TNIK expression inhibitor and application thereof

Abstract

The invention discloses an expression inhibitor of TNIK (Tumor Necrosis Induced Killer) and application thereof. The TNIK expression inhibitor is selected from at least one of a to e: a, siTNIK-1 with a nucleotide sequence as shown in SEQ ID NO: 1; b, siTNIK-2 with a nucleotide sequence as shown in SEQ ID NO: 2; c, siTNIK-3 of which the nucleotide sequence is as shown in SEQ ID NO: 3; d, shTNIK-1 with a nucleotide sequence as shown in SEQ ID NO: 4; and e, shTNIK-2 with a nucleotide sequence as shown in SEQ ID NO: 5. The compound can effectively inhibit the expression of TNIK in colorectal cancer drug-resistant cells and reverse the drug resistance of the colorectal cancer drug-resistant cells to 5FU. The TNIK expression inhibitor is combined with 5FU to overcome the drug resistance of colorectal cancer chemotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and biomedicine, and in particular to a TNIK (TRAF2-and NCK-interacting kinase) expression inhibitor and application thereof. Background Art

[0002] 5-Fluorouracil (5FU) is the first-line drug of choice for chemotherapy. However, since patients are prone to develop congenital or acquired resistance to 5FU, the therapeutic effect may be severely hindered and tumors are prone to recurrence and metastasis.

[0003] Therefore, solving the problem of how to improve the therapeutic efficacy of 5FU and reverse 5FU resistance is an important challenge in clinical practice. Summary of the Invention

[0004] The present invention provides a TNIK expression inhibitor and its application, which can effectively inhibit the expression of TNIK in colorectal cancer resistant cells, leading to S phase cell cycle arrest of colorectal cancer resistant cells, inhibiting the proliferation of colorectal cancer resistant cells, and at the same time inducing colorectal cancer resistant apoptosis, inhibiting the migration ability of colorectal cancer cells, reducing the expression of SOX2 in colorectal cancer resistant cells, inhibiting the stemness of colorectal cancer resistant cells, thereby inhibiting the Wnt / β-catenin signaling pathway of colorectal cancer resistant cells and reversing the resistance of colorectal cancer resistant cells to 5FU.

[0005] The technical solutions provided by the present invention are as follows:

[0006] The present invention provides a TNIK expression inhibitor, wherein the TNIK expression inhibitor is selected from at least one of a to e:

[0007] a. siTNIK-1 whose nucleotide sequence is shown in SEQ ID NO: 1;

[0008] b. siTNIK-2 whose nucleotide sequence is shown in SEQ ID NO: 2;

[0009] c. siTNIK-3, whose nucleotide sequence is shown in SEQ ID NO: 3;

[0010] d. shTNIK-1 having a nucleotide sequence as shown in SEQ ID NO: 4;

[0011] e. shTNIK-2 whose nucleotide sequence is shown in SEQ ID NO: 5.

[0012] siTNIK-1:5'-GAAGCTCTTGGTTACGACA-3' (SEQ ID NO: 1);

[0013] siTNIK-2:5'-GCTACGAGTTTACTATCTT-3' (SEQ ID NO: 2);

[0014] siTNIK-3:5'-GGACCATATTGATAGAACA-3' (SEQ ID NO: 3);

[0015] shTNIK-1:5'-CCATCTCATATTCAGGGCAAT-3' (SEQ ID NO: 4);

[0016] shTNIK-2:5'-GACAGTTTCAGCGGCAGTATT-3' (SEQ ID NO: 5);

[0017] In some embodiments, the TNIK expression inhibitor is chemically synthesized.

[0018] The present invention also provides a delivery system for a TNIK expression inhibitor, comprising the TNIK expression inhibitor and a carrier.

[0019] The present invention also provides a pharmaceutical composition comprising the TNIK expression inhibitor or the delivery system, and pharmaceutically acceptable excipients.

[0020] The present invention also provides use of the TNIK expression inhibitor, the delivery system, and the pharmaceutical composition in preparing drugs for preventing and / or treating cancer.

[0021] More specifically, the TNIK expression inhibitor can significantly enhance the inhibitory effect of 5FU on the proliferation of colorectal cancer resistant cells;

[0022] The TNIK expression inhibitor can significantly arrest the colorectal cancer drug-resistant cell cycle at the S phase;

[0023] The TNIK expression inhibitor can significantly increase 5FU-induced cell apoptosis;

[0024] The TNIK expression inhibitor can significantly inhibit the migration and invasion ability of colorectal cancer resistant cells;

[0025] The TNIK expression inhibitor can significantly reduce the expression of SOX2 in colorectal cancer resistant cells and inhibit the stemness of colorectal cancer resistant cells.

[0026] On this basis, the TNIK expression inhibitor can reverse the resistance of colorectal cancer resistant cells to 5FU by inhibiting the Wnt / β-catenin signaling pathway of colorectal cancer resistant cells.

[0027] In some embodiments, a combination drug comprising the TNIK expression inhibitor and 5FU is provided for use in the preparation of a drug for preventing and / or treating cancer, including colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, choriocarcinoma, malignant hydatidiform mole, head and neck squamous cell carcinoma, skin cancer, liver cancer, and bladder cancer.

[0028] Beneficial Effects: The present invention designs and synthesizes a TNIK expression inhibitor (e.g., siRNA, shRNA) that can effectively inhibit TNIK expression in colorectal cancer-resistant cells, leading to S-phase arrest in the colorectal cancer cell cycle and inhibiting the proliferation of colorectal cancer-resistant cells. It also induces apoptosis in colorectal cancer-resistant cells, inhibits the migration and invasion of colorectal cancer-resistant cells, reduces SOX2 expression in colorectal cancer-resistant cells, inhibits the stemness of colorectal cancer-resistant cells, thereby inhibiting the Wnt / β-catenin signaling pathway in colorectal cancer-resistant cells and reversing the resistance of colorectal cancer-resistant cells to 5FU. TNIK can become a potential therapeutic target for reversing 5FU resistance. The combination of TNIK expression inhibitors and 5FU provides a new research direction and treatment strategy for overcoming chemotherapy resistance in colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The expression of TNIK in HCT8-5FUR cells transfected with siRNA; A: qRT-PCR detection of TNIK mRNA expression in HCT8-5FUR cells transfected with three siRNA sequences of TNIK; B: Western blot detection of TNIK protein expression in HCT8-5FUR cells; C: grayscale analysis of TNIK protein.

[0030] Figure 2 Figure 3. Effects of different concentrations of 5FU on the viability of siRNA-transfected HCT8-5FUR cells. HCT8-5FUR cells were incubated with 5FU at concentrations of 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mM, and cell viability was assessed using CCK8 assay (*p < 0.05, **p < 0.01).

[0031] Figure 3 Figure 5 shows the colony formation results of HCT8-5FUR cells transfected with siRNA and treated with 5FU. Twenty-four hours after siRNA transfection, 5,000 cells were plated per well in a 6-well plate and cultured in complete medium containing 0.05 mM 5FU. After 10 days, the cells were stained with crystal violet and photographed.

[0032] Figure 4Figure 5: Effects of 5FU treatment on the proliferation of siRNA-transfected HCT8-5FUR and EdU-555 cells. A: HCT8-5FUR cells transfected with siRNA were incubated with 0.1 mM 5FU for 48 hours, stained with Hoechst 33342 and EdU, and the effect of 5FU on cell proliferation was observed under a fluorescence microscope. Hoechst 33342 is blue fluorescence; EdU is red fluorescence. B: Fluorescence images under the microscope were counted using Photoshop software, and a bar graph of the proportion of EdU-positive cells was plotted using Origin software. (*p < 0.05, **p < 0.01)

[0033] Figure 5 The effect of 5FU treatment on the cell cycle of HCT8-5FUR cells transfected with siRNA was shown. After siRNA transfection, HCT8-5FUR cells were treated with 5FU for 48 hours, and cell cycle distribution was assessed by flow cytometry. Cycle distribution data were first obtained using Modfit software, organized into tables, and then processed using Origin to plot histograms. (**p<0.01, ***p<0.001)

[0034] Figure 6 The effect of 5FU treatment on apoptosis in HCT8-5FUR cells transfected with siRNA was shown. After siRNA transfection, HCT8-5FUR cells were treated with 5FU for 48 hours. The intensity of the cells was double-stained with PI and Annexin V, and apoptosis was assessed by flow cytometry. Q1: Necrotic or mechanically damaged cells; Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Normal cells.

[0035] Figure 7 To establish a TNIK stable knockdown cell line; HCT8-5FUR cells were infected with lentivirus and screened with 4 μg / mL puromycin. TNIK mRNA expression levels were detected using qRT-PCR. (***p<0.001)

[0036] Figure 8 Western blot analysis of apoptosis-related protein expression; A: Western blot analysis of Bcl2, Bax, cleaved-caspase3, and cleaved-PARP1 protein expression. B: Grayscale analysis of Bcl2, Bax, cleaved-caspase3, and cleaved-PARP1 protein expression. (*p<0.05, **p<0.01)

[0037] Figure 9Figure 3: Effects of TNIK knockdown by shRNA lentiviral infection on HCT8-5FUR cell migration and invasion. A: After lentiviral infection of HCT8-5FUR cells to generate a TNIK stably knocked-down cell line, a wound wound healing assay was performed to assess cell migration. B: Wound wound area was obtained using Image J software, and data were collated and plotted using Origin software to plot wound healing percentages. C: After lentiviral infection of HCT8-5FUR cells to generate a TNIK stably knocked-down cell line, a Transwell Matrigel invasion assay was performed to assess cell invasion. D: Data were collated and plotted using Origin software to plot a bar graph. (**p < 0.01, ***p < 0.001)

[0038] Figure 10 Western blot analysis of the effect of shRNA lentiviral infection and TNIK knockdown on EMT-related proteins in HCT8-5FUR cells. A: After lentiviral infection of HCT8-5FUR cells to generate a TNIK stable knockdown cell line, changes in E-cadherin, N-cadherin, and Vimentin protein levels were detected by Western blot. B: Grayscale analysis of E-cadherin, N-cadherin, and Vimentin proteins. (**p<0.01, ***p<0.001)

[0039] Figure 11 Western blot analysis of the effect of TNIK inhibition on the Wnt / β-catenin signaling pathway in HCT8-5FUR cells. A: Changes in c-Myc, cyclinD1, and Survivin protein levels were detected by Western blot 72 hours after siRNA transfection in HCT8-5FUR cells. B: Grayscale analysis of c-Myc, cyclinD1, and Survivin proteins. (*p<0.05, ***p<0.001)

[0040] Figure 12 To investigate the effect of TNIK inhibition on the characteristics of HCT8-5FUR cancer stem cells; A: qRT-PCR analysis of SOX2 mRNA expression in HCT8-5FUR cells 48 hours after siRNA transfection. B: Western blot analysis of SOX2 protein expression in HCT8-5FUR cells 72 hours after siRNA transfection. C: Grayscale analysis of SOX2 protein. (**p < 0.01, ***p < 0.001)

[0041] Figure 13 This is a schematic diagram showing how inhibiting TNIK can reverse the resistance of colorectal cancer cells to 5FU. DETAILED DESCRIPTION

[0042] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0043] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application.

[0044] This application first systematically analyzed the HCT8-5FUR cell line model through CCK8 assay, cell clone formation assay and flow cytometry apoptosis assay. The results of CCK8 assay showed that the sensitivity of HCT8-5FUR resistant cells to 5FU was significantly reduced compared with the parental cells HCT8. 50 The expression of Wnt / β-catenin in HCT8-5FUR cells was significantly increased, indicating that the drug-resistant cells HCT8-5FUR have obvious resistance to 5FU. Cell clone formation experiments further confirmed that the clone formation ability of drug-resistant cells HCT8-5FUR after 5FU treatment was significantly higher than that of parental cells HCT8, supporting its drug-resistant phenotype. In addition, the results of flow cytometry detection of cell apoptosis showed that the apoptosis rate of drug-resistant cells HCT8-5FUR after 5FU treatment was significantly lower than that of parental cells HCT8, further verifying its drug-resistant characteristics. Western blot experimental results showed that compared with parental cells HCT8, the expression of key molecules of the Wnt / β-catenin pathway (β-catenin, c-Myc, etc.) in drug-resistant cells HCT8-5FUR was significantly increased, indicating that the pathway is overactivated in HCT8-5FUR cells. This finding suggests that abnormal activation of the Wnt / β-catenin signaling pathway may be involved in regulating the resistance of colorectal cancer cells to 5FU, laying an important molecular foundation for subsequent research. This application discovered that TNIK plays an important role in the Wnt / β-catenin signaling pathway, and confirmed through qRT-PCR and Western blot experiments that TNIK is highly expressed in colorectal cancer resistant cells, providing theoretical support for further research on the role of TNIK in drug resistance.

[0045] Example 1: Synthesis of siTNIK-1, siTNIK-2, and siTNIK-3

[0046] 1.siRNA design:

[0047] 1) Use the NCBI (National Center for Biotechnology Information) website to search for TNIK and obtain the FASTA format sequence.

[0048] 2) Use the DSIR website (DSIR Home page) to input the TNIK gene and FASTA format sequence, select siRNA 21nt, click "Run analysis", and select the top-ranked sequences for synthesis.

[0049] The final designed nucleotide sequences are shown as siTNIK-1 in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0050] 2. siRNA synthesis:

[0051] The solid-phase synthesis method (phosphoramidite triester method) is adopted, with a solid phase carrier (such as controlled pore glass beads) as the support. By gradually coupling phosphoramidite monomers (nucleotides with protective groups), the steps of deprotection, coupling, capping and oxidation are completed in sequence to finally synthesize the target sequence.

[0052] 1) Carrier fixation: The first nucleotide is fixed on the solid phase carrier through the connecting arm.

[0053] 2) a-cycle synthesis (the following steps need to be repeated for each nucleotide added)

[0054] b Deprotection: Use an acidic reagent (such as trichloroacetic acid) to remove the 5'-terminal DMT protecting group

[0055] c Coupling: Add phosphoramidite monomer (containing 5'-DMT protecting group) and activator (tetrazole) to form phosphite triester bond with nucleotide on the carrier

[0056] c Capping: Use acetic anhydride to block unreacted hydroxyl groups and reduce by-products

[0057] d Oxidation: Use iodine solution to oxidize the phosphite bond to a stable phosphotriester bond

[0058] 3) Cleavage and deprotection: Use ammonia or methylamine to cleave the connection between the nucleic acid chain and the carrier and remove the base protecting groups (such as benzoyl and isobutyryl)

[0059] 4) Purification: Purify the target product by HPLC or PAGE.

[0060] Example 2: Using siTNIK-1, siTNIK-2, siTNIK-3, shTNIK-1, and shTNIK-2 prepared in Example 1 to inhibit TNIK and reverse the drug resistance of colorectal cancer 5FU-resistant cells

[0061] 1 Experimental Materials

[0062] 1.1 Preparation of 5FU-resistant colorectal cancer cells

[0063] The human colorectal cancer cell line HCT8 and 5-fluorouracil-resistant HCT8 (HCT8-5FUR) cells used in this experiment were kindly donated by Professor Song Zhenbo's laboratory at Northeast Normal University.

[0064] HEK293T cells were used for lentiviral packaging, and HCT8-5FUR cells were infected with the virus solution to obtain the HCT8-5FUR cell TNIK stably knocked-down cell line.

[0065] 1.2 Experimental reagents and equipment

[0066]

[0067]

[0068]

[0069] 2 Experimental methods

[0070] 2.1 Human colorectal cancer cell culture

[0071] (1) Preparation of reagents

[0072] 1) RPMI-1640 complete medium: RPMI-1640 basal solution (containing 1% double antibody) supplemented with 10% fetal bovine serum

[0073] 2) PBS: 4 g NaCl, 0.72 g Na2HPO4, 0.1 g KCl, and 0.12 g KH2PO4 were dissolved in 500 mL ddH2O. The pH was measured with a pH meter to be within the range of pH 7.2-7.4.

[0074] 3) 0.25% Trypsin: Dissolve 0.5 g trypsin powder and 0.04 g EDTA in 200 mL of sterile, cooled PBS and stir until dissolved. Filter through a 0.22 μm microporous filter membrane and aliquot into 200 mL glass bottles for later use. Store at 4°C.

[0075] (2) Cell recovery

[0076] 1) Take out the reagents needed for cell treatment and heat them in a 37°C water bath.

[0077] 2) According to the size of the culture dish, aspirate the corresponding culture medium in the clean bench, add 6mL RPMI-

[0078] 1640 complete medium: add 3 mL of RPMI-1640 complete medium to a 6 cm culture dish.

[0079] 3) Thaw the cryopreserved tube of cells to be revived in warm water at about 37°C; centrifuge at room temperature at 1000 rpm for 3 minutes and discard the upper layer of cryopreserved solution.

[0080] 4) Pipette 1 mL of fresh RPMI-1640 complete medium into the cryovial and gently resuspend the cells.

[0081] 5) Inoculate the cell suspension into a culture dish, shake it evenly using the "M" and "8" methods, and place it in an incubator (37°C, 5%

[0082] CO2) and cultured for 24 h.

[0083] (3) Cell passage

[0084] 1) Same as cell recovery step 1).

[0085] 2) In a clean bench, discard the remaining culture medium and add 2 mL of PBS to the cell culture dish along the wall. Repeat the washing process three times.

[0086] 3) Discard the third PBS and add 1 mL of prepared trypsin solution. Shake well and place in an incubator for digestion (3 minutes for HCT8 and 5 minutes for HCT8-5FUR). When cells become rounded under a microscope, digestion is complete. Place in a clean bench and add 3 mL of RPMI-1640 complete medium to terminate digestion.

[0087] 4) Gently blow the cells to form a single-cell suspension until no cells adhere to the culture dish and the cell clusters are evenly dispersed in the solution. At this time, you can observe that the bottom of the cell culture dish changes from frosted to transparent, indicating that the cells have been completely digested. Transfer the single-cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 3 minutes.

[0088] 5) After centrifugation, discard the supernatant medium and add 1 mL of RPMI-1640 complete medium to prepare a single-cell suspension. Transfer the suspension to a new culture dish and shake well using the "R" or "8" method. Place the suspension in an incubator (37°C, 5% CO2) and continue culturing.

[0089] (4) Cell cryopreservation

[0090] 1) Same as cell passaging step 1).

[0091] 2) Same as cell passaging step 2).

[0092] 3) Same as cell passaging step 3).

[0093] 4) Same as cell passaging step 4).

[0094] 5) After centrifugation, discard the supernatant medium and resuspend the cells in 600 μL of RPMI-1640 complete medium to make a single-cell suspension. Add 300 μL of serum and 100 μL of DMSO to the cryovial to achieve a complete medium: serum: DMSO ratio of 6:3:1.

[0095] 6) After mixing, place the cell cryopreservation tube in a freezing box and freeze it in a -80°C refrigerator. After 2-3 days, transfer it to a liquid nitrogen tank for storage.

[0096] (5) Cell transfection

[0097] Take each well of a six-well plate as an example:

[0098] 1) Cultivate HCT8-5FUR cells until they are in good condition. Wash and digest the cells as in steps 1), 2), and 3).

[0099] 2) Collect the cell pellet by centrifugation to prepare a single cell suspension, similar to the cell passaging step 4)

[0100] 3) Place an appropriate volume of cell suspension in a 6-well plate and allow the cell suspension to reach a density of 60%-70% after 24 hours.

[0101] 4) When HCT8-5FUR cells grow to 60%-70% confluency, replace the serum-free medium and starve the cells for 4-6 hours.

[0102] 5) Prepare transfection mixtures A and B. Solution A: 50 μL serum-free, antibody-free RPMI-1640 medium, 2 μL liposome 2000, and 4 μL siNC or siTNIK (final concentration: 20 μM). Mix by pipetting. Solution B: 50 μL serum-free, antibody-free RPMI-1640 medium. Mix by pipetting and let stand at room temperature for 5 minutes. (The final transfection concentration of siRNA during transfection is 100 nM. The volume of siRNA to be pipetted will be determined based on the final concentration.)

[0103] 6) Add solution B to solution A to make a mixture of solution AB. Let it stand at room temperature for 20 minutes, then add it dropwise to a 6-well plate.

[0104] 7) Incubate in an incubator for 5 minutes, then add an appropriate amount of serum-free and antibody-free RPMI-1640 medium to a volume of 800 μL.

[0105] 8) After 6-8 hours, replace the culture medium with 2 mL of RPMI-1640 complete medium and continue culturing. Perform subsequent cell experiments according to the experimental purpose.

[0106] Cell clone formation assay

[0107] 1) Use cell clone survival assays to verify that HCT8-5FUR is drug-resistant: Culture HCT8 and HCT8-5FUR cells, and when cells are in good condition and have an appropriate density, process the cells. Wash and digest the cells using the same procedures as described in cell passaging 1), 2), and 3).

[0108] 2) Collect the cell pellet by centrifugation and obtain a single cell suspension as in step 4).

[0109] 3) The steps are the same as the cell counting steps, and finally 5×10 3 cells.

[0110] 4) Mix the cells in the 6-well plate using the "M" and "8" techniques alternately. Place the cells in an incubator and culture. When two to three colonies have formed, add 2.5 mL of 10% RPMI-1640 medium or a 5FU solution prepared in 10% RPMI-1640 medium at the appropriate concentration. Culture the cells in a 37°C incubator for 10 days.

[0111] 5) After 10 days, observe under a microscope, discard the original culture medium, wash three times with pre-cooled PBS, add 500-1000 μL of 4% paraformaldehyde to each well, and fix at room temperature for 15 minutes.

[0112] 6) Discard the fixative and wash with PBS. Add 200 μL of crystal violet to each well and stain for 15 minutes at room temperature in the dark. Rinse again with PBS until the background is clear. Remove the lid of the 6-well plate and invert it on a piece of white paper for photography.

[0113] 2.2 Cell Counting

[0114] After trypsinization, terminate the digestion of cells with 1 mL of fresh PRMI-1640 complete medium. Mix thoroughly with a pipette to create a single-cell suspension. Transfer an appropriate amount of the single-cell suspension to a new 1.5 mL centrifuge tube and dilute to a total volume of 1 mL with complete medium. Count the cells by taking 80 μL from the new centrifuge tube. Cover the hemocytometer with a coverslip, then pipette 20 μL of the mixed cell suspension and add it to the hemocytometer. Do not remove the coverslip during the addition process. Let the suspension stand for a while before counting under an inverted microscope.

[0115] The calculation formula is as follows: number of cells / mL = total number of cells in eight large squares / 8×10 4 .

[0116] 2.3 CCK8 Experiment

[0117] The CCK8 assay was used to analyze the effects of different concentrations of 5FU on the cell activity of siNC / siRNA.

[0118] 1) Cultivate HCT8-5FUR cells. When the HCT8-5FUR cells are in good condition and the density reaches about 80%, perform the washing and digestion steps in the same manner as cell passaging steps 1), 2), and 3).

[0119] 2) Collect the cell pellet by centrifugation and obtain a single cell suspension as in step 4 of cell passaging.

[0120] 3) Take an appropriate volume of the suspension and inoculate it into a 6-well plate so that the cell density reaches 50%-60% after 24 hours.

[0121] 4) The cell transfection steps are the same as above.

[0122] 5) 24 hours after transfection, trypsinize the cells, terminate the digestion with the culture medium, obtain the cell suspension by centrifugation, and re-inoculate into a 96-well plate.

[0123] 6) Take 50 μL of cell suspension, adjust the volume to 1 mL, pipette and mix, then count the cells in the cell suspension using a hemocytometer. Calculate the total number of cells required and the volume of cell suspension required. Add 7×10 cells per well of a 96-well plate. 3 cells.

[0124] 7) After observation under a microscope, the 96-well plate was placed in an incubator for 24 hours. After the time was up, 150 μL RPMI-1640 was added to each well.

[0125] Basal culture medium or a series of concentration gradient solutions (0, 0.2, 0.4, 0.8, 1.6,

[0126] 3.2 mM), and then placed in a 37°C incubator for 24 h and 48 h.

[0127] 8) After 24 and 48 hours, discard the original culture medium and prepare CCK8 diluent (CCK8:RPMI-1640 basal medium at a ratio of 1:9) in the dark. Add 100 μL of CCK8 diluent to each well, wrap the 96-well plate with tin foil, and incubate in an incubator for 1 hour.

[0128] 9) After incubation, measure the absorbance at 450 nm using a microplate reader, record the data, and calculate the cell viability using the formula: viability = (experimental group - blank group) / (control group - blank group).

[0129] 2.4 Cell clone formation experiment

[0130] 1) HCT8-5FUR cells in good growth condition were seeded into 6-well plates so that the cell density reached 50%-60% on the next day.

[0131] 2) Cell transfection: the same steps as above.

[0132] 3) 24 hours after transfection, trypsinize the cells again to collect the cell suspension and perform cell colony formation experiments.

[0133] 2.5 EdU-555 cell proliferation assay

[0134] 1) Cultivate HCT8-5FUR cells and process them when they are in good condition. Wash and digest the cells in the same way as in cell passaging step 1).

[0135] 2), 3).

[0136] 2) Collect the cell pellet by centrifugation and obtain a single cell suspension in the same manner as in step 4 of cell passaging.

[0137] 3) Place an appropriate volume of cell suspension in a 6-well plate so that the density reaches 60% after 24 hours.

[0138] 4) Perform cell transfection experiments on the next day, following the same steps as in 2.1.2.2.

[0139] 5) After 24 hours, the transfected cells were digested with complete medium to terminate the digestion. The cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cell pellet was collected.

[0140] 6) Take 50 μL of cell suspension and count them. 12×10 cells per well of a 24-well plate 4 cells.

[0141] 7) After culturing in the incubator for 24 hours, 300 μL of serum-free RPMI-1640 medium or a series of 5FU concentration gradient solutions prepared with serum-free RPMI-1640 medium was added to each well and cultured in the incubator for 48 hours.

[0142] 8) Perform EdU detection 48 hours after drug treatment; prepare 2× EdU working solution. Preheat the EdU working solution to 37°C before use. Discard 150 μL of culture medium from the 24-well plate and then add an equal volume (150 μL) of 2× EdU working solution to a final EdU concentration of 1×. Continue incubation for 4 hours.

[0143] 9) Discard the culture medium, add 100 μL of 4% paraformaldehyde, and fix at room temperature for 15 minutes.

[0144] 10) Discard the 4% paraformaldehyde and wash each well three times with 200 μL of washing solution (3% BSA in PBS), each time for 3-5 minutes.

[0145] 11) Discard the wash solution, add 100 μL of permeabilization solution (0.3% Triton X-100 in PBS) to each well, incubate at room temperature for 15 minutes, discard the permeabilization solution, and wash three times with 200 μL of wash solution per well, each time for 3-5 minutes.

[0146] 12) Prepare the Click reaction solution (using one well of a 24-well plate as an example): add 107.5 μL of Click Reaction Buffer, 5 μL of CuSO4, 0.25 μL of Azide 555, and 12.5 μL of Click Additive Solution. Add the corresponding volumes of solution in this order and use within 15 minutes.

[0147] 13) Discard the wash buffer and add 125 μL of Click reaction solution to each well. Gently shake to ensure the reaction mixture evenly covers the cells. Incubate at room temperature in the dark for 30 minutes.

[0148] 14) Discard the Click reaction solution and wash three times with 200 μL of wash buffer, each time for 3-5 minutes. Prepare 1× Hoechst 33342, discard the wash buffer, and incubate at room temperature in the dark for 10 minutes.

[0149] 15) Discard the 1× Hoechst 33342 and wash each well three times with 100 μL of washing buffer, each wash for 3-5 minutes. Photograph under a fluorescence microscope.

[0150] 2.6 Flow cytometry detection of cell apoptosis

[0151] 1) Cultivate HCT8-5FUR cells and wait until the cells are in good condition. Wash and digest the cells in the same way as in cell passage steps 1) and 2).

[0152] 3).

[0153] 2) Collect the cell pellet by centrifugation and obtain a single cell suspension in the same manner as in step 4 of cell passaging.

[0154] 3) Place an appropriate volume of cell suspension in a 6-well plate so that the density reaches 60% after 24 hours.

[0155] 4) Carry out cell transfection experiment on the next day.

[0156] 5) After 24 hours, the transfected cells were digested with complete medium to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the precipitated cells were collected.

[0157] 6) Plate an appropriate volume of the cell suspension onto a 6-well plate and incubate in a 37°C incubator for 24 hours. Discard the original culture medium and add 1 mL of serum-free culture medium or 5FU solution of the appropriate concentration to each well. Incubate in a 37°C incubator for 48 hours.

[0158] 7) After 48 hours, the cells were collected together with the supernatant to prepare a single-cell suspension.

[0159] 8) Stain with PI and Annexin V (Yishen apoptosis kit).

[0160] 9) Flow cytometer was used for detection within 1 hour. The experimental results were analyzed using Flwojo software and a scatter plot was drawn.

[0161] 2.7 Flow cytometry cell cycle assay

[0162] 1) Cultivate HCT8-5FUR cells and wait until the cells are in good condition. Wash and digest the cells in the same way as in cell passage steps 1) and 2).

[0163] 3).

[0164] 2) Collect the cell pellet by centrifugation and obtain a single cell suspension in the same manner as in step 4 of cell passaging.

[0165] 3) Place an appropriate volume of cell suspension in a 6-well plate so that the density reaches 60% after 24 hours.

[0166] 4) Carry out cell transfection experiment on the next day.

[0167] 5) After 24 hours, the transfected cells were digested with complete medium to terminate the digestion. The cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the precipitated cells were collected.

[0168] 6) Take an appropriate volume of cell suspension and plate it on a 6-well plate and culture in an incubator for 24 hours.

[0169] 7) Discard the original culture medium of the 6-well plate, add 1.5 mL of serum-free RPMI-1640 medium or 5FU solution prepared with serum-free RPMI-1640 medium to each well, and culture in an incubator for 48 hours.

[0170] 8) After 48 hours, wash the cells 2-3 times with pre-cooled PBS, digest with trypsin, terminate digestion with complete medium, and centrifuge at 1000 rpm.

[0171] After 5 min, the supernatant was discarded and the precipitated cells were collected to prepare a single cell suspension and stained with PI (KGI Cycle Kit).

[0172] 9) Prepare cell suspension, count on a hemocytometer, and adjust the cell concentration to 1×10 6 / mL,

[0173] 10) Take 1 mL of the cell suspension, centrifuge it, remove the supernatant, add 500 μL of 70% cold ethanol to the cells for fixation, and store at 4°C overnight.

[0174] 11) Wash the cells treated overnight three times with pre-cooled PBS, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 500 μL of the previously prepared PI / RNase A (Keygen Cycle Kit) staining solution to the cell pellet, and incubate at room temperature in the dark for 30 minutes.

[0175] 12) Flow cytometry was used for detection within 1 hour. The experimental data were analyzed using Modfit software and cell cycle histograms were drawn using Origin.

[0176] 2.8 Construction of shTNIK Stable Knockdown Cell Line

[0177] (1) Preparation of TNIK stable knockdown plasmid

[0178] 1) shRNA design:

[0179] a) Using NCBI ( National Center for Biotechnology Information ) website, search for TNIK, and obtain the FASTA format sequence.

[0180] b) Using DSIR ( DSIR Home page ) website, input the TNIK gene and FASTA format sequence, select shRNA, click "Run analysis", select the top-ranked SS sequence with a high score as the target (nucleotide sequence is shTNIK-1 shown in SEQ ID NO: 4 and shTNIK-2 shown in SEQ ID NO: 5), and add sticky ends with restriction enzyme cutting sites to the 5' and 3' ends of the primers.

[0181] 2) shRNA synthesis:

[0182] a) PCR amplification of target gene: Use high-fidelity PrimeSTAR enzyme to amplify the target gene. The reaction system and conditions are as follows:

[0183]

[0184]

[0185] PCR reaction (30 cycles):

[0186]

[0187] b) Agarose gel detection of amplification effect and recovery of DNA fragments

[0188] ① Prepare 1% agarose gel, add the PCR product to the agarose gel, and run the gel at 80V constant voltage for 30 minutes;

[0189] For UV gel imaging, cut the target band with a knife and place it in a 1.5 mL EP tube;

[0190] ② Weigh and add 300 μL of Buffer B2 per 100 mg of agarose gel into the EP tube;

[0191] ③Preheat the water bath to 50℃ in advance, place the agarose gel with Buffer B2 in a 50℃ water bath and heat for 10 minutes, inverting the EP tube several times during this period to fully dissolve the gel;

[0192] ④ Transfer the dissolved liquid to the adsorption column and centrifuge at 12000 rpm at room temperature for 30 seconds; discard the solution in the lower collection tube after centrifugation;

[0193] ⑤ Add 300 μL of Buffer B2 to the adsorption column, centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the liquid in the lower collection tube;

[0194] ⑥ Add 20 μL of preheated wash solution or ddH2O to the adsorption column, centrifuge at 12000 rpm for 30 seconds, discard the liquid in the collection tube, and return it to the adsorption column;

[0195] ⑦Repeat step 7;

[0196] ⑧Put the empty collection tube and adsorption column into the centrifuge and centrifuge at 12000 rpm for 1 min;

[0197] ⑨ Add 20 μL of preheated Elution Buffer to the center of the adsorption membrane, let it stand at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute;

[0198] ⑩ Pipette the liquid in the EP tube and add it back to the adsorption column to improve the recovery efficiency. Centrifuge again at 12000rpm for 1min. Pipette 1μL DNA solution and measure the DNA concentration using Nanodrop

[0199] c) Vector linearization

[0200] Add according to the following reaction system, bathe in 37℃ water for 30min, digest with enzymes and run on gel, recover vector fragments on gel and measure concentration using Nanodrop.

[0201]

[0202]

[0203] d) Homologous recombination

[0204] Add the target gene fragment and linearized vector to a centrifuge tube at a molar ratio of 3:1 for ligation. Generally, 100 ng of linearized expression vector is added, and the target gene fragment is added in an amount equal to 300 x the number of base pairs of the target gene / the number of base pairs of the linearized expression vector (in ng). The ligation reaction system (20 uL) is as follows:

[0205]

[0206] The above ligation solution was ligated at 16℃ overnight

[0207] e) Conversion

[0208] ①After taking out the DH5α competent cells from the -80℃ refrigerator, they should be immediately placed on ice to thaw. The competent cells should be packaged gently to reduce mechanical damage.

[0209] ② After the competent medium is thawed, aliquot it into 50 μL volumes per tube (20 μL is sufficient for plasmid transformation). After aliquoting, add the ligation product in an amount no more than 1 / 10 of the competent medium volume (currently add 5 μL of ligation product) and place it on ice for 20-30 minutes.

[0210] ③ Heat shock at 42℃ for 90 seconds (this time must be very strict), and immediately put it on ice for 2-3 minutes after heat shock;

[0211] In a clean bench, add 500 μL LB medium (note that it must be antibiotic-free LB medium) and gently invert it 3-5 times;

[0212] ④Incubate at 37°C, 230 rpm, shaking for 45-60 min;

[0213] ⑤ Apply the bacterial solution to a solid plate of the corresponding resistance, spread it evenly, and then incubate the plate upside down at 37°C for 12-16 hours; f) PCR identification of bacterial solution

[0214] Pick the transformants grown on the plate and resuspend them in 10 μl of LB culture medium. Take 1 μl as a template for colony PCR identification. The reaction system and PCR cycling conditions are as follows:

[0215] ① Bacterial liquid PCR identification system:

[0216]

[0217] ② Bacterial liquid PCR identification procedure

[0218]

[0219] g) Sequencing

[0220] h) Plasmid extraction

[0221] ① After successful sequencing, the bacterial solution was amplified and the plasmid was extracted and purified using the Novezan FastPure EndoFree Plasmid Mini Plus Kit. The colonies verified as positive clones by colony PCR were picked and placed in LB medium with ampicillin added. The culture was placed in a 37°C constant temperature incubator with shaking for 16 hours.

[0222] ② After the bacterial culture has fully grown, centrifuge at 4500 rpm for 10 minutes at room temperature to collect the cells. Take 5-15 mL of the overnight culture and centrifuge at 12,000 rpm (13,400 × g) for 1 minute to collect the cells. Aspirate as much of the remaining supernatant as possible.

[0223] ③ Add 500 μL of Buffer P1 (check whether RNase A Solution has been added first), vortex until the cells are completely resuspended, immediately add 500 μL of Buffer P2, gently invert up and down 8-10 times to mix, and let it stand at room temperature for 3 minutes.

[0224] ④ Add 500 μL of Buffer N3 and immediately and gently invert the tube 12-15 times until the blue color disappears completely and a white flocculent precipitate appears. Incubate at room temperature for 10 min. Centrifuge at 12,000 rpm (13,400 × g) for 10 min. Transfer the supernatant (approximately 1.5 mL) to a 5 mL centrifuge tube (self-provided). Add 0.3 times the volume of supernatant (approximately 450 μL) of isopropanol and mix thoroughly by inverting the tube 10-15 times.

[0225] ⑤ Column equilibration: Place FastPure DNA Mini Columns III in 2 mL Collection Tubes. Add 200 μL of Buffer QB to the FastPure DNA Mini Columns III and centrifuge at 12,000 rpm (13,400 × g) for 1 minute. Discard the filtrate and set aside. ⑥ Transfer the mixture from step 5 to the adsorption column and centrifuge at 12,000 rpm (13,400 × g) for 1 minute. Discard the filtrate.

[0226] Repeat step ⑤ until all the mixed solution is loaded onto the column.

[0227] ⑦ Add 500 μL of Buffer PW1 to the adsorption column, centrifuge at 12,000 rpm (13,400 × g) for 1 min, discard the filtrate, add 600 μL of Buffer PW2 (please check whether anhydrous ethanol has been added) to the adsorption column, centrifuge at 12,000 rpm (13,400 × g) for 1 min, and discard the filtrate.

[0228] ⑧Repeat step ⑦.

[0229] 9. Return the adsorption column to the collection tube and centrifuge at 12,000 rpm (13,400 × g) for 2 minutes to dry the column. Place the column in a new 1.5 mL centrifuge tube (prepare your own) and add 60-200 μL of Endotoxin-free Elution Buffer to the center of the column membrane. Let stand at room temperature for 1 minute. Centrifuge at 12,000 rpm (13,400 × g) for 1 minute and discard the column.

[0230] ⑩The extracted plasmid DNA was stored at -30~-15℃.

[0231] (2) Packaging

[0232] 1) Resuscitate HEK293T cells and culture them in fresh DMEM complete medium in a 100 mm culture dish. When the HEK293T cells in the culture dish grow to 90% density, subculture them into a 60 mm culture dish and continue culturing for transfection.

[0233] 2) When the HEK293T cells in the 60 mm culture dish grew to 80%, they were starved and cultured for 1-2 h using serum-free and dual antibody-free DMEM medium.

[0234] 3) Prepare the packaging system of Lipo2000, shTNIK, psPAX2, pMD2G, and serum-free DMEM. Taking a 60 mm culture dish as an example, the packaging system is as follows:

[0235]

[0236] 4) Transfection:

[0237] a Take a 60mm culture dish as an example: prepare a mixture of liposomes and three plasmids in a 1.5mL centrifuge tube. Liquid A is 200μL serum-free and antibody-free DMEM medium, and liquid B includes 200μL of the same medium, 2500ng knockdown plasmid, 1850ng

[0238] psPAX2, 650 ng of pMD2G plasmid and 5 μL of liposomes were mixed by gently pipetting and allowed to stand at room temperature for 5 min.

[0239] b. Transfer the mixed solution in tube B to tube A, mix gently and evenly, and let it stand at room temperature for 20 minutes.

[0240] c. Add the transfection mixture dropwise to the HEK293T cells. Incubate in the incubator for 5 minutes to improve transfection efficiency.

[0241] d. Add 1750 μL of serum-free and dual-antibody-free DMEM medium to a 60 mm culture dish and incubate in an incubator for 6-8 hours. After 6-8 hours, replace with complete DMEM medium and continue incubation for 48-72 hours.

[0242] 5) Collect the cell culture medium containing the virus released after HEK293T cell packaging at 48 h and 72 h respectively. Centrifuge the collected culture medium at 1000 rpm for 5 min, filter the virus liquid through a 0.48 μm microporous filter membrane, and package it. Store it in a refrigerator at -80°C until use to avoid repeated freezing and thawing.

[0243] (3) Viral fluid infection

[0244] 1) Wait until cells are in good condition. Wash and digest the cells in the same manner as in steps 1), 2), and 3).

[0245] 2) Collect the cell pellet by centrifugation and obtain a single cell suspension in the same manner as in step 4 of cell passaging.

[0246] 3) Place an appropriate volume of cell suspension in a 24-well plate so that the density reaches 60% after 24 hours.

[0247] 4) Virus infection was performed in a 24-well plate. The collected aliquoted virus solution was taken out from the -80°C freezer, slowly thawed on ice, and the virus solution was mixed with fresh complete medium containing 20% FBS in a 1:1 ratio.

[0248] 5) Take out the HCT8-5FUR cells to be infected, discard the old culture medium, wash with PBS, and add the above mixture to each well.

[0249] 300 μL was added to a 24-well plate.

[0250] 6) 48 hours after virus infection, the expression of green fluorescent protein (the knockdown plasmid carries a green fluorescent protein tag) can be observed under a fluorescence microscope to observe the infection efficiency.

[0251] (4) Puromycin screening of stable knockdown cell lines

[0252] 1) After 48 h, discard the culture medium containing the virus solution, wash with PBS, and continue screening with complete medium containing 6.0 μg / mL puromycin for 7 days. During this period, replace the fresh screening medium every 2 days. If the cell state is not good, replace it with complete medium without puromycin once in the middle. After one day of incubation, add complete medium containing puromycin again.

[0253] 2) The cells that survived the screening in the 24-well plate for one week were digested and inoculated into 6-well plates for further culture.

[0254] When the density reaches 90%, the cells are digested and subcultured, and transferred to 60mm culture dishes. When the cells grow to 90% density again, part of them are preserved as seeds and part of them are expanded.

[0255] 3) To verify the knockdown efficiency, total RNA was extracted from the cells successfully screened as described above, and the knockdown efficiency was verified by real-time quantitative PCR (qRT-PCR). The successfully knocked-down HCT8-5FUR cells were cultured for subsequent experiments.

[0256] 2.9 Cell scratch assay

[0257] 1) Mark the bottom of a 6-well plate by drawing a line. HCT8-5FUR, shTNIK-1, and shTNIK-2 cells in good growth condition were digested and passaged into the 6-well plate for culture until the cells were confluent.

[0258] 2) Use a 200 μL pipette tip to draw a straight line on the bottom of the dish along the ruler, then discard the original culture medium and wash with PBS to remove floating dead cells and cells attached to the horizontal line.

[0259] 3) Add 1.5 mL of serum-free culture medium, take pictures under an inverted microscope, and use this as the 0 h of cell migration for statistical analysis.

[0260] 3) The culture medium was then added to a 6-well plate, cultured for 48 h, and photographed under an inverted microscope.

[0261] 4) Count the cell migration area at 48 h and analyze the results at 0 h and 48 h.

[0262] 2.10 Transwell invasion assay

[0263] 1) Matrigel treatment: The night before the invasion experiment, seal the pre-packed Matrigel (10 mg / mL) centrifuge tube with sealing film, remove it from -20°C and slowly thaw it at 4°C.

[0264] 2) Dilute Matrigel to a final concentration of 1 mg / mL with 4°C pre-cooled serum-free medium. Add 80 μL of Matrigel dilution vertically to the center of the upper chamber of the chamber and incubate in a cell culture incubator for 2-4 hours to allow the gel to solidify.

[0265] 3) The desired experimental cells were digested with trypsin, digested with complete medium, centrifuged, washed with sterile PBS, and resuspended in medium without FBS. The cells were counted to a final concentration of 4 × 10 5 pcs / mL

[0266] 4) Add 600 μL of 10% FBS complete medium to the lower chamber and 100 μL of the cell suspension to the lower chamber. Incubate in an incubator for 48 hours. Note: After 2 hours of incubation, remove the cells and observe under a microscope for bubbles. Remove any bubbles if necessary.

[0267] 5) After 48 hours, remove the chamber with forceps, remove the culture medium, rinse twice with PBS, add 100ul of 4% paraformaldehyde to the upper chamber and 600ul of 4% paraformaldehyde to the lower chamber respectively, and fix at room temperature for 30-40 minutes.

[0268] 6) Aspirate the fixative, rinse the cells 2-3 times with PBS, add 600 μl of 1% crystal violet staining solution, and stain at room temperature in the dark for 15-20 minutes.

[0269] 7) Wash the chamber with PBS and gently wipe off the cells in the upper chamber with a cotton swab. Observe, photograph, and count the cells under an inverted microscope.

[0270] 2.11 Western blot experiment

[0271] 1) After the SDS-PAGE gel has solidified (approximately 20 minutes), rinse the gel plate with ddH2O. Then, assemble the electrophoresis tank and add 500 mL of 1× Running buffer. Immerse the glass gel plate in 1× Running buffer. Carefully remove the comb vertically and flush the gel wells with a pipette. Set a constant voltage of 60 V and run for 20 minutes.

[0272] 2) After 20 minutes, add 2.2 μL of protein marker (the size of the marker is determined by the band size of the target protein) and a series of protein samples (the protein concentration is measured before loading).

[0273] 3) Run electrophoresis at a constant voltage of 80V until all protein marker bands have moved from the stacking gel to the separation gel. Then switch to a constant voltage of 120V and continue. The time should be adjusted according to the molecular weight of the target protein.

[0274] 4) Measure the desired band size and position with a comb, cut a piece of PVDF membrane to the appropriate size, and mark the front and back sides and the order of spotting by cutting off the top left corner. Soak the cut PVDF membrane in methanol to activate it.

[0275] 5) Turn off the electrophoresis apparatus, remove the glass gel plate, and use a tool to tilt the short glass plate. Then, cut off the top layer of concentrated gel. Place the remaining lower layer of separating gel on a transfer cassette. Soak the cassette in transfer buffer and cover it with PVDF membrane. Use a hard card or other tool to remove any bubbles between the PVDF membrane and the gel. Once there are no bubbles, secure the cassette in place and place it in the electrophoresis tank. Add 1x transfer buffer and place the tank in an ice-water bath. Transfer the membrane at a constant current of 250 nA. The transfer time will vary depending on the size of the target band.

[0276] 6) After the transfer time is up, place the PVDF membrane in a sealed box containing 5% skim milk prepared in advance and incubate at room temperature for 1.5 hours.

[0277] 7) After the blocking time is up, recycle the blocking solution and rinse the PVDF membrane three times with TBST, each time using a shaker at the fast setting for 8 minutes. Cut the PVDF membrane according to the desired band size and place the corresponding PVDF membrane in the corresponding primary antibody (the antibody is diluted with 1× TBST; the specific antibody dilution ratio is shown in Appendix 1). After completion, incubate in a shaker at 4°C overnight.

[0278] 8) The next morning, after the primary antibody incubation is complete, recover the primary antibody and store it in a 4°C refrigerator for short-term use. For longer-term use, store it in a -20°C refrigerator. Wash the membrane three times, using a shaker at the fast setting for 8 minutes each time.

[0279] 9) After washing, add the corresponding secondary antibody to the corresponding incubation box and incubate at room temperature for 1 hour.

[0280] 10) After secondary antibody incubation, recover the secondary antibody and store it in a 4°C refrigerator for short-term use or in a -20°C refrigerator for long-term use. Wash the membrane three times, using a shaker at the fast setting for 8 minutes each time.

[0281] 11) After cleaning, prepare the required ECL colorimetric solution in a 1:1 ratio, then immerse the PVDF membrane in the prepared ECL colorimetric solution, expose with a Tanon 5200 scanner, and save the photo.

[0282] 2.12 Statistics

[0283] Experimental data are presented as mean ± standard deviation. Two-group comparisons were performed using a two-tailed T test, and multiple-group comparisons were performed using one-way analysis of variance. The significance level was set at: *p < 0.05, **p < 0.01, ***p < 0.001.

[0284] 3 Experimental results

[0285] 3.1 Knockdown of TNIK enhances the sensitivity of colorectal cancer resistant cells to 5FU

[0286] TNIK was initially studied for its promotion of migration and proliferation. Recent studies have shown that the kinase activity of TNIK plays a role in cancer, metabolic diseases, and neurotransmission. In addition, during tumor progression, TNIK can help promote tumorigenesis, drive cell invasion, and promote drug resistance. [89,90,117] . In the second chapter of this application, it was found that compared with the parent cell HCT8, the mRNA and protein levels of TNIK in the drug-resistant cell HCT8-5FUR were significantly increased. In order to verify whether inhibiting TNIK can increase the sensitivity of HCT8-5FUR to 5FU, this application reduced the expression level of TNIK in HCT8-5FUR cells by transfecting siRNA, and detected the knockdown efficiency by qRT-PCR. The results showed that compared with the control group transfected with non-specific siRNA (siNC), the expression of TNIK in HCT8-5FUR cells was significantly reduced after transfection with siTNIK-1, siTNIK-2, and siTNIK-3, among which the relative expression level of siTNIK-1 decreased by 76.7%, the relative expression level of siTNIK-2 decreased by 77.7%, and the relative expression level of siTNIK-3 decreased by 72.8% ( Figure 1 Based on the siRNA knockdown efficiency, siTNIK-1 and siTNIK-2 were selected for subsequent studies. To further verify the TNIK knockdown effect, protein was extracted 72 hours after transfection. Western blot analysis showed that siRNA transfection significantly reduced the protein expression level of TNIK in HCT8-5FUR cells compared with the siNC group ( Figure 1 Middle B).

[0287] In order to verify whether knocking down TNIK can reverse the resistance of colorectal cancer resistant cells to 5FU, this application transfected siNC, siTNIK-1, and siTNIK-2, and then performed a CCK8 experiment. Set a 0mM, 0.1mM, 0.2mM, 0.4mM, 0.8mM, 1.6mM and 3.2mM 5FU concentration gradient, treat for 48h, incubate with CCK8, and use a microplate reader to detect the effect of 5FU on cytotoxicity under different concentration gradients. The experimental results showed that compared with the control group siNC, HCT8-5FUR cells transfected with siTNIK-1 and siTNIK-2 were more sensitive to 5FU, and as the drug concentration increased, 5FU was more toxic to HCT8-5FU cells transfected with siTNIK-1 and siTNIK-2 ( Figure 2 ).

[0288] In order to further verify that knocking down TNIK can increase the sensitivity of colorectal cancer resistant cells to 5FU, the present application conducted a cell clone survival experiment and an EdU-555 cell proliferation detection experiment to detect the effect of 5FU on the proliferation of HCT8-5FUR cells transfected with siRNA. After 24 hours of transfection with siRNA, 5000 cells per well in a 6-well plate were cultured with complete medium containing 0.05mM 5FU, and photographed after 10 days of crystal violet staining. First, the results of cell clone formation showed that compared with the control group transfected with siNC, the proliferation of HCT8-5FUR cells was inhibited to a certain extent after treatment with 0.05mM5FU, but the cell proliferation of HCT8-5FUR cells transfected with siTNIK-1 and siTNIK-2 was significantly inhibited when treated with 0.05mM5FU. ( Figure 3 ) Secondly, the results of the EdU-555 cell proliferation assay showed that the proportion of EdU-positive cells in HCT8-5FUR cells transfected with siTNIK-1 and siTNIK-2 was significantly reduced when treated with 5FU compared to the control group transfected with siNC ( Figure 4 The above results indicate that inhibition of TNIK can make HCT8-5FUR cells more sensitive to 5FU.

[0289] 3.2 Knockdown of TNIK enhances 5FU-induced cell cycle arrest in drug-resistant colorectal cancer

[0290] The cell cycle plays an important role in cell proliferation, growth, and survival. In cancer, this normal cell cycle control is often disrupted, leading to unlimited cell proliferation, which is an important factor in the occurrence of cancer.

[118] . Given that cell cycle disorder is one of the main characteristics of cancer, this application speculates that inhibiting TNIK will affect the cell cycle of colorectal cancer resistant cells. In order to explore the effect of TNIK on the cell cycle of colorectal cancer resistant cells, this application transfected HCT8-5FUR cells with siNC, siTNIK-1, and siTNIK-2. After 24 hours, the cells were digested and plated. After the cells expanded, the cells were cultured with RPMI-1640 complete medium containing or without 5FU. After 48 hours of treatment, they were tested on the machine. Before the test, the cells need to be collected first, fixed with 70% cold ethanol, and then the cell cycle of colorectal cancer resistant cells is measured by flow cytometry. The experimental results show that ( Figure 5), compared with the control group, the proportion of HCT8-5FUR cells in the S phase increased from 29% to 37% after 5FU treatment. Furthermore, the proportion of HCT8-5FUR cells in the S phase significantly increased after 5FU treatment in HCT8-5FUR cells transfected with siTNIK-1. The proportion of HCT8-5FUR cells in the S phase increased from 17% to 60% in HCT8-5FUR cells transfected with siTNIK-1, and from 16% to 57% in HCT8-5FUR cells transfected with siTNIK-2. This enhanced the S phase arrest of the colorectal cancer-resistant cells induced by 5FU treatment, hindering the normal progression of the cell cycle and thus inhibiting the proliferation of colorectal cancer-resistant cells.

[0291] 3.3 Knockdown of TNIK enhances 5FU-induced apoptosis in drug-resistant colorectal cancer cells

[0292] The results of previous studies have shown that the proliferation of colorectal cancer-resistant cells transfected with siTNIK-1 and siTNIK-2 is significantly inhibited, and it has been found that inhibiting TNIK can enhance the S phase cell cycle arrest induced by 5FU. In order to explore its effect on the viability of colorectal cancer-resistant cells, this application detected the apoptosis of colorectal cancer cells in different experimental groups by flow cytometry. This application used serum-free RPMI-1640 culture medium containing or not containing 5FU to culture HCT8-5FUR cells transfected with siNC, siTNIK-1, and siTNIK-2, respectively. After 48 hours of treatment, when the cell samples were collected, the dead cells would float in the culture medium, so the cells in the culture medium needed to be centrifuged and collected for detection on a flow cytometer.

[0293] The experimental results show that ( Figure 6 ), after treatment with 5FU, the apoptosis rate of HCT8-5FUR cells transfected with siNC increased by 6.64%, the apoptosis rate of HCT8-5FUR cells transfected with siTNIK-1 increased by 13.74%, and the apoptosis rate of HCT8-5FUR cells transfected with siTNIK-2 increased by 12.37%. This experimental result shows that inhibiting TNIK can enhance the apoptosis of colorectal cancer resistant cells induced by 5FU, which is consistent with the results of CCK8, cell clone formation and EdU cell proliferation experiments in this application, confirming the hypothesis of this application.

[0294] Subsequently, the present application handed over the TNIK sequence to Qingke Biotechnology Company to construct and synthesize two lentiviral interference plasmids, which the present application named shTNIK-1 and sh-TNIK-2. After HEK293T cell packaging, the virus solution infected HCT8-5FUR cells for 48 hours. Since the vector plko.1-copGFP-PURO of the present application carries a puromycin screening tag, continuous screening was performed with the minimum lethal concentration of puromycin 4ug / mL, and the fluorescence was observed under a fluorescence microscope to preliminarily judge the knockdown efficiency. Subsequently, a qRT-PCR experiment was performed to detect the knockdown efficiency. The experimental results showed that ( Figure 7 ), both lentiviral interference plasmids shTNIK-1 and sh-TNIK-2 were able to significantly inhibit the expression of TNIK in HCT8-5FUR cells, with the knockdown efficiency of the lentiviral plasmid shTNIK-1 being 77.25% and that of the lentiviral plasmid shTNIK-2 being 73.29%, meeting the experimental requirements.

[0295] Then, this application selected the TNIK knockdown HCT8-5FUR stable cell line, and after 5FU treatment, used Western blot experiments to detect the expression of Bcl2 protein family, apoptosis signaling pathway related proteins, and apoptosis execution proteins caspase3 and PARP1 protein. Figure 8 As shown, compared with the control group, after 5FU treatment of TNIK knockdown HCT8-5FUR stable cell lines (shTNIK-1, shTNIK-2), the expression of anti-apoptotic protein Bcl2 was significantly downregulated, and the expression of pro-apoptotic protein Bax was significantly upregulated, indicating that the cell apoptosis pathway was activated, and the apoptosis execution protein caspase3 was further activated to be converted into active cleaved-caspase3. The expression level of cleaved-caspase3 was significantly increased, and the activated caspase3 was able to further cut PARP to make it active cleaved-PARP1. Finally, in the TNIK knockdown HCT8-5FUR cells, the expression level of cleaved-PARP1 was significantly increased after 5FU treatment, which is consistent with the results of the flow cytometry test of cell apoptosis in the previous application. The above experimental results show that in the TNIK knockdown HCT8-5FUR stable cell line, the level of cell apoptosis is significantly increased after treatment with 5FU.

[0296] 3.4 Knockdown of TNIK inhibits the migration and invasion of HCT8-5FUR cells

[0297] The previous application has studied the effect of TNIK on the proliferation, survival and apoptosis of HCT8-5FUR cells. Next, we will further explore the effect of TNIK on the migration and invasion ability of HCT8-5FUR cells. This application uses the TNIK knockdown HCT8-5FUR stable cell line for research. The cells are seeded in 6-well plates and the scratch test is performed after the cells are fully grown. The pictures are taken under the microscope at 0h and 48h. The results of the cell scratch test show that ( Figure 9 Middle A), compared with the control group, inhibition of TNIK can significantly inhibit the migration ability of HCT8-5FUR cells. The results of Transwell matrix gel invasion assay showed that ( Figure 9 Middle C), TNIK inhibition significantly inhibited the ability of HCT8-5FUR cells to pass through Matrigel compared with the control group.

[0298] Epithelial-mesenchymal transition (EMT) refers to the process in which epithelial cells lose polarity and intercellular connections and transform into invasive mesenchymal cells. This process plays an important role in physiological and pathological processes such as wound healing and tumor metastasis. In addition, a large amount of evidence shows that the EMT process can participate in cancer drug resistance. Therefore, this application uses Western blot experiments to detect changes in the expression of EMT-related marker proteins. The experimental results show that ( Figure 10 ), TNIK inhibition significantly upregulated the epithelial marker protein E-cadherin and significantly downregulated N-cadherin and Vimentin. These results indicate that inhibiting TNIK expression can inhibit epithelial-mesenchymal transition and the migration of HCT8-5FUR cells and play a role in reversing drug resistance.

[0299] 3.5 Knockdown of TNIK can inhibit the Wnt / β-catenin signaling pathway in colorectal cancer resistant cells

[0300] The Wnt / β-catenin signaling pathway is a key regulatory pathway in the development and progression of colorectal cancer. Aberrant activation of the Wnt / β-catenin signaling pathway not only promotes colorectal cancer progression but is also closely associated with the development of chemoradiotherapy resistance. Recent studies have shown that the Wnt / β-catenin signaling pathway can mediate colorectal cancer drug resistance through the presence of highly metastatic cancer stem cells, regulation of non-coding RNAs, and the tumor microenvironment. Preliminary experimental results showed that compared with the parental HCT8 cells, the expression of β-catenin, a key molecule in the Wnt / β-catenin pathway, and its downstream target c-Myc were significantly increased in the drug-resistant HCT8-5FUR cells, indicating that 5FU resistance in HCT8-5FUR cells is associated with aberrant activation of the Wnt / β-catenin signaling pathway. Previous studies have shown that TRAF2 and NCK-interacting kinase (TNIK) can bind to TCF4 and β-catenin to form a complex, regulate Wnt signaling, and is expected to block the Wnt / β-catenin signaling pathway even in colorectal cancer cells with adenomatous polyposis coli (APC) gene mutations, becoming a promising therapeutic target for colorectal cancer.

[0301] In order to explore the effect of inhibiting TNIK on the Wnt / β-catenin signaling pathway in drug-resistant HCT8-5FUR cells, the present invention transfected nonspecific siRNA, as well as siTNIK-1 and siTNIK-2 into drug-resistant HCT8-5FUR cells. After 72 hours of transfection, samples were collected and proteins were extracted. Western blot experiments were performed to detect changes in the protein levels of cycinD1, c-Myc, and Survivin, the classic downstream targets of the Wnt / β-catenin signaling pathway. The experimental results showed that ( Figure 11 ), compared with HCT8-5FUR cells transfected with nonspecific siRNA, cyclinD1, c-Myc and Survivin proteins in HCT8-5FUR cells transfected with siTNIK-1 and si-TNIK-2 were significantly downregulated. This experimental result shows that inhibiting TNIK can inhibit the Wnt / β-catenin signaling pathway in colorectal cancer resistant cells, thereby reversing drug resistance.

[0302] This application has previously confirmed that inhibition of TNIK and c-Myc are significantly downregulated in colorectal cancer resistant cells. To explore whether c-Myc can further regulate SOX2, this application was verified by qRT-PCR and Western blot. Figure 12 After inhibiting TNIK, SOX2 was significantly downregulated at both the mRNA and protein levels, inhibiting the stemness of colorectal cancer-resistant cells, thereby enhancing the sensitivity of colorectal cancer-resistant cells to 5FU.

[0303] The present invention studies the ability of inhibiting TNIK to increase the sensitivity of 5FU-resistant colorectal cancer cells to 5FU and explores its molecular mechanism through in vitro experiments.

[0304] This application uses two methods, small interfering RNA (siRNA) and short hairpin RNA (shRNA), to inhibit the expression of TNIK at the molecular level. First, this application observed through CCK8 experiments that 5FU-resistant colorectal cancer cells were more sensitive to 5FU by inhibiting TNIK; then, to further prove this, this application conducted cell clone formation experiments and EdU cell proliferation experiments, and found that 5FU treatment after inhibiting TNIK could significantly inhibit the proliferation of 5FU-resistant colorectal cancer cells. Based on the characteristics of cancer cells that the cell cycle is disrupted and cells proliferate indefinitely, this application then studied the effect of inhibiting TNIK on the cycle of drug-resistant colorectal cancer cells. The results showed that 5FU treatment of TNIK-knockdown colorectal cancer-resistant cells can significantly arrest the colorectal cancer-resistant cell cycle in the S phase. Then this application tested cell apoptosis. The experimental results showed that inhibiting TNIK can increase 5FU-induced cell apoptosis. Western blot experiments detected the expression of apoptosis-related proteins, and the experimental results also supported this conclusion. Then, this application studied the effect of TNIK on the migration and invasion ability of colorectal cancer resistant cells. The experimental results showed that inhibiting TNIK can significantly inhibit the migration and invasion ability of colorectal cancer cells, and Western blot experiments confirmed that inhibiting TNIK can inhibit the EMT process. Finally, through Western blot experiments, its molecular mechanism was studied, and it was found that by inhibiting TNIK, its classic downstream target genes such as cyclinD1, c-Myc, Survivin and other proteins were significantly downregulated, and the Wnt / β-catenin signaling pathway of colorectal cancer resistant cells was inhibited. In addition, many literatures reported that c-Myc can regulate the greening factor SOX2 related to cancer stem cells. The research of this application found that inhibiting TNIK can significantly reduce the expression of SOX2 in colorectal cancer resistant cells and inhibit the stemness of colorectal cancer resistant cells. In summary, inhibiting TNIK reverses the resistance of colorectal cancer resistant cells to 5FU by inhibiting the Wnt / β-catenin signaling pathway ( Figure 13The Wnt / β-catenin signaling pathway is abnormally activated in drug-resistant cells. Knockdown of TNIK downregulates the expression of downstream target genes such as cyclinD1, Survivin, and c-Myc, blocking the Wnt / β-catenin pathway and reducing SOX2 expression, thereby weakening cancer stem cell characteristics and increasing cell sensitivity to 5FU. Therefore, TNIK could be a potential therapeutic target for reversing 5FU resistance. The combination of TNIK inhibitors and 5FU provides a new research direction and treatment strategy for overcoming chemotherapy resistance in colorectal cancer.

[0305] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A TNIK expression inhibitor, wherein the TNIK expression inhibitor is at least one selected from a to e: a. siTNIK-1 whose nucleotide sequence is shown in SEQ ID NO: 1; b. siTNIK-2 whose nucleotide sequence is shown in SEQ ID NO: 2; c. siTNIK-3, whose nucleotide sequence is shown in SEQ ID NO: 3; d. shTNIK-1 having a nucleotide sequence as shown in SEQ ID NO: 4; e. shTNIK-2 whose nucleotide sequence is shown in SEQ ID NO:

5.

2. The TNIK expression inhibitor according to claim 1, characterized in that The TNIK expression inhibitor is chemically synthesized. 3 . A delivery system for a TNIK expression inhibitor, comprising the TNIK expression inhibitor according to any one of claims 1 to 2 and a vector. 4 . A pharmaceutical composition comprising the TNIK expression inhibitor according to any one of claims 1 to 2 or the delivery system according to claim 3 , and a pharmaceutically acceptable excipient.

5. Use of the TNIK expression inhibitor according to any one of claims 1 to 2, the delivery system according to claim 3, and the pharmaceutical composition according to claim 4 in the preparation of a drug for preventing and / or treating cancer.

6. The use according to claim 5, characterized in that The TNIK expression inhibitor can significantly enhance the inhibitory effect of 5FU on the proliferation of colorectal cancer resistant cells; And / or, the TNIK expression inhibitor can significantly arrest the colorectal cancer drug-resistant cell cycle at the S phase; And / or, the TNIK expression inhibitor can significantly increase 5FU-induced cell apoptosis; And / or, the TNIK expression inhibitor can significantly inhibit the migration and invasion ability of colorectal cancer resistant cells; And / or, the TNIK expression inhibitor can significantly reduce the expression of SOX2 in colorectal cancer resistant cells and inhibit the stemness of colorectal cancer resistant cells.

7. The use according to claim 5 or 6, characterized in that The TNIK expression inhibitor can reverse the resistance of colorectal cancer resistant cells to 5FU by inhibiting the Wnt / β-catenin signaling pathway of colorectal cancer resistant cells.

8. The use according to claim 5, characterized in that Use of a combined drug comprising the TNIK expression inhibitor and 5FU in preparing a drug for preventing and / or treating cancer.

9. The use according to claim 5, characterized in that The cancers include colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, choriocarcinoma, malignant hydatidiform mole, head and neck squamous cell carcinoma, skin cancer, liver cancer, and bladder cancer.

Images