Application of ENO1 K71R mutant as target spot in screening preparations for regulating and controlling glycolysis of colon cancer cells and improving tumor microenvironment

Abstract

The invention provides application of an ENO1K71R mutant as a target spot in screening preparations for regulating and controlling glycolysis of colon cancer cells and improving tumor microenvironment, and belongs to the technical field of biological medicine and molecular biology. The research on the colon cancer cells which stably knock down PCSK9 finds that PCSK9 achieves the regulation effect on lactic acid metabolism in the colon cancer cells by regulating ENO1 lactic acid modification, the blank of ENO1 protein lactic acid modification and the regulation effect of the ENO1 protein lactic acid modification in tumor progression is filled, a lactic acid modification site is verified by constructing an ENO1K71R mutation experience, and the result shows that the PCSK9 can be used for regulating the lactic acid metabolism in the colon cancer cells. After ENO1 (K71) point mutation, ENO1 lactic acid can be inhibited, a new site is provided for ENO1 lactic acid modification, and a theoretical basis is also provided for developing novel tumor targeted drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and in particular relates to the application of ENO1 K71R mutant as a target in screening and regulating colon cancer cell glycolysis and improving tumor microenvironment preparations. Background Art

[0002] ENO1, also known as 2-phospho-D-glycerate hydrolase, has been shown to be a key enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate during glycolysis. It is also a multifunctional oncoprotein that is present on the cell surface and in the cytoplasm. The function of ENO1 in the glycolytic pathway affects the regulation of cellular energy, maintains tumor proliferation, and inhibits cancer cell apoptosis. In addition, ENO1 can enable tumors to escape growth inhibition and help tumor immune escape. ENO1 acts as a plasminogen receptor on the cell surface and promotes cancer invasion and metastasis by inducing angiogenesis. The overexpression of ENO1 in multiple cancer types and its localization on the tumor surface make it a good prognostic and diagnostic cancer biomarker and a tumor treatment target worthy of study. However, there are no relevant studies on the lactylation modification of ENO1 protein and the regulatory role of this modification in tumor progression.

[0003] Lactic acid can regulate transcription levels and protein activity through lactylation modification, thereby affecting the progression of malignant tumors. Research and exploration of the regulatory mechanism of lactylation modification can help people systematically understand the occurrence and mechanism of lactylation modification in malignant tumors and other diseases, and provide a theoretical basis for the development of new tumor-targeted drugs. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an application of ENO1 K71R mutant as a target in screening and regulating colon cancer cell glycolysis and improving tumor microenvironment preparations. The present invention studies colon cancer cells with stable knockdown of PCSK9 and finds that PCSK9 regulates lactic acid metabolism in colon cancer cells by regulating ENO1 lactylation modification, filling the gap in ENO1 protein lactylation modification and the regulatory role of this modification in tumor progression. The lactylation modification site is verified by constructing ENO1K71R mutant. The results show that ENO1 (K71) point mutation can inhibit ENO1 lactylation, providing a new site for ENO1 lactylation modification, and also providing a theoretical basis for the development of new tumor targeted drugs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of an ENO1 K71R mutant as a target in screening a preparation for regulating glycolysis of colon cancer cells. The ENO1 K71R mutant is a mutant obtained by mutating the ENO1 K71 site from lysine to arginine, and a substance capable of mutating the ENO1 K71 site from lysine to arginine is screened.

[0007] Preferably, the NCBI accession number of ENO1 is NM_001428.5.

[0008] Preferably, the regulating glycolysis of colon cancer cells is inhibiting glycolysis of colon cancer cells.

[0009] The present invention provides the use of ENO1 K71R mutant as a target in screening a preparation for improving tumor microenvironment.

[0010] Preferably, the improving the tumor microenvironment improves the tumor microenvironment by inhibiting glycolysis of colon cancer cells.

[0011] The present invention also provides the use of an agent causing ENO1 K71R mutation in preparing a medicine for treating colon cancer.

[0012] Preferably, the reagent comprises a mutant plasmid.

[0013] Preferably, the ENO1 K71R mutation is a mutation of the ENO1 K71 site from lysine to arginine.

[0014] Preferably, the NCBI accession number of ENO1 is NM_001428.5.

[0015] Preferably, the colon cancer cells are HCT116 or HT-29.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides an application of the ENO1 K71R mutant as a target in screening preparations for regulating glycolysis of colon cancer cells and improving the tumor microenvironment. The present invention performs proteomics and lactylation modification omics tests and analyses on colon cancer cells with stable knockdown of PCSK9, and finds that knockdown of PCSK9 reduces the lactylation modification levels of the glycolytic enzymes ENO1, ALDOA, and PGK1. Through lactic acid modification verification, the results showed that down-regulating PCSK9 reduced the lactic acid modification level of ENO1, and had no significant effect on the lactic acid modification of ALDOA and PGK1; in addition, down-regulating PCSK9 reduced the lactic acid production of colon cancer cells and inhibited the enzyme activity of ENO1; according to the lactic acid modification omics, it was found that ENO1 was lactic acid modified at the K71 site, and the ENO1 (K71R) mutant was constructed by point mutation. It was found that ENO1 (K71) point mutation could inhibit ENO1 lactic acid. It can be seen that the ENO1 K71 site is the lactic acid modification site of ENO1. Through further research, it was found that ENO1 delactation can inhibit the enzyme activity of ENO1 and the lactic acid production level in colon cancer cells. The results showed that inhibiting PCSK9 can inhibit the lactic acid modification of ENO1 protein, thereby inhibiting the glycolysis of colon cancer cells and improving the tumor microenvironment. The present invention fills the gap in the lactic acid modification of ENO1 protein and the regulatory role of this modification in tumor progression, and also provides a new site for ENO1 lactic acid modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the KEGG enrichment analysis diagram of protein lactylation modification in colon cancer cells with stable knockdown of PCSK9;

[0018] Figure 2 To down-regulate the effect of PCSK9 on ENO1 lactylation modification in colon cancer cells;

[0019] Figure 3 To down-regulate the effect of PCSK9 on ENO1 enzyme activity, A shows the knockdown of PCSK9 level by siRNA transient transfection technology, and B shows the effect of knockdown of PCSK9 on ENO1 enzyme activity in colon cancer cells;

[0020] Figure 4 The effect of ENO1 K71R on the lactylation level of ENO1;

[0021] Figure 5 Figure 3 is the effect of ENO1 delactation modification on ENO1 enzyme activity and lactate production level, where A is the effect of ENO1 delactation on ENO1 enzyme activity, and B is the effect of ENO1 delactation on lactate production in colon cancer cells. DETAILED DESCRIPTION

[0022] The present invention provides an application of an ENO1 K71R mutant as a target in screening a preparation for regulating glycolysis of colon cancer cells. The ENO1 K71R mutant is a mutant obtained by mutating the ENO1 K71 site from lysine to arginine, and a substance capable of mutating the ENO1 K71 site from lysine to arginine is screened.

[0023] In the present invention, the ENO1 K71R mutant is an ENO1 overexpression plasmid and an ENO1 point mutation plasmid constructed by Shanghai Aibosi Biotechnology; the NCBI accession number of the ENO1 is NM_001428.5 。

[0024] In the present invention, the regulation of colon cancer cell glycolysis is to inhibit colon cancer cell glycolysis.

[0025] The present invention provides the use of ENO1 K71R mutant as a target in screening a preparation for improving tumor microenvironment.

[0026] In the present invention, the ENO1 K71R mutant is an ENO1 overexpression plasmid and an ENO1 point mutation plasmid commissioned by Shanghai Abbott Biotechnology to achieve ENO1 overexpression or point mutation of tyrosine at position ENO1 (K71) to achieve the purpose of de-lactation modification; the improvement of the tumor microenvironment improves the tumor microenvironment by inhibiting glycolysis of colon cancer cells.

[0027] The present invention also provides the use of an agent causing ENO1 K71R mutation in preparing a medicine for treating colon cancer.

[0028] In the present invention, the reagent includes a mutant plasmid, which is an ENO1 point mutation plasmid commissioned by Shanghai Aibosi Biological to construct; the ENO1 K71R mutation is to mutate the ENO1 K71 site from lysine to arginine, and the NCBI accession number of the ENO1 is NM_001428.5 。

[0029] In the present invention, the colon cancer cells are HCT116 or HT-29.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1 Functional analysis of differentially modified proteins by lactylation

[0032] 1. Plasmid transient transfection

[0033] (1) Plasmid aliquoting: The ENO1 point mutation plasmid was aliquoted to avoid repeated freezing and thawing and stored at -20°C;

[0034] (2) Colon cancer cells HCT-116 and HT-29 (both purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were digested with trypsin and resuspended, and the cell concentration was diluted to 2×10 5 cells / mL, 2 mL per well was seeded in a six-well plate;

[0035] (3) After culturing in a cell culture incubator at 37°C and 5% CO2 for 24 h, the medium was removed when the cell growth reached 50% to 70%, and the cells were washed with serum-free McCoy's 5A medium (containing 10% FBS), and 1.5 mL of serum-free medium was added to each well;

[0036] (4) Dissolve 4 μg of plasmid in 250 μL Opti-mem and mix by gently pipetting; add 8 μL of lipofectamine 2000 dropwise into 250 μL Opti-mem, mix well, and let stand for 5 min;

[0037] (5) Mix Opti-mem containing lipofectamine 2000 and plasmid and let stand for 20 minutes;

[0038] (6) Add the plasmid complex dropwise into a six-well plate, shake the six-well plate to mix evenly, place it in a cell culture incubator at 37° C., 5% CO 2 for 4 h, and then replace it with complete culture medium for subsequent experiments.

[0039] 2. Lentiviral Transfection

[0040] (1) HCT-116 and HT-29 cells that were in good growth condition were digested with trypsin and diluted to 2×10 5 cells / mL, seeded in 24-well plates and cultured overnight in an incubator;

[0041] (2) When the cell confluence rate reached 30% to 50%, the culture medium was removed and 250 μL of fresh culture medium (containing 10% fetal bovine serum) was added to each well using the 1 / 2 small volume infection method. After 4 h of infection with lentivirus, the volume was replenished to 500 μL with fresh culture medium.

[0042] (3) 24 h after infection, the culture medium was removed and fresh McCoy's 5A medium was added for culture;

[0043] (4) 72 hours after infection, the expression efficiency was detected by observing fluorescence. When the infection efficiency reached 80%, puromycin was used to select and culture stable cell lines.

[0044] Colon cancer cells with stable knockdown of PCSK9 were obtained by transient plasmid transfection and lentiviral transfection. Proteomics and lactomic tests (commissioned by Hangzhou Jingjie Biotechnology Co., Ltd.) were performed on colon cancer cells with stable knockdown of PCSK9. It was found that lactomic modified differentially expressed proteins were involved in cellular metabolic physiological processes such as carbohydrate metabolism and glucose metabolism. KEGG enrichment analysis showed that colon cancer cells with knockdown of PCSK9 were significantly enriched in the glycolysis pathway ( Figure 1 ). In addition, knockdown of PCSK9 reduced the lactylation modification levels of alpha-enolase (ENO1), aldolase A (ALDOA), and phosphoglycerate kinase 1 (PGK1). The lactylation modification of three glycolytic enzymes ENO1, ALDOA, and PGK1 was verified and the modification sites were further analyzed.

[0045] Example 2 Effect of down-regulating PCSK9 on the lactic acid modification level of ENO1 in colon cancer cells To explore the regulatory mechanism of PCSK9 on colon cancer glycolysis and verify the regulatory effect of PCSK9 on ENO1 lactic acid modification, the present invention uses IP antibody ENO1 for immunoprecipitation and western blot experiments. Among them, the irrelevant sequence gene silencing group (commissioned by Shanghai Aibosi Biotechnology Co., Ltd. to design) is the NC group, and the PCSK9 gene silencing group (commissioned by Shanghai Aibosi Biotechnology Co., Ltd. to design) is the KD group.

[0046] 1. Co-immunoprecipitation experiment

[0047] (1) Proteins from colon cancer cells HCT116 and HT-29 were extracted using lysis buffer, and the protein concentrations were measured using the BCA method;

[0048] (2) Removal of specific binding: Collect 500 μg of protein, add 1 μg of common IgG of the same species as the IgG used in immunoprecipitation and 20 μL of ProA+GAgrarose, mix well, and shake slowly at 4°C for 1 hour. Centrifuge the sample at 2500 rpm for 5 minutes and carefully collect the supernatant into a new centrifuge tube;

[0049] (3) Mix 1 μg of IP antibody and 500 μg of protein sample and incubate overnight at 4°C. Add 20 μL of ProA+GAgrarose to the incubated sample and incubate in a cell culture incubator at 37°C and 5% CO2 for 2 h;

[0050] (4) Wash the cultured samples with 1 mL of PBS, centrifuge at 2500 rpm for 5 min, and discard the supernatant. Repeat 4±1 times. After the last wash, add loading buffer and incubate at 100°C for 5 min.

[0051] 2. Western Blotting

[0052] (1) Prepare the glass plate and the glue rack in advance. The glass plate needs to be washed with distilled water and dried.

[0053] (2) SDS-PAGE preparation: Prepare separation gel according to the molecular weight of the target protein using the SDS-PAGE gel preparation kit (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.);

[0054] (3) Glue preparation: Prepare the separation gel according to the instructions, slowly add it to the glass plate using a pipette, add 2 mL of isopropanol to remove bubbles and seal the gel. After the lower layer of gel solidifies, pour out the isopropanol and absorb the remaining liquid with absorbent paper, use a pipette to add the prepared upper layer of gel solution, inject carefully to avoid bubbles, and insert the comb teeth;

[0055] (4) Sample loading: After the upper gel has solidified, add electrophoresis solution to the electrophoresis tank, remove the comb, and add samples to the wells. When loading samples, add markers to the wells on both sides of the protein sample;

[0056] (5) Electrophoresis: Select appropriate voltage and electrophoresis time according to the molecular weight of the sample protein;

[0057] (6) Transfer: Place the PVDF membrane in methanol solution in advance to activate it. After the electrophoresis is completed, open the gel plate and cut the gel. Place the PVDF membrane and gel in the transfer clip in the order of positive and negative poles to avoid bubbles between the gel and the membrane. Place the transfer clip in the transfer tank, add the transfer solution, and transfer the membrane at a constant current of 250mA for 90 minutes. The transfer must be performed in an ice tank;

[0058] (7) Blocking: After the transfer, the PVDF membrane was placed in 5% skimmed milk powder and blocked on a shaker at 18-25°C for 1.5 h;

[0059] (8) Incubation with primary antibody: Wash the blocked PVDF membrane with TBST three times, 5 to 10 minutes each time. Prepare the primary antibody of appropriate concentration according to the instructions, then place the PVDF membrane in the diluted primary antibody and incubate overnight at 4°C;

[0060] (9) Incubation with secondary antibody: The primary antibody was recovered the next day and stored in a refrigerator at 4°C. The membrane was washed with TBST three times, 10 min each time, and the secondary antibody of the corresponding species was prepared using secondary antibody diluent. The membrane was incubated on a shaker at 18-25°C for 2 h.

[0061] (10) Color development: Wash the membrane three times with TBST. Take ECL chemiluminescent color development solution, mix solution A and solution B in a 1:1 ratio, and use a pipette to mix well before use. Place the PVDF membrane in a gel imager, add the color development solution to expose, save the image, and use ImageJ software to analyze the gray value. The results are shown in Figure 2 .

[0062] Depend on Figure 2 It can be seen that after downregulating PCSK9, the protein lactylation level in colon cancer cells HCT116 and HT-29 was significantly decreased. Immunoprecipitation and western blot experiments were performed using IP antibody ENO1. The results showed that compared with the irrelevant sequence gene silencing group (NC group), the KD group had no effect on the ENO1 protein level after knocking down PCSK9, while the ENO1 lactylation level decreased. Knocking down PCSK9 inhibited the ENO1 lactylation modification level.

[0063] Example 3 Effect of downregulating PCSK9 on ENO1 enzyme activity in colon cancer cells

[0064] In order to further study the effect of PCSK9 on ENO1 function, the present invention set up a control group and an experimental group for colon cancer cells HCT116 and HT-29, wherein the control group was an irrelevant sequence gene silencing group (NC group), and the experimental group was a PCSK9 gene silencing group (KD group), and the ENO1 enzyme activity of each group was measured. The results are shown in Figure 3 .

[0065] ENO1 enzyme activity assay: (1) Sample preparation: Use a scraper to scrape the adherent cells and centrifuge at 4°C, 500 rpm for 10 minutes. Rinse the sample twice with PBS and add extraction buffer. Incubate on ice for 20 minutes. Centrifuge at 4°C, 16,000 rpm for 20 minutes and transfer the supernatant to a new EP tube;

[0066] (2) Use the BCA kit (purchased from Shanghai Biotech Biotechnology Co., Ltd.) to determine the protein concentration. Take out all reagents in advance, equilibrate the reagent temperature to room temperature, dilute the sample to the required protein concentration using 1x incubation buffer, and add 50 μL of sample to each well. Incubate at room temperature for 2 h;

[0067] (3) Each well was washed twice with 300 μL 1× washing solution by aspiration;

[0068] (4) Add 200 μL of 1× active reagent to each well and measure the absorbance at 340 nm.

[0069] Depend on Figure 3 It can be seen that compared with the control group, the ENO1 enzyme activity decreased after knocking down PCSK9. Therefore, downregulating PCSK9 in colon cancer cells can inhibit the ENO1 enzyme activity.

[0070] Example 4K71 is the ENO1 lactylation modification site

[0071] According to the results of lactylation modification analysis, the present invention mutated the ENO1 K71 site from K to R (lysine to arginine) to construct an ENO1 point mutation plasmid (commissioned to Shanghai Abbott Biotechnology Co., Ltd. for construction) for cell transfection to simulate the delactated state of the protein. The specific method is shown in Example 1.

[0072] Colon cancer HCT116 cells and HT-29 cells were transfected with overexpression plasmids of wild-type ENO1 (WT group) and mutant ENO1 (MUT group), respectively. The results showed that ( Figure 4 ), compared with the WT group, the level of ENO1 lactylation modification in the MUT group was reduced, indicating that the ENO1 K71R mutant caused the delactylation of ENO1 and the K71 site was the lactylation modification site of ENO1.

[0073] Example 5 Effect of down-regulating ENO1 lactylation on ENO1 enzyme activity and lactate production level in colon cancer cells

[0074] Numerous studies have shown that ENO1 is regulated by different mechanisms, thereby regulating cancer progression by affecting the glycolytic pathway. In addition to transcriptional regulation, mRNA alternative splicing, protein-protein interactions, and PTMs also play an important role in the regulation of ENO1. For example, UNC-51-like kinases 1 / 2 (2UNC-51-like kinases 1and 2, ULK1 / 2) reduce the enzymatic activity of ENO1 by phosphorylating it. In colorectal cancer, the loss of P300 reduces the level of lysine 2-hydroxyisobutyrylation on ENO1, inhibiting the enzymatic activity of ENO1, thereby affecting glycolysis. Other modifications, including acetylation and methylation, can regulate the function of ENO1, and the levels of these modifications in cancer cells are significantly higher than those in normal cells.

[0075] In order to verify the effect of lactylation modification on the function of ENO1, the present invention transfected overexpression wild-type ENO1 plasmid (WT group) and mutant ENO1 plasmid (MUT group) into colon cancer cells respectively. The transfection method was carried out according to the method in Example 1, and the effect of lactylation modification level on ENO1 enzyme activity was detected. The results are shown in FIG. Figure 5 .

[0076] Lactate level determination: (1) Collect 1×10 6 Colon cancer cells HCT-116 and HT-29 were placed in microtubes;

[0077] (2) Centrifuge at 300 rpm for 2 min at 4°C and remove the supernatant;

[0078] (3) Add 300 μL of cold PBS to wash the cells, pipette, centrifuge at 300 rpm for 2 min, and remove the supernatant;

[0079] (4) Add 300 μL of 0.1% Triton-X, vortex for 1 min, and centrifuge at 8000 rpm for 5 min to prepare cell lysate;

[0080] (5) Add the solution to an ultrafiltration tube, centrifuge at 12000 rpm for 10 min to remove protein, and aspirate the supernatant to obtain the sample to be tested;

[0081] (6) Measure the protein concentration of the sample using the BCA method. Add 80 μL of working solution and 20 μL of sample to each well of a 96-well plate, incubate at 37°C for 30 min, and use an ELISA reader to measure the absorbance at 450 nm.

[0082] (7) Use the lactate standard in the kit to draw a standard curve and calculate the lactate level of the sample based on the standard curve.

[0083] Depend on Figure 5 It can be seen that compared with the WT group, the ENO1 enzyme activity in the MUT group was significantly reduced ( Figure 5 A in the figure), and the lactate production level in colon cancer cells was significantly reduced ( Figure 5 B). It can be seen that delactation of ENO1 can reduce ENO1 enzyme activity and lactate production levels, suggesting that ENO1 lactylation modification promotes glycolysis in colon cancer cells.

[0084] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of ENO1 K71R mutant as a target in screening agents for regulating glycolysis in colon cancer cells, characterized in that: The ENO1 K71R mutant is a mutant obtained by mutating the ENO1 K71 site from lysine to arginine, and a substance capable of mutating the ENO1 K71 site from lysine to arginine is screened.

2. The use according to claim 1, characterized in that: The NCBI accession number of ENO1 is NM_001428.

5.

3. The use according to claim 1, characterized in that: The regulating of colon cancer cell glycolysis is to inhibit the glycolysis level of colon cancer cells.

4. Application of ENO1 K71R mutant as a target in screening preparations that improve tumor microenvironment.

5. The use according to claim 4, characterized in that: The improvement of the tumor microenvironment improves the tumor microenvironment by inhibiting glycolysis of colon cancer cells.

6. Use of an agent that causes ENO1 K71R mutation in the preparation of a drug for treating colon cancer.

7. The use according to claim 6, characterized in that: The reagents include a mutant plasmid.

8. The use according to claim 6, characterized in that: The ENO1 K71R mutation is to mutate the ENO1 K71 site from lysine to arginine.

9. The use according to claim 6, characterized in that: The NCBI accession number of ENO1 is NM_001428.

5.

10. The use according to claim 6, characterized in that: The colon cancer cells are HCT116 or HT-29.

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