Application of uckl1 in diagnosis, treatment and prognosis evaluation of colorectal cancer

Abstract

The application provides application of UCKL1 gene or protein in preparation and / or screening of drugs for treating colorectal cancer, and application of reagents for detecting UCKL1 gene and / or protein level in preparation of a colorectal cancer detection kit or a colorectal cancer curative effect evaluation kit or a colorectal cancer prognosis evaluation kit. The application innovatively illustrates that UCKL1 is highly expressed in colorectal cancer tissues, and the expression level of UCKL1 is significantly related to the prognosis of a colorectal cancer patient. Further experiments prove that knockdown of UCKL1 can reduce the survival rate of colorectal cancer cell lines HCT116 and RKO in vitro, inhibit the colony formation ability and migration, and inhibit the growth of colorectal cancer cells in nude mice in vivo. It is also found that knockdown of UCKL1 reduces the stability of NRF2 protein, makes colorectal cancer cells produce ferroptosis by inhibiting the NRF2-SLC7A11 pathway, and the combination of targeted inhibition of UCKL1 and GPX4 inhibitor has a synergistic antitumor effect. The application provides a new target for diagnosis, treatment and prognosis evaluation of colorectal cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of UCKL1 in diagnosis, treatment and prognosis evaluation of colorectal cancer. BACKGROUND

[0002] Colorectal cancer is a common malignant tumor in the gastrointestinal tract, and its morbidity and mortality rate ranks second only to gastric cancer, esophageal cancer and primary liver cancer among digestive system malignant tumors. Among all tumors, the morbidity of colorectal cancer is about 10.0%, ranking third in the world; the mortality rate is about 9.4%, ranking second in the world. For the treatment of colorectal cancer, the current main methods are surgery and chemotherapy. Chemotherapy drugs include 5-Fu, oxaliplatin, irinotecan, etc.; in addition, there are targeted drugs such as EGFR inhibitors, and immunotherapy drugs such as PD1 / PDL1 inhibitors.

[0003] Although various treatment methods including surgery, radiotherapy and chemotherapy are available, problems such as tumor recurrence and drug resistance are prone to occur, which brings great pain to patients. Therefore, it is of great significance to further study the mechanism of occurrence and development of colorectal cancer, find potential targets that can affect the growth of colorectal cancer, and develop anti-tumor drugs and explore new treatment methods. It is urgent to find new treatment targets and develop new treatment drugs. SUMMARY

[0004] Based on this, the purpose of the present application is to provide the application of UCKL1 in diagnosis, treatment and prognosis evaluation of colorectal cancer. UCKL1 is highly expressed in colorectal cancer tissues, and inhibition of its expression can inhibit the growth and migration of colorectal cancer cells.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows.

[0006] The application of UCKL1 gene or protein in the preparation and / or screening of drugs for treating colorectal cancer.

[0007] The application of the reagent for inhibiting the expression of UCKL1 gene and / or the UCKL1 protein neutralizing agent in the preparation of drugs for treating colorectal cancer.

[0008] In some embodiments, the reagent for inhibiting the expression of UCKL1 gene is selected from at least one of shRNA1 as shown in SEQ ID NO. 5 and shRNA2 as shown in SEQ ID NO. 6.

[0009] In some embodiments, the drug can induce ferroptosis of colorectal cancer cells.

[0010] In some embodiments, the drug can inhibit the NRF2-SLC7A11 signal pathway.

[0011] The application also provides use of a composition in preparation of a medicament for treating colorectal cancer, wherein the composition comprises an agent for inhibiting expression of UCKL1 gene and a GPX4 inhibitor.

[0012] In some embodiments, the agent for inhibiting expression of UCKL1 gene is selected from at least one of shRNA1 as shown in SEQ ID NO. 5 and shRNA2 as shown in SEQ ID NO. 6; and / or, the GPX4 inhibitor is selected from at least one of RSL3 and ML162.

[0013] The application also provides a medicament for treating colorectal cancer, wherein the medicament comprises an agent for inhibiting expression of UCKL1 gene and / or an UCKL1 protein neutralizing agent, and a pharmaceutically acceptable excipient.

[0014] In some embodiments, the agent for inhibiting expression of UCKL1 gene is selected from at least one of shRNA1 as shown in SEQ ID NO. 5 and shRNA2 as shown in SEQ ID NO. 6.

[0015] In some embodiments, the medicament further comprises a GPX4 inhibitor.

[0016] In some embodiments, the GPX4 inhibitor is selected from at least one of RSL3 and ML162.

[0017] The application also provides use of an agent for detecting UCKL1 gene and / or protein level in preparation of a colorectal cancer detection kit or a colorectal cancer efficacy evaluation kit or a colorectal cancer prognosis evaluation kit.

[0018] In some embodiments, the agent for detecting UCKL1 gene level comprises an upstream primer as shown in SEQ ID NO. 1 and a downstream primer as shown in SEQ ID NO. 2.

[0019] The present application innovatively discovers that UCKL1 gene is related to the diagnosis, treatment and prognosis evaluation of colorectal cancer. UCKL1 is highly expressed in colorectal cancer tissues, and patients with high expression of UCKL1 have shorter overall survival, and the expression level of UCKL1 is significantly related to the prognosis of patients with colorectal cancer. Further experiments prove that knocking down UCKL1 can reduce the survival rate of colorectal cancer cell lines HCT116 and RKO in vitro, inhibit the colony formation ability and migration, and inhibit the growth of colorectal cancer cells in nude mice. And it is also found that knocking down UCKL1 reduces the stability of NRF2 protein, inhibits the NRF2-SLC7A11 pathway to make colorectal cancer cells produce ferroptosis, and the combination of targeting inhibition of UCKL1 and GPX4 inhibitor has a synergistic antitumor effect. The present application provides a new target for the diagnosis, treatment and prognosis evaluation of colorectal cancer, and provides a new idea for the development of anti-colorectal cancer drugs and the exploration of new treatment methods. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 UCKL1 expression in normal colon cells and colorectal cancer cells.

[0021] Figure 2 UCKL1 is highly expressed in colorectal cancer tissues.

[0022] Figure 3 UCKL1 high expression is related to poor prognosis of patients with colorectal cancer.

[0023] Figure 4 Dox can effectively reduce the expression of UCKL1 in HCT116-shUCKL1 and RKO-shUCKL1.

[0024] Figure 5 UCKL1 shRNA resistant plasmid can reverse the decrease of protein expression and cell survival rate caused by UCKL1 shRNA.

[0025] Figure 6 HCT116 and RKO cell growth curves when UCKL1 is knocked down or not.

[0026] Figure 7 Knocking down UCKL1 reduces the colony formation ability of colorectal cancer cells.

[0027] Figure 8 Knocking down UCKL1 inhibits the migration of colorectal cancer cells.

[0028] Figure 9 Knocking down UCKL1 inhibits the growth of colorectal cancer cells in nude mice.

[0029] Figure 10The iron death inhibitor and the antioxidant can remedy the decrease in cell survival rate caused by knocking down UCKL1.

[0030] Figure 11 Knocking down UCKL1 increases lipid peroxidation in colorectal cancer cells.

[0031] Figure 12 Knocking down UCKL1 increases the total ROS level in colorectal cancer cells.

[0032] Figure 13 Knocking down UCKL1 can increase the content of GSSG and NADP+ in colorectal cancer cells, and increase the ratio of GSSG to GSH and NADP+ to NADPH.

[0033] Figure 14 Knocking down UCKL1 and GPX4 inhibitor have a synergistic tumor inhibition effect.

[0034] Figure 15 Knocking down UCKL1 and GPX4 inhibitor can synergistically increase the ROS and lipid peroxidation levels in colorectal cancer cells.

[0035] Figure 16 Knocking down UCKL1 and RSL3 can synergistically inhibit the growth of colorectal cancer cells in nude mice. DETAILED DESCRIPTION

[0036] The experimental methods not specified in the following examples of the present application are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available.

[0037] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0038] The terms "comprising" and "having" and any variations thereof in the present application are intended to cover non-exclusive inclusion. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but optionally also includes steps not listed, or optionally also includes other steps inherent to the process, method, product or equipment.

[0039] In the present application, "a plurality of" refers to two or more. "And / or", which describes the association relationship of associated objects, means that there can be three relationships, for example, A and / or B can mean: A exists alone, A and B exist together, and B exists alone. The character " / " generally represents that the associated objects before and after it are in an "or" relationship.

[0040] The following is described in connection with specific embodiments.

[0041] Example 1 Expression analysis of UCKL1 in colorectal cancer

[0042] 1. Experimental materials

[0043] 1.1 Cell source

[0044] Normal colon cells CCD-18Co, colorectal cancer cell lines DLD1, HCT116, RKO, LOVO, SW480, SW620, HCT8, HCT15, HT29 were purchased from ATCC (American Type Culture Collections).

[0045] 1.2 Tumor tissue sample source

[0046] Colorectal cancer and paracancerous tissue samples used for quantitative real-time PCR (RT-PCR) and Western blot were from the Sixth Affiliated Hospital of Sun Yat-sen University. Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of Sun Yat-sen University.

[0047] 2. Methods

[0048] 2.1 Tissue RNA extraction

[0049] The extraction steps of colorectal cancer and paracancerous tissue RNA are as follows:

[0050] (1) Take the appropriate amount of tissue from the intestinal cancer and paracancerous tissue samples, place it in a 2ml EP tube, add 400μl Trizol, and then place a steel ball. Place the EP tube containing the tissue in a tissue homogenizer to break up the tissue. After breaking up the tissue, there should be no obvious tissue pieces left.(2) Add 600μl Trizol to the broken-up sample, then add 200μl chloroform, shake vigorously by hand for 15s, and then let stand at room temperature for 3min.(3) After standing, centrifuge at 4℃, 1200rpm for 15min. After centrifugation, the sample will be clearly layered.(4) Carefully transfer the upper liquid containing the RNA to a new 1.5ml EP tube, then add 500μl isopropanol, mix well, and let stand at room temperature for 10min.(5) Centrifuge again at 4℃, 1200rpm for 10min. After centrifugation, a white RNA precipitate will be visible.(6) Discard the supernatant, add 1ml 75% ethanol for washing, then centrifuge at 4℃, 1200rpm for 10min, and discard the supernatant.(7) Dry the obtained RNA precipitate at room temperature for about 2min, then add an appropriate amount of DEPC water to dissolve.(8) Use a Nanodrop to measure the concentration of the extracted RNA and the values of 260 / 280 and 260 / 230.

[0051] 2.2 Reverse transcription of RNA to cDNA

[0052] 2.2.1 Purification of RNA

[0053] (1) According to the measured RNA concentration, calculate the volume of RNA required for reverse transcription of 2000ng of RNA, then add DEPC water to 9μl. Then add 1.1μl 10×DNase I buffer and 1μl DNase I, mix gently by blowing.(2) Place the sample in a PCR instrument, and perform genomic DNA removal at 37℃ for 45min and 75℃ for 10min. Immediately after the reaction is complete, take the sample out and place it on ice for 5min.

[0054] 2.2.2 Synthesis of cDNA

[0055] (1) Take the purified RNA sample described above, and add 4μl 5×PrimerScript RT Master Mix and 5μl ddH2O.(2) Place in a PCR instrument, and perform cDNA synthesis at 37℃ for 15min and 85℃ for 5s. After the reaction is complete, perform RT-PCR or store the cDNA in a -20℃ refrigerator.

[0056] 2.3 Real-time fluorescent quantitative PCR

[0057] (1) Configure the reaction system as shown in Table 1 below, with a total reaction system of 15μl:

[0058] Table 1 PCR reaction system

[0059]

[0060] The sequence of the detection primer used is as follows:

[0061] UCKL1: Forward Primer: 5'-GGCCGCACACAACAACTTC-3' (SEQ ID NO. 1); Reverse Primer: 5'-GGGCACCTTGACACTCTTCC-3' (SEQ ID NO. 2);

[0062] β-actin: Forward Primer: 5'-GATCATTGCTCCTCCTGAGC-3' (SEQ ID NO. 3); Reverse Primer: 5'-ACTCCTGCTTGCTGATCCAC-3' (SEQ ID NO. 4).

[0063] (2) Real-time fluorescent quantitative PCR reaction was performed according to the following procedure:

[0064] Hold Stage: 95°C, 30 s; PCR Stage: 95°C, 5 s; 60°C, 30 s. 40 cycles. Melt Curve Stage: 95°C, 15 s; 60°C, 1 min; 95°C, 15 s.

[0065] (3) Taking β-actin as the internal reference, the relative expression amount of the gene was calculated according to the measured Ct value according to the 2-ΔΔCt method.

[0066] 2.4 Tissue protein extraction

[0067] (1) Place the intestinal cancer and paracancer tissue blocks of appropriate size in a 2 ml EP tube, put in a steel ball, then add 400 μl of lysis buffer, and place the sample EP tube into the tissue homogenizer for tissue crushing. (2) Use an ultrasonic disrupter to crush the collected tissue homogenate, and place the sample on ice during crushing. (3) After crushing is completed, centrifuge the sample in a centrifuge at a temperature of 4°C and a speed of 12000 rpm for 30 min. (4) Absorb the supernatant into a new 1.5 ml EP tube, and perform protein quantification, sample preparation, or store in a -80°C refrigerator.

[0068] 2.5 Protein sample preparation

[0069] 2.5.1 Protein quantification

[0070] BCA kit was used for protein quantification, and the method was as follows:

[0071] (1) Preparation of BCA reagent working solution: 200 μl of BCA working solution is required for each protein sample. Calculate the total amount of BCA working solution required according to the amount of protein to be measured, then mix BCA-A reagent and BCA-B reagent at a ratio of 50:1.

[0072] (2) Preparation of protein standard curve: Take 1.5 ml EP tubes and add each reagent according to the following Table 2 to prepare the protein standard curve. The concentration of the protein standard used is 1 mg / ml.

[0073] Table 2 Preparation of protein standard curve

[0074]

[0075] Mix each group in Table 2 above with BCA working solution (200 μl), then add to a 96-well plate, incubate at 37°C for 30 min, then use a microplate reader to measure the absorbance at 562 nm. According to the absorbance and the corresponding protein concentration, a protein standard curve is drawn.

[0076] (3) Determination of the concentration of the protein to be measured: After diluting the protein 5 times, add 200 μl of BCA working solution, incubate at 37°C for 30 min, then measure the absorbance at 562 nm. According to the standard curve drawn, the concentration of the protein to be measured is calculated.

[0077] 2.5.2 Sample preparation

[0078] According to the measured protein concentration, calculate the volume required to take 20 μg of protein and the amount of water required. Add water, 6x SDS loading buffer, and sample protein to a pre-cooled 1.5 ml EP tube in sequence, denature the protein in a 95°C water bath for 5 min, then immediately place it on ice to cool.

[0079] 2.6 Western blot

[0080] 2.6.1 Preparation of SDS-PAGE gel

[0081] (1) This experiment mainly uses 8% separating gel, which is prepared according to the following Table 3

[0082] Table 3 8% separating gel formula

[0083]

[0084] (2) After preparing the separating gel, add it to the gel preparation glass plate, then press the gel with water, then prepare the concentrating gel according to the following Table 4:

[0085] Table 4 Concentrating gel formula

[0086]

[0087] (3) After the gel is solidified, discard the supernatant, add the concentrated gel, and insert the comb. Avoid generating air bubbles when inserting the comb.

[0088] (4) After the gel is solidified, perform SDS-PAGE gel electrophoresis or store in a 4°C refrigerator for subsequent use.

[0089] 2.6.2 SDS-PAGE gel electrophoresis

[0090] (1) Place the gel plate in the electrophoresis tank, add the electrophoresis solution, and ensure that the inner tank liquid level is higher than the outer tank. (2) Sample loading: slowly add the prepared protein sample to the sample loading well, and add protein Marker on both sides of the sample. (3) Electrophoresis: set the voltage to 80V for electrophoresis, and after the protein enters the separation gel, increase the voltage to 100V for continued electrophoresis until completion.

[0091] 2.6.3 Transferring

[0092] Before transferring, soak the PVDF membrane in methanol to activate it. Then, place the sponge pad, 3 layers of filter paper, the gel, 3 layers of filter paper, and the sponge pad in the order of sponge pad, 3 layers of filter paper, gel, 3 layers of filter paper, and sponge pad on the black plate of the transferring clamp, and make sure to remove the air bubbles between the PVDF membrane and the gel. After fixing the transferring device, insert it into the electric transfer tank, and then pour the pre-cooled electric transfer solution for electric transfer, with a voltage setting of 100V and a time of 90min.

[0093] 2.6.4 Blocking

[0094] After the transferring is completed, take out the PVDF membrane, and then place it in the blocking solution (100ml 1xTBST + 5.0g skimmed milk powder) for incubation at room temperature for 1h.

[0095] 2.6.5 Primary antibody incubation

[0096] After the blocking is completed, add the diluted primary antibody, and the antibody dilution is determined according to the instructions. Then, place it in the 4°C refrigerator on the shaker for overnight incubation.

[0097] 2.6.6 Secondary antibody incubation

[0098] (1) After the overnight incubation of the primary antibody, add 1xTBST, and then place it on the shaker at 200rpm to wash away the residual primary antibody, for 5min each time, for a total of 3 times. (2) After the membrane washing is completed, add the diluted secondary antibody of the corresponding species, and incubate it on the shaker at room temperature for 1h. (3) After the secondary antibody incubation is completed, wash the membrane with 1xTBST for 3 times, for 10min each time.

[0099] 2.6.7 Development

[0100] (1) Prepare ECL-A and ECL-B solutions. ECL-A solution: Luminol (250 mM) 50 μl, PC Acid (900 mM) 22 μl, Tris-HCL (pH 8.5) 500 μl, H2O 4.5 ml. ECL-B solution: 30% H2O2 3 μl, Tris-HCL (pH 8.5) 500 μl, H2O 4.5 ml.

[0101] (2) Mix equal amounts of ECL-A and B solutions to prepare the developing solution. Incubate the PVDF membrane in the developing solution, and then develop it in the Bio-Rad chemiluminescence imaging system.

[0102] 2.6.8 Protein band analysis

[0103] Scan and analyze the protein bands using Image J software.

[0104] 2.7 Immunohistochemistry

[0105] (1) Incubate the tissue sections in an oven at 65°C for 3 h. (2) Soak the sections in xylene for 10 min, then soak them in fresh xylene for another 10 min, and repeat this process for a total of three times. (3) Soak the sections in anhydrous ethanol for 5 min, then soak them in 95% ethanol for 5 min, and finally soak them in 75% ethanol for 5 min. (4) Wash the sections in PBS twice. (5) Place the sections in EDTA antigen retrieval solution, and boil them in a microwave oven for 10 min. After natural cooling, wash the sections in PBS. (6) Soak the sections in 3% hydrogen peroxide solution for 15 min to block endogenous peroxidase, and then wash them in PBS for 5 min three times. (7) Add an appropriate amount of 3% BSA to the tissue, and seal it at room temperature for 30 min. (8) Discard the blocking solution, and add the prepared primary antibody to the tissue, and place it in a wet box at 4°C overnight. (9) After washing in PBS, add the secondary antibody, and incubate it at room temperature for 50 min. (10) After washing in PBS, add the DAB developing solution, and incubate it at room temperature for 1-3 min. (11) Add hematoxylin to the tissue to perform nuclear staining, and incubate it at room temperature for 5 min, and then rinse the residual dye with running water. Then soak the sections in hydrochloric alcohol for 2 min, and rinse them with running water to decolorize. (12) Soak the sections in 75% ethanol, 85% ethanol, and anhydrous ethanol, respectively, and then dehydrate and clear them in xylene. After removing the sections, dry them, and seal them with neutral resin. (13) Place the prepared slides in a slide scanner to scan them.

[0106] 2.8 Statistical methods

[0107] Statistical analysis and statistical graphs were performed using GraphPad prism 8 software. Quantitative data conforming to normal distribution were expressed as mean ± standard deviation, and the comparison of means of two groups of samples used t test; the comparison among multiple groups of samples used One-way ANOVA. Quantitative data not conforming to normal distribution used rank sum test. P<0.05 was considered statistically significant.

[0108] 3. Experimental results

[0109] (1) The expression level of UCKL1 in colorectal cancer cell lines is higher than that in normal colon cells

[0110] The expression of UCKL1 in normal colon cells and 9 colorectal cancer cell lines including DLD1, RKO, etc. was detected by Western blot, and the results showed that compared with normal colon cells CCD-18Co, UCKL1 was highly expressed in colorectal cancer cells Figure 1 ).

[0111] (2) UCKL1 expression is increased in colorectal cancer tissues

[0112] To further verify the expression of UCKL1 in colorectal cancer, we obtained colorectal cancer and paracancer tissue samples from the clinic, and detected the clinical samples by RT-PCR and Western blot. The results showed that compared with paracancer tissues, the mRNA level of UCKL1 in colorectal cancer tissues was significantly increased Figure 2 A), and the protein expression was also significantly increased Figure 2 B). Immunohistochemical staining of colorectal cancer tissues and normal tissues showed that the expression of UCKL1 in colorectal cancer tissues was significantly higher than that in normal tissues Figure 2 C).

[0113] (3) High expression of UCKL1 is associated with poor prognosis of colorectal cancer patients

[0114] UCKL1 is highly expressed in colorectal cancer tissues, and then we further explored the relationship between the expression level of UCKL1 and the survival prognosis of colorectal cancer patients. By analyzing the TCGA database, we found that patients with high expression of UCKL1 had shorter overall survival, and the expression level of UCKL1 was significantly related to the prognosis of colorectal cancer patients Figure 3 ).

[0115] The above results show that UCKL1 has gene amplification, increased mRNA expression and other gene abnormalities in colorectal cancer; the mRNA and protein of UCKL1 are highly expressed in colorectal cancer tissues. High expression of UCKL1 is associated with poor prognosis of colorectal cancer patients.

[0116] Example 2 Effect of UCKL1 on colorectal cancer cell growth in vitro and in vivo

[0117] 1. Materials

[0118] 1.1 Cell source

[0119] Colorectal cancer cell lines HCT116, RKO and human renal epithelial cell line 293T were purchased from ATCC (American Type Culture Collections)

[0120] 2. Experimental methods

[0121] 2.1 Cell culture

[0122] Human intestinal cancer cell lines HCT116 and RKO were cultured in RPMI-1640 medium containing 10% FBS, and human renal epithelial cell line 293T was cultured in DMEM medium containing 10% FBS. They were grown in a constant temperature incubator at 37°C, 5% CO2, 95% humidity, and the cell growth condition was observed under an inverted microscope. When the cell density was about 90%, the cells were digested and subcultured.

[0123] 2.2 Knockdown plasmid construction

[0124] 2.2.1 Vector digestion

[0125] (1) This part uses pLKO-tet-on vector, which belongs to Tetracycline / Doxycycline inducible expression vector. When Tetracycline / Doxycycline is present, shRNA can be expressed. Select AgeI and EcoRI as the two enzyme digestion sites, and prepare the enzyme digestion reaction system according to the following Table 5.

[0126] Table 5 pLKO-tet-on vector digestion reaction system

[0127]

[0128] (2) The enzyme digestion system was placed at 37°C for 15 min.

[0129] 2.2.2 Electrophoresis and recovery of enzyme digestion products

[0130] (1) 0.8% agarose gel was prepared in 1x TAE solution and placed in the electrophoresis tank containing TAE buffer.(2) The enzyme digestion products were mixed with DNA loading buffer and added to the gel loading well, and electrophoresis separation was performed at 110 V.(3) After electrophoresis, the gel was taken out and placed under the ultraviolet gel cutter to observe, and the gel piece containing DNA was cut and transferred to a 1.5 ml EP tube.(4) The weight of the gel was measured, and buffer QG was added at a ratio of 300 μl per 100 mg of gel, and the gel was dissolved in a 50°C water bath, and vortexed every 2-3 min during the period to accelerate dissolution.(5) The sample was transferred to the column, centrifuged at 13000 rpm for 1 min, and the filtrate was discarded.(6) 0.5 ml of buffer QG was added, followed by centrifugation at 13000 rpm for 1 min, and the filtrate was discarded.(7) The empty column was centrifuged at 13000 rpm for 1 min to remove as much residual liquid as possible.(8) After standing at room temperature for 2 min, an appropriate amount of sterile water was added, and stood for 1 min.(9) Centrifuged at 13000 rpm for 1 min, and the liquid was collected, and the concentration of the obtained plasmid was determined using Nanodrop.

[0131] 2.2.3 shRNA oligo design

[0132] The UCKL1 shRNA oligo sequence used in this study is:

[0133] shUCKL1-1: 5'-CGCACACAACAACTTCAACTT-3' (SEQ ID NO. 5);

[0134] shUCKL1-2: 5'-GCCCATTTATGACTTCACCAC-3' (SEQ ID NO. 6).

[0135] 2.2.4 Oligo annealing

[0136] (1) The shRNA oligo was briefly centrifuged to collect it at the bottom of the tube, water was added to a concentration of 100 μM according to the instructions, and the oligo was dissolved by gentle vortexing.(2) 11.25 μl of each shRNA oligo was taken, 2.5 μl of 10x annealing buffer was added, and the total system was 25 μl.(3) Place in a 95°C PCR instrument for 5 min, then turn off the power and cool slowly.(4) Mix the oligo obtained in the previous step with 0.5x annealing buffer at a ratio of 1:400, such as 1 μl oligo plus 399 μl 0.5x annealing buffer.(5) The annealed shRNA was connected to the vector or stored at -20°C.

[0137] 2.2.5 Connection

[0138] (1) Use T4 ligase to connect shRNA Oligo and the digested vector, the reaction system is shown in Table 6 as follows:

[0139] Table 6 Connection reaction system

[0140]

[0141] (2) Place the reaction system in 16℃ overnight connection.

[0142] 2.2.6 Transformation

[0143] (1) Take 100 μl competent cells and place on ice to thaw, add 10 μl connected plasmid to the competent cells and mix, place on ice for 30 min. (2) Heat shock in 42℃ water bath for 60 s, quickly transfer to ice for 3 min. (3) Add 1 ml LB medium without antibiotics, shake at 37℃, 200 rpm for 1 h. (4) Centrifuge at 5000 rpm for 5 min, discard the supernatant, resuspend the bacterial body with 100 μl LB medium, then add all to the LB culture plate containing antibiotics and evenly spread, invert in the 37℃ incubator and culture for 12-16 h. (5) Pick single colonies and add to the LB medium containing antibiotics, shake at 37℃, 200 rpm, then send to Huada Gene Company for sequencing.

[0144] 2.3 Construction of UCKL1 induced knockdown cell line

[0145] 2.3.1 Lentivirus packaging

[0146] (1) 293T cells were seeded in 10 cm dish and incubated in cell incubator overnight. (2) Two 1.5 ml EP tubes were taken, 0.5 ml serum-free medium was added into each tube. UCKL1 shRNA 10 μg, pMD2.G 5 μg, psPAX2 5 μg (i.e. UCKL1 shRNA, pMD2.G, psPAX2 in the ratio of 2:1:1) were added into one tube, and PEI 30 μl was added into the other tube, and left for 5 min. (3) The medium containing PEI was added into the tube containing plasmid, and mixed gently, and left for 30 min. (4) The transfection mixture was added dropwise into 293T cells, and make sure that the transfection mixture was evenly distributed, and then the cells were incubated in cell incubator. (5) After 24 h, the medium was changed, and 10 ml fresh medium containing serum was added. (6) After 24 h, the medium containing lentivirus was collected, and 10 ml fresh medium containing 10% FBS was added into 293T cells. (7) After 24 h, the medium containing lentivirus was collected again, and the collected medium was filtered with 0.45 μm filter membrane, and the virus solution could be used immediately for infecting cells to construct stable transfectants, or stored at -80 °C for later use.

[0147] 2.3.2 Lentivirus infection of cells and screening of stable transfectants

[0148] (1) The cells to be infected were seeded in a six-well plate and incubated in a cell incubator. (2) When the cell density was about 60%, 2 ml virus solution was added, and 2 μl Polybrene and 200 μl FBS were added, and the cells were incubated in a cell incubator. (3) After 24 h, the medium was discarded, and the cells were infected with 2 ml virus solution again, and 2 μl Polybrene and 200 μl FBS were added, and the cells were incubated in a cell incubator. (4) After 24 h, the cells were passaged at a ratio of 1:5, and incubated in a cell incubator. (5) After the cells adhered, Puromycin was added for screening, and untransfected cells were used as a control, and the screening time was about 1 week. (6) When the cells could grow stably in the medium containing Puromycin, RNA was extracted, and the knockdown efficiency was detected by RT-PCR, and when the expression of UCKL1 mRNA was significantly decreased, the cells were expanded and stored for later use.

[0149] 2.5 Construction of UCKL1 shRNA resistant plasmid

[0150] 2.5.1 Design of site-directed mutagenesis primers

[0151] According to the shRNA sequence, the codon targeted by the shRNA was replaced by a synonymous substitution, and a primer containing a point mutation was designed. In this experiment, the primer sequence of the UCKL1 shRNA resistant plasmid was as follows:

[0152] shUCKL1-1 resistant Forward: 5'-GCGCAGAATAATTTCAATTTCGACCACCCAGATGCC-3' (SEQ ID NO. 7);

[0153] shUCKL1-1 resistant Reverse: 5'-GAAATTGAAATTATTCTGCGCGGCCTGTTCCTGCTG-3' (SEQ ID NO. 8);

[0154] shUCKL1-2 resistant Forward: 5'-CCGATCTAGGAGTTCACCACACACAGCCGGAAGAAG-3' (SEQ ID NO. 9);

[0155] shUCKL1-2 resistant Reverse: 5'-TGTGGTGAACTCCTAGATCGGCACCTTGACACTCTT-3' (SEQ ID NO. 10).

[0156] 2.5.2 Amplification of target plasmid

[0157] (1) The amplification system was prepared according to Table 7 below.

[0158] Table 7 Amplification reaction system

[0159]

[0160] (1) Perform PCR reaction. The reaction was performed according to the following procedure:

[0161] Pre-denaturation: 95°C, 30s; denaturation: 95°C, 15s; annealing: 65°C, 15s; extension: 72°C, 50s; re-extension: 72°C, 5min, wherein the denaturation, annealing and extension are 30 cycles.

[0162] 2.5.3 Removal of template plasmid

[0163] (1) After amplification of the target plasmid, the template plasmid needs to be removed, and the reaction system is prepared according to Table 8 below.

[0164] Table 8 Template plasmid removal reaction system

[0165]

[0166] (2) Gently blow and mix, react at 37°C for 2h.

[0167] (3) After the reaction is completed, the reaction product is subjected to agarose gel electrophoresis, and the gel is recovered to obtain a linear target product.

[0168] 2.5.4 Recombination

[0169] (1) The recombination reaction system was prepared according to Table 9 below.

[0170] Table 9 Recombination reaction system

[0171]

[0172] (2) Gently mix by blowing, place in a PCR instrument at 37°C for 30 min, and immediately take out and place on ice after the reaction is completed.

[0173] 2.5.5 Transformation

[0174] The same as above.

[0175] 2.6 RT-PCR

[0176] The same as Example 1

[0177] 2.7 Western blot

[0178] The same as Example 1.

[0179] 2.8 Cell growth curve drawing

[0180] Trypsinize cells in the logarithmic growth phase, and inoculate into a 24-well plate at a density of 10,000 cells per well. The next day, take 3-well cells for digestion and counting, and then take 3-well cells for counting every day, continuously determine the number of cells for 7 days, and draw a cell growth curve.

[0181] 2.9 Colony formation experiment

[0182] (1) Trypsinize cells in the logarithmic growth phase, and inoculate into a 24-well plate at a density of 10,000 cells per well. The next day, take 3-well cells for digestion and counting, and then take 3-well cells for counting every day, continuously determine the number of cells for 7 days, and draw a cell growth curve.

[0183] 2.10 Scratch test

[0184] Cell scratch was made using ibidi insert, the specific steps are as follows: (1) Place ibidi insert into a twelve-well plate, take logarithmic growth phase cells for digestion, centrifugation, resuspend the cells, and adjust the density to 2.0-5.0 x 10 5 / ml.(2) Take 100 μl of cell suspension and add it to each chamber of ibidi, and place it in the incubator for culture.(3) After the cells adhere, gently remove the ibidi chamber with tweezers, discard the culture medium, wash twice with PBS to remove unattached cells and cell debris, add fresh 1% FBS-containing medium, then take a photo under a microscope, and record the time point as 0h.(4) Observe after 24h, 48h, take a photo under a microscope to record the scratch healing situation.(5) Use Image J software to quantify the scratch healing situation.

[0185] 2.11 Transwell experiment

[0186] (1) HCT116, RKO shUCKL1 knockdown group cells were induced with Dox for 3 days before the experiment, then the knockdown group and the control group cells were digested, centrifuged, and resuspended with medium without FBS, and the cell density was adjusted to 5.0 x 10 5 / ml.(2) Place the Transwell chamber in a 24-well plate, take 200 μl of cell suspension and add it to the inner chamber of the Transwell chamber, add 500 μl of medium containing 5% FBS to the outer chamber, then place it in the cell culture incubator for culture.(3) After 24-48h, discard the culture medium in the culture plate, wash gently with PBS for 2 times, then add 200 μl to the inner chamber and 500 μl of 4% paraformaldehyde to the outer chamber for fixation, and stand for 20 min.(4) Discard the 4% paraformaldehyde, add 1% crystal violet for staining, and stand for 30 min.

[0187] (5) Wash the chamber with PBS for 2 times, then place the chamber in a cell culture dish and take a photo under a microscope, and use ImageJ software to count the migrated cells.

[0188] 2.12 CCK8 method

[0189] After the cells are seeded in a 96-well plate and treated with drugs or other experimental methods, discard the culture medium, add fresh medium containing 10% CCK8, place it in the incubator for 1-2h, use the enzyme marker to measure the absorbance at 450nm wavelength, and calculate the cell survival rate (cell survival rate = experimental group absorbance / control group absorbance).

[0190] 2.13 Subcutaneous tumor formation experiment in nude mice

[0191] (1) Select 4-6 weeks old BALB / c-nu nude mice for experiment. (2) Trypsinize the cells in logarithmic growth phase, centrifuge at 1000 rpm for 5 min, wash with PBS for 2 times, resuspend the cells, adjust the cell density to 3.0 x 10 7 / ml. (3) Inject 100 μl of cell suspension subcutaneously into the back of the mouse hind limb, and wait for subcutaneous tumor formation. (4) The UCKL1 knockdown group of mice is given drinking water containing 2 mg / ml Dox, and the drinking water of the non-knockdown group does not contain Dox. (5) The tumor size is measured every 3 days, and the tumor size is calculated according to the volume = length x width x width / 2. (6) About 4 weeks, the mice are euthanized, the tumor is removed and weighed, and the RNA and protein of the tumor tissue are extracted for analysis, part of the tissue is fixed with 4% paraformaldehyde and embedded, sectioned, and immunohistochemical staining is performed.

[0192] 2.14 Immunohistochemistry

[0193] The same as Example 1.

[0194] 2.14 Statistical methods

[0195] Statistical analysis and statistical graphs were performed using GraphPad prism 8 software. Quantitative data conforming to normal distribution were expressed as mean ± standard deviation, and the comparison of means of two groups of samples used t test; the comparison between multiple groups of samples used One-way ANOVA; the comparison between growth curves used Two-way ANOVA. Quantitative data not conforming to normal distribution used rank sum test. P<0.05 was considered statistically significant.

[0196] 3. Experimental results

[0197] (1) UCKL1 shRNA stable cell strain knockdown efficiency detection

[0198] To study the role of UCKL1 in the occurrence and development of colorectal cancer, we used the method of knocking down UCKL1 in colorectal cancer cell lines to observe the effect of UCKL1 knockdown on tumor cell growth. By detecting the expression of UCKL1 in various colorectal cancer cell lines, Figure 4), we selected HCT116 and RKO cell lines with relatively high expression of UCKL1 for knockdown experiments. Using lentiviral vectors, we constructed stable cell lines that express UCKL1 shRNA after induction by doxycycline (Dox). We detected the knockdown efficiency by RT-PCR and Western blot. The RT-PCR results showed that after the addition of Dox, both shRNAs effectively reduced the expression of UCKL1 mRNA. The knockdown efficiency of the four stable cell lines HCT116-shUCKL1-1, HCT116-shUCKL1-2, RKO-shUCKL1-1, and RKO-shUCKL1-2 was about 80% after Dox induction Figure 4 A; B). Western blot results showed that after the addition of Dox, the expression of UCKL1 protein in HCT116-shUCKL-1, HCT116-shUCKL1-2, RKO shUCKL1-1, and RKO-shUCKL1-2 was significantly reduced Figure 4 C; D).

[0199] (2) Transfection of UCKL1 shRNA resistant plasmid to exclude shRNA off-target effects

[0200] To exclude the off-target effects of UCKL1 shRNA, we designed UCKL1 shRNA-1resistant and UCKL1 shRNA-2resistant plasmids, which were then transfected into HCT116-shUCKL1-1 and HCT116-shUCKL1-2 stable cell lines, respectively. Western blot was used to detect the expression of UCKL1 protein, and CCK8 was used to detect cell survival rate. Western blot results showed that UCKL1 shRNA-1resistant and UCKL1 shRNA-2resistant plasmids were correctly expressed and not degraded by UCKL1 shRNA Figure 5 A). CCK8 results showed that the knockdown of UCKL1 reduced cell survival rate, while the cell survival rate of the group transfected with UCKL1 shRNA resistant plasmid did not change significantly Figure 5 B).

[0201] (3) Knockdown of UCKL1 slows the growth rate of colorectal cancer cells

[0202] HCT116-shUCKL1-1, HCT116-shUCKL1-2 and RKO-shUCKL1-1, RKO shUCKL1-2 cell lines were induced by Dox to knock down UCKL1, and the cell growth rate was determined by counting the number of cells every day for 7 consecutive days, while the control group without Dox (without knocking down UCKL1) was used as a control. The growth curve shows that after knocking down UCKL1, the growth rate of colorectal cancer cell line HCT116 is significantly slowed down, and there is no significant difference in the number of cells in the first 3 days, but from the 4th day, the number of cells in the UCKL1 knockdown group is significantly less than that in the control group Figure 6 A). Similarly, knocking down UCKL1 in RKO cells also slows down the cell growth rate Figure 6 B).

[0203] (4) Knocking down UCKL1 inhibits the colony formation ability of colorectal cancer cells

[0204] We compared the colony formation ability of UCKL1 knockdown group and normal group, and the results showed that after knocking down UCKL1, the number of colonies formed by HCT116 and RKO cells was significantly reduced Figure 7 A; B), knocking down UCKL1 significantly weakened the colony formation ability of colorectal cancer cells.

[0205] (5) Knocking down UCKL1 can inhibit the migration of colorectal cancer cells

[0206] We further used scratch test and Transwell test to verify the effect of UCKL1 on cell migration. The scratch effect was made by ibidi plug-in, and the time point of pulling out the plug-in was recorded as 0h, and the cells were observed and photographed at 0h and 48h, respectively. The results showed that after 48h of scratching, the healing area of the scratch in the UCKL1 knockdown group was significantly smaller than that in the UCKL1 non-knockdown group: in HCT116 cells, the non-knockdown UCKL1 group healed about 80% at 48h, and the knockdown UCKL1 group healed about 50% Figure 8 A); for RKO cells, the scratch healing area decreased from about 98% to about 60% after knocking down UCKL1 Figure 8 B). At the same time, we also used Transwell test to verify the effect of UCKL1 on the migration ability of colorectal cancer cells. The results showed that the number of RKO cells migrated after knocking down UCKL1 was significantly less than that of the non-knockdown group Figure 8 C). The above results show that knocking down UCKL1 can inhibit the migration ability of colorectal cancer cells.

[0207] (6) Knocking down UCKL1 can inhibit the proliferation of colorectal cancer cells in nude mice

[0208] In vitro experiments showed that knockdown of UCKL1 could inhibit the proliferation and migration of colorectal cancer cells. Subsequently, we investigated whether UCKL1 affected the growth of colorectal cancer cells in vivo through subcutaneous tumor experiments in nude mice. We inoculated RKO-shUCKL1-1 and RKO-shUCKL1-2 stable cell lines into nude mice subcutaneously, and the experimental group achieved knockdown of UCKL1 by adding Dox (2 mg / ml) to the drinking water of the animals, while the control group did not contain Dox in the drinking water. The size of the transplanted tumors was measured every three days, and the observation was continued for about one month. After the experiment, the transplanted tumors were collected, the tumors were weighed, and the RNA and protein of the tumor tissue were extracted for analysis of the expression of UCKL1. The size of the tumors in nude mice was measured continuously, and statistical analysis was performed,

[0209] We found that the growth rate of tumors in the UCKL1 knockdown group was significantly lower than that in the control group ( Figure 9 A). After knockdown of UCKL1, the weight and size of the tumors collected at the end were significantly smaller than those in the non-knockdown group ( Figure 9 B; C). The RNA and total protein of the tumor tissue of the animals were extracted, and RT-PCR and Western blot were used to detect the expression of UCKL1. The RT-PCR results showed that in the mice given drinking water containing Dox (2 mg / ml), the UCKL1 mRNA was significantly down-regulated ( Figure 9 D). The Western blot results also confirmed that the expression of UCKL1 in the UCKL1 knockdown group of mice was significantly reduced ( Figure 9 E). Immunohistochemical staining of the tumor tissue of nude mice showed that in the Dox-fed group, the expression of UCKL1 was significantly reduced, and the positive rate of Ki67 was significantly lower than that in the non-Dox-fed group ( Figure 9 F).

[0210] The above results show that knockdown of UCKL1 can reduce the survival rate of colorectal cancer cell lines HCT116 and RKO in vitro, inhibit colony formation ability and migration; knockdown of UCKL1 can inhibit the growth of colorectal cancer cells in vivo.

[0211] Example 3 UCKL1 regulates ferroptosis of colorectal cancer cells

[0212] 1. Experimental materials

[0213] 1.1 Cell source

[0214] The same as in Example 2.

[0215] 2. Experimental method

[0216] 2.1 Cell culture

[0217] The same as in Example 2.

[0218] 2.2 Detection of lipid peroxidation

[0219] Lipid peroxidation was detected using the C11 BODIPY 581 / 591 fluorescent probe. Specifically...

[0220] The steps are as follows: (1) Seed cells into a six-well plate, and after adhesion, perform appropriate drug or other treatments. (2) Dilute C11 BODIPY 581 / 591 dye with serum-free medium to a final concentration of 5 μM. (3) Take the cells to be tested, discard the culture medium, wash once with PBS, add 1 ml of diluted C11 BODIPY 581 / 591 to each well, and incubate in a cell culture incubator for 30 min. (4) After incubation, discard the dye, wash once with PBS, then digest the cells with trypsin, and centrifuge at 1000 rpm for 5 min. (5) Discard the liquid, add PBS to wash again, centrifuge, and collect the cells. (6) Resuspend the cells in 300-500 μl PBS, filter into a flow cytometer, and then use a flow cytometer for detection, with the detection channel being FITC. (7) Analyze the flow cytometry results using FlowJo software and calculate the lipid peroxidation rate of each group.

[0221] 2.3 ROS detection

[0222] The intracellular ROS level was detected using a ROS detection kit. The specific steps are as follows: (1) Cells were seeded into six-well plates and subjected to appropriate drugs or other treatments after adhesion. (2) The DCFH-DA fluorescent probe was diluted with serum-free medium at a ratio of 1:1000. (3) The cells to be tested were taken, the original medium was discarded, and the cells were washed once with PBS. Then, 1 ml of diluted DCFH-DA was added to each well and incubated in a cell culture incubator for 30 min. (4) After incubation, the dye was discarded, the cells were washed once with PBS, and then the cells were digested with trypsin and centrifuged at 1000 rpm for 5 min. (5) The liquid was discarded, PBS was added and the cells were washed again, centrifuged, and the cells were collected. (6) The cells were resuspended in 300-500 μl of PBS, filtered into flow cytometry tubes, and then detected by flow cytometer with the FITC detection channel set. (7) The flow cytometry results were analyzed using FlowJo software, and the fluorescence intensity of each group was calculated.

[0223] 2.4 GSSG Content Detection

[0224] GSH and GSSG detection kit was used to detect the content of GSSG in cells, and the specific steps were as follows: (1) the cells were inoculated into six-well plates, and the corresponding drugs or other treatments were carried out after adhering. (2) The cells to be detected were taken, the culture medium was discarded, and PBS was washed once. After trypsin digestion, the cells were collected by centrifugation. (3) 30 μl of protein removal reagent was added, and vortexed for 30 s. (4) The sample was quickly frozen in liquid nitrogen, and then thawed in a 37°C water bath. Repeat 2 times. (5) Stand for 5 min on ice, centrifuge at 4°C, 10000 rpm for 10 min. The supernatant was transferred to a new 1.5 ml EP tube for total glutathione detection. (6) Dilute the above sample 10 times, add GSH removal auxiliary liquid at a ratio of 5:1, vortex well. Then add GSH removal reagent at a ratio of 25:1, mix well and react at 25°C for 1 h. This is the GSSG detection sample. (7) Add 150 μl of glutathione detection reagent to every 10 μl of sample, mix well and incubate at 25°C for 5 min. (8) Add 50 μl of NADPH solution (0.5 mg / ml), mix well and react for 25 min, then use the enzyme marker to detect the absorbance at 412 nm.

[0225] 2.5 NADP+ and NADP+ / NADPH detection

[0226] NADP+ / NADPH detection kit was used to detect NADP+ in cells, and the specific steps were as follows: (1) The cells were inoculated into six-well plates, and the corresponding drug treatment was carried out after adhering. (2) The cells to be detected were taken, the culture medium was discarded, and PBS was washed twice. 200 μl of NADP+ / NADPH extraction reagent was added to each well, and the cells were lysed on ice. (3) Centrifuge at 4°C, 12000 rpm for 10 min, take the supernatant as the sample for detection of total NADP+ and NADPH. (4) Take 100 μl of the above sample, incubate at 60°C for 30 min to decompose NADP+, and obtain the NADP detection sample. (5) Take 50 μl of the sample, add 100 μl of G6PD working solution, and react at 37°C for 10 min in the dark. (6) Add 10 μl of chromogenic solution, react at 37°C for 20 min, then use the enzyme marker to detect the absorbance at 450 nm.

[0227] 2.6 Statistical method

[0228] GraphPad prism 8 software was used for statistical analysis and statistical graph. The quantitative data conforming to normal distribution were represented by mean ± standard deviation, and the comparison of means of two groups of samples used t test; the comparison among multiple groups of samples used One-way ANOVA. The quantitative data not conforming to normal distribution used rank sum test. P<0.05 was considered statistically significant.

[0229] 3. Experimental results

[0230] (1) Ferroptosis inhibitors and antioxidants can rescue the decrease of cell viability caused by knockdown of UCKL1 in colorectal cancer cells

[0231] Knockdown of UCKL1 can cause decrease of cell viability in colorectal cancer cells, so we investigated which kind of cell death was involved in this process. After knockdown of UCKL1 in HCT116 cells, we added apoptosis inhibitor Z-VAD-FMK (Z-VAD), necrosis inhibitor Necrostatin-1 (Nec-1) and ferroptosis inhibitor Ferrostatin-1 (Fer-1) to observe whether they had a rescue effect. We found that apoptosis inhibitor Z-VAD and necrosis inhibitor Nec-1 could not effectively reverse the decrease of cell viability caused by knockdown of UCKL1, while ferroptosis inhibitor Fer-1 could increase the cell viability of UCKL1 knockdown group to some extent, increasing the cell viability from about 50% to about 80%. Figure 10 Since ferroptosis is caused by lipid peroxidation of unsaturated fatty acids on the cell membrane, we also used antioxidant N-acetyl-L-cysteine (NAC) for rescue, and the results showed that antioxidant NAC could also reverse the decrease of cell viability caused by knockdown of UCKL1

[0232] (2) Knockdown of UCKL1 can increase lipid peroxidation in colorectal cancer cells

[0233] Ferroptosis inhibitors and antioxidants can rescue the decrease of cell viability caused by knockdown of UCKL1 in colorectal cancer cells, which suggests that ferroptosis may occur in cells after knockdown of UCKL1. Therefore, we detected the ferroptosis marker-lipid peroxidation after knockdown of UCKL1. C11 BODIPY 581 / 591 is a fluorescent probe for detecting cell lipid peroxidation, which can reflect the ferroptosis of cells by flow cytometry or fluorescence microscope after incubation with cells. After staining cells with C11 BODIPY 581 / 591, we analyzed them by flow cytometry and found that the level of lipid peroxidation in colorectal cancer cells significantly increased after knockdown of UCKL1, with the lipid peroxidation rate of HCT116-shUCKL1-1, HCT116-shUCKL1-2 and RKO-shUCKL1-1, RKO-shUCKL1-2 groups increasing to about 15% Figure 11 A; B). This result indicates that UCKL1 regulates the ferroptosis process of colorectal cancer cells, and knockdown of UCKL1 promotes the increase of cell ferroptosis.

[0234] (3) Knockdown of UCKL1 increases total ROS levels in colorectal cancer cells.

[0235] In addition to detecting changes in lipid ROS, we also measured the total ROS level in cells to more comprehensively reflect ferroptosis. The total ROS level in cells was detected and analyzed using flow cytometry with the DCFH-DA fluorescent probe. The results showed that in HCT116-shUCKL1-1 cells, the ROS level in the UCKL1 knockdown group was approximately 3 times that of the non-knockdown group, and in HCT116-shUCKL1-2 cells, the ROS level in the UCKL1 knockdown group was approximately 2.6 times that of the non-knockdown group. Figure 12 A). Similarly, after UCKL1 knockdown, ROS in RKO cells increased significantly, with ROS in both the RKO-shUCKL1-1 and RKO-shUCKL1-2 knockdown groups increasing by approximately 2.3-fold. Figure 12 B). This indicates that knocking down UCKL1 can put colorectal cancer cells in a state of oxidative stress and cause lipid peroxidation, thus inducing ferroptosis.

[0236] (4) Knockdown of UCKL1 can increase the levels of oxidized glutathione (GSSG) and NADP+ in colorectal cancer cells.

[0237] To further verify that tumor cells were in a high ROS state after UCKL1 knockdown, we measured the relative content of GSSG in the cells and calculated the ratio of oxidized glutathione to reduced glutathione (GSSG / GSH). An increase in GSSG or the GSSG / GSH ratio reflects an increase in ROS in the cells. The results showed that after UCKL1 knockdown, the GSSG content in HCT116 cells increased by approximately 1.5 times, and in RKO cells by 1.5–1.8 times. Figure 13 A; B). Regarding GSSG / GSH, the UCKL1 knockdown group in HCT116 cells was approximately 1.5 times that of the control group, while the GSSG / GSH ratio in the UCKL1 knockdown group of RKO cells was significantly lower.

[0238] It also showed an increase, approximately 1.6 times that of the control group. Figure 13 C; D). We also examined the relative content of NADP+ in cells and calculated the NADP+ / NADPH ratio. NADP+ is also an indicator of intracellular redox levels; increases in NADP+ and the NADP+ / NADPH ratio indicate increased ROS levels in cells. The results showed that knocking down UCKL1 increased the NADP+ content in both HCT116 and RKO cells (C; D). Figure 13 E; F), and NADP+ / NADPH also increased (E; F), Figure 13G; H). The above results further confirmed that after knocking down UCKL1, a high ROS state was present in colorectal cancer cells.

[0239] The above results show that knocking down UCKL1 induces ferroptosis in colorectal cancer cells, which is manifested in that after knocking down UCKL1, the cell lipid peroxidation increases, the total ROS level in the cell increases, the GSSG and GSSG / GSH values in the cell increase, and the NADP+ and NADP+ / NADPH values increase; the decrease in cell survival rate after knocking down UCKL1 can be remedied by ferroptosis inhibitors and antioxidants.

[0240] Further experiments confirmed that after knocking down UCKL1, the expression of SLC7A11 in colorectal cancer cells was down-regulated, and overexpression of SLC7A11 could remedy the decrease in cell survival rate caused by knocking down UCKL1, and reduce the high ROS and lipid peroxidation levels in the cell. UCKL1 regulates the expression of SLC7A11 by affecting the stability of NRF2 protein, and after knocking down UCKL1, the stability of NRF2 protein decreases, the expression decreases, thereby inhibiting the expression of downstream gene SLC7A11. Overexpression of NRF2 can remedy the decrease in cell survival rate caused by knocking down UCKL1, and inhibit the increase of ROS and lipid peroxidation in the cell (specific data omitted).

[0241] Example 4 Synergistic inhibition of the growth of colorectal cancer cells by targeting UCKL1 in combination with GPX4 inhibitors

[0242] In the ferroptosis-related pathway, SLC7A11 and GPX4 are two key proteins for inhibiting ferroptosis, and UCKL1 acts on one of the two inhibitors, SLC7A11. This embodiment studies whether the combination of targeted inhibition of UCKL1 and GPX4 inhibitors can further increase ferroptosis and play a synergistic tumor inhibition effect.

[0243] 1. Experimental materials

[0244] The cell source is the same as in Example 2.

[0245] 2. Experimental method

[0246] The cell culture is the same as in Example 2. The lipid peroxidation detection is the same as in Example 3. The ROS detection is the same as in Example 3. The CCK8 experiment is the same as in Example 2. The immunohistochemistry is the same as in Example 1.

[0247] Statistical method

[0248] Statistical analysis and statistical graphs were performed using GraphPad prism 8 software. Quantitative data that met the normal distribution were expressed as mean ± standard deviation, and the comparison of the means of two groups of samples used t test; the comparison among multiple groups of samples used One-way ANOVA; the comparison between growth curves used Two-way ANOVA. Quantitative data that did not obey the normal distribution used rank sum test. P<0.05 was considered statistically significant.

[0249] 3. Experimental results

[0250] (1) Knockdown of UCKL1 combined with GPX4 inhibitor can synergistically reduce the in vitro survival rate of colorectal cancer cells

[0251] Knockdown of UCKL1 can promote ferroptosis of colorectal cancer cells by down-regulating SLC7A11 expression, and SLC7A11 and GPX4 are two key inhibitors in the process of ferroptosis. We speculate that targeting UCKL1 combined with GPX4 inhibitor has a synergistic effect on inhibiting tumors. To verify this inference, we used knockdown of UCKL1 combined with GPX4 inhibitor RSL3 or ML162 to observe the effect of their combination on colorectal cancer cells. First, CCK8 was used to detect the survival rate of cells treated with RSL3 alone and after combination. In HCT116 cells, 2 μM RSL3 was used to treat cells for 24 h, and the survival rate of cells was about 85%. When UCKL1 was knocked down and combined with RSL3, the survival rate of cells was only about 7% ( Figure 14 A). Similarly, in RKO cells, the survival rate of cells was greatly reduced when UCKL1 was knocked down and combined with RSL3 ( Figure 14 B), which was lower than that of the group with only UCKL1 knocked down and lower than that of the group with only RSL3. At the same time, we selected another inhibitor of GPX4, ML162, to conduct experiments. CCK8 results also showed that the combination of knockdown of UCKL1 and GPX4 inhibitor could more greatly inhibit tumor growth. In HCT116 cells, 5 μM ML162 was used to treat cells for 24 h, and the survival rate of cells was about 90%. After knockdown of UCKL1, the survival rate of cells decreased to about 10% after treatment with 5 μM ML162 ( Figure 14 C). In RKO cells, there was the same trend, and the combination of knockdown of UCKL1 and ML162 greatly reduced the survival rate of tumor cells ( Figure 14 D).

[0252] (2) Knockdown of UCKL1 combined with GPX4 inhibitor can synergistically increase ROS and lipid peroxidation in colorectal cancer cells

[0253] Next, we detected the ROS and lipid peroxidation levels in cells after the combination of knockdown of UCKL1 and GPX4 inhibitors to reflect the situation of ferroptosis in cells. After DCFH-DA incubation of cells, the flow cytometry analysis results showed that the ROS level was significantly increased in HCT116 and RKO cells after the combination of knockdown of UCKL1 and RSL3 (5 μM, 4 h) Figure 15 A; B). Similarly, the combination of knockdown of UCKL1 and RSL3 (5 μM, 4 h) also significantly increased the lipid peroxidation level of cells Figure 15 C; D). We also observed the ROS and lipid peroxidation in cells after the combination of GPX4 inhibitor ML162 and knockdown of UCKL1. The results also showed that the combination of the two could significantly increase the ROS Figure 15 E; F) and lipid peroxidation level Figure 15 G; H) in colorectal cancer cells. This indicates that the combination of knockdown of UCKL1 and GPX4 inhibitors can synergistically increase the ferroptosis of colorectal cancer cells.

[0254] (3) The combination of targeting UCKL1 and GPX4 inhibitors can synergistically inhibit the growth of colorectal cancer cells in nude mice

[0255] Finally, we verified the synergistic tumor inhibition effect of knockdown of UCKL1 and GPX4 inhibitors in nude mice. We inoculated HCT116-shUCKL1-1 stable cell line into the subcutaneous tumor of nude mice, and achieved the purpose of knockdown of UCKL1 by adding Dox (2 mg / ml) in the drinking water of animals. RSL3 (50 mg / kg) was injected intratumorally on the 7th and 12th day after tumor formation, and the size of transplanted tumor was measured every three days for about one month, and the growth rate of tumor was analyzed. After the end of the experiment, the transplanted tumor was collected, the tumor was weighed, and the expression of UCKL1, SLC7A11 and lipid peroxidation marker 4-HNE in tumor tissue was analyzed by immunohistochemistry. The size of tumor in nude mice was measured continuously, and the statistical analysis was performed, and we found that the growth rate of tumor in the combination of knockdown of UCKL1 and RSL3 group was significantly slower than that in the control group, the group of only knockdown of UCKL1 and the group of only RSL3 Figure 16 A). The tumor was weighed, and the tumor weight in the group of only knockdown of UCKL1 and the group of only RSL3 was less than that in the control group, and the tumor weight in the combination of knockdown of UCKL1 and RSL3 group was the lightest Figure 16 B), the volume was the smallest Figure 16 C). The RNA in tumor tissue in each group was extracted, and the expression of UCKL1 and SLC7A11 was detected by RT-PCR, and the results showed that the expression of UCKL1 mRNA in tumor tissue in the Dox feeding group was significantly reduced Figure 16 D), and the expression of SLC7A11 mRNA was also significantly decreasedFigure 16 E) Immunohistochemical analysis of each group of tumor tissues showed that UCKL1 expression was reduced in the Dox group, and SLC7A11 expression was also down-regulated; in the UCKL1 knockdown group and the RSL3 combination group, the lipid peroxidation marker 4-HNE staining was most obvious Figure 16 F).

[0256] The above results show that the combination of UCKL1 knockdown and GPX4 inhibitor can synergistically inhibit the growth of colorectal cancer cells in vitro, increase the ROS and lipid peroxidation levels in the cells; the combination of UCKL1 knockdown and GPX4 inhibitor can synergistically inhibit the growth of colorectal cancer cells in nude mice.

[0257] Each of the technical features of the above-described embodiments can be combined arbitrarily, and to make the description concise, each of the technical features in the above embodiments is not described in all possible combinations, however, as long as the combination of these technical features does not exist contradictory, it should be considered as the scope of the present disclosure.

[0258] The above-described embodiments only express several embodiments of the present application, which are described in detail and in detail, but should not be construed as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Use of an agent that inhibits the expression of the UCKL1 gene for the manufacture of a medicament for the treatment of colorectal cancer, characterized in that, The agent for inhibiting expression of the UCKL1 gene is at least one selected from shRNA1 as shown in SEQ ID NO. 5 and shRNA2 as shown in SEQ ID NO.

6.

2. Use according to claim 1, wherein The drug can induce ferroptosis in colorectal cancer cells.

3. The use according to claim 1, wherein The drug can inhibit the NRF2-SLC7A11 signaling pathway.

4. Use of a composition for the manufacture of a medicament for the treatment of colorectal cancer, characterized in that, The composition comprises an agent for inhibiting expression of the UCKL1 gene and a GPX4 inhibitor. The agent for inhibiting expression of the UCKL1 gene is at least one selected from shRNA1 as shown in SEQ ID NO. 5 and shRNA2 as shown in SEQ ID NO.

6. The GPX4 inhibitor is at least one selected from RSL3 and ML162.

5. A medicament for treating colorectal cancer, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The drug comprises an agent for inhibiting expression of the UCKL1 gene and a GPX4 inhibitor, and a pharmaceutically acceptable excipient. The agent for inhibiting expression of the UCKL1 gene is at least one selected from shRNA1 as shown in SEQ ID NO. 5 and shRNA2 as shown in SEQ ID NO.

6. The GPX4 inhibitor is at least one selected from RSL3 and ML162.

6. Use of an agent for detecting the level of UCKL1 gene and / or protein in the preparation of a colorectal cancer detection kit, a colorectal cancer therapeutic effect evaluation kit or a colorectal cancer prognosis evaluation kit.

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