Application of FMNL1 malonylation modification in inhibition of ccRCC transfer

Abstract

The invention relates to the technical field of biology, discloses application of FMNL1 malonylation modification in inhibition of ccRCC transfer, and inhibits ccRCC transfer and invasion by promoting the FMNL1 malonylation modification level. The malonylation modification site of the FMNL1 is located at the 852 site of the FH2 functional structure domain; the malonylation modification level of lysine at the 852 site of the FMNL1FH2 functional structural domain is promoted, and migration and invasion of ccRCC are inhibited. According to the application of the FMNL1 malonylation modification in inhibition of ccRCC metastasis, in-vivo and in-vitro tests verify that the FMNL1 malonylation modification level can inhibit ccRCC invasion and metastasis, so that FMNL1 malonylation is a key target spot for regulating ccRCC invasion and metastasis, and based on the target spot, drugs for inhibiting renal clear cancer cell metastasis can be researched and developed.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of FMNL1 malonylation modification in inhibiting ccRCC transfer. Background Technology

[0002] Clear cell renal cell carcinoma (ccRCC) is a malignant tumor originating from renal tubular epithelial cells, accounting for approximately 80% of renal cell carcinomas, and metastasis is the leading cause of death. Fatty acid metabolism reprogramming is an important pathological feature of clear cell renal cell carcinoma, and lipid droplet accumulation accelerates ccRCC progression. A 2023 Nat Rev Urol review systematically described the research progress on fatty acid metabolism reprogramming in ccRCC, pointing out that various key lipid metabolism enzyme genes could serve as potential therapeutic targets. Even though numerous studies have revealed that lipid metabolism reprogramming is one of the key factors in the development and progression of ccRCC, an effective therapeutic target has yet to be found.

[0003] Exploring the regulation of tumor-related proteins by small lipid metabolites through lipid acylation modification is currently a hot research topic in the field of oncology. Malonyl-CoA, a lipid metabolism intermediate, acts as a "molecular switch" for fatty acid catabolism and synthesis. On one hand, the conversion of acetyl-CoA to malonyl-CoA under ACC1 catalysis is the rate-limiting initiation step of fatty acid synthesis; on the other hand, malonyl-CoA can also act as a small metabolic molecule to inhibit CPT1α, the rate-limiting enzyme for fatty acid β-oxidation, effectively suppressing fatty acid catabolism. In 2011, malonyl-CoA was first identified as a precursor to K-mal modification, mediating novel lysine K-mal modification. This modification currently plays an important role in the metastasis of hepatocellular carcinoma (HCC) and the development of colorectal cancer (CRC), but has not yet been reported in ccRCC, where lipid metabolism reprogramming is significant.

[0004] In recent years, the development of mass spectrometry has led to the identification of various novel protein acylation modifications, further enriching the regulatory network of post-translational protein modifications. Since its identification in 2011, malonylation has seen relatively little systematic research in cancer compared to other concurrently identified modifications such as succinylation and crotonylation. Given the characteristics of lipid metabolism reprogramming in clear cell renal cell carcinoma and the role of malonyl-CoA in lipid metabolism, exploring the mechanism of action of K-malyl modification in renal cell carcinoma progression will contribute to a deeper understanding of the molecular mechanisms by which lipid metabolism influences the malignant progression of renal cell carcinoma, and may provide new strategies and targets for renal cell carcinoma treatment. Summary of the Invention

[0005] The purpose of this invention is to provide the application of FMNL1 malonylation modification in inhibiting ccRCC metastasis. In vivo and in vitro experiments have verified that the level of FMNL1 malonylation modification can inhibit the invasion and metastasis of ccRCC. Therefore, FMNL1 malonylation is a key target for regulating the invasion and metastasis of ccRCC. Based on this target, drugs that inhibit the metastasis of renal clear cell carcinoma cells can be developed.

[0006] To achieve the above objectives, the present invention provides the application of FMNL1 malonylation modification in inhibiting ccRCC transfer, thereby promoting the level of FMNL1 malonylation modification and inhibiting the migration and invasion of ccRCC.

[0007] Furthermore, the FMNL1 malonylation modification site is located at position 852 of the FH2 functional domain; promoting the malonylation modification level of lysine at position 852 of the FMNL1 FH2 functional domain inhibits the migration and invasion of ccRCC.

[0008] Furthermore, it promotes the malonylation modification level of FMNL1, inhibits the formation of FH2 homodimer, and suppresses the polymerization and rearrangement of F-actin.

[0009] Furthermore, it promotes the malonylation modification level of FMNL1 and inhibits the interaction between FMNL1 and invasive pseudopodia regulators.

[0010] The interaction between Cortactin and Fascin inhibits the recruitment of Cortactin and Fascin at the site of invasive pseudopodia, thereby inhibiting the formation of invasive pseudopodia in ccRCCs and thus suppressing the migration and invasion of ccRCCs.

[0011] Furthermore, FMNL1 malonylation inhibits the expression of MMPs, thereby suppressing the migration and invasion of ccRCCs.

[0012] To achieve the above objectives, the present invention also provides the application of the FMNL1 852 site as a target in the preparation of drugs that inhibit the metastasis of renal clear cell carcinomas.

[0013] To achieve the above objectives, the present invention also provides the application of the FMNL1 852 site as a target in screening drugs that inhibit the metastasis of clear cell renal cells.

[0014] To achieve the above objectives, the present invention also provides a product, wherein the active ingredient of the product is used to promote the level of FMNL1 malonylation modification, and the application of the product is one of the following:

[0015] ① Inhibit the migration and invasion of ccRCC;

[0016] ②Inhibit the formation of FH2 homodimers;

[0017] ③ Inhibit the polymerization and rearrangement of F-actin;

[0018] ④ Inhibit the interaction between FMNL1 and the invasive pseudopodia regulators Cortactin and Fascin;

[0019] ⑤ Inhibit the recruitment of Cortactin and Fascin at invasive pseudopodia sites;

[0020] ⑥ Inhibit the formation of invasive pseudopodia in ccRCC;

[0021] ⑦ Inhibit the expression of MMPs.

[0022] To achieve the above objectives, the present invention also provides an inhibitor that inhibits the activity of demalonylation modifying enzymes, increases the level of FMNL1 malonylation modification, and inhibits the migration and invasion of ccRCC.

[0023] Furthermore, the demalonylated modifying enzyme is SIRT5.

[0024] The advantages and positive effects of the FMNL1 malonylation modification described in this invention in inhibiting ccRCC transfer are as follows:

[0025] 1. This invention reveals for the first time the mechanism by which lipid metabolism intermediates regulate ccRCC metastasis through K-mal modification, establishes a central regulatory model of lipid metabolites-protein post-translational modification-tumor metastasis, and defines an important way in which metabolites regulate ccRCC metastasis through non-metabolic pathways. This helps to comprehensively and deeply understand the molecular mechanism by which lipid metabolism and acylation modification affect the occurrence and development of ccRCC from a new perspective, and further expands people's understanding of the role of K-mal modification in tumor occurrence and development.

[0026] 2. This invention first explores the impact of lipid metabolism reprogramming on ccRCC metastasis from the perspective of lipid acylation modification, enriching the regulatory network of protein post-translational modification in ccRCC progression; secondly, it systematically investigates the impact and molecular mechanism of FMNL1 on renal cell carcinoma metastasis from the perspective of novel K-mal modification, expanding the understanding of cytoskeleton regulation of renal cell carcinoma metastasis, and revealing that the cytoskeleton protein FMNL1 is expected to become a potential new target for the treatment of ccRCC metastasis.

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0028] Figure 1 In this embodiment of the invention, heat map analysis is used to determine the overall K-mal modification level of proteins in cancer tissue (C) and para-tumor tissue (P).

[0029] Figure 2 The following are GO clustering and volcano plot analyses of the K-mal modified group in the ccRCC sample of this invention, where A is the GO clustering analysis of significantly different modified proteins in the K-mal modified group between cancer and adjacent normal tissue (p<0.05); and B is the volcano plot of significantly different modified proteins in the K-mal modified group.

[0030] Figure 3 A shows HEK 293T cells transiently transfected with EV and pcDNA3.1-FMNL1-3×Flag, followed by K-mal pan-antibody detection of FMNL1 K-mal modification; B shows 786-O cell lines stably expressing EV and FMNL1-Flag, followed by K-mal pan-antibody detection of FMNL1 K-mal modification after cell lysis; C shows Caki-1 cell lines stably expressing EV and FMNL1-Flag, followed by K-mal pan-antibody detection of FMNL1 K-mal modification after cell lysis; D shows whole-cell lysate of 786-O parental cells, using a combination of FMNL1 antibody and Protein A / G with magnetic beads to enrich endogenous FMNL1 protein, with homologous IgG as a negative control, and K-mal pan-antibody detection of FMNL1 K-mal modification; E shows whole-cell lysate of Caki-1 parental cells, using a combination of FMNL1 antibody and Protein A / G with magnetic beads. A / G affinity enrichment of endogenous FMNL1 protein, homologous IgG as negative control, and K-mal pan-antibody detection of FMNL1 K-mal modification;

[0031] Figure 4 In Figure A, HEK 293T cells were transiently transfected with pLenti-puro, pLenti-FMNL1-3×Flag, and pLenti-FMNL1 K852R-3×Flag, and FMNL1 K-mal modification was detected by K-mal pan-antibody. In Figure B, 786-O cell lines stably expressing EV, FMNL1-3×Flag, and FMNL1 K852R-3×Flag were lysed, and FMNL1 K-mal modification was detected by K-mal pan-antibody. In Figure C, Caki-1 cell lines stably expressing EV, FMNL1-3×Flag, and FMNL1 K852R-3×Flag were lysed, and FMNL1 K-mal modification was detected by K-mal pan-antibody.

[0032] Figure 5A represents the impact of FMNL1 expression levels on patient survival analyzed using the TISDB online database; B represents the protein expression levels of FMNL1 in ccRCC clinical samples and adjacent tissues analyzed using the CPTAC database; C represents the abundance of K-mal modification of FMNL1 in ccRCC cancer and adjacent tissues analyzed using the K-mal modification group; D represents the relative analysis of FMNL1 K-mal modification levels and FMNL1 protein expression levels in the CPTAC database.

[0033] Figure 6 In Figure A, a stable 786-O cell line expressing EV, FMNL1 wild type-3×Flag, FMNL1 K852R-3×Flag, and FMNL1 K852E-3×Flag was constructed using lentiviral packaging, and the expression level of FMNL1-Flag was detected by Western blotting. In Figure B, the migration and invasion abilities of the 786-O stable cell line were detected by Transwell migration / invasion assays with and without matrix gel coating. In Figure C, a stable Caki-1 cell line expressing EV, FMNL1 wild type-3×Flag, FMNL1 K852R-3×Flag, and FMNL1 K852E-3×Flag was constructed using lentiviral packaging, and the expression level of FMNL1-Flag was detected by Western blotting. In Figure D, the migration and invasion abilities of the Caki-1 stable cell line were detected by Transwell migration / invasion assays with and without matrix gel coating.

[0034] Figure 7 A shows the sequence analysis of human and mouse FMNL1 from the CLUSTAL Omega website, with conserved K-mal modification sites marked in red. The mouse K847 site corresponds to the human K852 site. B shows the mouse Rencaluci cell line stably expressing EV, mFMNL1 wild type-3×Flag, mFMNL1 K847R-3×Flag, and mFMNL1 K847E-3×Flag, constructed using lentiviral packaging, and the expression level of mFMNL1-Flag was detected by Western blotting. C shows the mFMNL1 expression level detected by K-mal pan-antibody in the Rencaluci cell line stably expressing EV, mFMNL1-3×Flag, and mFMNL1 K847R-3×Flag after cell lysis. K-mal modification; D is a map of lung metastases in the tail vein lung metastasis model; E is the luminescence signal of the luciferase substrate in lung metastasis detected by in vivo imaging on day 21 of the nude mouse tail vein lung metastasis model, with signal intensity representing lung tumor burden; F is the statistical analysis of the peak value of the luciferase substrate luminescence signal using IndiGo software.

[0035] Figure 8Image A shows the analysis of FMNL1-Flag dimer levels in the 786-O stable cell line using glutaraldehyde cross-linking combined with Western blotting; Image B shows the quantification of FMNL1-Flag dimer levels using image grayscale analysis; Image C shows the analysis of FMNL1-Flag dimer levels in the Caki-1 stable cell line using glutaraldehyde cross-linking combined with Western blotting; Image D shows the quantification of FMNL1-Flag dimer levels using image grayscale analysis; Image E shows the analysis of native FMNL1-Flag dimer levels using native PAGE; Image F shows the quantification of FMNL1-Flag dimer levels using image grayscale analysis; Image G shows the detection of FMNL1 K-mal modification using K-mal pan-antibody after treating stable FMNL1-overexpressing wild-type 786-O cells with 0 μM, 5 μM, and 10 μM fatty acid synthase inhibitor Orlistat concentration gradients for 24 h; Image H shows the detection of FMNL1 K-mal modification using 0 μM, 5 μM, and 10 μM K-mal pan-antibody. After treating FMNL1 wild-type 786-O cells with Orlistat concentration gradients for 24 h, the dimer level of FMNL1-Flag was analyzed by glutaraldehyde cross-linking combined with Western blotting. In step I, whole-cell lysates of stably overexpressing FMNL1 wild-type cells were co-incubated with 0 μM, 20 μM, 50 μM, and 100 μM Mal-CoA at 37℃ for 1 h, and the FMNL1-Flag dimer level was detected. The remaining samples were used to detect FMNL1 K-mal levels. In step J, FMNL1 wild-type 786-O cells were treated with 0 μM, 5 μM, 10 μM, and 20 μM Mal-CoA concentration gradients for 24 h, and the dimer level of FMNL1-Flag was analyzed by glutaraldehyde cross-linking combined with Western blotting.

[0036] Figure 9Image A shows the separation of G-actin and F-actin components in the 786-O stable cell line using the G-actin / F-actin In Vivo Assay Biochem Kit, combined with Western blotting analysis to determine the levels of G-actin and F-actin. Image B shows the quantification of the F-actin / G-actin ratio using image grayscale analysis. Image C shows the separation of G-actin and F-actin components in the Caki-1 stable cell line using the G-actin / F-actin In Vivo Assay Biochem Kit, combined with Western blotting analysis to determine the levels of G-actin and F-actin. Image D shows the quantification of the F-actin / G-actin ratio using image grayscale analysis. Image E shows the FMNL1 K-mal level detected by K-mal pan-antibody after transient transfection of HEK 293T cells with pcDNA3.1-FMNL1-3×Flag for 24 h, followed by treatment with SM or Orlistat for 24 h. Image F shows the transient transfection of HEK 293T cells with Empty. After transfection with vector and pcDNA3.1-FMNL1-3×Flag for 24 h, the cells were treated with 10 μM Orlistat for 24 h. The levels of G-actin and F-actin in the cells were analyzed by immunoblotting.

[0037] Figure 10 In Figure A, HeLa cells were transiently transfected with pLenti-puro, pLenti-FMNL1-3×Flag, pLenti-FMNL1K852R-3×Flag, and pLenti-FMNL1 K852E-3×Flag for 48 hours. Cells were then fixed and subjected to immunofluorescence staining. FMNL1-Flag was labeled with mouse Flag antibody (red), F-actin was labeled with actin tracker Green dye (green), and cell nuclei were labeled with DAPI dye (blue). Figure B shows immunofluorescence experiments using the 786-O stable cell line. FMNL1-Flag was labeled with mouse Flag antibody (red), F-actin was labeled with actin tracker Green dye (green), and cell nuclei were labeled with DAPI dye (blue). Figure C shows the quantitative analysis of the F-actin green fluorescence signal intensity in representative images from Image J. Figure D shows representative fluorescence images with and without F-actin rearrangement, and the percentage of cells with F-actin rearrangement in the acquired field of view was statistically analyzed.

[0038] Figure 11Image A shows the cell invasion ability of cells treated with SMIFH2, an F-actin polymerization inhibitor targeting the Formin FH2 domain, for 24 hours; Image B shows the quantitative statistical analysis of invasive cells in Image J; Image C shows the cell invasion ability of cells treated with LAT-B, an F-actin polymerization inhibitor, for 24 hours; Image D shows the quantitative statistical analysis of invasive cells in Image J.

[0039] Figure 12 Image A shows the knockdown of endogenous FMNL1 protein in 786-O cells using the pLKO.1 system, with Western blotting used to detect the knockdown effects of shRNA#1 and shRNA#2 on FMNL1 protein and the expression level of Cortactin. Image B shows the expression level of Cortactin in stable 786-O cell lines analyzed by Western blotting. Image C shows the knockdown of endogenous FMNL1 protein in Caki-1 cells using the pLKO.1 system, with Western blotting used to detect the knockdown effects of shRNA#1 and shRNA#2 on FMNL1 protein and the expression level of Cortactin. Image D shows the expression level of Cortactin in stable Caki-1 cell lines analyzed by Western blotting. Image E shows the re-exogenous overexpression of empty vector, FMNL1 wild type-3×Flag, and FMNL1 in 786-O cells with endogenous FMNL1 knockdown. K852R-3×Flag and FMNL1K852E-3×Flag were used to detect the expression level of Cortactin by immunoblotting. F shows the immunofluorescence experiment performed using the 786-O cell line stably expressing wild-type FMNL1. The mouse Flag antibody labeled FMNL1-Flag (red), the actin tracker Green dye labeled F-actin (green), the rabbit Cortactin antibody labeled Cortactin protein (far-infrared), and the cell nucleus labeled with DAPI dye (blue). G shows the immunofluorescence experiment performed using the 786-O cell line stably expressing wild-type FMNL1. The mouse Flag antibody labeled FMNL1-Flag (red), the rabbit Fascin antibody labeled Fascin protein (green), and the cell nucleus labeled with DAPI dye (blue). H shows the co-localization signal of FMNL1-Flag, F-actin, and Cortactin analyzed by Image J. I shows the co-localization signal of FMNL1-Flag and Fascin analyzed by Image J.

[0040] Figure 13A shows the immunoprecipitation technique used to analyze the interaction between FMNL1-Flag and Fascin in the 786-O stable cell line; B shows the immunoprecipitation technique used to analyze the interaction between FMNL1-Flag and Cortactin in the 786-O stable cell line; C shows the adjacent junction assay (PLA) used to analyze the interaction between Flag-Fascin and Flag-Cortactin in the 786-O stable cell line, with red dotted fluorescent signals indicating positive interactions. Cell nuclei were labeled with DAPI dye, and fluorescence images were acquired using a Zeiss LSM 880 confocal microscope; D shows the statistical analysis of the number of dotted signals in single cells using Image J.

[0041] Figure 14 In Figure A, immunofluorescence analysis shows the co-localization of FMNL1-Flag and Fascin in the 786-O stable cell line. FMNL1-Flag is labeled with a mouse-derived Flag antibody (red), Fascin protein is labeled with a rabbit-derived Fascin antibody (green), and the cell nucleus is labeled with DAPI dye (blue). Figure B shows the percentage of Fascin-recruiting positive cells. Figure C shows the co-localization of FMNL1-Flag and Cortactin in the 786-O stable cell line, with white arrows indicating Cortactin aggregation at pseudopodia. Figure D shows the percentage of Cortactin-recruiting positive cells.

[0042] Figure 15 In Figure A, the expression levels of MMP2 and MMP9 in the stable 786-O cell line were detected by Western blotting; in Figure B, the expression level of MMP2 was quantified by Image J grayscale analysis; in Figure C, the expression level of MMP9 was quantified by Image J grayscale analysis; in Figure D, the cell invasion ability was detected after treating 786-O cells with the MMPs inhibitor GM6001 for 24 h; and in Figure E, the number of invasive cells was quantified by Image J and statistically analyzed.

[0043] Figure 16A shows the adjoint linkage assay (PLA) analysis of Flag-SIRT5 interaction in the stable 786-O cell line. Red dotted fluorescent signals indicate positive interaction signals. DAPI dye was used to label cell nuclei, and ImageJ was used to quantify and statistically analyze the number of dotted signals per cell. B shows the detection of exogenous FMNL1-SIRT5 interaction in the 786-O cell line stably expressing EV and FMNL1-Flag. C shows the detection of endogenous FMNL1-SIRT5 interaction in the whole-cell lysate of 786O parental cells. D shows the detection of endogenous FMNL1-SIRT5 interaction in the whole-cell lysate of Caki-1 parental cells. E shows the detection of FMNL1 K-mal levels using K-mal pan-antibody after co-transfection of HEK 293T cells with pLenti-puro, pLenti-FMNL1-3×Flag, and pLV3-CMV-puro, pLV3-SIRT5-3×Flag. F shows the 786-O cell line... In FMNL1 wild-type stable cells, after transient transfection of emptyvector or pLV3-SIRT5-3×Flag using the Lipo8000 transfection system, the FMNL1 K-mal level was detected by K-mal pan-antibody; in G-type 786-O parental cells, After transient transfection of the SIRT5-targeted knockdown siRNA mix, the FMNL1 K-mal level was detected by K-mal pan-antibody.

[0044] Figure 17 In Figure A, the FMNL1 K-mal level was detected by K-mal pan-antibody after treating 786-O stable cell lines expressing EV and FMNL1-Flag with CoCl2 for 24 h; in Figure B, the FMNL1 K-mal level was detected by K-mal pan-antibody after culturing 786-O stable cell lines expressing EV and FMNL1-Flag in a hypoxic incubator (1% O2) and a normoxic incubator (20% O2) for 9 h or 12 h; and in Figure C, the FMNL1 K-mal level was detected by K-mal pan-antibody after culturing Caki-1 stable cell lines expressing EV and FMNL1-Flag in a hypoxic incubator (1% O2) and a normoxic incubator (20% O2) for 9 h or 12 h.

[0045] Figure 18In Figure A, the stable 786-O cell line expressing FMNL1-Flag was treated with CoCl2. The FMNL1 K-mal level was detected by K-mal pan-antibody, and the interaction level of exogenous FMNL1-SIRT5 was detected by SIRT5 antibody. In Figure B, the parental 786-O cells were cultured in a hypoxic incubator (1% O2) and a normoxic incubator (20% O2) for 8 h or 12 h, respectively. The FMNL1 K-mal level was detected by K-mal pan-antibody, and the interaction level of endogenous FMNL1-SIRT5 was detected by SIRT5 antibody. In Figure C, the parental Caki-1 cells were cultured in a hypoxic incubator (1% O2) and a normoxic incubator (20% O2) for 8 h or 12 h, respectively. The FMNL1 K-mal level was detected by K-mal pan-antibody, and the interaction level of endogenous FMNL1-SIRT5 was detected by SIRT5 antibody.

[0046] Figure 19 In case A, parental 786-O cells were cultured in a hypoxic incubator (1% O2) for 0 h, 2 h, and 4 h, respectively. The level of FMNL1 endogenous dimer was analyzed by glutaraldehyde cross-linking assay combined with Western blotting. In case B, 786-O cells stably expressing EV and wild-type FMNL1 were cultured in a hypoxic incubator (1% O2) for 0 h, 4 h, and 12 h, respectively. The level of FMNL1 K-mal was detected by immunoprecipitation, and the level of FMNL1 exogenous dimer was analyzed by glutaraldehyde cross-linking assay. In case C, 786-O cells stably expressing wild-type FMNL1 were pretreated with the HIF1α inhibitor PX for 1 h, followed by continuous treatment with CoCl2 for 24 h. The level of FMNL1 K-mal was detected by K-mal pan-antibody, and the level of exogenous FMNL1-Fascin interaction was detected by Fascin antibody.

[0047] Figure 20In section A, 786-O cell lines with stable FMNL1 knockout were constructed using lentiviral packaging after endogenous knockout of FMNL1 using the pLKO.1 system, and the knockout effect was detected by Western blotting. Section B showed the effect of hypoxia on cell invasion by culturing the stable FMNL1 knockout cell lines from section A in a hypoxic incubator (1% O2) and a normoxic incubator (20% O2) for 12 hours. Section C used ImageJ to quantify the number of invasive cells. Section D showed the re-expression of exogenous empty vector, FMNL1 wild type, FMNL1 K852R, and FMNL1K852E in cells with endogenous FMNL1 knockout using shRNA#1 and shRNA#2, and the expression level of exogenously expressed FMNL1 was detected by Western blotting. Section E showed the stable 786-O cell lines from section D, with endogenous FMNL1 knockout and exogenous replenishment in the shRNA#1 group, incubated in a hypoxic incubator (1% O2) and a normoxic incubator (20% O2) for 12 hours. The cells were cultured in a hypoxic incubator (1% O2) for 12 h, and the effect of hypoxia on cell invasion ability was analyzed by Transwell invasion assay. F represents the 786-O stable cell line of group #2 with FMNL1 endogenous knockdown and exogenous replenishment in D placed in a hypoxic incubator (1% O2) and a normoxic incubator (20% O2) for 12 h, respectively. The effect of hypoxia on cell invasion ability was analyzed by Transwell invasion assay. G represents the number of invasive cells in E by Image J quantitative analysis, and H represents the number of invasive cells in F by Image J quantitative analysis. Detailed Implementation

[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0049] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0050] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0051] The human renal cell carcinoma lines 786-O, Caki-1, HEK293T, and HeLa used in this experiment were all purchased from the cell bank of the Shanghai Branch of the Chinese Academy of Sciences.

[0052] All laboratory animals used in this experiment were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and subsequently housed at the Laboratory Animal Center of Xuzhou Medical University. The animals involved in this experiment were 6-8 week old female BALB / c nude mice, weighing 20-25g, and SPF grade. The animals were strictly housed and the subsequent experimental procedures were carried out in accordance with the animal center's regulations.

[0053] Example

[0054] I. The levels of FMNL1 K-mal in cancerous tissue of ccRCC clinical samples were significantly lower than those in adjacent normal tissue:

[0055] 1. The overall K-mal level of proteins in ccRCC was significantly lower than that in adjacent normal tissue:

[0056] Numerous studies have shown that lipid metabolism reprogramming drives the occurrence and development of ccRCC, accompanied by the abnormal accumulation or consumption of acyl-CoA. Previous studies have found that malonyl-CoA levels in ccRCC are significantly lower than in adjacent normal tissues. As a precursor to novel lysine malonylation modification (K-mal), malonyl-CoA suggests that differential malonylation modification levels in ccRCC may closely regulate its malignant progression. Therefore, further K-mal modification proteomics analysis was performed on three clinical ccRCC samples and adjacent normal tissues, revealing a significant downregulation of overall K-mal levels in ccRCC proteins, consistent with the previous lipidomics findings showing a decreasing trend in malonyl-CoA content. Figure 1 The results suggest that K-mal modification may play an important role in ccRCC's perception of lipid metabolism abnormalities and its malignant progression.

[0057] 2. FMNL1 K-mal levels were significantly downregulated in ccRCC:

[0058] To further clarify which pathways are mainly regulated by K-mal modification in ccRCC, GO cluster analysis was performed on differentially modified proteins in cancer and adjacent normal tissues in the ccRCC K-mal modification group. This revealed a significant enrichment of actin-binding proteins (CMBs). Figure 2 Volcano plot analysis revealed that, compared to adjacent normal tissue, the K-mal modification level of FMNL1, a member of the Formins family, was most significantly downregulated in ccRCC cancer tissue. Figure 2 (B) suggests that FMNL1 K-mal modification may play a key regulatory role in the progression of ccRCC.

[0059] In summary, our previous laboratory research found that the Mal-CoA content and K-mal modification level in ccRCC were significantly lower than those in adjacent normal tissues, while the K-mal level of the cytoskeletal protein FMNL1 was most significantly downregulated in cancerous tissues. This suggests that FMNL1 may be a key K-mal modification target that regulates the malignant progression of renal cell carcinoma. Subsequent experiments will focus on the K-mal modification target FMNL1 system.

[0060] II. K-mal modification occurs at the FMNL1 K852 site:

[0061] 1. FMNL1 is modified by K-mal:

[0062] Based on the K-mal modification proteomics analysis results of ccRCC samples, subsequent research focused on FMNL1, the cytoskeletal protein with the most significant downregulation of K-mal levels in ccRCC. First, in vitro verification was conducted to confirm the presence of K-mal modification. A eukaryotic expression plasmid FMNL1 fused with a Flag tag was constructed. This plasmid was then transiently transfected into HEK 293T cells or used to construct stable 786-O and Caki-1 cell lines overexpressing exogenous FMNL1. Immunoprecipitation was used to enrich FMNL1 protein with an affinity antibody conjugated to agarose beads. K-mal modification was detected using a pan-K-mal antibody. Figure 3 (AC), the results showed that FMNL1 was modified by K-mal, and the FMNL1 antibody endogenous IP experiment ( Figure 3 The results from DE further confirmed the presence of K-mal modification in FMNL1.

[0063] 2. FMNL1 K-mal modification occurs at site K852 of its FH2 functional domain:

[0064] After confirming the presence of K-mal modification in FMNL1, the next step was to identify the main sites of K-mal modification. Mass spectrometry results showed that the K-mal modification in FMNL1 occurs at the K852 site of its main functional domain, FH2. Furthermore, NCBI multi-species sequence alignment analysis indicated that this site is highly conserved across different species, suggesting the importance of K-mal modification at this site. Using point mutagenesis, the lysine (K) at the K852 site was mutated to arginine (R), which has a similar amino acid structure and charge but cannot undergo K-mal modification. IP experiments (…) Figure 4 By comparing the changes in K-mal modification levels of wild-type FMNL1 and FMNL1 K852R, it was found that the K-mal modification level of FMNL1 was significantly reduced after the K852R mutation, indicating that the K852 site is its main K-mal modification site.

[0065] III. FMNL1 K-mal modification significantly inhibits the migration, invasion, and metastasis of renal cell carcinoma:

[0066] 1. FMNL1 maintains a low K-mal modification state in ccRCC to exert its oncogenic function:

[0067] To preliminarily clarify the potential function of FMNL1 in the development and progression of ccRCC, bioinformatics analysis was conducted using the online databases CPTAC and TISDB to examine the expression level of FMNL1 protein in ccRCC and adjacent normal tissues and its relationship with patient prognosis. The results showed that FMNL1 was significantly highly expressed in ccRCC, and high FMNL1 expression was negatively correlated with patient prognosis, suggesting that it primarily functions as an oncogene. Figure 5 (AB). K-mal modification proteomics analysis of FMNL1 K-mal modification levels relative to CPCAC protein levels showed that FMNL1 was in a very low K-mal modification state in ccRCC ( Figure 5 The results (CD) suggest that FMNL1 in ccRCC maintains a low level of K-mal modification and plays a role in promoting cancer.

[0068] 2. FMNL1 K-mal modification significantly inhibited the migration and invasion of 786-O and Caki-1:

[0069] To investigate the effects of FMNL1 K-mal modification on renal cell carcinoma migration and invasion, stable cell lines of 786-O and Caki-1 expressing EV, FMNL1 WT-3×Flag, FMNL1 K852 R-3×Flag, and FMNL1 K852 E-3×Flag were constructed using a lentiviral system. Western blotting was used to verify the expression levels of exogenous Flag in cell lines 786-O and Caki-1. Figure 6 (A, C). Transwell assays were performed in chambers with and without matrix gel coating to detect the differences in migration and invasion rates of renal cell carcinoma lines 786-O and Caki-1. The results showed that overexpression of wild-type FMNL1 slightly promoted the migration and invasion of both 786-O and Caki-1 cells compared to uncoated cells. Using FMNL1 wild-type cells as a control, the effects of K-mal modification on 786-O (… Figure 6 (B) and Caki-1 ( Figure 6The study investigated the effects of FMNL1 K852R (simulating demalonylation modification) on renal cell carcinoma cells. FMNL1 K852E (simulating K-mal modification) significantly promoted the migration and invasion of renal cell carcinoma cells, while FMNL1 K852E (simulating K-mal modification) significantly inhibited their migration and invasion. This aligns with hypotheses based on K-mal modification proteomics analysis of clinical renal cell carcinoma samples. In summary, in vitro analysis validated that FMNL1 K-mal modification significantly inhibits the migration and invasion of renal cell carcinoma cells 786-O and Caki-1.

[0070] 3. In vivo validation of FMNL1 K-mal significantly inhibiting renal cell carcinoma metastasis:

[0071] To further clarify the impact of FMNL1 K-mal modification on renal cell carcinoma metastasis, a murine FMNL1 mutant plasmid was constructed. Sequence alignment confirmed that the murine FMNL1 K847 site corresponds to the human FMNL1 K852 site. Figure 7 (A); Furthermore, a murine renal cell carcinoma Renca cell line was constructed, and immunoprecipitation technology was used to verify that K-mal modification was significantly inhibited after the KR mutation at the K847 site. Figure 7 (Central BC). Next, at a rate of 600,000 cells per nude mouse, 8-9 mice were slowly injected into the nude mouse tail vein to construct a nude mouse tail vein lung metastasis model. In vivo imaging was performed weekly to observe lung metastasis. At the experimental endpoint on day 21, after recording the in vivo imaging data, the mice were euthanized with CO2, and their lungs were harvested, photographed, and fixed. The results were combined with in vivo imaging and analysis of ex vivo lung metastases. Figure 7 In a study conducted in the FMNL1 K847E group (in DF), it was found that compared with the wild type, the lung tumor burden was significantly increased in the FMNL1WT group, while the lung tumor burden was relatively low in the FMNL1 K847E group. The difference was statistically significant, indicating that FMNL1 K-mal modification significantly inhibited the metastasis of renal cell carcinoma.

[0072] IV. FMNL1 K-mal modification significantly inhibits the formation of FH2 homodimers:

[0073] FMNL1 is a member of the microfilament nucleosynthetic protein family. This family has two characteristic formin homology (FH) domains, FH1 and FH2, with FH2 being the most important and conserved domain responsible for F-actin polymerization. Intracellularly, two FMNL1 monomers interact through the FH2 domain to form a stable dimer, mediating G-actin nucleation and F-actin polymerization and elongation, regulating physiological processes such as cell adhesion and motility, and pseudopodia formation. Literature reports that phosphorylation of the FH2 domain of other forminimin members, such as DAAM1, affects its dimer formation, thereby regulating its biological functions. To investigate whether FMNL1 K-mal modification affects homodimer formation, a glutaraldehyde cross-linking method was used (…). Figure 8 (AD) and Native-PAGE experiments ( Figure 8 The dimer levels of FMNL1 wild-type, K852R mutant, and K852E mutant were detected by EF. The results showed that compared with FMNL1 wild-type, K852R significantly promoted FMNL1 dimerization, while K852E inhibited FMNL1 dimer formation.

[0074] Since K-mal modification depends on the level of its substrate Mal-CoA, to eliminate the interference of KE mutation charge mimicking other acylation modifications (such as succinylation), the effects of K-mal modification on FMNL1 biological function were further confirmed using the fatty acid synthase inhibitor Orlistat and the Mal-CoA drug induction system. On the one hand, Orlistat concentration gradient treatment was used to block fatty acid synthesis, thereby achieving intracellular Mal-CoA accumulation and inducing an increase in FMNL1 K-mal. Figure 8 On the one hand, GH was added, and on the other hand, whole-cell lysis buffer was co-incubated with gradient concentrations of Mal-CoA at 37°C for 1 hour. Figure 8 Cells were treated with a direct concentration gradient of either I or Mal-CoA. Figure 8 The levels of FMNL1-K-mal modification induced by Orlistat or Mal-CoA increased, and the FMNL1 dimer level was detected by glutaraldehyde cross-linking assay. The results showed that as the FMNL1 K-mal level was upregulated by Orlistat or Mal-CoA gradient, the FMNL1 dimer level was downregulated by a gradient. These results indicate that FMNL1 K-mal modification significantly inhibits the formation of its homodimer, potentially interfering with the biological function of F-actin.

[0075] V. FMNL1 K-mal modification significantly inhibits the polymerization and rearrangement of F-actin:

[0076] 1. FMNL1 K-mal inhibits the polymerization of F-actin:

[0077] Existing research indicates that Forminin family proteins mediate F-actin polymerization and elongation through FH1 and FH2 domains. Previous results clearly show that K-mal modification of FMNL1 significantly inhibits the formation of its own dimer. The next step is to investigate whether K-mal modification affects its F-actin polymerization function. Following the instructions of the G-actin / F-actin separation kit, G-actin and F-actin were separated by ultracentrifugation at 100,000g. The separation results of G-actin / F-actin components in stable 786-O and Caki-1 cell lines showed that FMNL1-Flag was mainly present in the F-actin component, consistent with its status as an Actin-binding protein, indicating that the separation results are highly reliable. Compared to wild-type FMNL1 cells, F-actin was significantly reduced in FMNL1K852E cells, while no significant increase was observed in K852R cells. Figure 9 The reason for the low K-mal modification level of FMNL1 in renal cell carcinoma cells (AD) is that the FMNL1 wild-type cells are already in a low K-mal modification state, and the intracellular FMNL1 dimer is sufficient to meet the biological process of F-actin polymerization. Meanwhile, in HEK 293T cells, exogenous overexpression of wild-type FMNL1 and Orlistat-induced increase in FMNL1 K-mal modification levels, along with analysis of F-actin polymerization function, revealed that exogenous overexpression of wild-type FMNL1 significantly promoted F-actin polymerization, and after Orlistat treatment upregulated FMNL1 K-mal modification levels, F-actin levels were significantly reduced. Figure 9 (EF). In summary, FMNL1 K-mal modification significantly inhibits its F-actin polymerization function.

[0078] 2. FMNL1 K-mal inhibits F-actin rearrangement:

[0079] Cellular deformation, movement, and invasion are usually accompanied by cytoskeleton rearrangement and the formation of lamellar or filopodia. To investigate whether FMNL1 K-mal modification interferes with F-actin rearrangement, HeLa cells were transiently transfected, and changes in F-actin were analyzed by immunofluorescence. The results showed that FMNL1 wild-type and K852R mutant cells were more prone to forming filopodia-like structures from bundles of F-actin at the cell's leading edge, while this structure was almost absent in EV and K852E cells. Figure 10(A). Further results from the 786-O stable cell line showed that, in addition to the relative decrease in F-actin fluorescence signal in K852E cells, consistent with previous G-actin / F-actin separation results, it was also found that in FMNL1 wild-type and FMNL1 K852R cells overexpressing F-actin tended to accumulate on one side of the cell membrane, forming a plate-like pseudopodia-like structure. Figure 10 In BC cells, F-actin was uniformly distributed throughout the cell in EV and K852E cells. Statistical analysis of F-actin rearrangement-positive 786-O cells revealed that, compared to wild-type cells, K852E cells significantly inhibited F-actin rearrangement. Figure 10 (D). In summary, the results show that FMNL1 K-mal modification significantly inhibits the rearrangement of F-actin, suggesting that FMNL1 demalonylation is more conducive to stabilizing F-actin and promoting its reconstruction to form a pseudopodia-like structure.

[0080] 3. FMNL1 demalonylation promotes cell invasion in response to its F-actin polymerization function:

[0081] The above results showed that FMNL1 malonylation significantly inhibited F-actin polymerization and rearrangement. To investigate whether the effect of FMNL1 malonylation on renal cell carcinoma migration and invasion depends on its F-actin polymerization and rearrangement function, cells were treated with the widely used F-actin polymerization inhibitor LAT-B and the small molecule inhibitor SMIFH2 targeting the FH2 domain of Formins family proteins. Transwell results showed that, compared with the untreated group, the differences in cell invasion phenotype between the LAT-B or SMIFH2 treatment groups disappeared. Figure 11 The results (AD) indicate that the demalonylation modification of FMNL1 promotes the migration and invasion of renal cell carcinoma cells, which depends on the F-actin polymerization function of its FH2 domain.

[0082] VI. FMNL1 K-mal modification significantly inhibits the formation of invasive pseudopodia:

[0083] 1. FMNL1 K-mal modification may be involved in regulating the formation of aggressive pseudopodia in renal cell carcinoma:

[0084] Cortactin is a key upstream regulator of invasive pseudopodia. To investigate whether FMNL1 and its K-mal modification are involved in the formation of invasive pseudopodia, we first knocked down endogenous FMNL1 in 786-O and Caki-1 using the pLKO.1 plasmid system. We found that Cortactin protein levels were significantly reduced, and Cortactin expression was increased in cells overexpressing wild-type FMNL1 and K852R, with a more pronounced trend in K852R. Conversely, overexpression of K852E resulted in decreased Cortactin protein levels. Figure 12 The above trend was further validated in cell lines with endogenous FMNL1 knockdown followed by exogenous replenishment of EV, FMNL1 WT, FMNL1, K852R, and FMNL1 K852E. Figure 12 (E). Since invasive pseudopodia are mainly composed of branched F-actin and bundled F-actin, and cytoskeletal regulatory factors Cortactin and Fascin mediate the formation and stability of branched and bundled F-actin, respectively, Cortactin and Fascin were selected as indicators for detecting invasive pseudopodia. Immunofluorescence analysis showed that FMNL1 co-localized with Fascin and Cortactin. It formed punctate co-localization signals with Fascin at the pseudopodia site, while the co-localization signals with Cortactin and F-actin were mainly located at the pseudopodia protrusions on one side of the cell membrane. Figure 12 (FI), in summary, this suggests that FMNL1 is involved in regulating the formation of invasive pseudopodia.

[0085] 2. FMNL1 K-mal modification significantly inhibits its interaction with Cortactin and Fascin:

[0086] To determine whether FMNL1 interacts with Fascin and Cortactin, IP experiments were conducted. Figure 13 Experiments A and B) and PLA ( Figure 13 C and D) demonstrate that FMNL1 interacts with both Fascin and Cortactin, and this interaction is significantly weakened in K852E mutant cells. This indicates that FMNL1 K-mal significantly inhibits its interaction with the invasive pseudopodia regulators Fascin and Cortactin.

[0087] 3. FMNL1 K-mal modification significantly inhibited the recruitment of cortactin and fatsin at invasive pseudopodia:

[0088] The above results clearly indicate that FMNL1 K-mal inhibits its interaction with Fascin and Cortactin. Immunofluorescence analysis was then performed to determine whether K-mal modification affected the accumulation of Fascin and Cortactin at sites of invasive pseudopodia. Firstly, in the 786-O cell line co-stained with FMNL1-Flag and Fascin, punctate colocalization signals with Fascin were evident in overexpression of wild-type FMNL1 and the K852R mutant, while in the K852E mutant, the punctate colocalization signal was significantly reduced. Figure 14 (A, B). Secondly, in the 786-O cell line co-stained with FMNL1-Flag and Cortactin, compared with wild-type cells overexpressing FMNL1, the colocalization signal at the leading edge protrusion of the cell membrane of the K852R mutant was stronger, while that of the K852E mutant was significantly weakened. Figure 14 (C, D). The results in summary indicate that FMNL1 malonylation inhibits its co-localization with Cortactin and Fascin, that is, it inhibits the recruitment of Fascin and Cortactin at pseudopodia.

[0089] 4. FMNL1 K-mal modification significantly inhibits MMP expression:

[0090] Since the invasive pseudopodia-mediated degradation of the extracellular matrix by cancer cells requires the secretion of matrix metalloproteinases (MMPs), the expression levels of MMP2 and MMP9 were further examined. The results showed that overexpression of FMNL1 wild-type and the K852R mutant both increased the protein levels of MMP2 and MMP9 to some extent, while no upregulation of MMP2 and MMP9 was observed in the K852E mutant. Figure 15 (A, B). Simultaneously, treatment of the 786-O stable cell line with the MMPs inhibitor GM6001 revealed that, compared to the untreated group, the promoting effect of FMNL1 demalonylation on renal cell carcinoma migration and invasion disappeared. Figure 15 (C and D) indicate that its effect on renal cell carcinoma cell migration and invasion depends on the formation of invasive pseudopodia. In summary, these results suggest that FMNL1 K-mal modification participates in regulating the formation of invasive pseudopodia in renal cell carcinoma. Its demalonylation modification facilitates its co-localization and interaction with Fascin and Cortactin, recruiting Fascin and Cortactin to the pseudopodia site, promoting F-actin rearrangement, maintaining F-actin stability, and jointly mediating the formation of invasive pseudopodia, ultimately promoting the migration, invasion, and metastasis of renal cell carcinoma.

[0091] 7. SIRT5 is a demalonylation modifying enzyme of FMNL1:

[0092] Because previous clinical samples of renal cell carcinoma showed that the K-mal modification level of FMNL1 was significantly lower than that of adjacent normal tissue, and that the demalonylation state was crucial for its pro-cancer function, this study will explore the upstream factors regulating FMNL1 malonylation. Existing research has identified SIRT5 as the main demalonylation enzyme, followed by SIRT311, but its modifying enzymes have not been reported, and some studies suggest that malonylation is a non-enzyme-dependent process. Considering that SIRT5, as a demalonylation enzyme, may participate in regulating the K-mal level of FMNL1, since enzymes usually need to interact with proteins to function, we will first explore the upstream factors regulating FMNL1 malonylation through PLA (…). Figure 16 IP experiments (both internal and external sources) Figure 16 (BD) studies have confirmed the interaction between FMNL1 and SIRT5, suggesting that SIRT5 may be a demalonylation modifier of FMNL1. Next, HEK293T cells were transiently co-transfected with FMNL1 WT-3×Flag and SIRT5-3×Flag, and changes in FMNL1 K-mal levels were detected. It was found that, on the basis of FMNL1 overexpression, SIRT5 overexpression significantly reduced the K-mal modification level of FMNL1. Figure 16 (E). Secondly, transient overexpression of SIRT5 in the 786-O stable cell line overexpressing wild-type FMNL1 yielded similar results. Figure 16 Finally, endogenous SIRT5 was knocked down using siRNA, and changes in FMNL1 K-mal modification levels were detected. The results showed that after knocking down the demodifying enzyme SIRT5, FMNL1 K-mal modification levels significantly increased (F). Figure 16 (G). The results above indicate that SIRT5 mediates the demalonylation modification of FMNL1.

[0093] 8. Hypoxia-regulated FMNL1 K-mal modification level:

[0094] 1. Hypoxia-induced significant downregulation of FMNL1 K-mal modification levels:

[0095] Numerous studies have shown that VHL mutation is an important molecular pathological feature of clear cell renal cell carcinoma, accompanied by elevated HIF-1α levels, indicating that hypoxia plays a crucial role in the development and progression of clear cell renal cell carcinoma. Based on this, this study aimed to investigate whether hypoxia participates in regulating FMNL1 K-mal modification. Using a hypoxic incubator and CoCl2 to simulate a hypoxic cellular environment, changes in FMNL1 K-mal modification levels were detected by intracellular incubation (IP) assays. The results showed that CoCl2-induced hypoxia, as well as 786-O 12h 1% O2 hypoxia induction and Caki-1 9h 1% O2 hypoxia induction, significantly downregulated FMNL1 K-mal modification levels. Figure 17The results (AC) indicate that hypoxia induction can significantly downregulate the FMNL1 K-mal modification level in renal cell carcinoma cells.

[0096] 2. Hypoxia-induced enhancement of the interaction between FMNL1 and the demodified enzyme SIRT5:

[0097] Previous studies confirmed that SIRT5 is a demalonylated modifying enzyme of FMNL1, suggesting that hypoxia might affect the interaction between FMNL1 and SIRT5, further regulating FMNL1 K-mal levels. Therefore, further in vitro immunoassay (IP) experiments were conducted to investigate the effect of hypoxia on the interaction between FMNL1 and SIRT5. Intrinsic and exogenous IP results from both the 786-O and Caki-1 renal cell carcinoma lines showed that compared to cells cultured under normal oxygen (20%), hypoxia (1%) induction significantly increased the interaction between FMNL1 and SIRT5. Figure 18 (AC). In summary, hypoxia reduces the K-mal modification level of FMNL1 by upregulating the interaction between FMNL1 and the demodifying enzyme SIRT5.

[0098] 3. Hypoxia promotes the formation of FMNL1 homodimers and their interaction with Fascin:

[0099] The above results suggest that hypoxia is a positive regulator of FMNL1 demalonylation modification. Next, we will investigate whether its regulation of FMNL1 K-mal under hypoxia affects its biological function. First, since the formation of its own dimer is crucial to its downstream functions, we primarily examined whether hypoxia affected dimer formation. The results showed that after hypoxia induction, the levels of both endogenous and exogenous dimers significantly increased. Figure 19 (A and B) correspond to the trend of decreasing K-mal modification levels. Simultaneously, in the CoCl2 system, using the HIF1α inhibitor PX to inhibit hypoxia revealed that hypoxia-induced decrease in FMNL1 K-mal upregulates the interaction between FMNL1 and the invasive pseudopodia regulator Fascin, while PX treatment only partially reversed this trend. Figure 19 The result (C) indicates that the hypoxia-induced decrease in FMNL1 K-mal may not be dependent on HIF1α.

[0100] 4. Hypoxia promotes renal cell carcinoma invasion dependent on FMNL1 K852 site demalonylation modification:

[0101] Existing research shows that hypoxia is a key factor promoting cancer metastasis. This study first investigated whether hypoxia-promoted cell migration and invasion depended on FMNL1 using a shRNA knockdown system. Transwell invasion assays revealed that under normoxic culture, FMNL1 knockdown slightly inhibited the invasive ability of 786-O cells, consistent with previous phenotypic trends. Compared to normoxic culture, hypoxia-induced invasion of control PLKO.1 empty vector cells significantly increased. However, knockdown of endogenous FMNL1 did not significantly promote cell invasion, indicating that hypoxia-promoted 786-O invasion depends on FMNL1. Figure 20 (AC). Next, we investigated whether hypoxia-promoted cell invasion depended on changes in the K-mal modification level at the FMNL1 K852 site. Based on endogenous FMNL1 knockdown, exogenous complementation of EV, FMNL1 WT, FMNL1 K852R, and FMNL1 K852E cells was performed to construct two stable knockdown-complementation cell lines (AC). Figure 20 The results showed that hypoxia significantly promoted 786-O invasion only in cells replenished with FMNL1 WT, while in cells replenished with EV, FMNL1K852R, and FMNL1 K852E, hypoxia treatment had little or no effect on promoting invasion. Figure 20 (EH). The results in summary indicate that hypoxia promotes renal cell carcinoma migration and invasion in dependence on demalonylation modification at the FMNL1 K852 site. The data suggest that hypoxia induces demalonylation modification at the FMNL1 K852 site by upregulating the interaction between FMNL1 and the demodification enzyme SIRT5, thereby promoting the migration and invasion of renal cell carcinomas.

[0102] Therefore, this invention utilizes the above-mentioned FMNL1 malonylation modification in inhibiting ccRCC metastasis. In vivo and in vitro experiments have verified that the level of FMNL1 malonylation modification can inhibit the invasion and metastasis of ccRCC. Therefore, FMNL1 malonylation is a key target for regulating the invasion and metastasis of ccRCC. Based on this target, drugs that inhibit the metastasis of renal clear cell carcinoma cells can be developed.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of FMNL1 malonylation modification in inhibiting ccRCC transfer: promoting the level of FMNL1 malonylation modification and inhibiting the migration and invasion of ccRCC.

2. The application according to claim 1, characterized in that: The FMNL1 malonylation modification site is located at position 852 of the FH2 functional domain; promoting the malonylation modification level of lysine at position 852 of the FMNL1 FH2 functional domain inhibits the migration and invasion of ccRCC.

3. The application according to claim 1, characterized in that: It promotes the malonylation modification level of FMNL1, inhibits the formation of FH2 homodimer, and inhibits the polymerization and rearrangement of F-actin.

4. The application according to claim 1, characterized in that: It promotes the malonylation modification level of FMNL1, inhibits the interaction between FMNL1 and the invasive pseudopodia regulators Cortactin and Fascin, inhibits the recruitment of Cortactin and Fascin at the invasive pseudopodia site, and thus inhibits the formation of invasive pseudopodia in ccRCC, thereby inhibiting the migration and invasion of ccRCC.

5. The application according to claim 1, characterized in that: FMNL1 malonylation inhibits the expression of MMPs and suppresses the migration and invasion of ccRCC.

6. Application of FMNL1 852 site as a target in the preparation of drugs to inhibit the metastasis of renal clear cell carcinoma.

7. Application of FMNL1 852 site as a target in screening drugs to inhibit the metastasis of clear cell renal cells.

8. A product, characterized in that: The active ingredient of the product is used to promote the malonylation modification level of FMNL1, and the application of the product is one of the following: ① Inhibit the migration and invasion of ccRCC; ②Inhibit the formation of FH2 homodimers; ③ Inhibit the polymerization and rearrangement of F-actin; ④ Inhibit the interaction between FMNL1 and the invasive pseudopodia regulators Cortactin and Fascin; ⑤ Inhibit the recruitment of Cortactin and Fascin at invasive pseudopodia sites; ⑥ Inhibit the formation of invasive pseudopodia in ccRCC; ⑦ Inhibit the expression of MMPs.

9. An inhibitor, characterized in that: It inhibits the activity of demalonylation modifying enzymes, increases the level of FMNL1 malonylation modification, and inhibits the migration and invasion of ccRCC.

10. The inhibitor according to claim 9, characterized in that: The demalonylation modifying enzyme is SIRT5.

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