Application of mitochondrial pyruvic acid transporter 2 in preparation of medicine for treating liver cancer

Abstract

The invention relates to the field of biological medicines, in particular to an effect of mitochondrial pyruvate carrier 2 (MPC2) in proliferation, drug resistance and progress of liver cancer and an application of the MPC2 in preparation of a medicine for treating the liver cancer. According to the invention, a molecular target spot MPC2 for promoting proliferation, drug resistance and progress of liver cancer is found, and can be used as a new therapeutic target spot applied to treatment of liver cancer. The target MPC2 is provided, the expression quantity of the target MPC2 is reduced by inhibiting the function of the MPC2 or by means of gene editing and the like, the target MPC2 is applied to liver cancer treatment, and a new thought is provided for precise treatment of liver cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to the role of mitochondrial pyruvate carrier 2 (MPC2) in promoting liver cancer proliferation, drug resistance and progression, and its use as a target in the preparation of drugs for treating liver cancer. Background Art

[0002] Currently, surgical treatment is considered the first choice for radical treatment of liver cancer. For some patients with advanced HCC who cannot tolerate surgical treatment, researchers have turned their attention to molecular targeted therapy and immunotherapy. Since the emergence of immune checkpoint inhibitor (ICI)-based therapy, the prognosis of patients with advanced HCC has improved, but only about 30% of patients have objective remission, and the 3-year overall survival (OS) rate is far below 50% (LLOVET JM, PINYOL R, KELLEY RK, EL-KHOUEIRY A, REEVES HL, WANG XW, GORES GJ, VILLANUEVA A. Molecular pathogenesis and systemic therapies for hepatocellular carcinoma [J]. NatCancer, 2022, 3(4): 386-401.). Since liver cancer is one of the most heterogeneous tumors, the effectiveness of therapeutic targets is an important factor in achieving precise treatment and improving treatment response rate.

[0003] The mitochondrial pyruvate carrier (MPC), located in the inner mitochondrial membrane, transports pyruvate from the cytoplasm to the mitochondrial matrix, where it participates in the tricarboxylic acid cycle, gluconeogenesis, and metabolic processes such as lipids and amino acids, providing energy for the body. Therefore, the MPC may regulate energy metabolism by regulating the flux of pyruvate into the mitochondrial matrix (HERZIG S, RAEMY E, MONTESSUIT S, VEUTHEY JL, ZAMBONI N, WESTERMANN B, KUNJIE R, MARTINOU JC. Identification and functional expression of the mitochondrial pyruvate carrier [J]. Science, 2012, 337(6090): 93-96.). The mammalian mitochondrial pyruvate transporter is composed of two proteins, MPC1 and MPC2, which form a heteromeric complex within the mitochondrial inner membrane and are both required for the activity and stability of the MPC complex (BRICKER DK, TAYLOR EB, SCHELL JC, ORSAK T, BOUTRON A, CHEN YC, COX JE, CARDON CM, VAN VRANKEN JG, DEPHOUREN, REDIN C, BOUDINA S, GYGI SP, BRIVET M, THUMMEL CS, RUTTER J. Amitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans [J]. Science, 2012, 337(6090): 96-100.).Scientists from Baylor College of Medicine in the United States revealed that the energy source required by androgen receptor-driven prostate cancer cells depends on mitochondrial pyruvate oxidation. Inhibiting MPC can effectively inhibit the level of oxidative phosphorylation in cells, suggesting that MPC may be a potential therapeutic target for prostate cancer (BADER DA, HARTIG SM, PUTLURI V, FOLEY C, HAMILTON MP, SMITH EA, SAHA PK, PANIGRAHI A, WALKER C, ZONG L, MARTINI-STOICA H, CHEN R, RAJAPAKSHEK, COARFA C, SREEKUMAR A, MITSIADES N, BANKSON JA, ITTMANN MM, O'MALLEY BW, PUTLURI N, MCGUIRE S E. Mitochondrial pyruvate import is a metabolicvulnerability in androgen receptor-driven prostate cancer [J]. Nat Metab,2019, 1(1): 70-85.). In addition, in a mouse model with MPC2 deficiency in the liver, researchers observed that gluconeogenesis was impaired to a certain extent, and hepatocytes partially compensated for the metabolic abnormalities caused by MPC2 deficiency through changes in amino acid metabolism (including the pyruvate-alanine cycle) (MCCOMMIS KS, CHEN Z, FU X, MCDONALD WG, COLCA JR, KLETZIEN RF, BURGESS SC, FINCK B N. Loss of Mitochondrial Pyruvate Carrier2 in the Liver Leads to Defects in Gluconeogenesis and Compensation via Pyruvate-Alanine Cycling [J]. Cell Metab, 2015, 22(4): 682-694.). However, there are currently no reports on the role of MPC2 in liver cancer, nor any studies on MPC2 as a therapeutic target for liver cancer. Summary of the Invention

[0004] The purpose of the present invention is to provide a molecular target, mitochondrial pyruvate carrier 2 (MPC2), which promotes liver cancer proliferation and drug resistance and thus liver cancer progression. It can be used as a new therapeutic target in liver cancer treatment.

[0005] Previous studies in this study revealed that mitochondrial pyruvate transporter 2 (MPC2) is highly expressed in liver cancer tissues. Single-cell sequencing data analysis confirmed that MPC2 is highly expressed in liver cancer cells. Real-time quantitative PCR, Western blot, and immunohistochemistry confirmed that both the MPC2 gene and protein levels were significantly elevated in liver cancer tissues, suggesting a role in liver cancer progression. To investigate the effects of MPC2 on liver cancer cells, stably knocked-down MPC2 cell lines were constructed using the human liver cancer cell lines HUH7 and PLC, respectively. Cell proliferation experiments demonstrated a positive correlation between MPC2 expression and liver cancer cell proliferation. Furthermore, MPC2 mediates resistance to lenvatinib in liver cancer cells. MPC2-overexpressing cells exhibited significantly higher IC50s for lenvatinib than cells with low MPC2 expression. Furthermore, MPC2-overexpressing cells exhibited significantly higher IC50s for 5-fluorouracil and oxaliplatin than cells with low MPC2 expression. The combined use of MPC2 inhibitors and lenvatinib can enhance the killing ability of lenvatinib against liver cancer cells.

[0006] In the in vivo animal model, the liver orthotopic tumor-bearing model: Hepatocellular carcinoma was formed by high-pressure injection of the tail vein with a CTNNB1 gene mutation plasmid using the Sleeping Beauty system. When AAV-shMPC2 was administered to knock down MPC2 for intervention treatment, the liver tumor burden of the mice was significantly lower than that of the control group. The subcutaneous tumor-bearing experiment in mice: C57BL / 6 mice were inoculated with liver cancer cells to construct a subcutaneous tumor-bearing model. MPC2 targeted inhibitors were administered. The tumor size, tumor weight, and tumor growth rate of the inhibitor group mice were significantly lower than those of the control group, indicating that MPC2 regulates the growth of liver cancer in the in vivo model. Therefore, the present invention believes that MPC2 promotes the progression of liver cancer by regulating the proliferation and drug resistance of liver cancer cells. Targeting MPC2 can be used as a new target for the treatment of liver cancer.

[0007] The first aspect of the present invention provides a molecular target, MPC2, which promotes liver cancer proliferation, drug resistance and progression.

[0008] The specific information of MPC2 is as follows: the gene information encoding this protein is Gene ID: 25874; the existing aliases are: BRP44, SLC54A2, and the encoding gene is located at: 1q24.2.

[0009] The second aspect of the present invention provides the use of an agent that blocks the function of MPC2 or inhibits the expression of MPC2 in the preparation of a drug for treating liver cancer.

[0010] Furthermore, the blocking of MPC2 function or inhibition of MPC2 expression is an agent that blocks the function of MPC2 or regulates the expression and / or secretion level of MPC2, including but not limited to MPC2 inhibitors, MPC2 antibodies, siRNA or shRNA of the MPC2 gene, microRNA that inhibits MPC2 gene expression, or plasmids or viral expression vectors.

[0011] In one embodiment of the present invention, the agent that blocks the function of MPC2 or inhibits the expression of MPC2 is UK5099.

[0012] In one embodiment of the present invention, the agent for blocking the function of MPC2 or inhibiting the expression of MPC2 is shRNA of the MPC2 gene, and its nucleotide sequence is shown in SEQ ID NO.3.

[0013] Furthermore, the application is for single or combined use.

[0014] Furthermore, the combination therapy is a combination of an agent that blocks the function of MPC2 or inhibits the expression of MPC2 and an anti-liver cancer drug.

[0015] Furthermore, the anti-liver cancer drugs include targeted drugs such as sorafenib, lenvatinib, regorafenib, apatinib, anlotinib; 5-FU, cisplatin, doxorubicin (ADM), oxaliplatin, etc., as well as monoclonal antibodies including immune checkpoint inhibitors such as atezolizumab and bevacizumab.

[0016] Furthermore, the drug for treating liver cancer is a drug that inhibits the growth and progression of liver cancer.

[0017] The third aspect of the present invention provides the use of an agent that blocks the function of MPC2 or inhibits the expression of MPC2 in the preparation of an anti-liver cancer drug sensitizer.

[0018] Furthermore, the reagent that blocks the function of MPC2 or inhibits the expression of MPC2 can enhance the therapeutic effect of anti-liver cancer drugs in killing tumors.

[0019] The advantages of the present invention are:

[0020] This study discovered a molecular target, MPC2, that promotes liver cancer proliferation, drug resistance, and progression. This target could be used as a new therapeutic target for liver cancer treatment. This study proposes using MPC2 to treat liver cancer by blocking its function or regulating its expression and / or secretion, providing new insights into the precision treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Detection of MPC2 expression in HCC and adjacent tissues; (A) Real-time fluorescence quantitative PCR detection of gene expression levels; (B) Western blot detection of protein expression levels; (C) Immunohistochemistry detection of MPC2 levels and quantitative analysis.

[0022] Figure 2 . The biological behavior of MPC2 affecting liver cancer proliferation: CCK8 experiment was used to detect the effect of MPC2 on the proliferation ability of liver cancer cells.

[0023] Figure 3 CCK8 assay detects MPC2-mediated lenvatinib resistance; (A) Resistance curve of liver cancer cell line; (B) Resistance curve of liver cancer cell line with stable expression of MPC2 knockdown; (C) Effect of combined treatment with an MPC2 inhibitor and lenvatinib on the survival rate of the HUH7 liver cancer cell line; (D) Effect of combined treatment with an MPC2 inhibitor and lenvatinib on the survival rate of the PLC liver cancer cell line.

[0024] Figure 4 (A) Effect of 5-fluorouracil on the survival rate of G2 and G2-shMPC2 liver cancer cell lines; (B) Effect of oxaliplatin on the survival rate of G2 and G2-shMPC2 liver cancer cell lines.

[0025] Figure 5 (A) Correlation between MPC2 and 5-fluorouracil resistance in liver cancer cell lines; (B) Correlation between MPC2 and oxaliplatin resistance in liver cancer cell lines

[0026] Figure 6 Subcutaneous tumor-bearing experiment in mice; (A) Schematic diagram of model establishment; (B) Gross image of in situ liver cancer; (C) Liver-to-body ratio curve.

[0027] Figure 7 Subcutaneous tumor-bearing experiment in C57BL / 6 mice; (A) Gross image of subcutaneous tumor; (B) Subcutaneous tumor growth curve. DETAILED DESCRIPTION

[0028] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0029] Example 1: MPC2 is highly expressed in liver cancer tissue

[0030] This study obtained a validation cohort of liver cancer tissue samples from the Oriental Hepatobiliary Surgery Hospital. Informed consent was obtained from the patients and approved by the Ethics Committee of the Oriental Hepatobiliary Surgery Hospital. In this example, HCC and adjacent adjacent tissue samples from 12 of these patients were analyzed using polymerase chain reaction (PCR) and Western blot assays to measure MPC2 levels.

[0031] This study was approved by the Ethics Committee of Oriental Hepatobiliary Surgery Hospital and included tissue microarrays of 262 patients with HCC and adjacent adjacent tissue samples.

[0032] (1) Reagents and materials

[0033] 1. Western and IP cell lysate (Biyuntian Biotechnology, P0013) were purchased from Biyuntian;

[0034] 2. PMSF (Biyuntian Biotechnology, ST506) was purchased from Biyuntian;

[0035] 3. BCA protein quantification kit (Pierce), purchased from Thermo Fisher;

[0036] 4.OLFM4 (D1E4M) XP® Rabbit mAb #14369, purchased from CST.

[0037] 5.HiScript II Reverse Transcriptase (Novozymes, R201), purchased from Novozymes;

[0038] 6. FastStart Universal SYBR Green Master (Roche, 04913850001), purchased from Roche.

[0039] 7. MPC2 (D4I7G) Rabbit mAb #46141, purchased from CST.

[0040] 8. MPC2 Antibody (OACA09748) was purchased from Aviva.

[0041] (II) Real-time fluorescence quantitative PCR experimental steps

[0042] 1. Primer Design: Primers are short DNA sequences used to amplify the target DNA fragment. Two primers are placed at either end of the target sequence and are typically 18-24 base pairs long. The primer sequences must be complementary to the target sequence to ensure binding and amplification specificity. The MPC2 primer sequences are: F: 5'- GGGACCTTTTCCTCACGTCC-3' (SEQ ID NO. 1), R: 5'- CTTTATCGAGGAGCCGGTGG-3' (SEQ ID NO. 2).

[0043] 2. RNA Extraction: 1) Sample Preparation: Add 1 ml of Trizol to 50-100 mg of tissue and homogenize on ice. Lyse at room temperature for 5-10 minutes. 2) Chloroform Extraction: Add 200 μl of chloroform to every 1 ml of Trizol. Cap the tube and mix vigorously by inverting for approximately 15 seconds. Incubate at room temperature for 2-3 minutes. After centrifugation at 12,000 g for 15 minutes at 2-8°C, the solution is separated into three layers: aqueous phase (RNA), middle layer, and organic phase (DNA, protein, etc.) from top to bottom. Transfer the aqueous phase to a new EP tube. 3) Isopropanol precipitation: Add 500 μl of isopropanol to every 1 ml of Trizol, mix thoroughly by inversion, incubate at room temperature for 10 minutes, and centrifuge at 12,000 g for 10 minutes at 2-8°C. The resulting white precipitate is the RNA precipitate. 4) Ethanol wash: Discard the supernatant, add 1 ml of 75% ethanol (per 1 ml of Trizol) to wash the RNA precipitate, and centrifuge at 7,500 g for 5 minutes at 2-8°C. 5) Dissolve: Discard the supernatant, dry the RNA precipitate in a fume hood for 5-10 minutes, and resuspend the precipitate in about 50 μl of enzyme-free water to obtain the RNA solution.

[0044] 3. Reverse Transcription: Before the PCR reaction begins, the DNA template must be heat denatured to unwind the double-stranded DNA into single-stranded DNA for primer binding. Using the HiScript II Reverse Transcriptase Kit, heat denaturation steps at 37°C and 85°C for 15 minutes are performed to generate cDNA.

[0045] 4. Real-time fluorescence quantitative PCR: In a 20-ul reaction system, purified cDNA (20 ng) was used as a template. Water (1:3 volume ratio of water to cDNA), 1 ul of primer, and 5 ul of FastStart Universal SYBR Green Master reagent were added. PCR was performed using a Roche LC96 LightCycler real-time fluorescence quantitative PCR instrument.

[0046] (III) Western Blot Experimental Steps

[0047] 1. Preparation of Cell Lysate: Take 1g of fresh-frozen liver cancer sample, add the appropriate amount of Western Blot and IP cell lysis buffer, and lyse using a homogenizer. Next, sonicate the cells on ice (using a sonicator at 30% intensity, 3-5 times for 3-5 seconds each, with 1-second intervals). After sonication, centrifuge the mixture at 4°C for 15 minutes at 12,000 rpm. Transfer the supernatant to a fresh EP tube to obtain the cell lysate.

[0048] 2. Protein quantification (BCA assay): Take a 96-well plate and add 25 μl of each protein standard to wells 1-8. The concentrations are expressed in μg / μl and are 0.00, 0.125, 0.25, 0.5, 0.75, 1.00, 1.25, and 1.50, respectively. Add 5 μl of the cell lysate to be tested and 20 μl of deionized water to the remaining wells. Then, add 200 μl of BCA protein colorimetric solution to each well containing the protein standard and cell lysate to be tested. This colorimetric solution must be prepared fresh by mixing Solution A and Solution B from the BCA kit in a 50:1 ratio. Then, place the 96-well plate in a light-proof incubator at 37°C for 15-20 minutes. After incubation, the protein standard and samples will develop a purple color of varying shades, depending on the protein concentration. After color development, place the 96-well plate in a microplate reader and measure the absorbance. A standard curve is constructed using the concentrations and corresponding absorbances of the protein standards to calculate the protein concentration corresponding to the absorbance of each sample. After quantification, 40 μg of protein is taken from each sample and one-third of the volume of 4× SDS is added. Heat the sample at 100°C for 5 minutes using a honeycomb heater to denature the protein. After denaturation, place the sample in an ice bath for 2 minutes. The sample can then be subjected to electrophoresis or stored refrigerated.

[0049] 3. Protein Electrophoresis: Prepare a 10% SDS-PAGE discontinuous electrophoresis gel (consisting of a lower separating gel and an upper stacking gel) and install it in a vertical electrophoresis tank. Add 1x protein electrophoresis buffer and load the sample and protein marker into each lane. After loading, run the electrophoresis at a constant voltage of 80V for approximately 1 hour to allow the proteins to run through the upper gel and into the lower gel. Then, change the voltage to 120V until the bromophenol blue line at the protein front reaches the bottom of the separating gel, at which point the electrophoresis is stopped.

[0050] 4. Transfer and Blocking: After electrophoresis, remove the gel and rinse with deionized water. Place the gel on the NC membrane (being careful not to trap any air bubbles). Place a layer of filter paper above and below the gel and NC membrane. Install the membrane in a wet transfer apparatus, add sufficient electrotransfer buffer, and transfer the membrane at a constant current of 0.23A for 90 minutes. Remove the NC membrane and soak it in 5% BSA blocking buffer at room temperature on a shaker for 1 hour.

[0051] 5. Antibody Incubation and Scanning: After blocking, wash the NC membrane three times with 1× TBST (5 minutes each). Depending on the molecular weight being detected, cut the NC membrane and place it in a dark, light-proof incubation box. Add the corresponding primary antibody and incubate on a shaker at 4°C overnight (or for 2 hours at room temperature). After incubation, wash the membrane three times with 1× TBST (5 minutes each). Then, add the corresponding fluorescent secondary antibody based on the primary antibody species and incubate on a shaker at room temperature for 1 hour. After incubation, wash the membrane three times with 1× TBST (5 minutes each). Then, detect on an Odyssey fluorescence scanner.

[0052] (IV) HE and immunohistochemical staining

[0053] 1. HE staining: 1) Before starting the experiment, bake the sections in a 60°C oven for one hour. Then, rehydrate them in the following order: soak in xylene for 10 minutes (three times) → soak in 100% ethanol for 5 minutes → soak in 95% ethanol for 5 minutes → soak in 85% ethanol for 5 minutes → soak in 75% ethanol for 5 minutes → soak in ddH2O for 5 minutes. 2) Counterstain the nuclei with hematoxylin for 10 minutes, then wash off the excess hematoxylin on the sections in ddH2O. 3) Soak the sections in hydrochloric acid-ethanol for 1 second and immediately remove them. This step can be repeated once. This step can slightly remove the hematoxylin dye. 4) Rinse the sections with tap water for about 30 minutes to reverse the blue. The longer the rinse time, the darker the blue. After the anti-blueing is completed, remove the slices and drain the water; 5) Dehydrate the slices in the following order: 75% ethanol, 5 minutes → 85% ethanol, 5 minutes → 95% ethanol, 10 minutes → replace with fresh 95% ethanol, 10 minutes; 6) Stain with eosin for about 20 seconds. The eosin solution can be recovered after staining. If the solution is newly prepared, the staining time can be adjusted appropriately; 7) Dehydrate in the following order: 95% ethanol 5 times → replace with fresh 95% ethanol 5 times → 100% ethanol for 10 minutes → 100% ethanol for 10 minutes → treat the slices with carbolic acid for 5 minutes → treat the slices in xylene for 10 minutes, and continue to treat with fresh xylene solution for another 5 minutes; 8) Add 50ul of neutral resin, cover with a clean coverslip, and scan and photograph the slices after thorough drying.

[0054] 2. MPC2 immunohistochemical staining: 1) oven-dry the sections at 60 degrees for one hour and then dewax them (same steps as in step 1); 2) soak them in 3% H2O2 methanol solution for 20 minutes to inactivate endogenous peroxidase, and then wash them three times with double-distilled water; 3) then perform antigen retrieval using acid or alkali repair, and cool naturally after completion; 4) block the sections with 1% BSA blocking solution and incubate them at 37 degrees for 30 minutes; 5) then add the primary antibody (diluted at a ratio of 1:200) to the tissue part of the slide and incubate them at 4 degrees overnight; 6) the next day, wash the tissue sections three times with PBS for 3 minutes each time, then add the rabbit secondary antibody and incubate at 37 degrees for 30 minutes; 7) wash the sections with PBS three times for 3 minutes each time, then develop the color with DAB for 2 minutes, terminate the reaction with double-distilled water, and then wash them three more times; 8) counterstain with hematoxylin for 10 minutes, differentiate with hydrochloric acid and ethanol, and counter-blue with running water for 20 9) Then put the slides on the machine for dehydration (same as step 1); 10) Finally, seal the slides with resin and scan them.

[0055] The results showed that when real-time fluorescence quantitative PCR and western blot protein level detection were performed on fresh frozen liver cancer samples, the expression of MPC2 in cancer tissue was significantly higher than that in adjacent tissues, indicating that MPC2 may have important biological functions in tumors ( Figure 1 A. Figure 1 B). The results of immunohistochemical staining showed (see Figure 1 C) MPC2 expression in liver cancer tissues was significantly higher than that in normal tissues.

[0056] Example 2: MPC2 promotes liver cancer cell proliferation and MPC2 knockdown or MPC2 inhibitor treatment slows proliferation in liver cancer cell lines

[0057] (1) Reagents and materials

[0058] 1. The cell viability assay reagent used was the CellCounting Kit-8 assay (CCK-8 kit) developed by Dojindo.

[0059] 2. UK5099 (PF-1005023), Catalog No. S5317, purchased from Selleck;

[0060] 3. HUH7 and PLC cell lines were purchased from Synbiotics (Shanghai) Biotechnology Co., Ltd.;

[0061] 4. MPC2 knockdown lentivirus was purchased from Heyuan (Shanghai) Biotechnology Co., Ltd.; the shRNA sequence is shown in SEQ ID NO.3:

[0062]

[0063] (II) Experimental steps

[0064] 1. Knockdown cell line construction: HUH7 and PLC cells were infected with viral supernatant and 1 μg / ml Polybrene was added to improve infection efficiency. 1 μg / ml puromycin was added to select cells stably expressing MPC2 knockdown to obtain HUH7-shMPC2 and PLC-shMPC2 cell lines.

[0065] 2. Cell Pretreatment: HUH7, HUH7-shMPC2, PLC, and PLC-shMPC2 cells in logarithmic growth phase were washed with physiological saline, digested, counted, and density adjusted. The corresponding cells were evenly distributed in a 96-well plate at approximately 4000 cells / well and cultured in a 37°C incubator overnight.

[0066] 3. After cells adhered, culture HUH7 and PLC hepatoma cells in DMEM high-glucose medium. Monitor cell proliferation over a time course (0 h, 12 h, and 24 h), with four replicate wells set up for each time period. After the treatment period, add 100 μl of CCK8 assay solution (CCK8 stock solution mixed with serum-free DMEM at a volume ratio of 1:9, prepared immediately before use) to each well. Continue incubating in the incubator for 1 hour, then measure absorbance at 450 nm using a microplate reader.

[0067] 4. After cells adhered, HUH7 and PLC liver cancer cells were cultured with the MPC2 inhibitor UK5099 and normal culture medium. Cell proliferation was monitored over a time course (0 h, 12 h, and 24 h), with four replicate wells set up for each time period. After the treatment period, the drug-containing culture medium was discarded, and 100 μl of CCK8 detection solution (CCK8 stock solution mixed with serum-free DMEM at a volume ratio of 1:9, prepared immediately before use) was added to each well. After incubation for another 1 hour, the absorbance at 450 nm was measured using a microplate reader. The absorbance at each drug concentration was divided by the absorbance at zero concentration to determine the percentage of cells surviving at that concentration.

[0068] The results showed that compared with the CTRL group, after MPC2 knockdown in the liver cancer cell line, cell proliferation slowed down. At the same time, after using the MPC2 inhibitor, the proliferation of the liver cancer cell line slowed down, and there was a significant statistical difference in the quantitative counting results between the two groups. This indicates that MPC2 can affect the proliferation ability of liver cancer cells ( Figure 2 A-2D).

[0069] Example 3: Using inhibitors to target MPC2 or knocking down MPC2 to enhance the killing ability of lenvatinib on liver cancer cells

[0070] (1) Reagents and materials

[0071] 1. The cell viability assay reagent used was the CellCounting Kit-8 assay (CCK-8 kit) developed by Dojindo.

[0072] 2. Lenvatinib, Catalog No. S1164, purchased from Selleck;

[0073] 3. The cell line source and construction method are the same as in Example 2.

[0074] (II) Experimental steps

[0075] 1. Cell pretreatment: HUH7 and HUH7-shMPC2 cells in logarithmic phase growth were washed with physiological saline, digested, counted, and density adjusted. The corresponding cells were evenly distributed in a 96-well plate at approximately 4000 cells / well and cultured in a 37°C incubator overnight.

[0076] 2. After cells adhered, hepatocellular carcinoma cells were treated with increasing concentrations of lenvatinib (0 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, and 50 μM), with four replicate wells set up for each concentration. After 24 hours of treatment, the drug-containing culture medium was discarded and 100 μl of CCK8 assay solution (CCK8 stock solution mixed with serum-free DMEM at a volume ratio of 1:9, prepared immediately before use) was added to each well. After incubation for another hour, the absorbance at 450 nm was measured using a microplate reader. The absorbance at each drug concentration was divided by the absorbance at zero concentration to determine the percentage of cells surviving at that concentration. A nonlinear regression curve was then constructed using the percentage of cells surviving at each concentration. The 24-hour half-inhibitory concentration (IC50) of the drug was calculated as: survival rate = minimum survival rate + (maximum survival rate - minimum survival rate) / (1 + 10 (LogIC50 - drug concentration) × Hill Slope).

[0077] 3. After cells adhered, an MPC2 inhibitor was combined with lenvatinib (CTRL group, MPC2 inhibitor 50 μM group, lenvatinib 5 μM group, and MPC2 inhibitor + lenvatinib group). Four replicate wells were set up for each concentration. After 24 hours of treatment, the drug-containing culture medium was discarded and 100 μl of CCK8 detection solution (CCK8 stock solution mixed with serum-free DMEM at a volume ratio of 1:9, prepared immediately before use) was added to each well. After incubation for another hour, the absorbance at a wavelength of 450 nm was measured using a microplate reader. The absorbance at each drug concentration was divided by the absorbance at zero concentration to determine the percentage of cells surviving at that concentration.

[0078] like Figure 3 As shown, there was no significant difference in the effect of MPC2 inhibitors or lenvatinib alone on the survival rate of liver cancer cells, but the combined use of the two significantly decreased the survival rate of liver cancer cells. At the same time, there were significant differences in the cell survival inhibition rate of lenvatinib in the PLC and PLC-shMPC2 cell lines. The PLC cell line had high MPC2 expression and a higher IC50 value (22.35μM), while the knockdown cell line had low MPC2 expression and a lower IC50 value (13.10μM). This suggests that using inhibitors to target MPC2 or knocking down MPC2 can enhance the killing ability of lenvatinib against liver cancer cells.

[0079] Example 4: Knockdown of MPC2 enhances the killing ability of 5-fluorouracil and oxaliplatin against liver cancer cells

[0080] 1. The cell viability assay reagent used was the CellCounting Kit-8 assay (CCK-8 kit) developed by Dojindo.

[0081] 2. G2 cell line was purchased from Synuclein (Shanghai) Biotechnology Co., Ltd.;

[0082] 3. MPC2 knockdown lentivirus was purchased from Heyuan (Shanghai) Biotechnology Co., Ltd.;

[0083] 4.5-FU (5-Fluorouracil), Catalog No. S1224, purchased from Selleck;

[0084] 5. Oxaliplatin, Catalog No. S1224, purchased from Selleck.

[0085] (II) Experimental steps

[0086] 1. Knockdown cell line construction: Infect G2 cells with viral supernatant and add 1ug / ml Polybrene to improve infection efficiency. Add 1ug / mL puromycin to select cells stably expressing MPC2 knockdown to obtain the G2-shMPC2 cell line.

[0087] 2. Cell pretreatment: G2 and G2-shMPC2 cells in logarithmic growth phase were washed with physiological saline, digested and counted, and density adjusted. The corresponding cells were evenly distributed in a 96-well plate at approximately 4000 cells / well and cultured in a 37°C incubator overnight.

[0088] 3. After cells adhere, treat liver cancer cells with increasing concentrations of 5-fluorouracil and oxaliplatin (0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM), with four replicate wells set up for each concentration. After 24 hours of treatment, discard the drug-containing culture medium and add 100 μL of CCK8 detection solution (CCK8 stock solution mixed with serum-free DMEM at a volume ratio of 1:9, prepared immediately before use) to each well. Continue incubating in the incubator for 1 hour, and then measure the absorbance at 450 nm using a microplate reader. Divide the absorbance value at each drug concentration by the absorbance value at 0 concentration to obtain the survival rate of cells at that concentration; then use nonlinear regression to draw a regression curve based on the survival rate of cells at each concentration. Survival rate = minimum survival rate + (maximum survival rate - minimum survival rate) / (1 + 10 (LogIC50 - drug concentration) × HillSlope), and the 24-hour half-inhibitory concentration (IC50) of the drug on the cells can be calculated.

[0089] like Figure 4 As shown in Figure 2, there are significant differences in the cell survival inhibition rates of 5-fluorouracil and oxaliplatin against G2 and G2-shMPC2 cell lines. The G2 cell line has a higher expression of MPC2 and a higher IC50 value of 5-fluorouracil (0.03 μM), while the knockdown cell line has a lower expression of MPC2 and a lower IC50 value (0.008 μM), suggesting that knockdown of MPC2 enhances the killing ability of 5-fluorouracil against liver cancer cells (see Figure 4 A). Meanwhile, the IC50 value of G2 cell line to oxaliplatin was higher (9.31 μM), while the IC50 value of knockdown cell line to MPC2 was lower (4.59 μM), which suggested that knockdown of MPC2 enhanced the killing ability of oxaliplatin to liver cancer cells (see Figure 4 B).

[0090] Example 5: GDSC database data analysis verifies that MPC2 expression is positively correlated with drug resistance in liver cancer cells

[0091] (1) Required documents

[0092] Enter the GDSC database, enter the download interface, and download the required files:

[0093] 1. Cell_Lines_Details: All cell line annotation information

[0094] 2. GDSC2_fitted_dose_response_27Oct23: IC50 and AUC values for all cell lines and drug combinations

[0095] 3. Cell_line_RMA_proc_basalExp.txt: Gene expression data of cell line (gene ID is ENSMBL) compressed package

[0096] (2) Analysis steps

[0097] 1. Screening liver cancer cell lines in EXCEL based on cell line annotation information

[0098] 2. Screen out cell lines treated with 5-fluorouracil and oxaliplatin based on cell line and drug data

[0099] 3. Organize the target gene expression data based on the cell line gene expression data

[0100] 4. Complete the data merge in RStudio according to the following code:

[0101] HCC=readxl::read_xlsx("HCC.xlsx")

[0102] fu=readxl::read_xlsx("5-fu.xlsx")

[0103] Oxa=readxl::read_xlsx("oxa.xlsx")

[0104] total=merge(HCC,fu,by='CELL_LINE_NAME')

[0105] total2=merge(HCC,Oxa,by='CELL_LINE_NAME')

[0106] write.table(total, file="total.txt",sep="\t",quote=F,row.names=F)

[0107] write.table(total2, file="total2.txt",sep="\t",quote=F,row.names=F)

[0108] MPC2=readxl::read_xlsx("HCC MPC2.xlsx")

[0109] write.table(MPC2, file="MPC2.txt",sep="\t",quote=F,row.names=F)

[0110] 5. Use GraphPad to draw a scatter plot of the gene and IC50, perform linear fitting, and calculate the correlation coefficient (Pearson). R>0 indicates a positive correlation. The higher the gene expression, the larger the IC50, indicating that the gene is highly expressed and resistant.

[0111] like Figure 5 As shown, MPC2 was positively correlated with 5-fluorouracil resistance and was statistically significant (see Figure 5 A). MPC2 was positively correlated with oxaliplatin resistance and was statistically significant (see Figure 5 B).

[0112] Example 6: Animal experiments showed that knocking down MPC2 inhibited the growth of hepatocellular carcinoma

[0113] (1) Reagents and materials

[0114] 1. 4-week-old male C57BL / 6 mice weighing approximately 18 g were purchased from Nanjing Jicui Pharmaceutical Co., Ltd.

[0115] 2. Adenovirus AAV-shMPC2 was purchased from Shanghai Heyuan Biotechnology Co., Ltd., and the shRNA sequence used was the same as above.

[0116] (II) Experimental steps

[0117] 1. The experiment was divided into two groups, AAV-CTRL and AAV-shMPC2. First, the Sleeping Beauty system was used to establish an orthotopic liver cancer model in mice by high-pressure injection of three plasmids (pT3-EF5a-S45Y-Ctnnb1, pT3-EF5a-hMet and Sleeping Beauty transposon) through the tail vein (see Figure 6 A).

[0118] 2. Take 20 C57BL / 6 mice and divide them evenly into two groups of 10 mice each, based on body weight. Inject the mixture of pT3-EF5a-S45Y-Ctnnb1, pT3-EF5a-hMet, and the Sleeping Beauty transposon into the mice via the tail vein over 10 seconds. Ensure that the plasmid suspension is thoroughly mixed before use. Insert the needle at an angle to prevent fluid from leaking. After injection, apply pressure to the injection site with a cotton swab for 1 minute.

[0119] 3. Two weeks after inoculation, adeno-associated virus (AAV) was injected via the tail vein to establish a liver-specific MPC2 knockdown model. Dissolve the AAV in saline and inject 150 μl of the virus into the mouse tail vein at a viral load of 2e+11.

[0120] 4. Six weeks after inoculation, mice were sacrificed, their livers carefully dissected, and photographed for preservation. Mouse and liver weights were measured. Some samples were soaked in 10% formalin and embedded in paraffin for immunohistochemistry.

[0121] like Figure 6 As shown, the number of orthotopic liver cancers in mice with MPC2 knockdown group was less than that in the control group (see Figure 6 B). The final liver weight of the mice in the MPC2 knockdown group was also significantly lighter than that in the control group CTRL, and this difference was statistically significant (see Figure 6 C). These results indicate that knocking down MPC2 expression can reduce the occurrence and progression of HCC in situ.

[0122] Example 7: Targeting MPC2 with an inhibitor significantly slows the growth of hepatocellular carcinoma

[0123] (1) Reagents and materials

[0124] 1. 6-week-old male C57BL / 6 mice weighing approximately 18 g were purchased from Nanjing Jicui Pharmaceutical Co., Ltd.

[0125] 2. The liver cancer cell line Hepa1-6 was purchased from Shanghai Biotechnology Co., Ltd.

[0126] (II) Experimental steps

[0127] 1. The experiment was divided into two groups: a control group and an MPC2 inhibitor group. First, Hepa1-6 cells were cultured and expanded. They were routinely trypsinized, washed twice with sterile saline, and the cell concentration was adjusted to 1×10 cells / mL using serum-free RPMI 1640. 7 cells / ml;

[0128] 2. Take 16 C57BL / 6 mice and divide them into 2 groups according to their body weight, with 8 mice in each group. Inject 100uL of Hepa1-6 cell suspension (2×10 6 cells). Before operation, be sure to mix the cell suspension and insert the needle at an angle to prevent liquid from flowing out. After injection, press the injection site with a cotton swab for 1 minute;

[0129] 3. Seven days after inoculation, MPC2 inhibitors were administered daily. The growth of the subcutaneous implanted tumors in C57BL / 6 mice was observed daily. The long and short diameters of the transplanted tumors were measured. The maximum long diameter (a) and short diameter (b) of the subcutaneous transplanted tumors in C57BL / 6 mice were recorded. The volume of the transplanted tumors was calculated as follows: Volume (mm 3 ) = b 2 ×a / 2, draw the growth curve of transplanted tumor;

[0130] 4. One week after inoculation, C57BL / 6 mice were sacrificed, and the implanted tumors were carefully removed and photographed. Tumor size and weight were measured. Some transplanted tumor samples were soaked in 10% formalin and embedded in paraffin for immunohistochemistry.

[0131] like Figure 7 As shown in Figure 2, the growth rate of subcutaneous tumors in mice treated with the MPC2 inhibitor group was slower than that in the control group (CTRL). The final tumor weight of the subcutaneous tumors in mice was also significantly lighter than that in the control group (CTRL), and this difference was statistically significant (see Figure 7 B). These results indicate that inhibiting MPC2 function can slow the growth of HCC cells subcutaneously in C57BL / 6 mice.

[0132] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of an agent that blocks MPC2 function or inhibits MPC2 expression in the preparation of a drug for treating liver cancer.

2. The use according to claim 1, characterized in that The blocking of MPC2 function or inhibition of MPC2 expression includes but is not limited to MPC2 inhibitors, MPC2 antibodies, siRNA or shRNA of MPC2 gene, microRNA that inhibits MPC2 gene expression, or plasmid or viral expression vectors.

3. The use according to claim 2, characterized in that The agent for blocking MPC2 function or inhibiting MPC2 expression is UK5099.

4. The use according to claim 2, characterized in that The reagent for blocking the function of MPC2 or inhibiting the expression of MPC2 is shRNA of the MPC2 gene, and its nucleotide sequence is shown in SEQ ID NO.

3.

5. The use according to claim 1, characterized in that The application is single or combined use.

6. The use according to claim 5, characterized in that The combined drug therapy is a combination of an agent that blocks the function of MPC2 or inhibits the expression of MPC2 and an anti-liver cancer drug.

7. The use according to claim 6, characterized in that The anti-liver cancer drugs include sorafenib, lenvatinib, regorafenib, apatinib, anlotinib, 5-FU, cisplatin, doxorubicin, oxaliplatin, atezolizumab, and bevacizumab.

8. The use according to claim 1, characterized in that The drug for treating liver cancer is a drug that inhibits the growth and progression of liver cancer.

9. Use of an agent that blocks MPC2 function or inhibits MPC2 expression in the preparation of an anti-liver cancer drug sensitizer, characterized in that: The anti-liver cancer drugs include sorafenib, lenvatinib, regorafenib, apatinib, anlotinib, 5-FU, cisplatin, doxorubicin, oxaliplatin, atezolizumab, and bevacizumab.

10. The use according to claim 9, characterized in that The reagent that blocks the function of MPC2 or inhibits the expression of MPC2 enhances the therapeutic effect of anti-liver cancer drugs in killing tumors.

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