3) other pathways: TIE-2 and DDR2. Clinically validated, lenvatinib demonstrates superior efficacy compared to sorafenib. Since 2018, lenvatinib has been recommended as the preferred first-line treatment for patients with advanced HCC. However, the emergence of drug resistance significantly diminishes its therapeutic efficacy. Therefore, there is an urgent need to further explore the underlying mechanisms of lenvatinib resistance, identify potential targets, and develop appropriate combination therapy strategies to overcome this resistance challenge and further improve the prognosis of HCC patients. Therefore, the development of effective drug resistance diagnostic markers is particularly critical for the detection of drug resistance, timely intervention and systemic treatment of hepatocellular carcinoma. Summary of the Invention

[0004] The inventors of the present invention used CRISPR-Cas9 whole-gene knockout technology to screen drug-resistant hepatocellular carcinoma cells under a simulated tumor microenvironment and discovered SMYD3, a key factor in lenvatinib resistance in hepatocellular carcinoma cells under a simulated in vivo immune microenvironment. Further RT-qPCR and Western blotting of normal tumor cells and drug-resistant cells revealed that SMYD3 expression was significantly increased in the drug-resistant hepatocellular carcinoma cell lines. Inhibition of SMYD3 with the specific inhibitor BCI-121 significantly reduced drug resistance. Simultaneously, combined analysis with RNA-Seq data from existing drug-resistant cell lines revealed that MELK is a key downstream target gene for SMYD3 in regulating receptor tyrosine kinase activity. Molecular docking, COIP, and binding site mutagenesis experiments confirmed that SMYD3 regulates MELK expression and trimethylation. Subsequently, receptor tyrosine kinase microarray screening identified IGF1R as a key receptor tyrosine kinase in regulating RTK activity in hepatocellular carcinoma cells through SMYD / MELK signaling. In addition, inhibition of SMYD3 and MELK using inhibitors and shRNA, respectively, as well as mutation of MELK methylation sites, can significantly reduce the activity of IGF1R and RTKs and the drug resistance level of hepatocellular carcinoma cells.

[0005] Based on this, the purpose of the present invention is to provide a TKI resistance marker for hepatocellular carcinoma and its application.

[0006] One of the purposes of the present invention is to provide the use of any one or more of the expression level of methyltransferase SMYD3, the expression level of MELK, MELK trimethylation, and IGF1R activity as TKI resistance markers for hepatocellular carcinoma.

[0007] Furthermore, the application is as a marker of lenvatinib resistance in hepatocellular carcinoma.

[0008] A second object of the present invention is to provide the use of the marker in the preparation of a product for diagnosing TKI resistance in hepatocellular carcinoma.

[0009] Furthermore, the application is the use of the marker in the preparation of a product for diagnosing lenvatinib resistance in hepatocellular carcinoma.

[0010] Furthermore, the product comprises a reagent for detecting the hepatocellular carcinoma TKI resistance marker.

[0011] Furthermore, the reagent includes specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting the TKI resistance marker of hepatocellular carcinoma.

[0012] A third object of the present invention is to provide a product for diagnosing TKI resistance in hepatocellular carcinoma, wherein the product comprises a reagent for detecting the abundance of the TKI resistance marker in hepatocellular carcinoma.

[0013] Furthermore, the product is a product for diagnosing lenvatinib resistance in hepatocellular carcinoma.

[0014] Furthermore, the reagent includes specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting the hepatocellular carcinoma TKI resistance marker.

[0015] A fourth object of the present invention is to provide an application of the hepatocellular carcinoma TKI resistance marker in constructing a computational model for predicting hepatocellular carcinoma TKI resistance.

[0016] Furthermore, the application is the application of the hepatocellular carcinoma TKI resistance marker in constructing a computational model for predicting lenvatinib resistance in hepatocellular carcinoma.

[0017] A fifth object of the present invention is to provide a system for predicting TKI resistance in hepatocellular carcinoma, comprising:

[0018] (1) A nucleic acid sample separation unit and a tissue sample immunohistochemistry processing unit, used to separate nucleic acid samples from patient samples and process tissue section samples;

[0019] (2) a detection unit, configured to perform relative abundance detection on nucleic acid samples and tissue section samples separated from patient samples to obtain the abundance value results of the aforementioned hepatocellular carcinoma TKI resistance markers;

[0020] (3) a data processing unit, configured to import the obtained relative abundance values ​​of the aforementioned hepatocellular carcinoma TKI markers into a risk warning system to obtain a predicted value;

[0021] (4) A result determination unit, configured to compare the predicted value obtained by the data processing unit with the set diagnostic value; and to determine whether the subject individual is a TKI-resistant or sensitive hepatocellular carcinoma individual by comparing the obtained relative abundance value with a predetermined critical value.

[0022] The sixth object of the present invention is to provide a method for improving TKI resistance in hepatocellular carcinoma, which comprises using biotechnology means to downregulate the expression level of SMYD3 and / or MELK in cells, and / or inhibit MELK trimethylation, and / or reduce IGF1R activity, thereby improving TKI resistance in hepatocellular carcinoma.

[0023] Furthermore, the method is to use shRNA, inhibitors or clinical drugs to knock out or inhibit SMYD3 and / or MELK, and / or mutate MELK methylation sites to reduce IGF1R activity, thereby improving TKI resistance in hepatocellular carcinoma.

[0024] Beneficial effects of the present invention: This invention reveals for the first time the direct correlation between the expression level of the methyltransferase SMYD3, the expression level of MELK, MELK trimethylation, and IGF1R activity and lenvatinib resistance in hepatocellular carcinoma. Through systematic experimental verification, it is confirmed that SMYD3 is significantly overexpressed in lenvatinib-resistant hepatocellular carcinoma cell lines and can regulate the expression and trimethylation of MELK, thereby activating IGF1R activity and ultimately leading to lenvatinib resistance in hepatocellular carcinoma. This discovery fills a gap in this field and provides a new direction for the study of the resistance mechanism of hepatocellular carcinoma. The present invention also provides a new method for detecting lenvatinib resistance in hepatocellular carcinoma, which uses mature and widely used technical means such as RT-qPCR, immunohistochemistry, and Western Blot to accurately detect the expression levels of SMYD3 and MELK, the methylation status of MELK, and the activity level of IGF1R in patient samples. These detection indicators can accurately reflect the patient's resistance to lenvatinib, provide a powerful tool for clinical diagnosis, and can be used to guide timely adjustment of treatment strategies, with important social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the key drug-resistant gene SMYD3 induced by CRISPR-Cas9 whole-gene screening of M2TAM supernatant culture in liver cancer drug-resistant cells. Figure 1 A is the cell model and flow chart of CRISPR-Cas9 whole-genome screening; Figure 1 B is the key drug resistance factor SMYD3 analyzed by CRISPR-Cas9 whole-genome screening.

[0026] Figure 2 The effect of SMYD3 on drug resistance was verified in liver cancer cells using CCK8, cell cloning, and flow cytometry. Figure 2 A is the CCK8 test result, indicating that BCI-121 (SMYD3 specific inhibitor, labeled as SMYD3i) treatment significantly inhibited the proliferation of drug-resistant liver cancer cells; Figure 2 BC is the result of cell clone sphere experiment, which shows that BCI-121 treatment significantly inhibits the formation of drug-resistant liver cancer cell clone spheres; Figure 2 D is the result of flow cytometry experiment, which shows that BCI-121 treatment significantly promotes the apoptosis of drug-resistant liver cancer cells.

[0027] Figure 3 In vivo animal experiments verified that blocking SMYD3 significantly inhibited the progression of drug-resistant tumors. Figure 3 A is a photo of a subcutaneously transplanted tumor. Figure 3 B is the statistical result of subcutaneous transplanted tumor weight. Figure 3 C is the volume statistics of subcutaneously transplanted tumors.

[0028] Figure 4 The downstream target gene MELK of SMYD3 was screened. Figure 4 A is the RT-qPCR result, showing that BCI-121 treatment significantly inhibited the gene expression of MELK in drug-resistant liver cancer cells; Figure 4 B is the result of ChIP-Seq data analysis, indicating that SMYD3-induced H3K4me3 regulates the gene transcription of MELK; Figure 4 C is the WB result, showing that BCI-121 treatment significantly inhibited the protein expression levels of H3K4me3 and MELK in drug-resistant liver cancer cells; Figure 4 D is the WB result, showing that H3K4me3 inhibitor treatment significantly reduced the protein expression level of MELK in drug-resistant liver cancer cells.

[0029] Figure 5 Molecular docking and co-IP verified that SMYD3 targeted and bound to MELK protein to regulate its activity. Figure 5 A is the result of molecular docking experiment, showing that SMYD3 can target and bind to MELK protein; Figure 5 B is the result of CO-IP experiment, showing that SMYD3 can target and bind to MELK protein.

[0030] Figure 6 The effect of mutations in MELK predicted methylation sites on MELK trimethylation. Figure 6 A is the result of mass spectrometry analysis of the methylation site sequence of MELK; Figure 6 B is the WB analysis result of MELK methylation site sequence.

[0031] Figure 7 CCK8, cell cloning, and flow cytometry were used to verify the effect of MELK knockout on drug resistance of liver cancer cells. Figure 7 A is the CCK8 detection result, indicating that shRNA knockdown of MELK significantly inhibits the proliferation of drug-resistant liver cancer cells; Figure 7 B is the result of cell clone sphere experiment, which shows that shRNA knockdown of MELK significantly inhibits the formation of drug-resistant liver cancer cell clone spheres; Figure 7 C is the result of flow cytometry experiment, showing that shRNA knockdown of MELK significantly promotes the apoptosis of drug-resistant liver cancer cells.

[0032] Figure 8 CCK8, cell cloning, and flow cytometry were used to verify the effect of MELK methylation site mutations on drug resistance of liver cancer cells. Figure 8 A is the CCK8 test result, indicating that the MELK methylation site mutation significantly inhibits the proliferation of drug-resistant liver cancer cells; Figure 8BC is the result of cell clone sphere experiment, which shows that MELK methylation site mutation significantly inhibits the formation of drug-resistant liver cancer cell clone spheres; Figure 8 D is the result of flow cytometry experiment, which shows that the MELK methylation site mutation significantly promotes the apoptosis of drug-resistant liver cancer cells.

[0033] Figure 9 The results of the human phosphorylated receptor tyrosine kinase array showed that MELK is a key regulatory factor for IGF1R phosphorylation.

[0034] Figure 10 The activity of the receptor tyrosine kinase IGF1R was detected by WB. The results showed that the inhibition of SMYD3 and MELK significantly reduced the activity of IGF1R.

[0035] Figure 11 ELISA was used to verify the effects of SMYD3 and MELK inhibition and mutation on RTKs activity. Figure 11 A is the result of the SMYD3 and MELK inhibition experiment, which shows that the inhibition of SMYD3 and MELK significantly reduced the activity of RTKs; Figure 11 B is the result of the mutation experiment of MELK methylation site, which shows that the mutation of MELK methylation site significantly reduces the activity of RTKs.

[0036] Figure 12 The expression of SMYD3 and MELK in patients with lenvatinib-resistant liver cancer was detected using clinical samples. The results showed that SMYD3 and MELK were significantly overexpressed in patients with lenvatinib-resistant liver cancer. DETAILED DESCRIPTION

[0037] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following examples are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above-mentioned contents of the present invention are encompassed within the scope that the present invention is intended to protect. It should be noted that, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental reagents in the examples, unless otherwise specified, can all be obtained commercially. The experimental methods in the examples, unless otherwise specified, are all conventional methods.

[0038] 1. Sources of various tumor cells, consumables, and reagents used in the examples:

[0039] Human hepatocellular carcinoma cell lines HCCLM3 and HepG2 can be purchased from ATCC (USA) or the China Center for Type Culture Collection (CCTCC). Drug-resistant hepatocellular carcinoma cell lines were screened by culturing in lenvatinib-containing medium and then tested for drug resistance (LR M3 and LR G2).

[0040] Lenvatinib was purchased from Selleck, and the RAN extraction kit was purchased from Tiangen. RNA reverse transcription kits and TBPremix Ex Taq fluorescent quantitative PCR kits were purchased from Foregene. DMSO and PBS solutions were purchased from Shanghai Shenggong Biotechnology Co., Ltd. Fetal bovine serum was produced by Gibico. RPMI 1640 and DMEM culture media were produced by Gibico. Penicillin-streptomycin solution was produced by HyClone. EDTA trypsin was produced by Solebol. Various cell culture dishes, 6-well plates, 12-well plates, and 96-well plates were produced by Corning Incorporated in the United States. CCK-8 was produced by Biomake. All other reagents, unless otherwise specified, are conventional reagents in the field and can be obtained commercially.

[0041] 2. Experimental methods

[0042] 2.1 CRISPR-Cas9 whole-genome screening

[0043] 2.1.1 CRISPR-Cas9 lentiviral transfection of HCCLM3 cells

[0044] (1) The day before the viral infection experiment, prepare the target cells into a cell suspension and adjust the density to 105 cells / mL. Then, inoculate them into a 24-well plate. The number of cells to be inoculated depends on the growth rate of different cell types. Generally, it is necessary to ensure that the cell confluency is between 50% and 70% when the virus is infected the next day.

[0045] (2) On the day of the virus infection experiment, the GFP reference virus was taken out of the -80°C refrigerator, thawed in an ice bath, and the virus was diluted; the original culture medium in the 24-well plate was aspirated and replaced with freshly prepared culture medium (the appropriate LV-Assistant concentration was screened in the range of 1 to 10 μg / mL through preliminary experiments in advance), and the virus was added according to different MOI values ​​(such as MOI values ​​of 1, 5, 10, 50, and 100) and cultured.

[0046] (3) 24 hours after infection, replace the culture medium with fresh medium (without virus and LV-Assistant) and continue culturing. 72 hours after infection, observe GFP fluorescence under a fluorescence microscope to determine the infection efficiency. The infection conditions and MOI values ​​corresponding to wells with an infection efficiency of approximately 80% and good cell growth status were selected as the basis for the formal experiment.

[0047] (4) Digest the liver cancer cells and seed them in a 24-well plate at a density of approximately 50,000 cells per 500 μl. Shake well and count the cells before transfection. Select two wells of equal size as a group. Ensure at least two groups, one for CCK-8 detection and the other for expansion culture.

[0048] (5) The volume of lentivirus required for transfection should be calculated based on the cell count before transfection. The MOI value for transfection should be 15. It should be noted that the virus needs to be thawed in an ice bath and the approximate amount to be used should be dispensed into small tubes to avoid repeated freezing and thawing of the mother solution that affects its efficacy.

[0049] (6) After adding the calculated amount of virus to the transfection group, infect for 24 hours, change the medium, replace it with complete medium without virus, and continue culturing.

[0050] (7) After 72 hours of infection, observe the fluorescence or use other detection methods to determine the infection efficiency before conducting subsequent experiments. For viruses carrying the Puromycin resistance gene, replace with fresh complete culture medium containing Puromycin and screen for stably transfected cell lines. The working concentration of Puromycin for different cells is different (the final concentration range is 1-10μg / mL). In addition, please set up an uninfected screening control (wild-type cells that have not been infected with the virus) and add equal amounts of Puromycin at equal concentrations. Replace the complete culture medium containing Puromycin every 2-3 days until all cells in the uninfected screening control group are killed by Puromycin, and then proceed to the next step according to the experimental requirements. Puromycin is selected at 5μg / ml

[0051] (8) Select one of the groups to perform CCK-8 cell survival test. The non-transfected group should ideally have a death rate close to 100% compared to the transfected group. The transfected group in the other group of cells can be digested for amplification and culture.

[0052] 2.1.2 Sample preparation:

[0053] After obtaining cells transfected with lentivirus, they were inoculated into six-well plates, and the amount of cells in each well was ensured to be the same. At the same time, the following experimental wells were set up: adding supernatant containing serine (Ser) and administering lenvatinib, adding supernatant lacking Ser and administering lenvatinib, and only adding supernatant containing Ser. Two replicates were made for each well to ensure screening and standby. After 48 hours of culture, some cells were digested with trypsin and passaged. After repeated operation for about 14 days, the cells were digested with trypsin and centrifuged. After washing with PBS, they were centrifuged again and the PBS was discarded to retain the cell pellet, which was stored in a -80°C environment and sent for testing on dry ice.

[0054] 2.1.3 Library construction:

[0055] In order to ensure that the gRNA sequence in the sample is fully amplified, sufficient genomic DNA should be fully extracted from 300X to 500X number of cells according to the size of the gRNA library. Take 2 to 3ug of genomic DNA for each reaction for amplification (set a sufficient number of reaction tubes until the genomic DNA is completely consumed), introduce spacer bases of different lengths and partial sequencing adapters, and introduce complete sequencing adapters and indexes through the second round. After purification, the Illumina platform high-throughput sequencing library is obtained. The constructed library is preliminarily quantitatively diluted using Qubit3.0, and the insertsize and nucleic acid concentration of the library are detected using Agilent 2100. After mixing, NanoQC is used to accurately quantify the effective concentration of the mixed library to ensure the accuracy of the concentration on the machine and the data output.

[0056] 2.1.4 Bioinformatics analysis

[0057] After sequencing is completed and raw data with sufficient sequencing depth is obtained, bioinformatics analysis begins. This process mainly includes the following stages:

[0058] (1) gRNA sequence extraction and data quality control: extract the data containing gRNA sequences from the sequencing data according to the library construction method, retain the 20nt target recognition related core sequence, obtain the fastq file of the gRNA core sequence, and perform data quantity statistics and quality assessment to ensure that it meets the requirements of subsequent analysis;

[0059] (2) gRNA alignment and number statistics: The data after quality control are used for alignment, and the alignment rate, gRNA sequencing depth, and gRNA and gene coverage are evaluated. Only when the requirements are met can the next step of analysis be carried out.

[0060] (3) Genetic differences between samples: MAGeCK software was used to count gRNAs and genes after alignment, and the counting results were standardized to compare the differences in the number of gRNAs and genes between samples.

[0061] (4) Enrichment analysis: GSEA analysis was performed on the difference comparison results, mainly including KEGG / GO enrichment, and the enrichment results were visualized.

[0062] (5) Gene annotation: Genes with significant differences are annotated. The annotation information includes the COSMIC database and published gRNA screen articles.

[0063] 2.2 Western blotting (WB)

[0064] (1) Total protein extraction: The proteins required for Western blotting (WB) were obtained from liver cancer cells cultured in 6-well plates and then processed according to the previous method. The specific steps for extraction are as follows:

[0065] ① Take out the liver cancer cells with different treatments from the incubator, discard the culture medium, and wash them three times with pre-cooled PBS.

[0066] ② Add 100 μL of RAPI lysis buffer containing 10% PMSF to each well, place the culture dish on ice for 30 minutes, scrape the liver cancer cells with a cell scraper, and collect them into a 1.5 mL EP tube.

[0067] ③ Place the EP tube on ice and vortex it once every 10 minutes for a total of three times.

[0068] ④ Place the EP tube in a high-speed refrigerated centrifuge and centrifuge at 4°C, 12,000 rpm for 30 minutes to remove cell debris, collect the supernatant, and store it in a -20°C refrigerator.

[0069] (2) Determination of protein concentration: Prepare BCA protein quantification working solution according to the instructions.

[0070] ① Prepare BSA standard proteins at different concentrations: 0, 0.125, 0.25, 0.5, 1, 2, and 5 mg / mL.

[0071] ② Dilute the sample 10-fold with PBS, take 20 μL of sample and standard protein, add them to a 96-well plate respectively, and then add 100 μL of BCA protein quantification working solution.

[0072] ③ Place the 96-well plate in a 37°C oven and incubate for 30 minutes, then measure the absorbance at 595 nm.

[0073] ④ Based on the absorbance value and concentration of the standard protein, draw a standard curve, fit the linear regression equation, and calculate the protein concentration of the sample.

[0074] ⑤ Adjust the protein concentration of the sample to about 1 mg / mL, mix it with 5x loading buffer at a ratio of 4:1, boil it in boiling water for 10 minutes, and then store it in a refrigerator at -20℃.

[0075] (3) SDS-PAGE gel electrophoresis: To perform a Western blotting experiment, proteins must first be separated using SDS-PAGE gel electrophoresis. The gel electrophoresis process is as follows:

[0076] Prepare 12% separating gel and 5% stacking gel according to the table below, based on the desired gel percentage. Add each to the glass plates, seal the surface of the separating gel with methanol, and allow to solidify. TEMED must be added last.

[0077] Table 1 Separation gel configuration

[0078]

[0079]

[0080] Prepare 5% stacking gel according to the following configuration table. After mixing, immediately add it to the glass plate for gel preparation, insert a comb, and wait for it to solidify.

[0081] Table 2 Stacking gel configuration

[0082]

[0083] Add 40 μg / 40 μL of protein sample to each well into the gel, run at 60 V for half an hour, then switch to 120 V until the loading buffer reaches the bottom of the gel and stop electrophoresis.

[0084] (4) The separated proteins need to be transferred to a PVDF membrane. The transfer process is as follows:

[0085] ① Peel off the separation gel from the glass plate, cut off the unnecessary part with a gel cutting board, and balance the required part in the transfer buffer.

[0086] ② Activate a PVDF membrane of the same size as the separation gel in methanol for 15 seconds, and then equilibrate it in the transfer buffer.

[0087] ③ Place the sponge, filter paper, separation gel, PVDF membrane, filter paper and sponge on the transfer clamp in sequence, install the clamp and place it in the transfer tank.

[0088] ④ Transfer the membrane with a current of 200 mA for 2 hours, and then rinse with TBST.

[0089] Next, the PVDF membrane needs to be incubated with antibodies to detect the expression of the target protein. The antibody incubation process is as follows:

[0090] ① Block with 5% skim milk powder at room temperature and then wash with TBST.

[0091] ②Add primary antibody diluted with primary antibody diluent, incubate overnight at 4°C, and wash three times with TBST, each time for 5 minutes.

[0092] ③Add secondary antibody diluted with TBST, incubate at room temperature for 1 hour, and wash with TBST three times, 5 minutes each time.

[0093] Finally, develop the PVDF membrane with a developer solution to visualize the target protein bands. The development process is as follows: Prepare the developer solution according to the developer's instructions and immerse the PVDF membrane in it. Develop the PVDF membrane using a gel imaging system to visualize the target protein bands and record their position and intensity.

[0094] 2.3 Real-time fluorescence quantitative PCR

[0095] First, extract total RNA:

[0096] (1) Cell treatment and lysis:

[0097] ① Remove the cells from the well plate and remove the culture medium with a pipette.

[0098] ②Wash the cells twice with PBS buffer, adding 1 mL of PBS each time.

[0099] ③ Add 1 mL of Trizol lysis buffer, place the plate on an ice tray, and shake on a shaker for 8 minutes.

[0100] ④ Use a pipette to blow away the flocculent material in the lysate, then transfer it to a 1.5 mL centrifuge tube and freeze the centrifuge tube at -80℃.

[0101] (2) Phase separation and RNA precipitation:

[0102] ① Take out the frozen cell lysate from the -80℃ freezer, wait for it to completely dissolve, add 200μL of chloroform, shake vigorously 30 times to fully mix the two phases, and then let it stand at room temperature for 3 minutes.

[0103] ② Place the centrifuge tube at 4°C and centrifuge at 12,000 rpm for 15 minutes. The sample will then separate into an upper aqueous phase, an intermediate layer, and a lower organic phase.

[0104] ③ Add 500 μL of isopropanol to a new 1.5 mL centrifuge tube. Use a 200 μL pipette tip to slowly and accurately transfer the aqueous phase to the centrifuge tube containing isopropanol, taking care not to aspirate the interphase or the organic phase. The volume of the aqueous phase should be approximately 400-500 μL.

[0105] ④ Invert the centrifuge tube containing the aqueous phase and isopropanol several times to mix them evenly and leave them at room temperature for 10 minutes.

[0106] ⑤ Centrifuge the tube at 12,000 rpm for 10 minutes at 4°C. The RNA will settle at the bottom of the tube. Pour off the isopropanol layer, taking care not to disturb the RNA pellet.

[0107] (3) RNA precipitation and washing:

[0108] ① Dilute anhydrous ethanol to 70% concentration with RNase-Free ddH2O, add 1 mL of 70% ethanol to each centrifuge tube, and stir gently to allow the RNA precipitate to fully contact with the ethanol.

[0109] ② Place the centrifuge tube at 4℃ and centrifuge at 7500rpm for 5 minutes, and pour off the ethanol on the top layer.

[0110] ③ Repeat the previous step and wash the RNA precipitate with 70% ethanol again, centrifuge for 5 minutes, and pour off the ethanol.

[0111] ④ Place the centrifuge tube at 4°C and centrifuge at 7500 rpm for 5 minutes. Use a pipette to remove the remaining ethanol, trying not to absorb the RNA precipitate.

[0112] (4) Dissolution and measurement:

[0113] ① Open the cap of the centrifuge tube and place it in a fume hood to dry until the ethanol evaporates completely. The RNA precipitate will turn from white to transparent.

[0114] ②Add an appropriate amount of RNase-Free ddH2O (generally 30-50μL) and stir gently to completely dissolve the RNA precipitate.

[0115] Determination of RNA concentration:

[0116] (1) Prepare measuring instruments and clean them:

[0117] ①Open the Nanodrop 2000 ultra-micro-volume spectrophotometer software, select the "Nucleic Acid / RNA" interface, and wait for the instrument self-test to complete.

[0118] ② Use 2 μL of RNase-free water to clean the detection platform and repeat the operation three times.

[0119] ③ Add 1 μL of RNase-free water and click the "Blank" button to perform a blank correction. Repeat this operation three times. After the correction is completed, ensure that the concentration of the water is approximately 0 (μg / μL).

[0120] (2) RNA concentration and purity determination:

[0121] ① Take 1 μL of the RNA sample to be tested and add it to the detection platform. Click the "Measure" button to measure and record the RNA concentration (μg / μL).

[0122] ②Observe the A260 / A280 ratio of RNA, which reflects the purity of RNA. The ideal RNA sample should have an A260 / A280 ratio of 1.8-2.2.

[0123] (3) Result analysis and sample processing:

[0124] ① Based on the concentration and purity of the RNA, decide whether to perform a reverse transcription experiment or preserve the RNA sample.

[0125] ② Store RNA samples in a -80℃ freezer and generally use within 1 month.

[0126] Steps of real-time fluorescence quantitative PCR:

[0127] ① Take 200 μL of new RNase-free EP tubes and add 10 μL of reverse transcription mixed reagent (Foregene) to each EP tube, then add 1 μg of RNA template. Finally, add RNase-free water to a final volume of 20 μL.

[0128] ②Gently mix the liquid in the EP tube and place it in the PCR instrument to perform reverse transcription according to the reverse transcription program.

[0129] (3) RT-qPCR

[0130] ① Primer design and synthesis

[0131] The primer sequences are shown in the following table:

[0132] Table 3 Gene primer sequences

[0133]

[0134] ②Real-time qPCR amplification

[0135] Establish the real-time fluorescence quantitative PCR reaction system according to the following process:

[0136] ① Add the reagents to the eight tube strips in the following order. Keep all the operations on ice and perform 3 replicates for each sample:

[0137] Table 4 Real-time PCR system

[0138]

[0139] ②PCR cycle amplification conditions are as follows:

[0140] After the reaction, the results were analyzed using CFX manager software.

[0141] Table 5 Real-time PCR amplification program

[0142]

[0143] Relative quantitative analysis: using 2 -ΔΔCt The obtained data were analyzed by relative quantitative method.

[0144] 2.4 CCK-8 assay for cell viability

[0145] (1) 3000 cells / well were pre-plated in a 96-well plate. Six replicates were set for each concentration gradient. 100 μL PBS was added to each well around the cell wells to prevent evaporation from affecting the cells.

[0146] (2) Treat cells with different concentrations of drugs or transfected plasmids, set up at least 4 drug gradients and keep 0 concentration as the control group.

[0147] (3) Serum-free culture medium and CCK-8 reagent were mixed in a ratio of 10:1 to prepare the working solution.

[0148] (4) Aspirate the drug-containing culture medium and add 100 μL of CCK-8 working solution to each well (a blank control group containing no cells and no drug but only the working solution should be set up) and incubate in the dark for 2 hours.

[0149] (5) Finally, the absorbance was measured at a wavelength of 450 nm.

[0150] (6) Calculate cell viability.

[0151] 2.5 Cell cloning experiment

[0152] (1) HCCLM3 and HepG2 cells were seeded into 6-well plates, with 500 cells per well. 2 h before seeding, the plates were coated with Matrigel.

[0153] (2) After the cells adhere to the wall, the corresponding drug treatment is given.

[0154] (3) After 2 days of culture, remove the old culture medium and replace it with fresh culture medium, and continue culturing for 5-7 days.

[0155] (4) At the end of the culture, the old culture medium was aspirated, the cells were gently washed twice with PBS buffer, and fixed with 4% paraformaldehyde for 4 h. Then, 0.8 mL of crystal violet staining solution was added to each well and stained for 4 min.

[0156] (5) After staining, discard the crystal violet staining solution, wash twice with double-distilled water, and place at room temperature for 24 hours to allow the double-distilled water to evaporate completely. Then take pictures and count the number of cell colonies.

[0157] 2.6 Mass spectrometry detection

[0158] Protease cleavage:

[0159] (1) Protein enrichment was performed using MELK antibody, followed by electrophoresis and cutting of the target band. The target band was placed in an EP tube.

[0160] (2) Wash twice with ultrapure water by vortexing, 5 min each time.

[0161] (3) Mix 50 mM NH4HCO3 and ACN (i.e., acetonitrile) in a ratio of 1:1, decolorize by ultrasonication for 15 min, and remove the liquid by aspiration.

[0162] (4) Repeat this step until the solution and gel are colorless.

[0163] (5) Mix 50 mM NH4HCO3 and ACN (i.e., acetonitrile) in a ratio of 1:1 and wash once by vortexing.

[0164] (6) Add 100% acetonitrile and shake to dehydrate until the particles turn white. Remove the liquid by aspiration and vacuum dry for 5 min.

[0165] (7) Add 150 μl of 50 mM DTT solution to submerge the gelatin block, shake and mix until the gelatin block becomes swollen and transparent, and place in an oven at 56°C for 1 hour.

[0166] (8) Take out and cool to room temperature, aspirate dry, quickly add 150ul 100mM IAM solution to submerge the gel, and place in the dark for 45min.

[0167] (9) 25mM NH4HCO3, 50mM NH4HCO3 and ACN 1:1 mixture, 100% ACN wash once each,

[0168] (10) Dehydrate with acetonitrile until the particles turn white and vacuum dry for 5 min.

[0169] (11) Prepare the enzyme reaction solution: dilute 0.1 μg / μl of the enzyme stock solution with 25 mM NH4HCO3. Add enough enzyme solution to cover the volume of the swollen gel. Centrifuge briefly to allow the gel to fully contact the enzyme solution and incubate at 4°C for 30 min.

[0170] (12) After the solution is fully absorbed by the gel, remove the excess enzyme solution, add 25 mM NH4HCO3 to submerge the gel, and then add 20 μl more. Digest at 37°C overnight.

[0171] (13) Collect the enzyme solution, add 100 μl of 30% acetonitrile / 0.1% TFA to the original tube, sonicate for 15 min, and aspirate the solution. Add 100 μl of 60% acetonitrile / 0.1% TFA, sonicate for 15 min, and then add the previous solution. Combine and lyophilize.

[0172] Desalting with ZipTip C18:

[0173] (1) Take a sample and dissolve it thoroughly with 0.1% TFA.

[0174] (2) Rinse the tip 10 times with 50 μl of 60% ACN / 0.1% TFA.

[0175] (3) Wash the TIP 10 times with 10 μl of 0.1% TFA.

[0176] (4) Aspirate and expel the sample into the TIP 20 times to drain the liquid.

[0177] (5) Wash the TIP five times with 10 μl of 0.1% TFA.

[0178] (6) Elute the peptide fragments with 10 μl of 60% ACN and 0.1% TFA into a new EP tube and vacuum dry.

[0179] (7) Computer testing.

[0180] RPLC-MS:

[0181] The peptide was dissolved in 20 μl of dissolving solution (0.1% formic acid), vortexed thoroughly, centrifuged at 17000 x g, 4°C for 20 min, and the supernatant was transferred to a sample tube. 3 μl was aspirated for mass spectrometry identification.

[0182] Database analysis: PEAKS software was used to search the database for this sample.

[0183] Example 1 CRISPR-Cas9 whole-genome screening

[0184] CRISPR-Cas9 whole gene knockout technology was used to screen drug-resistant hepatocellular carcinoma cells in a simulated tumor microenvironment. The screening cell model and flow chart are shown in the figure. Figure 1 As shown in A, the screening process included CRISPR-Cas9 lentiviral transfection of HCCLM3 cells, sample preparation, library construction, and bioinformatics analysis. The results showed that SMYD3 was significantly increased in drug-resistant cell lines, indicating that SMYD3 is a potential key factor for lenvatinib resistance in hepatocellular carcinoma ( Figure 1 B).

[0185] Example 2: CCK8, cell cloning, and flow cytometry verification of the effect of SMYD3 on drug resistance

[0186] SMYD3 was inhibited by the specific inhibitor BCI-121 (SMYD3 specific inhibitor, labeled SMYD3i), and the effect of SMYD3 on drug resistance was verified by CCK8, cell cloning, and flow cytometry. CCK8 detection found that BCI-121 treatment significantly inhibited the proliferation of drug-resistant liver cancer cells ( Figure 2 A); Cell cloning experiments showed that BCI-121 treatment significantly inhibited the formation of drug-resistant liver cancer cell clones ( Figure 2 BC); Flow cytometry experiments showed that BCI-121 treatment significantly promoted the apoptosis of drug-resistant liver cancer cells ( Figure 2D) The results further indicate that SMYD3 is a key factor for lenvatinib resistance in hepatocellular carcinoma.

[0187] Example 3 In vivo animal experiments verify the effect of SMYD3 blockade on drug resistance

[0188] The effect of SMYD3 blockade on drug resistance was verified by in vivo animal experiments. The results showed that SMYD3 blockade significantly inhibited the progression of drug-resistant tumors ( Figure 3 AC).

[0189] Example 4 Screening and verification of downstream target genes of SMYD3

[0190] The data obtained from the CRISPR-Cas9 whole-genome screening were subjected to GO enrichment screening, and related genes affecting tyrosine kinase activity related to lenvatinib resistance were obtained: MELK, JAK3 and SLA. After correlation analysis including but not limited to prognosis and expression, MELK was finally found to be a potential key regulatory factor affecting tyrosine kinase activity and lenvatinib resistance.

[0191] Real-time fluorescence quantitative PCR results showed that the inhibition of SMYD3 significantly reduced the expression of MELK gene ( Figure 4 A).

[0192] ChIP-Seq data analysis results showed that SMYD3-induced H3K4me3 regulated the gene transcription of MELK ( Figure 4 B).

[0193] Immunoblotting experiments were performed to detect the expression of H3K4me3 and MELK proteins in drug-resistant cells. The results showed that BCI-121 treatment significantly inhibited the protein expression levels of H3K4me3 and MELK in drug-resistant liver cancer cells ( Figure 4 C), H3K4me3 inhibitor treatment significantly reduced the protein expression level of MELK in drug-resistant liver cancer cells ( Figure 4 D).

[0194] Molecular docking experiments and COIP experiments were used to further verify the relationship between SMYD3 and MELK. Molecular docking experiments showed that SMYD3 could target and bind to MELK protein ( Figure 5 A); CO-IP experiments showed that SMYD3 can target and bind to MELK protein ( Figure 5 B).

[0195] The methylation sites of MELK were detected by mass spectrometry, and the results showed that the sites where MELK lysine was methylated ( Figure 6A). MELK wild-type and trimethylation site mutant plasmids were constructed and co-transfected with SMYD3 overexpression plasmid into the drug-resistant liver cancer cell line LR M3. MELK trimethylation antibodies and pan-methylation antibodies were then used to detect the MELK. The results showed that compared with the wild-type, the trimethylation level of MELK in samples with mutant methylation sites was significantly reduced ( Figure 6 B), demonstrating that SMYD3 mediates MELK trimethylation.

[0196] Example 5: CCK8, cell cloning, and flow cytometry verification of the effect of MELK knockout on drug resistance in liver cancer cells

[0197] CCK8, cell cloning, and flow cytometry were used to verify the effect of MELK knockout on drug resistance in liver cancer cells. The CCK8 assay results showed that shRNA knockout of MELK significantly inhibited the proliferation of drug-resistant liver cancer cells ( Figure 7 A); Cell cloning sphere experiments showed that MELK shRNA knockout significantly inhibited the formation of drug-resistant liver cancer cell cloning spheres ( Figure 7 B); Flow cytometry experiments showed that shRNA knockdown of MELK significantly promoted apoptosis of drug-resistant liver cancer cells ( Figure 7 C).

[0198] Example 6: CCK8, cell cloning, and flow cytometry verification of the effect of MELK methylation site mutations on drug resistance in liver cancer cells

[0199] CCK8, cell cloning, and flow cytometry were used to verify the effect of MELK methylation site mutation on drug resistance in liver cancer cells. The results of the CCK8 experiment showed that MELK methylation site mutation significantly inhibited the proliferation of drug-resistant liver cancer cells ( Figure 8 A); Cell cloning experiments showed that MELK methylation site mutations significantly inhibited the formation of drug-resistant liver cancer cell clones ( Figure 8 BC); Flow cytometry results showed that MELK methylation site mutations significantly promoted apoptosis of drug-resistant liver cancer cells ( Figure 8 D).

[0200] Example 7 Detection of Human Phospho-Receptor Tyrosine Kinase Array

[0201] The Human Phospho-RTKArray Kit was used to detect the phosphorylation levels of 49 receptor tyrosine kinases. The specific experimental steps are as follows: The drug-resistant liver cancer cells were inoculated in a culture medium containing 10% fetal bovine serum and cultured. After the cells reached the optimal degree of fusion, the cells were lysed using array buffer. Subsequently, the array membrane was placed in a blocking buffer for 1 hour, 200 μg of cell lysate was added, and incubated at 4°C overnight. The next day, after thoroughly washing the array membrane, a phosphokinase antibody was added for incubation. Finally, the ChemiDoc Touch System chemiluminescence imaging system was used for exposure. To ensure the reliability of the experimental results, all experiments were repeated three times independently. The test results found that MELK is a key regulatory factor for IGF1R phosphorylation ( Figure 9 ).

[0202] Example 8 WB detection of receptor tyrosine kinase IGF1R activity

[0203] The activity of receptor tyrosine kinase IGF1R was detected by WB experiment. The results showed that the inhibition of SMYD3 and MELK significantly reduced the activity of IGF1R ( Figure 10 ).

[0204] Example 9 ELISA Verification of the Inhibition of SMYD3 and MELK and the Effect of Mutation on RTKs Activity

[0205] To verify whether the expression levels of SMYD3 and MELK and the mutation of MELK methylation sites affect the RTK enzyme activity of liver cancer resistant cells, ELISA was used to verify the effects of SMYD3 and MELK inhibition and mutation on RTKs activity. The experimental results showed that the inhibition of SMYD3 and MELK significantly reduced RTKs activity ( Figure 11 A); Mutation of MELK trimethylation sites significantly reduces RTKs activity ( Figure 11 B).

[0206] Example 10 Clinical Sample Detection of SMYD3 and MELK Expression in Lenvatinib-Resistant Liver Cancer Patients

[0207] Clinical samples were used to detect the expression of SMYD3 and MELK in patients with lenvatinib-resistant liver cancer. Tissue immunofluorescence results showed that SMYD3 and MELK were significantly overexpressed in patients with lenvatinib-resistant liver cancer ( Figure 12 ).

[0208] In summary, the present invention first discovered SMYD3, a key factor in resistance to the hepatocellular carcinoma targeted drug lenvatinib, by utilizing CRISPR-Cas9 whole-genome screening, molecular docking, CO-IP, and mass spectrometry technologies. Further analysis then revealed that the dysregulation of the activity of the receptor tyrosine kinase IGF1R is a key factor affecting drug resistance, and MELK is the key factor mediating the effect of SMYD3 on the activity of the receptor tyrosine kinase. SMYD3 interacts with MELK, mediating methylation modification (trimethylation) of the MELK lysine site, thereby enhancing the activity of the tyrosine kinase IGF1R. The detection of this site and the activation factor of IGF1R activity can be used as an accurate judgment of the resistance of hepatocellular carcinoma patients to lenvatinib, and thus the medication or combination of medications can be changed in advance to prolong the patient's survival. Therefore, the present invention has important theoretical significance and application prospects in the field of drug resistance detection and targeted therapy of hepatocellular carcinoma, and is expected to contribute to the development of clinical tumor diagnosis and treatment.

[0209] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of any one or more of the expression level of methyltransferase SMYD3, the expression level of MELK, MELK trimethylation, and IGF1R activity as markers of TKI resistance in hepatocellular carcinoma.

2. The use according to claim 1, characterized in that The application is as a marker for lenvatinib resistance in hepatocellular carcinoma.

3. Use of the marker according to claim 1 in the preparation of a product for diagnosing TKI resistance in hepatocellular carcinoma.

4. The use according to claim 3, characterized in that The application is the use of the marker described in claim 1 in the preparation of a product for diagnosing lenvatinib resistance in hepatocellular carcinoma.

5. A product for diagnosing TKI resistance in hepatocellular carcinoma, characterized in that: The product includes a reagent for detecting the abundance of the hepatocellular carcinoma TKI resistance marker according to claim 1.

6. The product according to claim 5, characterized in that The product described is for diagnosing lenvatinib resistance in hepatocellular carcinoma.

7. The product according to claim 5, characterized in that The reagents include specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting the hepatocellular carcinoma TKI resistance marker.

8. Use of the hepatocellular carcinoma TKI resistance marker according to claim 1 in constructing a computational model for predicting hepatocellular carcinoma TKI resistance.

9. The use according to claim 8, characterized in that The application is the application of the hepatocellular carcinoma TKI resistance marker according to claim 1 in constructing a computational model for predicting lenvatinib resistance in hepatocellular carcinoma.

10. A system for predicting TKI resistance in hepatocellular carcinoma, comprising: (1) A nucleic acid sample separation unit and a tissue sample immunohistochemistry processing unit, used to separate nucleic acid samples from patient samples and process tissue section samples; (2) a detection unit for performing relative abundance detection on nucleic acid samples and tissue section samples separated from patient samples to obtain the abundance value result of the hepatocellular carcinoma TKI resistance marker according to claim 1; (3) a data processing unit, configured to import the relative abundance value of the hepatocellular carcinoma TKI marker obtained as claimed in claim 1 into a risk warning system to obtain a predicted value; (4) a result determination unit, configured to compare the predicted value obtained by the data processing unit with the set diagnostic value; By comparing the obtained relative abundance value with a predetermined critical value, it is determined whether the subject individual is a hepatocellular carcinoma TKI-resistant individual or a hepatocellular carcinoma-sensitive individual.

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