Application of TRIM27 gene as target spot in screening medicine for treating mitochondrial autophagy abnormality related diseases

Abstract

The invention provides application of a TRIM27 gene as a target spot in screening of drugs for treating mitochondrial autophagy abnormality related diseases, and belongs to the technical field of biomedicine.The research finds that TRIM27 participates in regulation and control of mitochondrial functions; the TRIM27 gene silently weakens mitochondrial functions in cells, reduces mitochondrial membrane potential and activates mitochondrial autophagy, PINK1 protein and Parkin protein levels are up-regulated, a classical autophagy marker LC3B is up-regulated, mitochondrial autophagy can be activated by targeted inhibition of TRIM27 gene expression, and occurrence and development of mitochondrial autophagy abnormality related diseases are further interfered.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of the TRIM27 gene as a target in screening drugs for treating diseases related to abnormal mitochondrial autophagy. Background Art

[0002] Mitochondrial autophagy is a key mechanism for cells to maintain mitochondrial homeostasis. It ensures the stability of cellular energy metabolism and internal environment by accurately removing damaged or redundant mitochondria. However, abnormal mitochondrial autophagy is closely related to the occurrence and development of many major diseases such as neurodegenerative diseases, cardiovascular diseases, metabolic diseases, and tumors. In the process of tumor proliferation, energy consumption is huge, and the supply of energy is inseparable from mitochondrial energy supply. However, tumor cells, such as colorectal cancer (CRC) cells, are often accompanied by abnormalities in glycolipid metabolism. These abnormal changes are closely related to mitochondrial function, but the detailed mechanism of changes in mitochondrial function has not yet been clarified.

[0003] Currently, there are limited treatment options for diseases related to abnormal mitochondrial autophagy. Traditional treatments such as medication and surgery can only alleviate symptoms but cannot fundamentally correct the pathological mechanism of abnormal mitochondrial autophagy. Some existing therapeutic drugs have problems such as poor specificity, large side effects, and insignificant efficacy, making it difficult to meet clinical needs. For example, some drugs used to treat neurodegenerative diseases, although they can improve patient symptoms to a certain extent, long-term use can cause a variety of adverse reactions and cannot prevent the progression of the disease. Therefore, finding new therapeutic targets and developing specific and highly effective therapeutic drugs have become the focus and difficulty of current research. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an application of the TRIM27 gene as a target in screening drugs for treating diseases related to abnormal mitochondrial autophagy.

[0005] The present invention provides an application of the TRIM27 gene as a target in screening drugs for treating diseases related to abnormal mitochondrial autophagy, and screens substances that inhibit the expression of the TRIM27 gene.

[0006] Preferably, the disease associated with abnormal mitochondrial autophagy includes colorectal cancer.

[0007] The present invention provides the use of a TRIM27 gene expression inhibitor in the preparation of a medicament for treating diseases related to abnormal mitochondrial autophagy.

[0008] Preferably, the TRIM27 gene expression inhibitor includes LOXO292 or TRIM27 gene interfering RNA.

[0009] The present invention provides the use of a TRIM27 gene expression inhibitor in preparing a reagent for inhibiting abnormal mitochondrial autophagy cells.

[0010] Preferably, the cells with abnormal mitochondrial autophagy include HCT8 cells.

[0011] The present invention provides the use of a TRIM27 gene expression inhibitor in promoting the expression of mitophagy-related proteins.

[0012] Preferably, the mitophagy-related proteins include PINK1 protein and Parkin protein.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present study found that TRIM27 is involved in regulating mitochondrial function. TRIM27 gene silencing weakens mitochondrial function in cells, reduces mitochondrial membrane potential, and activates mitophagy: PINK1 and Parkin protein levels are upregulated, as is the classic autophagy marker LC3B. TRIM27 overexpression can enhance mitochondrial function in colorectal cancer cells, increase mitochondrial membrane potential, and inhibit mitophagy in colorectal cancer cells.

[0015] In addition, the TRIM27 targeted inhibitor LOXO-292 can also reduce mitochondrial membrane potential. In vivo experiments have shown that the TRIM27 targeted inhibitor LOXO-292 inhibits tumor growth. The research results of the present invention indicate that targeting TRIM27 can activate mitochondrial autophagy and thus interfere with the occurrence and development of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 To verify the results of TRIM27 gene silencing;

[0017] Figure 2 To verify the results of TRIM27 gene overexpression;

[0018] Figure 3 The effect of TRIM27 gene silencing on mitochondrial respiratory capacity in colorectal cancer cells (Seahorse XFe96 mitochondrial function assay);

[0019] Figure 4 The effect of TRIM27 gene overexpression on mitochondrial respiratory capacity in colorectal cancer cells (SeahorseXFe96 mitochondrial function assay);

[0020] Figure 5 Effects of TRIM27 gene silencing on mitochondrial membrane potential in colorectal cancer cells (JC-1 staining method);

[0021] Figure 6The effect of TRIM27 gene overexpression on mitochondrial membrane potential in colorectal cancer cells (JC-1 staining method);

[0022] Figure 7 To test the therapeutic effect of targeted inhibition of TRIM27 on colorectal cancer (subcutaneous tumor size) in vivo;

[0023] Figure 8 To verify the therapeutic effect of targeted inhibition of TRIM27 on colorectal cancer by in vivo experiments (Ki-67 staining method);

[0024] Figure 9 The TRIM27 inhibitor LOXO-292 treatment group showed changes in PINK1 expression;

[0025] Figure 10 The effect of LOXO-292 treatment on the mitochondrial membrane potential of subcutaneous colorectal cancer tumors (JC-1 staining) was analyzed by immunohistochemical staining.

[0026] Figure 11 Effects of TRIM27 gene silencing on the expression of PINK1, Parkin, and LC3B;

[0027] Figure 12 This is the effect of TRIM27 gene overexpression on the expression of PINK1, Parkin and LC3B. DETAILED DESCRIPTION

[0028] The present invention provides an application of the TRIM27 gene as a target in screening drugs for treating diseases related to abnormal mitochondrial autophagy, and screens substances that inhibit the expression of the TRIM27 gene.

[0029] The present invention does not specifically limit the specific types of the diseases associated with abnormal mitochondrial autophagy. In the specific implementation of the present invention, the diseases associated with abnormal mitochondrial autophagy include colorectal cancer.

[0030] The present invention also provides the use of a TRIM27 gene expression inhibitor in the preparation of a drug for treating diseases associated with abnormal mitochondrial autophagy. In the present invention, the TRIM27 gene expression inhibitor preferably includes LOXO292 or TRIM27 gene interfering RNA.

[0031] The present invention provides the use of a TRIM27 gene expression inhibitor in the preparation of a reagent for inhibiting abnormal mitochondrial autophagy in cells. In the present invention, the abnormal mitochondrial autophagy cells include HCT8 cells.

[0032] The present invention provides the use of a TRIM27 gene expression inhibitor in promoting the expression of mitophagy-related proteins.

[0033] In the present invention, the mitophagy-related proteins include PINK1 protein and Parkin protein.

[0034] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] 1) The cells used in the present invention are human colorectal cancer cell line HCT8 purchased from ATCC, USA.

[0036] 2) DMEM high glucose medium (containing double antibodies) was purchased from Jiangsu KeyGen Biotech Co., Ltd. with the catalog number KGL-1206-500.

[0037] 3) 1× phosphate buffered saline (1× PBS buffer) was purchased from VICMED, China, with the catalog number VC2001P.

[0038] 4) Trypsin cell digestion solution (containing EDTA, 0.25%) was purchased from Jiangsu KeyGen Biotech Co., Ltd. with the catalog number VC2005.

[0039] 5) BCA Protein Assay Kit was purchased from Jiangsu Kangrun Biotechnology Co., Ltd. with the catalog number E162-01.

[0040] 6) Anti-TRIM27 antibody (Anti-TRIM27 Polyclonal Antibody 12205-1-AP), anti-PINK1 antibody (Anti-PINK1 Polyclonal antibody 23274-1-AP), anti-LC3B antibody (Anti-LC3B Polyclonal antibody 18725-1-AP), and anti-GAPDH antibody (Anti-GAPDH Monoclonal antibody (1E6D9) 60004-1-1g) were all purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0041] 7) Selpercatinib (LOXO-292) was purchased from MCE (MedChemExpress), USA, with the product number HY-114370.

[0042] 8) TRIM27 siRNA (Regular siRNA Economic Package-A) was purchased from Suzhou Genetron Health Co., Ltd.

[0043] 9) OE-TRIM27 plasmid was provided by Wuhan Miaoling Biotechnology Co., Ltd.

[0044] 10) Lipofectamine TM2000 was purchased from Invitrogen, USA.

[0045] 11) SilentFect Lipid Reagent was purchased from Thermo Fisher Scientific, USA.

[0046] 12) ECL chemiluminescence substrate kit (ultrasensitive) was purchased from China Biosharp Company, catalog number: BL523A.

[0047] 13) Cell apoptosis mitochondrial membrane potential detection kit (JC-1) was purchased from Shanghai KeyGene Biotechnology, catalog number: KGA1904-100.

[0048] 14) RIPA lysis buffer (strong) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0049] 15) Rotenone / Antimycin A was purchased from Selleck, USA.

[0050] 16) Oligomycin A was purchased from Selleck, USA.

[0051] 17) FCCP was purchased from Selleck, USA.

[0052] 18) Seahorse XF culture medium, calibration solution, XFe96 probe card, and XFe96 cell culture microplate were purchased from Guangzhou Boshida Instrument Co., Ltd.

[0053] 19) BALB / C-Nude mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0054] Example 1

[0055] Verification of TRIM27 gene silencing efficiency (Western Blotting method)

[0056] 1. Experimental Procedure

[0057] 0.8×10 6HCT8 and HCT116 cells were seeded at a concentration of 1 cell / mL in a 60 mm cell culture dish and cultured at 37°C in 5% CO2. The next day, when the cells reached approximately 40-50% growth, siRNA transfection (TRIM27 siRNA) was performed. The original culture medium was discarded and 1.5 mL of serum-free culture medium was added. Prepare four 1.5 mL EP tubes (A, B, C, and D): Tube A: 750 μL of serum-free culture medium plus 18 μL of SilentFect Lipid Reagent; Tube B: 250 μL of serum-free culture medium plus 3 μL of siCTL (30 pmol); Tube C: 250 μL of serum-free culture medium plus 3 μL of siTRIM27#2 (30 pmol); and Tube D: 250 μL of serum-free culture medium plus 3 μL of siTRIM27#3 (30 pmol). Mix thoroughly and incubate at room temperature for 5 minutes. Add 250 μL of each of tubes AB, AC, and AD in a 1:1 ratio and incubate at room temperature for 20 minutes. Add the above mixture to a culture dish containing fresh medium to a total volume of 2 mL. Continue incubating at 37°C in a 5% CO2 incubator, changing the medium after 6–8 hours. Transfection was continued for 48 hours. Protein was extracted and TRIM27 protein expression was determined by western blotting; GAPDH was used as an internal control.

[0058] 2. Experimental Results

[0059] The results of TRIM27 gene silencing efficiency verification are as follows Figure 1 As shown, transfection of two siRNA fragments significantly reduced the protein expression level of TRIM27.

[0060] Example 2

[0061] Verification of TRIM27 gene overexpression efficiency (Western Blotting method)

[0062] 1. Experimental Procedure

[0063] 0.8×10 6 HCT8 and HCT116 cells were seeded into 60mm cell culture dishes at a concentration of 100 μg / mL and cultured at 37°C in 5% CO2. The next day, when the cells reached 70% to 80% of their growth, plasmid DNA transfection was performed. The original culture medium was discarded, and after washing twice with 1× PBS, 1.5 mL of serum-free culture medium was added. Three 1.5 mL EP tubes A, B, and C were prepared. Tube A contained 500 μL of serum-free culture medium and 12 μL of Lipofectamine. TM2000; 250 μL of serum-free medium plus 3 μg of empty vector plasmid in tube B; 250 μL of serum-free medium plus 3 μg of OE-TRIM27 plasmid in tube C, mix thoroughly, and incubate at room temperature for 5 minutes. 250 μL of each of tubes AB and AC, respectively, mix thoroughly in a 1:1 ratio and continue incubating at room temperature for 20 minutes. Add the above mixture to the culture dish that has been changed with medium to a total volume of 2 mL. Continue incubating at 37°C, 5% CO2 in a cell culture incubator, change the medium after 6-8 hours, and transfect for 48 hours. Protein extraction, and detection of TRIM27 protein expression levels by western blotting; GAPDH was used as an internal control.

[0064] 2. Experimental Results

[0065] The results of TRIM27 gene overexpression efficiency verification are as follows Figure 2 As shown, transfection of the overexpression plasmid significantly enhanced the protein expression level of TRIM27.

[0066] Example 3

[0067] Effects of TRIM27 gene changes on mitochondrial respiratory capacity in colorectal cancer cells (Seahorse XFe96 mitochondrial function assay)

[0068] 1. Experimental Procedure

[0069] HCT8 and HCT116 cells were transfected with different TRIM27 small interfering fragments or TRIM27 overexpression plasmids. After 48 hours, the cells were counted and 1×10 4Seed 80 μL of cells / well in a cell culture microplate. Let it sit on the benchtop for 1 hour to allow the cells to better distribute across the bottom of the wells. Then, place it in a cell culture incubator overnight. Simultaneously, add 200 μL of ddH2O to the hydrated microplate and place it in a CO2-free 37°C incubator overnight. Preheat the Seahorse XFe overnight. The next day, prepare the base reagents: 97 mL of XF base medium + 1 mL of L-glutamine (200 mM) + 1 mL of glucose solution (1.0 mmol / L) + 1 mL of sodium pyruvate (100 mmol / L). Filter through a 0.2 μM filter and preheat at 37°C. Change the plate's medium: Discard the ddH2O, add 200 μL of hydration solution, and place in a CO2-free 37°C incubator for 1 hour. Discard 60 μL of medium from the cell culture microplate and add 200 μL of basal medium. Discard 200 μL of medium, add 200 μL of basal medium, discard another 200 μL of medium, and finally add 160 μL of basal medium, bringing the final volume to 180 μL / well. Place the washed cell plate in a CO2-free 37°C incubator for 1 hour. Prepare the experimental drugs and add them to each well of the hydrated plate. Well A: 20 μL of 15 μmol / L oligomycin A; Well B: 22 μL of 5 μmol / L FCCP; Well C: 25 μL of 5 μmol / L rotenone / antimycin A. Expel air bubbles before adding the drugs. Onboarding: Add the names and record the order of sample addition. Place the drug-doped test plate and the Ufility Plate containing the calibration solution into the instrument first. After the 20-minute calibration period, replace the cell plate and continue the experiment. Data analysis was performed using Wave software.

[0070] 2. Experimental Results

[0071] The results of Seahorse energy metabolism analysis are as follows Figures 3 and 4 As shown in the results, when the TRIM27 gene was knocked down, the oxygen consumption rate of colorectal cancer tumor cells HCT8 and HCT116 decreased; while TRIM27 gene overexpression enhanced the oxygen consumption rate of HCT8 and HCT116 cells, indicating that TRIM27 is involved in regulating the mitochondrial function of colorectal cancer tumor cells.

[0072] Example 4

[0073] Effects of TRIM27 gene changes on mitochondrial membrane potential in colorectal cancer cells (JC-1 staining method)

[0074] 1. Experimental Procedure

[0075] HCT8 and HCT116 cells were transfected with different TRIM27 small interfering fragments or TRIM27 overexpression plasmids. After 48 h, the cells were counted and 2 × 10 4Cells were seeded in 12-well plates covered with coverslips and cultured for 24 hours before JC-1 staining: 4 μM JC-1 fluorescent dye was added to each well and incubated at 37°C in the dark for 20 minutes. The wells were washed three times with HBSS and fixed with 4% cell tissue fixative at room temperature for 30 minutes. The wells were then washed three times with PBS, each for 5 minutes. The wells were mounted with DAPI-containing mounting medium and allowed to dry. The wells were observed under an inverted fluorescence microscope, and photographs were taken of randomly selected fields of view.

[0076] 2. Experimental Results

[0077] The experimental results are as follows Figures 5 and 6 As shown, in the TRIM27 knockdown group, mitochondrial transmembrane potential (ΔΨm) decreased (green fluorescence increased, red fluorescence decreased); in the TRIM27 overexpression group, mitochondrial transmembrane potential (ΔΨm) increased (green fluorescence decreased, red fluorescence increased). This further verified that TRIM27 enhances mitochondrial function in colorectal cancer cells.

[0078] Example 5

[0079] In vivo experiments to detect the therapeutic effect of targeted inhibition of TRIM27 on colorectal cancer (subcutaneous tumor size)

[0080] 1. Experimental Procedure

[0081] HCT116 cells were collected at 5×10 7 The cells were resuspended in serum-free culture medium at a density of 100 μL. 100 μL of the above density liquid was inoculated into the left abdomen of 10 6-8 week old BALB / C-Nude mice. 3 Based on tumor size, mice were randomly divided into a control group (PBS) and a group treated with the TRIM27 inhibitor LOXO-292 (LOXO-292). The LOXO-292 group received 10 mg / kg intraperitoneal administration every two days, while the control group received an equal volume of PBS intraperitoneally for two weeks before the experiment was terminated. Mice were sacrificed by cervical dislocation, and tumors were harvested and photographed to record tumor size.

[0082] 2. Experimental Results

[0083] The experimental results are as follows Figure 7 As shown: Compared with the PBS group, the tumor volume and tumor weight of the TRIM27 inhibitor LOXO-292 treatment group were smaller and lighter, indicating that the TRIM27 inhibitor LOXO-292 has a good therapeutic effect on colorectal cancer.

[0084] Example 6

[0085] In vivo experiments verify the therapeutic effect of targeted inhibition of TRIM27 on colorectal cancer (Ki-67 staining method)

[0086] 1. Experimental Procedure

[0087] HCT116 cells were collected at 5×10 7 The cells were resuspended in serum-free culture medium at a density of 100 μL. 100 μL of the above density liquid was inoculated into the left abdomen of 10 6-8 week old BALB / C-Nude mice. 3According to the tumor size, the cells were randomly divided into a control group (PBS) and a TRIM27 inhibitor LOXO-292 treatment group (LOXO-292). Among them, the LOXO-292 group was intraperitoneally administered at 10 mg / kg once every two days; the control group was intraperitoneally injected with the same volume of PBS. The experiment was terminated after 2 weeks of continuous administration, and the mice were killed by cervical dislocation. The tumors were collected, fixed in 4% paraformaldehyde for three days, transferred to 75% ethanol, and stored at room temperature; dehydration: the tumor tissues were placed in 75% ethanol for 1 hour, 85% ethanol for 1 hour, and 95% ethanol at room temperature overnight; transparency: on the second day, new 95% ethanol was replaced for 1 hour, 100% ethanol twice, each for 30 minutes, and xylene twice, each for 30 minutes, to make the tissue translucent and amber; wax dipping: soft wax was prepared in a 70°C oven in advance, and the above-mentioned transparent tissue was immersed in it for 3 to 4 hours; wax block wrapping: the melted embedding wax was poured into the embedding mold, and the wax-soaked tissue block was quickly placed in it, and the prepared label was inserted. Wait for the wax to solidify and store at -20°C for later use. Sectioning: Section the paraffin tissue at a thickness of 4 μm. Place the sliced ​​paraffin tissue in a 37°C water bath, fully unfold the slices, and attach the slices to a poly-lysine-coated slide. Slide baking: Place the slide containing the tissue in a 60°C oven overnight. Dewaxing and hydration: Place the slices in xylene (I) and (II) for 30 min each, then place them in anhydrous ethanol (I), (II), 95% ethanol, 90% ethanol, 85% ethanol, 80% ethanol, 70% ethanol, and distilled water for 5 min each. Endogenous peroxidase removal: Cover the tissue with 3% H2O2, place in a humidified chamber, and incubate at room temperature in the dark for 10 min. Wash with 1× PBS three times, 5 min each time. Antigen retrieval: Use high temperature and high pressure retrieval - Pour enough citric acid (pH 6.0) Ensure that the repair solution covers the slices. After the liquid boils, put the slices in, cover the pot, and wait for the pressure cooker to start venting for 3 minutes to perform antigen repair; after the repair is completed and the pressure drops to normal pressure, open the pot lid, let the slices warm up at room temperature, and then wash them 3 times with 1× PBS, each time for 5 minutes; blocking: cover the slices with 1% goat serum blocking solution, incubate at room temperature, in a humidified box, and incubate in the dark for 30 minutes; primary antibody overnight: dilute Ki-67 with PBS at a ratio of 1:250, cover each slice with approximately 50μL, and place in a humidified box at 4°C overnight; the next day, let the humidified box warm up at room temperature for 30 minutes, and wash them 3 times with 1× PBS, each time for 5 minutes, to wash off the excess primary antibody; incubate with secondary antibody: select the corresponding secondary antibody according to the primary antibody, and refer to the secondary antibody instruction manual - incubation with enhancement solution Sections were incubated in a humidified chamber at room temperature for 20 minutes, then washed three times with 1× PBS (5 minutes each time). Antibody solution was then incubated in a humidified chamber at room temperature for 20 minutes, followed by three washes with 1× PBS (5 minutes each time). DAB staining: Prepare DNA staining solution by adding one drop of solution B to 1 mL of solution A, mix thoroughly, and prepare immediately before use. Add the solution to the sections and incubate in the dark for several minutes, depending on the condition of the sections, until a brown precipitate appears. Rinse the sections with distilled water to terminate the staining reaction. Hematoxylin counterstaining: After staining, rinse the sections thoroughly with distilled water and then soak in hematoxylin for 3-5 minutes. Hydrochloric acid acidification and lithium carbonate debluing: Aspirate the sections in 1% hydrochloric acid, draw several times, rinse any remaining liquid with distilled water, and place in saturated lithium carbonate for 1-2 minutes for debluing. Observe the staining under a microscope. Dehydrate and mount the sections: Dehydrate in the reverse order of dewaxing. Mount the sections with gum, observe under a light microscope, and photograph and analyze.

[0088] 2. Experimental Results

[0089] The results of tumor immunohistochemical staining were as follows Figure 8 As shown, compared with the PBS group, the number of Ki-67 positive cells in the TRIM27 inhibitor LOXO-292 treatment group was less, further verifying that the TRIM27 inhibitor LOIXO-292 has a good therapeutic effect on colorectal cancer.

[0090] Example 7

[0091] In vivo experiments verify the effect of targeted inhibition of TRIM27 on mitochondrial function in colorectal cancer tissues

[0092] 1. Experimental Procedure

[0093] HCT116 cells were collected at 5×10 7 The cells were resuspended in serum-free culture medium at a density of 100 μL. 100 μL of the above density liquid was inoculated into the left abdomen of 10 6-8 week old BALB / C-Nude mice. 3According to the tumor size, the cells were randomly divided into a control group (PBS) and a TRIM27 inhibitor LOXO-292 treatment group (LOXO-292). Among them, the LOXO-292 group was intraperitoneally administered at 10 mg / kg once every two days; the control group was intraperitoneally injected with the same volume of PBS. The experiment was terminated after 2 weeks of continuous administration, and the mice were killed by cervical dislocation. The tumors were collected, fixed in 4% paraformaldehyde for three days, transferred to 75% ethanol, and stored at room temperature; dehydration: the tumor tissues were placed in 75% ethanol for 1 hour, 85% ethanol for 1 hour, and 95% ethanol at room temperature overnight; transparency: on the second day, new 95% ethanol was replaced for 1 hour, 100% ethanol twice, each for 30 minutes, and xylene twice, each for 30 minutes, to make the tissue translucent and amber; wax dipping: soft wax was prepared in a 70℃ oven in advance, and the above-mentioned transparent tissue was immersed in it for 3-4 hours; wax block wrapping: the melted embedding wax was poured into the embedding mold, and the wax-soaked tissue block was quickly placed in it, and the label prepared in advance was inserted, and waited for 2-3 hours. Wax solidified and stored at -20°C until ready for use; Sectioning: Paraffin tissue was sectioned at 4 μm thickness. The cut paraffin tissue was placed in a 37°C water bath, fully unfolded with sections, and then mounted on poly-lysine-coated slides; Slide baking: The slides containing tissue were placed in a 60°C oven overnight; Dewaxing and hydration: Sections were placed in xylene (I) and (II) for 30 min each, followed by ethanol (I), (II), 95% ethanol, 90% ethanol, 85% ethanol, 80% ethanol, 70% ethanol, and distilled water for 5 min each; Endogenous peroxidase removal: 3% H2O2 was dripped onto the tissue, placed in a humidified chamber, and incubated at room temperature in the dark for 10 min; Washed three times with 1× PBS, 5 min each time; Antigen retrieval: High temperature and high pressure retrieval was performed by pouring enough EDTA (pH 8.0) Ensure that the repair solution covers the slices. After the liquid boils, add the slices and cover the pot. After the pressure cooker starts to vent, count for 3 minutes to perform antigen repair. After the repair is completed and the pressure drops to normal pressure, open the pot lid and let the slices warm up at room temperature. Wash them 3 times with 1× PBS, each time for 5 minutes. Block: Cover the slices with 1% goat serum blocking solution and incubate them in a humidified box at room temperature for 30 minutes in the dark. Primary antibody overnight: Dilute PINK1 1:250 with PBS, cover each slice with approximately 50 μL, and place in a humidified box at 4°C overnight. The next day, let the humidified box warm up at room temperature for 30 minutes. Wash them 3 times with 1× PBS, each time for 5 minutes, to wash off the excess primary antibody. Incubate with secondary antibody: Select the corresponding secondary antibody according to the primary antibody. Refer to the secondary antibody instruction manual - incubation with enhancement solution. Sections were incubated in a humidified chamber at room temperature for 20 minutes, then washed three times with 1× PBS (5 minutes each time). Antibody solution was then incubated in a humidified chamber at room temperature for 20 minutes, followed by three washes with 1× PBS (5 minutes each time). DAB staining: Prepare DNA staining solution by adding one drop of solution B to 1 mL of solution A, mix thoroughly, and prepare immediately before use. Add the solution to the sections and incubate in the dark for several minutes, depending on the condition of the sections, until a brown precipitate appears. Rinse the sections with distilled water to terminate the staining reaction. Hematoxylin counterstaining: After staining, rinse the sections thoroughly with distilled water and then soak in hematoxylin for 3-5 minutes. Hydrochloric acid acidification and lithium carbonate debluing: Aspirate the sections in 1% hydrochloric acid, draw several times, rinse any remaining liquid with distilled water, and place in saturated lithium carbonate for 1-2 minutes for debluing. Observe the staining under a microscope. Dehydrate and mount the sections: Dehydrate in the reverse order of dewaxing. Mount the sections with gum, observe under a light microscope, and photograph and analyze.

[0094] 2. Experimental Results

[0095] The results of tumor immunohistochemical staining were as follows Figure 9 As shown in the data, compared with the PBS group, the expression of PINK1 in the TRIM27 inhibitor LOXO-292 treatment group was significantly decreased, indicating that TRIM27 promotes the occurrence and development of colorectal cancer by enhancing mitochondrial function.

[0096] Example 8

[0097] Effect of targeted inhibition of TRIM27 on mitochondrial membrane potential in colorectal cancer cells (JC-1 staining)

[0098] 1. Experimental Procedure

[0099] HCT8 and HCT116 cells were plated at 2×10 4Cells were seeded in 12-well plates covered with coverslips and cultured for 24 hours. The cells were then treated with different concentrations (0, 200 nM) of the TRIM27 targeted inhibitor Selpercatinib (LOXO-292) for 48 hours. JC-1 staining was performed by adding 4 μM JC-1 fluorescent dye to each well, incubating at 37°C in the dark for 20 minutes, washing three times with HBSS, fixing with 4% cell tissue fixative at room temperature for 30 minutes, and washing three times with PBS for 5 minutes each. The slides were mounted with a mounting medium containing DAPI and allowed to dry. The slides were observed under an inverted fluorescence microscope, and photographed and recorded at random in different fields of view.

[0100] 2. Experimental Results

[0101] The test results are as follows Figure 10 As shown, after LOXO-292 treatment of cells for 48 h, the mitochondrial transmembrane potential (ΔΨm) decreased (green fluorescence increased, red fluorescence decreased), further clarifying that TRIM27 can enhance mitochondrial function in colorectal cancer cells.

[0102] Example 9

[0103] Exploring the mechanism by which TRIM27 enhances mitochondrial function in colorectal cancer cells HCT8 and HCT116

[0104] 1. Experimental Procedure

[0105] HCT8 and HCT116 cells were transfected with different TRIM27 small interfering fragments or TRIM27 overexpression plasmids. After 48 h, the cells were counted and 0.8 × 10 6 HCT8 cells were seeded at 400 μg / mL into 6-well plates and cultured at 37°C in 5% CO2. Proteins were extracted, and the expression levels of mitophagy-related proteins (PINK1, Parkin, LC3B) and TRIM27 were determined by western blotting; GAPDH was used as an internal control.

[0106] 2. Experimental Results

[0107] The test results are as follows Figure 11-12 As shown, silencing TRIM27 activates mitophagy (increased levels of PINK1, Parkin, and the classic autophagy marker LC3B), while overexpressing TRIM27 inhibits mitophagy. These results suggest that TRIM27 promotes the development and progression of colorectal cancer by inhibiting mitophagy and enhancing mitochondrial function. Targeted inhibition of TRIM27 can weaken mitochondrial function in tumor cells and potentially treat colorectal cancer.

[0108] It can be seen from the above examples that TRIM27 is involved in regulating mitochondrial function. Targeted inhibition of TRIM27 expression can activate mitophagy, thereby interfering with the occurrence and development of diseases related to abnormal mitophagy.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. The use of TRIM27 gene as a target in screening drugs for treating diseases related to abnormal mitochondrial autophagy, characterized in that: Screening for substances that inhibit TRIM27 gene expression.

2. The use according to claim 1, characterized in that The diseases associated with abnormal mitochondrial autophagy include colorectal cancer.

3. Application of TRIM27 gene expression inhibitors in the preparation of drugs for the treatment of diseases related to abnormal mitochondrial autophagy.

4. The use according to claim 3, characterized in that The TRIM27 gene expression inhibitor includes LOXO292 or TRIM27 gene interfering RNA.

5. Application of TRIM27 gene expression inhibitors in the preparation of reagents for inhibiting abnormal mitochondrial autophagy cells.

6. The use according to claim 5, characterized in that The cells with abnormal mitochondrial autophagy include HCT8 cells.

7. Application of TRIM27 gene expression inhibitors in promoting the expression of mitochondrial autophagy-related proteins.

8. The use according to claim 7, characterized in that The mitochondrial autophagy-related proteins include PINK1 protein and Parkin protein.

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