Application of MRPS10 inhibitor in diagnosis and treatment of pancreatic ductal adenocarcinoma

Abstract

The invention discloses application of an MRPS10 inhibitor in diagnosis and treatment of pancreatic ductal adenocarcinoma, and relates to the technical field of biological science. The preparation method of the MRPS10 inhibitor comprises experimental design verification and preparation of the MRPS10 inhibitor, and the experimental design verification comprises the following steps: S1, immunohistochemical staining: performing labeling, dewaxing, hydration, antigen repair, endogenous peroxidase blocking, non-specific staining blocking, primary antibody incubation, secondary antibody incubation, color development and sheet sealing on a paraffin tissue section; s2, cell culture: cell resuscitation, liquid change, passage and cryopreservation operations are included. According to the invention, the effect of MRPS10 in PDAC is systematically analyzed, and the potential of MRPS10 as a potential therapeutic target is disclosed; in combination with bioinformatics analysis and various experimental methods, the function of the MRPS10 in the PDAC is comprehensively verified.

Description

Technical Field

[0001] The present invention relates to the field of bioscience and technology, and in particular to the application of an MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma. Background Art

[0002] Pancreatic ductal adenocarcinoma is a common malignant tumor in the digestive tract. Due to its insidious onset, patients often reach the advanced stage at the time of diagnosis. Moreover, it has a high degree of malignancy, lacks effective treatment means, and the survival prognosis of patients is poor, which still poses a major challenge for clinicians. Therefore, in-depth research on the occurrence and development mechanism of pancreatic ductal adenocarcinoma, exploration of new early diagnostic markers and treatment targets, and providing a new theoretical basis for the early diagnosis and treatment of pancreatic cancer have important clinical significance.

[0003] Oxidative phosphorylation (OXPHOS) is a key process of cellular energy metabolism. Pyruvate enters the mitochondrial matrix through pyruvate translocase, undergoes the tricarboxylic acid cycle (TCA cycle) and the electron transport chain, and is finally oxidized to generate ATP, H2O and CO2. Related studies have shown that there are two main energy production pathways in PDAC: glycolysis and mitochondrial respiration. A study detected the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of six pancreatic cancer cell lines by a Seahorse apparatus and found that in all cells, the production of ATP depends more on OXPHOS than on glycolysis. This finding suggests that mitochondrial oxidative phosphorylation plays an important role in the energy metabolism of PDAC. In addition, clinical studies have shown that the risk of cancer in diabetic patients treated with metformin is reduced, the prognosis of tumor patients is improved, and the survival rate is increased, and its mechanism may be related to metformin inhibiting mitochondrial respiratory complex I. These studies further support the possibility of mitochondrial oxidative phosphorylation as a potential therapeutic target for PDAC.

[0004] As an indispensable semi-autonomous organelle in eukaryotic cells, mitochondria originated from the evolutionary process of aerobic bacteria. Mitochondrial ribosomes are assembled by mitochondrial ribosomal proteins and rRNA. The 13 proteins synthesized by mitochondrial ribosomes are all hydrophobic components of the oxidative phosphorylation system, which is crucial for cell energy production. In recent years, more and more studies have shown that MRPs play important roles in various tumors. For example, MRPs such as DAP3, MRPL41, and MRPS30 are involved in the regulation of apoptosis. MRPS10 is a component of the small subunit of mitochondrial ribosomes encoded by the nuclear genome, and it has been confirmed to be related to breast cancer and thyroid cancer. In this study, through preliminary bioinformatics analysis, it was found that the expression of MRPS10 was significantly increased in pancreatic ductal adenocarcinoma. Further survival analysis and protein interaction network analysis showed that the high expression of MRPS10 was closely related to the poor survival prognosis of PDAC patients, and the proteins interacting with MRPS10 have been confirmed to be closely related to the occurrence and development of various tumors. As a member of the mitochondrial ribosomal protein family, MRPS10 may play an important role in the occurrence and development of pancreatic ductal adenocarcinoma. Summary of the Invention

[0005] The object of the present invention is to solve the deficiencies in the prior art and propose an application of an MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] An application of an MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma, and the use of the MRPS10 inhibitor in the preparation of a drug for treating pancreatic ductal adenocarcinoma.

[0008] Preferably: The preparation method of the MRPS10 inhibitor includes experimental design verification and preparation of the MRPS10 inhibitor. Among them, the experimental design verification includes:

[0009] S1: Immunohistochemical staining: Perform steps of labeling, dewaxing, hydration, antigen repair, blocking of endogenous peroxidase, blocking of non-specific staining, primary antibody incubation, secondary antibody incubation, color development, and sealing on paraffin tissue sections;

[0010] S2: Cell culture: Include operations of cell resuscitation, medium change, subculture, and cryopreservation;

[0011] S3: Construct a stable knockdown cell line: Transfect cells with lentivirus, obtain knockdown group and control group cells by puromycin screening, and detect the gene and protein knockdown efficiency by qRT-PCR and Western-blot;

[0012] S4: RNA extraction and qRT-PCR: Extract cellular RNA, reverse transcribe it into cDNA, and perform real-time fluorescence quantitative PCR analysis;

[0013] S5: Western-blot: Extract cellular proteins, determine the concentration, and then perform SDS-PAGE electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and ECL luminescent solution development;

[0014] S6: CCK8 assay: Seed the cells in a 96-well plate, add the CCK8 working solution, and measure the OD values at different time points;

[0015] S7: EDU staining assay: Perform EDU staining on the cells and observe the proliferation situation;

[0016] S8: Cellular immunofluorescence: Fix, permeabilize, block, incubate with primary and secondary antibodies, perform DAPI staining, and mount the cells, then observe the fluorescence;

[0017] For the preparation of the MRPS10 inhibitor, based on the experimental results verified by bioinformatics analysis and experimental design, select effective small molecule compounds, polypeptides, or antibodies as the MRPS10 inhibitor.

[0018] Preferably, in the above S1, the immunohistochemical staining specifically is:

[0019] S11: After labeling the paraffin tissue sections, blow them with a hair dryer for 5 min; soak them in xylene I and II for 20 min each; soak them in absolute ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, and 75% ethanol for 5 min; wash them with pure water;

[0020] S12: Place the sections in the EDTA antigen retrieval solution that has been boiled in a pressure cooker beforehand, boil for 15 min, and then naturally cool to room temperature; wash them with pure water;

[0021] S13: Add 50 μl of endogenous peroxidase blocker to each section, incubate at room temperature in a humidified box in the dark for 20 min; wash with pure water; add 50 μl of non-specific staining blocker, incubate at room temperature in a humidified box in the dark for 30 min - 60 min;

[0022] S14: Discard the blocking solution, add the primary antibody, incubate at 4°C in a humidified box in the dark overnight;

[0023] S15: After rewarming at room temperature for 30 min, wash with pure water; add the secondary antibody, incubate at 25°C in a humidified box in the dark for 20 min;

[0024] S16: Wash with pure water; add 50 μl of streptavidin-peroxidase to each section, incubate in a humidified box in the dark at room temperature for 10 min;

[0025] S17: Wash with pure water; add 50 μl of 1× DAB working solution, rinse with tap water after 3 minutes of color development to terminate;

[0026] S18: Immerse in hematoxylin for 1 minute, rinse with running water for 2 minutes to turn blue; soak in hydrochloric acid alcohol and lithium carbonate for 2 seconds each; dehydrate the sections in 75% ethanol, 85% ethanol, 95% ethanol, and absolute ethanol for 5 minutes each; clear in xylene for 10 minutes;

[0027] S19: Mount the sections with gum and observe under a microscope.

[0028] Preferably, in S2, the cell culture is specifically as follows:

[0029] S21: Cell resuscitation. After taking the cells out of the liquid nitrogen tank, quickly warm them in a 37°C water bath for 1 minute. Transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 1 ml of medium to resuspend the cells, pipette evenly, and add them to a T25 culture flask pre-filled with 4 ml of complete DMEM medium. Culture in a 37°C incubator and observe the cell morphology after attachment;

[0030] S22: Cell medium change. Take the cells out of the 37°C incubator, observe the morphology under a microscope, discard the original medium, add 2 ml of sterile PBS to wash, then discard the PBS, add 5 ml of complete DMEM medium, and place it back in the incubator for continued culture;

[0031] S23: Cell passage. After the cells grow to more than 85% confluence, discard the original medium, wash twice with 2 ml of PBS, then discard the PBS, add 1 ml of trypsin, incubate in the incubator for 3 minutes, add 2 ml of complete medium to terminate digestion. Transfer the cell suspension to a 15 ml sterile centrifuge tube, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 2 ml of complete medium to resuspend the cell pellet, and distribute it to T25 culture flasks pre-filled with 4 ml of complete medium at a ratio of 1:2. Place the cells back in the incubator for continued culture;

[0032] S24: Cell cryopreservation. Take the cells out of the 37°C incubator, observe the morphology under a microscope, discard the original medium, add 2 ml of sterile PBS to wash, then discard the PBS, add 1 ml of trypsin, incubate in the incubator for 3 minutes, add 2 ml of complete medium to terminate digestion. Transfer the cell suspension to a 15 ml sterile centrifuge tube, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 1 ml of cell cryopreservation solution, resuspend the cell pellet and add it to a cell cryopreservation tube; place it at 4°C for 30 minutes, -20°C for 1 hour, -80°C overnight, and transfer the cells to the liquid nitrogen tank for storage the next day.

[0033] Preferably, in S3, the construction of a stable knockdown cell line using lentivirus is specifically as follows:

[0034] S31: Uniformly plate the cells in a 6-well plate. After the cells adhere, change the cell medium, add 1 ml of complete medium, add the knockdown lentivirus and the control lentivirus to the cell medium respectively. After 6 hours, supplement 1 ml of liquid. Change the cell medium the next day and observe the virus infection situation under a fluorescence microscope on the third day.

[0035] S32: Puromycin screening: Add puromycin to the cell medium on the third day to screen the cells infected with the virus. After one week of screening, the knockdown group cells and the control group cells can be obtained, and the gene and protein knockdown efficiency can be detected by qRT-PCR and Western-blot.

[0036] Preferably, in S4, RNA extraction and qRT-PCR are specifically as follows:

[0037] S41: RNA extraction: After digesting the cells, add 1 ml of Trizol and incubate on ice for 10 min. Then add 200 μl of chloroform and mix well until the solution becomes milky white. Let it stand on ice for 5 min and centrifuge at 12000 g for 15 min. Transfer 400 μl of the supernatant to a new RNase-free 1.5 ml centrifuge tube, add 400 μl of isopropanol and mix well. Let it stand on ice for 20 min. Centrifuge at 12000 g at 4 °C for 10 min. Discard the supernatant, add 400 μl of absolute ethanol to resuspend and wash the precipitate. Centrifuge at 7500 g for 5 min. Aspirate the ethanol, dry at room temperature for 5 min and add 20 μl of DEPC water. Gently pipette to dissolve and then measure the concentration and store at -80 °C in the refrigerator.

[0038] S42: Reverse transcription reaction. Calculate the volume required for 2 μg of RNA according to the measured concentration. Carry out the reverse transcription reaction according to the program: 37 °C for 15 min, 60 °C for 10 min, 95 °C for 3 min, 4 °C +∞. The resulting product is cDNA and is stored at -20 °C.

[0039] S43: Real-time fluorescence quantitative PCR analysis.

[0040] Preferably, in S5, Western-blot is specifically as follows:

[0041] S51: Protein extraction + BCA protein concentration determination: After digesting the cells, add 100 μl of RIPA lysis buffer, and add protease inhibitor, phosphatase inhibitor, and PMSF according to the ratio of 100:1; After shaking and lysing on ice for 30 min, centrifuge at 4°C and 12,000 rpm for 20 min; Take 80 μl of the supernatant, add 20 μl of protein loading buffer, boil at 99°C for 5 min, and store at -20°C after natural cooling; Dilute 4 μl of the supernatant by 5 times, add 200 μl of BCA working solution to each well, react at 37°C for 30 min, and measure the absorbance at 526 nm; Calculate the protein concentration according to the standard curve and adjust the loading amount;

[0042] S52: Prepare a gel with the corresponding concentration according to the instructions of the gel preparation kit; Fix the gel on the electrophoresis clamp and place it correctly in the electrophoresis tank, add freshly prepared electrophoresis buffer, remove the comb, load the sample according to the BCA measurement result, set the voltage to 80 V, and after the protein runs out of the upper gel, adjust the voltage to 120 V;

[0043] S53: After taking out the gel, place the gel and the membrane in the order of blackboard - gel - PVDF membrane - white board, place them correctly in the electrotransfer tank, add freshly prepared electrotransfer buffer and an ice brick, and set the current to 120 mA and the time to 1 minute for 1 kD protein; After electrotransfer, place the membrane in skim milk for blocking for 2 h; After blocking, wash it once with TBST and then incubate with the primary antibody at 4°C overnight;

[0044] S54: The next day, wash the membrane three times with TBST, 10 min each time; Incubate with the secondary antibody at room temperature for 2 h and then wash it three times with TBST, 10 min each time; Prepare the ECL luminescent solution for development.

[0045] Preferably: In the above S6, the CCK8 detection is specifically as follows:

[0046] One day in advance, count the cells and seed them in a 96-well plate. Add 10 μl of CCK8 working solution to every 100 μl of complete medium. Add the CCK8 working solution at the same time on the 1st, 3rd, and 5th days respectively, and then put the cells into the incubator for continuous culture for 2 h. Measure the OD value at 450 nm with an enzyme-labeled instrument; The value obtained by subtracting the absorbance of the blank group from the absorbance measured for each group of samples is used as statistical data.

[0047] Preferably: In the above S7, the EDU staining detection is specifically as follows:

[0048] S71: Seed each group of cells evenly into a six-well plate in advance. Prepare a 2x EDU staining solution: Dilute with complete medium at a ratio of 1:500 to prepare a 20 μM EDU working solution;

[0049] S72: Replace the medium of the cells in the six-well plate. After washing twice with PBS, first add 500 μL of complete medium containing 20 μM EDU to each well, and then supplement 500 μL of complete medium to each well to make the EDU concentration in the six-well plate 10 μM; continue to incubate the cells at 37 °C for 2 h;

[0050] S73: Remove the medium, wash twice with PBS, add 500 μL of 4% paraformaldehyde to each well to fix the cells for 15 min; after removing the paraformaldehyde, wash twice with PBS; add 1 mL of permeabilization solution to each well for 15 min; after removing the permeabilization solution, wash twice with PBS;

[0051] S74: Prepare the click solution, add 500 μL of the prepared Click working solution to each well, and incubate in the dark for 30 min; remove the reaction solution, and wash twice with PBS;

[0052] S75: Prepare the Hoechst solution with PBS at 1:1000, add 500 μL to each well, incubate in the dark for 10 min, after removing the solution, wash twice with PBS;

[0053] S76: Take pictures and observe under a fluorescence microscope.

[0054] Preferably: In the above S8, the cell immunofluorescence is specifically as follows:

[0055] S81: Clean the cell slide with sterile PBS and place it in a 12-well plate, and evenly plate the cells;

[0056] S82: After replacing the medium of the cells, add 1 mL of 4% paraformaldehyde to each well to fix the cells for 20 min, and wash twice with PBS; add 500 μL of permeabilization solution to each well to permeabilize the cells for 10 min, and wash twice with PBS; add 500 μL of ready-to-use goat serum to each well to block for 30 min;

[0057] S83: Incubate the primary antibody Ki67 overnight at 4 °C;

[0058] S84: Wash twice with PBS, incubate the fluorescent secondary antibody in the dark for 1 h; all subsequent operations need to be strictly in the dark. After washing twice with PBS, stain with DAPI for 3 min. After washing twice with PBS, seal the slide with nail polish and fix the slide with the fixing solution;

[0059] S85: Observe under a fluorescence microscope.

[0060] The beneficial effects of the present invention are as follows:

[0061] 1. The present invention systematically analyzes the role of MRPS10 in PDAC and reveals its potential as a potential therapeutic target; combining bioinformatics analysis and a variety of experimental methods, comprehensively verifies the function of MRPS10 in PDAC.

[0062] 2. The research results of the present invention provide new ideas for the early diagnosis and treatment of PDAC, which helps to improve the survival prognosis of patients; the inhibitors based on MRPS10 may become new therapeutic drugs and have broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is a box plot of the expression of MRPS10 of the present invention in normal and pancreatic ductal adenocarcinoma patients;

[0064] Figure 2 It is a survival analysis curve graph of the high-expression group and low-expression group of MRPS10 of the present invention;

[0065] Figure 3 It is a schematic diagram of the interaction between MRPS10 and other proteins analyzed by protein-protein interaction network analysis of the present invention;

[0066] Figure 4 It is a Venn diagram of the differential analysis and enrichment analysis of MRPS10 of the present invention;

[0067] Figure 5 It is a GO enrichment analysis graph of the genes obtained by taking the intersection of the differential genes and mitochondrial-related genes of the present invention;

[0068] Figure 6 It is a KEGG enrichment analysis graph of the present invention;

[0069] Figure 7 It is a display graph of the normal group in the study of the expression of MRPS10 in human pancreatic ductal adenocarcinoma tissues of the present invention;

[0070] Figure 8 It is a display graph of the pancreatic ductal adenocarcinoma group in the study of the expression of MRPS10 in human pancreatic ductal adenocarcinoma tissues of the present invention;

[0071] Figure 9 It is a statistical analysis result graph of immunohistochemical scores in the study of the expression of MRPS10 in human pancreatic ductal adenocarcinoma tissues of the present invention;

[0072] Figure 10 It is a graph of the viability changes of PANC-1 (A) and Capan-1 (B) normal cells and after knocking down MRPS10 detected by CCK8 at Day1, Day3 and Day5 respectively of the present invention;

[0073] Figure 11 It is a graph of the proliferation level changes of PANC-1 (A) and Capan-1 (B) normal cells and after knocking down MRPS10 detected by EDU of the present invention;

[0074] Figure 12This is a graph showing the changes in the expression levels of MKI67 in normal cells of PANC-1 (A) and Capan-1 (B) and after knocking down MRPS10 detected by qRT-PCR in the present invention;

[0075] Figure 13 This is a graph showing the changes in the expression levels of MT-ND1, MT-ND5, MT-CYB, and SDHA in normal cells of PANC-1 (A) and Capan-1 (B) and after knocking down MRPS10 detected by qRT-PCR in the present invention;

[0076] Figure 14 This is a graph showing the changes in ROS in normal cells of PANC-1 (A) and Capan-1 (B) and after knocking down MRPS10 in the present invention;

[0077] Figure 15 This is a graph showing the changes in ATP in normal cells of PANC-1 (A) and Capan-1 (B) and after knocking down MRPS10 in the present invention. Detailed implementation manners

[0078] The technical solutions of the present invention will be further described in detail below in conjunction with the specific implementation manners.

[0079] Example 1:

[0080] The application of an MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma is determined based on experiments, and the experiments include:

[0081] I. Use of databases

[0082] (1) Differential analysis

[0083] Download the RNA sequencing results of patients with pancreatic ductal adenocarcinoma (n = 177) and the normal group (n = 171) from the TCGA and GTEx databases, perform differential analysis using the limma package of R software, and draw a volcano plot of the visualization results of differential analysis using the ggrepel package.

[0084] (2) Use of the GEPIA2 database

[0085] Log in to the GEPIA2 website, click "General" in the drop-down list of "Expression Analysis", enter the gene name "MRPS10" in the search box, click "Search", and draw a box plot for the differential analysis of MRPS10 in various tumors. Then click "Expression DIY", select "Box Plot", enter the gene name "MRPS10" in the search box, and select the tumor type as PAAD (pancreatic ductal adenocarcinoma), click "Plot" to draw a box plot for the differential analysis of MRPS10 in pancreatic ductal adenocarcinoma. Then click "Survival Analysis", enter the gene name "MRPS10" in the search box, and select the tumor type as PAAD, click "Plot" to draw a survival analysis curve graph of MRPS10 in pancreatic ductal adenocarcinoma.

[0086] (3) Use of STRING database and Cytoscape software

[0087] Log in to the STRING database website, enter the protein name "MRPS10" in the "Protein Name" search box, select "Homo sapiens" for "Organisms", click "SEARCH", and draw a protein-protein interaction map (PPI) of MRPS10. Download the data in TSV format in "Exports" and import the data into the "Cytoscape" software for plotting.

[0088] (4) Use of Mitocarta database

[0089] Log in to the Mitocarta3.0 website, click "Human MitoCarta3.0" to download human mitochondrial-related genes (n = 1136). And use the VennDiagram package of R software to draw a Venn diagram of the intersection of differential genes in pancreatic ductal adenocarcinoma and mitochondrial-related genes.

[0090] (5) Enrichment analysis

[0091] Use the GO.db, clusterProfiler, and DOSE packages of R software to perform GO and KEGG enrichment analysis on the above-obtained intersection genes (n = 344), and draw bubble charts respectively.

[0092] II. Experimental methods

[0093] (1) Immunohistochemistry (IHC) staining:

[0094] 1) After labeling the paraffin tissue sections, blow them with a hair dryer for 5 min; soak them in xylene I and II for 20 min each; soak them in absolute ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, and 75% ethanol for 5 min. Wash them 3 times with pure water, 5 min each time.

[0095] 2) Place the sections in the EDTA antigen retrieval solution that has been pre-boiled in a pressure cooker. After boiling for 15 min, let it cool naturally to room temperature. Wash it 3 times with pure water, 5 min each time.

[0096] 3) Drop 50 μl of endogenous peroxidase blocker on each section, incubate at room temperature in a humidified box in the dark for 20 min; wash it 3 times with pure water, 5 min each time; drop 50 μl of non-specific staining blocker, incubate at room temperature in a humidified box in the dark for 30 - 60 min.

[0097] 4) Discard the blocking solution, drop the primary antibody (MRPS10 antibody: prepared with ready-to-use goat serum = 1:200), incubate at 4°C in a humidified box in the dark overnight.

[0098] 5) After rewarming at room temperature for 30 min, wash it 3 times with pure water, 5 min each time; drop the secondary antibody, incubate at 25°C in a humidified box in the dark for 20 min.

[0099] 6) Wash it 3 times with pure water, 5 min each time. Drop 50 μl of streptavidin-peroxidase on each section, incubate in a humidified box in the dark at room temperature for 10 min.

[0100] 7) Wash it 3 times with pure water, 5 min each time. Drop 50 μl of 1×DAB working solution, develop for 3 min and then rinse with tap water to terminate.

[0101] 8) Immerse it in hematoxylin for 1 min, rinse with running water to blue for 2 min; soak in hydrochloric acid alcohol and lithium carbonate for 2 s each; dehydrate the sections in 75% ethanol, 85% ethanol, 95% ethanol, and absolute ethanol for 5 min each; clear in xylene for 10 min.

[0102] 9) Mount with gum and observe under a microscope.

[0103] (2) Cell culture

[0104] 1) Cell resuscitation

[0105] After taking the cells out of the liquid nitrogen tank, quickly rewarm them in a 37°C water bath for 1 min. Transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 ml of medium to resuspend the cells, pipette evenly, and add them to a T25 culture flask containing 4 ml of DMEM complete medium in advance. Culture in an incubator at 37°C and observe the cell morphology after attachment;

[0106] 2) Cell medium replacement

[0107] Take out the cells from the 37°C incubator, observe the morphology under the microscope, discard the original culture medium, add 2 ml of sterile PBS for washing, then discard the PBS, add 5 ml of complete DMEM medium, and place it in the incubator for continued culture;

[0108] 3) Cell passage

[0109] After the cells grow to more than 85% confluence, discard the original culture medium, wash twice with 2 ml of PBS, then discard the PBS, add 1 ml of trypsin (0.25%), incubate in the incubator for 3 min, add 2 ml of complete medium to terminate digestion, transfer the cell suspension to a 15-ml sterile centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 2 ml of complete medium to resuspend the cell pellet, and distribute it to a T25 culture flask pre-added with 4 ml of complete medium at a ratio of 1:2, and place the cells in the incubator for continued culture;

[0110] 4) Cell cryopreservation

[0111] Take out the cells from the 37°C incubator, observe the morphology under the microscope, discard the original culture medium, add 2 ml of sterile PBS for washing, then discard the PBS, add 1 ml of trypsin (0.25%), incubate in the incubator for 3 min, add 2 ml of complete medium to terminate digestion, transfer the cell suspension to a 15-ml sterile centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 ml of cell cryopreservation solution, resuspend the cell pellet and add it to a cell cryopreservation tube; place it at 4°C for 30 min, at -20°C for 1 h, at -80°C overnight, and transfer the cells to a liquid nitrogen tank for storage the next day.

[0112] (3) Using lentivirus to construct a stable knockdown cell line

[0113] 1) Evenly plate the cells in a 6-well plate. After the cells adhere, perform cell medium replacement, add 1 ml of complete medium, add the knockdown lentivirus and the control lentivirus to the cell culture medium respectively, and replenish 1 ml of liquid after 6 hours; perform cell medium replacement the next day, and observe the virus infection situation under a fluorescence microscope on the third day;

[0114] 2) Puromycin screening: Add puromycin (2 μg / ml) to the cell culture medium on the third day to screen the cells infected with the virus. After screening for one week, the knockdown group cells and the control group cells can be obtained, and the gene and protein knockdown efficiencies are detected by qRT-PCR and Western-blot.

[0115] (4) RNA extraction and qRT-PCR

[0116] 1) RNA extraction: After digesting the cells, add 1 ml of Trizol and incubate on ice for 10 min; then add 200 μl of chloroform and mix well until the solution becomes milky white. Let it stand on ice for 5 min and centrifuge at 12,000 g for 15 min; transfer 400 μl of the supernatant to a new RNase-free 1.5 ml centrifuge tube, add 400 μl of isopropanol and mix well. Let it stand on ice for 20 min; centrifuge at 12,000 g at 4 °C for 10 min; discard the supernatant, add 400 μl of absolute ethanol to resuspend and wash the precipitate; centrifuge at 7,500 g for 5 min; aspirate the ethanol, dry at room temperature for 5 min and add 20 μl of DEPC water. After gently pipetting to dissolve, measure the concentration and store at -80 °C in the refrigerator;

[0117] 2) Reverse transcription reaction

[0118] Calculate the volume required for 2 μg of RNA according to the measured concentration. Prepare the reaction system according to the following table:

[0119]

[0120] Perform the reverse transcription reaction according to the program: 37 °C for 15 min, 60 °C for 10 min, 95 °C for 3 min, 4 °C +∞; the resulting product is cDNA and store at -20 °C.

[0121] 3) Real-time fluorescence quantitative PCR analysis

[0122] The primer sequences are shown in the following table:

[0123]

[0124]

[0125] Prepare the reaction system according to the following table:

[0126]

[0127] Perform the PCR reaction according to the program:

[0128]

[0129] (5) Western-blot

[0130] 1) Protein extraction + BCA protein concentration determination: After digesting the cells, add 100 ul of RIPA lysis buffer, and add protease inhibitor, phosphatase inhibitor, and PMSF according to the ratio of 100:1; shake and lyse on ice for 30 min, then centrifuge at 4°C and 12,000 rpm for 20 min; take 80 ul of the supernatant, add 20 ul of protein loading buffer, boil at 99°C for 5 min, cool naturally, and store at -20°C; dilute 4 ul of the supernatant by 5 times, add 200 ul of BCA working solution to each well, react at 37°C for 30 min, and measure the absorbance (OD) value at 526 nm; calculate the protein concentration according to the standard curve and adjust the loading amount.

[0131] 2) Prepare the gel with the corresponding concentration according to the gel preparation kit instructions; fix the gel on the electrophoresis clamp and place it correctly in the electrophoresis tank, add freshly prepared electrophoresis buffer, remove the comb, load the samples according to the BCA measurement results, set the voltage to 80 V, and after the protein runs out of the upper layer of the gel, adjust the voltage to 120 V.

[0132] 3) After taking out the gel, place the gel and the membrane in the order of blackboard - gel - PVDF membrane - white board, place them correctly in the electrotransfer tank, add freshly prepared electrotransfer buffer and ice bricks, and set the current to 120 mA and the time to 1 minute for 1 kD protein; after electrotransfer, place the membrane in skim milk for blocking for 2 h; after blocking, wash it once with TBST and then incubate with the primary antibody at 4°C overnight.

[0133] 4) The next day, wash the membrane three times with TBST, 10 min each time; incubate with the secondary antibody at room temperature for 2 h and then wash three times with TBST, 10 min each time; prepare the ECL luminescent solution for development.

[0134] (6) CCK8 assay

[0135] One day in advance, count the cells and seed them in a 96-well plate (about 3000 cells per well). Add 10 ul of CCK8 working solution to every 100 ul of complete medium. At the same time on the 1st, 3rd, and 5th days, add the CCK8 working solution and then put the cells back into the incubator for continued culture for 2 h. Measure the OD value at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The measured absorbance of each group of samples minus the value of the blank group (medium + CCK8 working solution) is used as the statistical data.

[0136] (7) EDU staining assay

[0137] 1) Seed each group of cells evenly into a six-well plate in advance. Prepare 2x EDU staining solution: Dilute with complete medium at a ratio of 1:500 to prepare 20 uM EDU working solution.

[0138] 2) Replace the medium in the six-well plate. After washing the cells twice with PBS, first add 500 μL of complete medium containing 20 μM EDU to each well, and then supplement each well with an additional 500 μL of complete medium to make the EDU concentration in the six-well plate 10 μM. Incubate the cells at 37 °C for an additional 2 h.

[0139] 3) Remove the medium. After washing twice with PBS, add 500 μL of 4% paraformaldehyde to each well to fix the cells for 15 min. After removing the paraformaldehyde, wash twice with PBS. Add 1 mL of permeabilization solution (0.3% Tritonx-100) to each well for 15 min. After removing the permeabilization solution, wash twice with PBS.

[0140] 4) Prepare the click solution according to the instructions. Add 500 μL of the prepared Click working solution to each well and incubate in the dark for 30 min. Remove the reaction solution and wash twice with PBS.

[0141] 5) Prepare the Hoechst solution by diluting it 1:1000 with PBS. Add 500 μL to each well and incubate in the dark for 10 min. After removing the solution, wash twice with PBS.

[0142] 6) Take pictures and observe under a fluorescence microscope.

[0143] (8) Cellular immunofluorescence

[0144] 1) Clean the cell slides with sterile PBS and place them in a 12-well plate. Seed the cells evenly.

[0145] 2) After replacing the cell medium, add 1 mL of 4% paraformaldehyde to each well to fix the cells for 20 min, and wash twice with PBS; add 500 μL of permeabilization solution (0.2% Tritonx-100) to each well to permeabilize the cells for 10 min, and wash twice with PBS; add 500 μL of ready-to-use goat serum to each well to block for 30 min.

[0146] 3) Incubate with the primary antibody Ki67 (1:200) overnight at 4 °C.

[0147] 4) Wash twice with PBS and incubate with the fluorescent secondary antibody for 1 h in the dark; all subsequent operations need to be strictly in the dark. After washing twice with PBS, stain with DAPI for 3 min. After washing twice with PBS, seal the slides with nail polish and fix the cell slides with the fixing solution.

[0148] 5) Observe under a fluorescence microscope.

[0149] Results:

[0150] 1. Bioinformatics analysis of MRPS10 in human pancreatic ductal adenocarcinoma.

[0151] 1.1 Pan-cancer analysis of MRPS10 using the GEPIA2 database revealed that MRPS10 was upregulated in various tumors, such as diffuse large B-cell lymphoma, glioblastoma, pancreatic ductal adenocarcinoma, gastric adenocarcinoma, etc. The box plot of the expression level ( Figure 1 ) also confirmed that the expression of MRPS10 was increased in pancreatic ductal adenocarcinoma compared with the normal group. The survival analysis curve ( Figure 2 ) indicated that patients with high expression of MRPS10 had poor survival prognosis. By analyzing the protein-protein interaction relationship of MRPS10 using the STRING database ( Figure 3 ), it was found that there was an interaction relationship between MRPS10 and proteins that have been reported to be related to various tumors.

[0152] 1.2 Differential analysis of the data of pancreatic ductal adenocarcinoma and normal group downloaded from the TCGA and GTEx databases was performed using R language (v4.2.2) to obtain 5,564 differential genes, and the results were visualized to obtain a volcano plot, showing that MRPS10 was highly expressed in pancreatic cancer. Taking the intersection of 1,136 human mitochondrial-related genes downloaded from the Mitocarta database with the differential genes, 328 differential genes related to human mitochondria in pancreatic ductal adenocarcinoma were obtained ( Figure 4 ). GO ( Figure 5 ) and KEGG ( Figure 6 ) enrichment analysis of these 328 genes found that the two enriched terms "energy derivation by oxidation of organic compounds" and "chemical carcinogenesis - reactive oxygen species" had the largest number of enriched genes and were statistically significant.

[0153] 2. Expression of MRPS10 in human pancreatic ductal adenocarcinoma tissues.

[0154] Immunohistochemical staining revealed that MRPS10 was highly expressed in human pancreatic ductal adenocarcinoma tissues, and it was proved to be statistically significant by the immunohistochemical scoring system. (P < 0.01).

[0155] 3. MRPS10 increased the proliferation level of pancreatic cancer cells.

[0156] 3.1 The cells were divided into two groups: shControl group and shMRPS10 group, and they were plated in 96-well plates at a density of 3,000 cells / well, and CCK8 assays were performed at 9:00 am on the first, third, and fifth days respectively. The results showed that the cell viability of PANC-1 and Capan-1 cells decreased significantly after MRPS10 knockdown ( Figure 10 ).

[0157] 3.2 Divide the cells into two groups: shControl group and shMRPS10 group. One day in advance, evenly plate the cells into a 6-well plate, and perform EDU cell proliferation assay the next day. The results show that the cell proliferation level of PANC-1 and Capan-1 cells is significantly decreased under the background of MRPS10 knockdown ( Figure 11 ).

[0158] 3.3 Extract the RNA of PANC-1 and Capan-1 cells in the shControl group and shMRPS10 group respectively, and use fluorescence quantitative qPCR to detect the expression level of MKI67. The results show that MRPS10 significantly affects the expression of MKI67, and MRPS10 increases the expression of MKI67 in pancreatic cancer cells ( Figure 12 ).

[0159] 4. MRPS10 significantly upregulates the mitochondrial function of pancreatic ductal adenocarcinoma cells.

[0160] 4.1 Divide PANC-1 and Capan-1 cells into two groups respectively: shControl group and shMRPS10 group. Extract the RNA of the two groups of cells and detect the expression levels of the key genes of the mitochondrial respiratory chain complex: MT-ND1, MT-ND5, MT-CYB and SDHA. The results show that after MRPS10 knockdown, the expression levels of these four genes in pancreatic cancer cells are significantly decreased ( Figure 13 ).

[0161] 4.2 Divide PANC-1 and Capan-1 cells into two groups respectively: shControl group and shMRPS10 group. Use a ROS detection kit to detect the changes in reactive oxygen species in the two groups of cells. The results show that after MRPS10 knockdown, the level of reactive oxygen species in pancreatic cancer cells is significantly decreased ( Figure 14 ).

[0162] 4.3 Divide PANC-1 and Capan-1 cells into two groups respectively: shControl group and shMRPS10 group. Use an ATP detection kit to detect the changes in the ATP levels of the two groups of cells. The results show that after MRPS10 knockdown, the ATP level of pancreatic cancer cells is significantly decreased ( Figure 15 ).

[0163] As described above, it is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered within the protection scope of the present invention.

Claims

1. Use of an MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma, characterized in that: Use of the MRPS10 inhibitor in preparing a drug for treating pancreatic ductal adenocarcinoma.

2. The use of a MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma according to claim 1, characterized in that: The method for preparing the MRPS10 inhibitor includes experimental design verification and preparing the MRPS10 inhibitor, wherein the experimental design verification includes: S1: Immunohistochemical staining: labeling, dewaxing, hydration, antigen retrieval, endogenous peroxidase blocking, nonspecific staining blocking, primary antibody incubation, secondary antibody incubation, color development and sealing steps for paraffin tissue sections; S2: Cell culture: including cell recovery, medium replacement, subculturing and freezing operations; S3: Construction of stable knockdown cell lines: Use lentivirus to transfect cells, select by puromycin to obtain knockdown group and control group cells, and use qRT-PCR and Western-blot to detect gene and protein knockdown efficiency; S4: RNA extraction and qRT-PCR: extract cellular RNA, reverse transcribe it into cDNA, and perform real-time fluorescence quantitative PCR analysis; S5: Western-blot: extract cell proteins, determine the concentration, and then perform SDS-PAGE electrophoresis, transfer, block, incubate with primary antibodies, incubate with secondary antibodies, and develop with ECL luminescent solution; S6: CCK8 detection: cells were seeded in a 96-well plate, CCK8 working solution was added, and the OD values ​​at different time points were measured; S7: EDU staining detection: EDU staining was performed on cells to observe proliferation; S8: Cell immunofluorescence: Fix, permeabilize, block, incubate with primary and secondary antibodies, stain with DAPI, and mount the cells to observe fluorescence; In the preparation of the MRPS10 inhibitor, effective small molecule compounds, peptides or antibodies are selected as the MRPS10 inhibitor according to the experimental results verified by bioinformatics analysis and experimental design.

3. Use of a MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma according to claim 2, characterized in that: In S1, immunohistochemical staining is specifically as follows: S11: After marking the paraffin tissue sections, blow them with a hair dryer for 5 minutes; soak them in xylene I and II for 20 minutes each; Absolute ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, and 75% ethanol for 5 min; rinse with pure water; S12: Place the slices in EDTA antigen retrieval solution that has been boiled in a pressure cooker in advance, boil for 15 minutes, and then cool naturally to room temperature; wash with pure water; S13: Add 50 μl of endogenous peroxidase blocker to each section, incubate at room temperature in a wet box away from light for 20 min; wash with pure water; add 50 μl of nonspecific staining blocker, incubate at room temperature in a wet box away from light for 30 min-60 min; S14: discard the blocking solution, add primary antibody, and incubate overnight at 4°C in a wet box away from light; S15: After rewarming at room temperature for 30 minutes, wash with pure water; add secondary antibody and incubate in a wet box in the dark at 25°C for 20 minutes; S16: Wash with pure water; add 50 μl of streptomycin-peroxidase to each slice and incubate in a humidified box at room temperature for 10 min in the dark; S17: Wash with pure water; add 50 μl of 1×DAB working solution, color for 3 min, rinse with tap water, and terminate; S18: Soak in hematoxylin for 1 min, rinse with running water to turn blue for 2 min; hydrochloric acid alcohol and lithium carbonate for 2 s each; dehydrate the slices with 75% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol for 5 min each; xylene for 10 min to make them transparent; S19: Seal the slides with gum and observe under a microscope.

4. The use of a MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma according to claim 2, characterized in that: In S2, cell culture specifically includes: S21: Cell recovery: After taking the cells out of the liquid nitrogen tank, quickly rewarm them in a 37°C water bath for 1 min, transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 ml of culture medium to resuspend the cells, pipet evenly, add them to a T25 culture flask that has been added with 4 ml of DMEM complete culture medium in advance, culture in a 37°C incubator, and observe the cell morphology after attachment; S22: Change the cell medium. Take the cells out of the 37°C incubator, observe their morphology under a microscope, discard the original culture medium, add 2 ml of sterile PBS to wash, discard the PBS, add 5 ml of DMEM complete culture medium, and continue culturing in the incubator; S23: Cell passaging: After the cells grow to a confluence of more than 85%, discard the original culture medium, wash twice with 2 ml PBS, discard the PBS, add 1 ml trypsin, incubate in the incubator for 3 min, add 2 ml complete culture medium to terminate digestion, transfer the cell suspension to a 15 ml sterile centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 2 ml complete culture medium to resuspend the cell pellet, distribute it in a 1:2 ratio into a T25 culture flask that has been added with 4 ml complete culture medium in advance, and place the cells in the incubator for continued culture; S24: For cell freezing, take the cells out of the 37°C incubator, observe the morphology under a microscope, discard the original culture medium, add 2 ml of sterile PBS for washing, discard the PBS, add 1 ml of trypsin, incubate in the incubator for 3 min, add 2 ml of complete culture medium to terminate digestion, transfer the cell suspension to a 15 ml sterile centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 ml of cell freezing solution, resuspend the cell pellet and add it to the cell freezing tube; place at 4°C for 30 min, -20°C for 1 h, -80°C overnight, and transfer the cells to a liquid nitrogen tank for storage the next day.

5. Use of a MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma according to claim 2, characterized in that: In S3, the use of lentivirus to construct a stable knockdown cell line is specifically as follows: S31: Cells were evenly plated in a 6-well plate. After the cells adhered to the wall, the cell culture medium was changed. 1 ml of complete culture medium was added. The knockdown lentivirus and the control lentivirus were added to the cell culture medium respectively. After 6 hours, 1 ml of the culture medium was added. The cell culture medium was changed on the second day. The virus infection was observed under a fluorescence microscope on the third day. S32: Puromycin screening: On the third day, puromycin was added to the cell culture medium to screen the virus-infected cells. After one week of screening, knockdown group cells and control group cells were obtained, and the gene and protein knockdown efficiency was detected by qRT-PCR and Western-blot.

6. Use of a MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma according to claim 2, characterized in that: In S4, RNA extraction and qRT-PCR are specifically as follows: S41: RNA extraction: After digesting the cells, add 1 ml of Trizol and incubate on ice for 10 min; then add 200 ul of chloroform and mix until the solution turns milky white, let stand on ice for 5 min, and centrifuge at 12000 g for 15 min; Transfer 400ul of supernatant to a new 1.5ml centrifuge tube without enzyme, add 400ul of isopropanol and mix, and place on ice for 20min; centrifuge at 12000g, 4℃ for 10min; discard the supernatant, add 400ul of anhydrous ethanol to resuspend and wash the precipitate; centrifuge at 7500g for 5min; aspirate the ethanol, dry at room temperature, wait for 5min, and add 20ul of DEPC water, gently blow to dissolve, measure the concentration, and store in a -80℃ refrigerator; S42: Reverse transcription reaction, calculate the required volume of 2ug RNA according to the measured concentration; perform reverse transcription reaction according to the procedure: 37℃15min, 60℃10min, 95℃3min, 4℃+∞; the obtained product is cDNA, which is stored at -20℃; S43: Real-time fluorescence quantitative PCR analysis.

7. Use of a MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma according to claim 2, characterized in that: In S5, Western-blot is specifically: S51: Protein extraction + BCA protein concentration determination: After digesting the cells, add 100ul RIPA lysis buffer, and add protease inhibitors, phosphatase inhibitors and PMSF according to the ratio of 100:1; shake and lyse on ice for 30min, centrifuge at 4℃, 12000rpm for 20min; take 80ul supernatant and add 20ul protein loading buffer, boil at 99℃ for 5min, cool naturally and store at -20℃; take 4ul supernatant and dilute it 5 times, add 200ul BCA working solution to each well, react at 37℃ for 30min, and measure the absorbance at 526nm; calculate the protein concentration according to the standard curve and adjust the loading amount; S52: Prepare gel of corresponding concentration according to the instructions of gel preparation kit; fix the gel on the electrophoresis clamp and place it correctly in the electrophoresis tank, add freshly prepared electrophoresis solution, take out the comb, load the sample according to the BCA test result, set the voltage to 80V, and adjust the voltage to 120V after the protein runs out of the upper gel; S53: After taking out the gel, place the gel and membrane in the order of blackboard-gel-PVDF membrane-white board, correctly place them in the electrotransfer tank, add freshly prepared electrotransfer solution and ice bricks, and set the current to 120mA and the time to 1 minute for 1kd protein; after the electrotransfer, place the membrane in skim milk for 2h; after the blocking, wash it with TBST and incubate it with the primary antibody at 4℃ overnight; S54: The next day, the membrane was washed three times with TBST, each time for 10 min; after incubation with secondary antibody at room temperature for 2 h, the membrane was washed three times with TBST, each time for 10 min; and ECL luminescent solution was prepared for development.

8. Use of a MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma according to claim 2, characterized in that: In S6, CCK8 detection is specifically: One day in advance, the cells were counted and seeded in a 96-well plate. 10ul of CCK8 working solution was added to every 100ul of complete culture medium. After adding CCK8 working solution at the same time on the 1st, 3rd, and 5th days, the cells were placed in the incubator and cultured for 2h. The OD value at 450nm was measured by a microplate reader. The absorbance of each group of samples minus the value of the blank group was used as statistical data.

9. Use of a MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma according to claim 2, characterized in that: In S7, the EDU staining detection is specifically as follows: S71: Evenly seed each group of cells into a six-well plate in advance and prepare 2x EDU staining solution: dilute 20uM EDU working solution with complete culture medium at 1:500; S72: Replace the medium of the cells in the six-well plate, wash twice with PBS, add 500 μl of complete medium containing 20 uM EDU to each well, and then add 500 μl of complete medium to each well to make the EDU concentration in the six-well plate 10 uM; continue to incubate the cells at 37°C for 2 h; S73: Remove the culture medium, wash twice with PBS, add 500 μL of 4% paraformaldehyde to each well to fix the cells for 15 min; remove the paraformaldehyde, wash twice with PBS; add 1 mL of permeabilization solution to each well for 15 min; remove the permeabilization solution, wash twice with PBS; S74: Prepare click solution, add 500 μl of prepared click working solution to each well, incubate for 30 min in the dark; remove the reaction solution and wash twice with PBS; S75: Prepare Hoechst solution with PBS 1:1000, add 500 μl to each well, incubate for 10 min in the dark, remove the solution, and wash twice with PBS; S76: Take photos and observe under a fluorescence microscope.

10. Use of a MRPS10 inhibitor in the diagnosis and treatment of pancreatic ductal adenocarcinoma according to claim 2, characterized in that: In said S8, the cell immunofluorescence is specifically: S81: Wash the cell slide with sterile PBS and place it in a 12-well plate, and spread the cells evenly; S82: After changing the cell medium, add 1 ml of 4% paraformaldehyde to each well to fix the cells for 20 min, wash twice with PBS; add 500 ul of permeabilization solution to each well to permeabilize the cells for 10 min, wash twice with PBS; add 500 ul of ready-to-use goat serum to each well to block for 30 min; S83: primary antibody Ki67 incubated overnight at 4°C; S84: Wash twice with PBS, incubate with fluorescent secondary antibody for 1 hour in the dark; all subsequent operations must be strictly protected from light. After washing twice with PBS, stain with DAPI for 3 minutes, wash twice with PBS, seal the slide with nail polish, and fix the slide with fixative; S85: Observation under fluorescence microscope.

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