Application of TRIM27 gene as target spot in screening medicine for treating abnormal lipid metabolism

Abstract

The invention provides an application of a TRIM27 gene as a target spot in screening of drugs for treating abnormal lipid metabolism, and belongs to the technical field of biomedicine.The research finds that the TRIM27 gene participates in regulation and control of colorectal cancer lipid metabolism reprogramming, overexpression of the TRIM27 gene can promote generation of lipid droplets in colorectal cancer tumor cells, and the effect of treating abnormal lipid metabolism is achieved. The silent TRIM27 gene can inhibit the generation of lipid droplets in colorectal cancer tumor cells.

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 abnormal lipid metabolism. Background Art

[0002] In recent years, with improvements in living standards and changes in dietary patterns, obesity has become a global health issue. Abnormal lipid metabolism is a primary cause of obesity. Abnormal lipid metabolism refers to a physiological and pathological process in which abnormalities in the synthesis, breakdown, digestion, absorption, and transport of lipids in the body result in excessive or insufficient lipid levels in various tissues, which in turn affects bodily functions. Factors that affect lipid metabolism include long-term high cholesterol, high saturated fatty acid levels, a high-calorie diet, genetic factors, apolipoprotein abnormalities, mental work, lack of exercise, and mental stress.

[0003] Abnormal lipid metabolism can lead to a range of serious diseases. For example, lipid deposition in the vascular endothelium can cause atherosclerosis, which in turn can lead to coronary heart disease (CHD) and peripheral vascular disease. Most patients are asymptomatic in the early stages, but in the late stages, it can lead to hypertension, arteriosclerosis, myocardial infarction, stroke, fatty liver, pancreatitis, and hyperuricemia. In addition, cholesterol measurements showing elevated serum cholesterol can be seen in fatty liver, liver tumors, hypothyroidism, diabetes, atherosclerosis, and nephrotic syndrome; decreased serum cholesterol can be seen in parenchymal liver lesions such as acute liver necrosis, cirrhosis, and hyperthyroidism. Elevated triglycerides are seen in diabetes, nephrotic syndrome, fatty liver, pancreatitis, and systemic lupus erythematosus; decreased triglycerides are commonly seen in hypothyroidism, adrenocortical insufficiency, and severely impaired liver function.

[0004] There is increasing evidence that obesity can affect the immune system's response to tumors through lipid metabolism reprogramming, promoting fatty acid metabolism in colorectal cancer cells to suppress anti-tumor immunity. Colorectal cancer is a common malignant tumor of the digestive tract. Abnormal lipid metabolism, as an important feature of tumor lipid metabolism reprogramming, is one of the core mechanisms of the occurrence and development of colorectal cancer, but its mechanism of action is unclear. Discovering and screening related genes that can effectively regulate lipid metabolism, exploring their role in the occurrence and development of colorectal cancer, and finding colorectal cancer-specific lipid metabolism regulatory genes as drug treatment targets or for the development of low-toxic and high-efficiency new drugs are key issues that need to be addressed urgently. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the use of TRIM27 gene as a target in screening drugs for treating abnormal lipid metabolism; the present invention's research found that TRIM27 gene is involved in regulating the reprogramming of lipid metabolism in colorectal cancer, overexpression of TRIM27 gene can promote the formation of lipid droplets in colorectal cancer tumor cells, and silencing of TRIM27 gene can inhibit the formation of lipid droplets in colorectal cancer tumor cells.

[0006] The present invention provides the use of TRIM27 gene as a target in screening drugs for treating abnormal lipid metabolism, and screens substances that inhibit the expression of TRIM27 gene.

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

[0008] Preferably, the lipid metabolism abnormalities diseases include obesity and colorectal cancer.

[0009] The present invention provides the use of a TRIM27 gene expression inhibitor in the preparation of a drug or reagent for inhibiting lipid droplet formation in colorectal cancer cells.

[0010] Preferably, the TRIM27 gene expression inhibitor includes LOXO292 or TRIM27 gene interfering RNA.

[0011] Preferably, the colorectal cancer cells include HCT116 and HCT8 cells.

[0012] The present invention provides the use of a TRIM27 gene expression inhibitor in preparing a reagent for inhibiting the expression of lipid metabolism-related genes.

[0013] Preferably, the lipid metabolism-related genes include FASN gene, SREBF1 gene and GPAM gene

[0014] Compared with the existing technology, the present invention has the following beneficial effects: The present invention has found that the TRIM27 gene is involved in regulating the reprogramming of lipid metabolism in colorectal cancer, overexpression of the TRIM27 gene can promote the formation of lipid droplets in colorectal cancer tumor cells, and silencing of the TRIM27 gene can inhibit the formation of lipid droplets in colorectal cancer tumor cells, significantly reducing the protein expression levels of lipid metabolism-related genes FASN, SREBF1, and GPAM. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 To verify the results of Western Blotting for the efficiency of TRIM27 gene silencing;

[0016] Figure 2 To verify the results of Western Blotting for the efficiency of TRIM27 gene overexpression;

[0017] Figure 3 The results of saturated Oil Red O staining after HCT116 and HCT8 cells were transfected with different TRIM27 gene small interfering fragments (to knock down TRIM27);

[0018] Figure 4 The results of saturated Oil Red O staining after TRIM27 overexpression plasmid (overexpressing TRIM27 gene) in HCT116 and HCT8 cells, respectively;

[0019] Figure 5 The results of saturated Oil Red O staining show the effect of TRIM27 targeted inhibitor LOXO-292 on lipid metabolism in colorectal cancer cells;

[0020] Figure 6 To test the effect of TRIM27 on lipid metabolism in colorectal cancer cells in vivo (saturated Oil Red O staining method);

[0021] Figure 7 To analyze the effect of TRIM27 overexpression on fatty acid (FA) content by non-targeted lipidomics;

[0022] Figure 8 To analyze the effect of TRIM27 overexpression on the content of monoglyceride (MG) by non-targeted lipidomics;

[0023] Figure 9 To analyze the effect of TRIM27 overexpression on diglycerol (DG) content by non-targeted lipidomics;

[0024] Figure 10 To analyze the effect of TRIM27 overexpression on triglyceride (TG) content by non-targeted lipidomics;

[0025] Figure 11 This is the effect of treating cells with the TRIM27 targeted inhibitor LOXO-292 on the protein expression levels of lipid metabolism-related genes FASN, SREBF1, and GPAM. DETAILED DESCRIPTION

[0026] 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.

[0027] The sources of reagents and biological materials used in the following examples are as follows:

[0028] 1) Human colorectal cancer cell lines HCT116 and HCT8 were purchased from ATCC, USA.

[0029] 2) DMEM high glucose medium (containing double antibodies) was purchased from Jiangsu KeyGen Biotech Co., Ltd. with the catalog number KGL-1206-500.

[0030] 3) 1× phosphate buffered saline (1× PBS buffer) was purchased from VICMED, China, with the catalog number VC2001P.

[0031] 4) Trypsin cell digestion solution (containing EDTA, 0.25%) was purchased from Jiangsu KeyGen Biotech Co., Ltd. with the catalog number VC2005.

[0032] 5) BCA protein concentration determination kit (BCA Protein Assay Kit) was purchased from Jiangsu Kangrun Biotechnology Co., Ltd., with the catalog number E162-01.

[0033] 6) Anti-TRIM27 antibody (Anti-TRIM27 Polyclonal Antibody 12205-1-AP), anti-FASN antibody (anti-FASN Polyclonal antibody 10624-2-AP), anti-SREBF1 antibody (Anti-SREBF1 Polyclonal antibody 14088-1-AP), anti-GPAM antibody (Anti-GPAM Recombinant antibody 241144H6), and anti-GAPDH antibody (Anti-GAPDH Monoclonal antibody (1E6D9) 60004-1-1g) were all purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0034] 7) Selpercatinib (LOXO-292) was purchased from MCE (MedChemExpress), USA, with the product number HY-114370.

[0035] 8) TRIM27 siRNA (Regular siRNA Economic Package-A) was purchased from Suzhou Genetron Health Co., Ltd.

[0036] 9) OE-TRIM27 plasmid was provided by Wuhan Miaoling Biotechnology Co., Ltd.

[0037] 10) Lipofectamine TM 2000 was purchased from Invitrogen, USA.

[0038] 11) SilentFect Lipid Reagent was purchased from Thermo Fisher Scientific, USA.

[0039] 12) ECL chemiluminescence substrate kit (ultrasensitive) was purchased from China Biosharp Company, catalog number: BL523A.

[0040] 13) Saturated Oil Red O staining solution was purchased from Beijing Solebow Technology Co., Ltd., catalog number: G1260.

[0041] 14) RIPA lysis buffer (strong) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0042] 15) Hematoxylin was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

[0043] 16) BALB / C-Nude mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0044] Example 1

[0045] TRIM27 gene silencing

[0046] 1. Experimental Procedure

[0047] 0.8×10 6 HCT116 and HCT8 cells were seeded at a concentration of 1 cell / mL in a 60 mm cell culture dish and cultured at 37°C and 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#1 (30 pmol); and Tube D: 250 μL of serum-free culture medium plus 3 μL of siTRIM27#2 (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 should continue for 48 hours. Protein was extracted and TRIM27 protein expression was determined by western blotting; GAPDH was used as an internal control.

[0048] 2. Experimental Results

[0049] 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.

[0050] Example 2

[0051] TRIM27 gene overexpression efficiency

[0052] 1. Experimental Procedure

[0053] 0.8×10 6 HCT116 and HCT8 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. TM 2000; 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.

[0054] 2. Experimental Results

[0055] 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.

[0056] Example 3

[0057] Effect of TRIM27 gene silencing on lipid metabolism in colorectal cancer cells (saturated Oil Red O staining)

[0058] 1. Experimental Procedure

[0059] HCT116 and HCT8 cells were transfected with different TRIM27 small interfering fragments (TRIM27 siRNA). After 48 hours, the cells were counted and 1×10 5Cells were seeded in a 6-well plate and cultured for 24 hours. A working solution of saturated Oil Red O staining solution and distilled water was prepared in a ratio of 3:2, mixed, and allowed to stand at room temperature for 5–10 minutes. Filtered before use. The culture medium was discarded, and the cells were washed once with PBS. Fixation was performed in 4% tissue cell fixative at room temperature for 10 minutes. The cells were washed twice with PBS, rinsed with 60% isopropanol, and stained with Oil Red O working solution for 10 minutes. Differentiation was performed with 60% isopropanol until the interstitial matrix was clear. The cells were washed twice with PBS and counterstained with hematoxylin for 1 minute. Excess hematoxylin was then washed away with PBS. The results were observed under a microscope, and photographs were taken of randomly selected fields.

[0060] 2. Experimental Results

[0061] The experimental results are as follows Figure 3 As shown, in the control group, lipid droplets in the cells appeared bright red, the nuclei appeared blue, and the interstitial tissue was colorless. After TRIM27 gene silencing, the number of lipid droplets was significantly reduced, indicating that TRIM27 gene knockdown inhibited intracellular lipid droplet synthesis.

[0062] Example 4

[0063] Effect of TRIM27 gene overexpression on lipid metabolism in colorectal cancer cells (saturated Oil Red O staining)

[0064] 1. Experimental Procedure

[0065] HCT116 and HCT8 cells were transfected with empty vector or TRIM27 overexpression plasmid (OE-TRIM27) respectively. 48 h after transfection, the cells were counted and 1×10 5 Cells were seeded in a 6-well plate and cultured for 24 hours. A working solution of saturated Oil Red O staining solution and distilled water was prepared in a ratio of 3:2, mixed, and allowed to stand at room temperature for 5–10 minutes. Filtered before use. The culture medium was discarded, and the cells were washed once with PBS. Fixation was performed in 4% tissue cell fixative at room temperature for 10 minutes. The cells were washed twice with PBS, rinsed with 60% isopropanol, and stained with Oil Red O working solution for 10 minutes. Differentiation was performed with 60% isopropanol until the interstitial matrix was clear. The cells were washed twice with PBS and counterstained with hematoxylin for 1 minute. Excess hematoxylin was then washed away with PBS. The results were observed under a microscope, and photographs were taken of randomly selected fields.

[0066] 2. Experimental Results

[0067] The experimental results are as follows Figure 4 As shown, in the control group, lipid droplets appeared bright red, the nuclei appeared blue, and the stroma was colorless. After transfection with an exogenous TRIM27 overexpression plasmid, the number of lipid droplets increased significantly, indicating that TRIM27 gene overexpression promotes lipid droplet synthesis in colorectal cancer cells.

[0068] Example 5

[0069] Effects of TRIM27 targeted inhibitors on lipid metabolism in colorectal cancer cells (saturated Oil Red O staining)

[0070] 1. Experimental Procedure

[0071] 0.8×10 5 HCT116 and HCT8 cells were seeded into 6-well plates at a concentration of 100 cells / mL and cultured at 37°C in 5% CO2. The next day, when the cells reached approximately 70% growth, they were treated with different concentrations (0 and 200 nM) of the TRIM27 inhibitor Selpercatinib (LOXO-292) for 48 hours. A working solution of saturated Oil Red O staining solution: distilled water was prepared in a ratio of 3:2, mixed, and allowed to stand at room temperature for 5-10 minutes. The solution was filtered before use. The culture medium was discarded, and the cells were washed once with PBS and fixed with 4% tissue cell fixative for 10 minutes at room temperature. The cells were then washed twice with PBS, rinsed with 60% isopropanol, and stained with Oil Red O working solution for 10 minutes. After differentiation with 60% isopropanol until the interstitial matrix was clear, the cells were washed twice with PBS, and counterstained with hematoxylin for 1 minute. Excess hematoxylin was then washed with PBS. The results were observed under a microscope, and photographs were taken of randomly selected fields.

[0072] 2. Experimental Results

[0073] The experimental results are as follows Figure 5 As shown, compared with the 0 nM group, the number of lipid droplets in colorectal cancer cells treated with 200 nM LOXO-292 for 48 hours was significantly reduced, indicating that targeted inhibition of TRIM27 can block the synthesis of lipid droplets in colorectal cancer cells.

[0074] Example 6

[0075] In vivo experiments verify the effect of TRIM27 on lipid metabolism in colorectal cancer cells (saturated Oil Red O staining)

[0076] 1. Experimental Procedure

[0077] Ten 6- to 8-week-old BALB / C-Nude male mice were randomly divided into two groups according to their body weight: control group (Vector) and TRIM27 overexpression group (OE-TRIM27). Cells in each group were expressed at 5×10 7 The cells were resuspended in serum-free culture medium at a density of 10 cells / mL and injected into each mouse at a density of 5×10 6 The subcutaneous tumors were inoculated with 100 μL of PBS. The experiment was terminated when a significant difference in tumor growth between the two groups appeared. The mice were sacrificed by cervical dislocation, and the tumors were collected, fixed in 4% paraformaldehyde, and sent to Wuhan Sevier Biotechnology Co., Ltd. for preparation of frozen sections and detection of lipid droplet formation.

[0078] 2. Experimental Results

[0079] The experimental results were consistent with those of in vitro experiments, such as Figure 6 As shown: Compared with the Vector group, the number of lipid droplets in the TRIM27 overexpression group was significantly increased, further proving that TRIM27 can promote lipid production in vivo.

[0080] Example 7

[0081] Non-targeted lipidomics analysis of the effect of TRIM27 on lipid metabolism in colorectal cancer cells

[0082] 1. Experimental Procedure

[0083] Six replicates of each control (Ctl) and HCT116 cells stably overexpressing TRIM27 (OE-TRIM27, OET) were prepared. When the cell confluence reached 80% to 90%, the cells were harvested using 0.25% trypsin-EDTA digestion and washed three times with pre-chilled 1× PBS. The cell pellets were shipped to Shanghai Biopharm Biotechnology Co., Ltd. for non-targeted lipidomics and data analysis.

[0084] 2. Experimental Results

[0085] The results of non-targeted lipidomics analysis are as follows Figures 7-10 As shown in the results, the fatty acid (FA) content of the TRIM27 overexpression group was significantly reduced, while the relative contents of monoglyceride (MG), diglycerol (DG), and triglyceride (TG) were significantly increased. These results further demonstrate the ability of TRIM27 to promote lipid accumulation in colorectal cancer cells.

[0086] Example 8

[0087] Effect of targeted inhibition of TRIM27 on the expression of lipogenic enzymes in colorectal cancer cells

[0088] 1. Experimental Procedure

[0089] 0.8×10 6 HCT116 cells were seeded at 400 μg / mL into 6-well plates and cultured at 37°C in 5% CO2. The next day, after the cells reached approximately 70% growth, they were treated with different concentrations (0 and 200 nM) of the TRIM27 inhibitor Selpercatinib (LOXO-292) for 48 hours. Proteins were extracted and the expression levels of the lipogenic enzymes FASN, SREBF1, and GPAM were determined by western blotting; GAPDH was used as an internal control.

[0090] 2. Experimental Results

[0091] The test results are as follows Figure 11 As shown in the results, LOXO-292 treatment of cells for 48 h significantly reduced the protein expression levels of lipid metabolism-related genes FASN, SREBF1, and GPAM, further clarifying that TRIM27 can promote lipid production in colorectal cancer.

[0092] It can be seen from the above examples that the TRIM27 gene can be used as a target in screening drugs for treating abnormal lipid metabolism; the present invention's research found that the TRIM27 gene is involved in regulating the reprogramming of lipid metabolism in colorectal cancer, overexpression of the TRIM27 gene can promote the formation of lipid droplets in colorectal cancer tumor cells, silencing the TRIM27 gene can inhibit the formation of lipid droplets in colorectal cancer tumor cells, and inhibiting the expression of the TRIM27 gene can significantly reduce lipid metabolism-related genes.

[0093] 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 abnormal lipid metabolism, characterized in that: Screening for substances that inhibit TRIM27 gene expression.

2. Application of TRIM27 gene expression inhibitors in the preparation of drugs for the treatment of lipid metabolism disorders.

3. The use according to claim 2, characterized in that The lipid metabolism abnormalities diseases include obesity and colorectal cancer.

4. Use of TRIM27 gene expression inhibitors in the preparation of drugs or reagents for inhibiting lipid droplet formation in colorectal cancer cells.

5. The use according to claim 4, characterized in that TRIM27 gene expression inhibitors include LOXO292 or TRIM27 gene interfering RNA.

6. The use according to claim 5, characterized in that The colorectal cancer cells include HCT116 and HCT8 cells.

7. Use of TRIM27 gene expression inhibitors in the preparation of reagents for inhibiting the expression of lipid metabolism-related genes.

8. The use according to claim 6, characterized in that The lipid metabolism-related genes include FASN gene, SREBF1 gene and GPAM gene.

Images