Predictive markers, kits and applications based on usp5 / hmgb1 signaling axis for colorectal cancer and drug screening methods

Abstract

The application belongs to the technical field of biological medicine, and discloses a colorectal cancer prediction marker, a kit and application based on a USP5 / HMGB1 signal axis and a drug screening method. The application provides application of USP5 as a target in preparation of a preparation for regulating degradation of HMGB1 protein. Through the target of USP5, the K48-linked ubiquitination-mediated degradation process of HMGB1 can be precisely intervened, so that the existing level of HMGB1 in colorectal cancer cells is regulated, and the application has an excellent application prospect in research and development of drugs for treating colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, and particularly relates to a predictive marker for colorectal cancer based on the USP5 / HMGB1 signal axis, a reagent kit, its application, and a drug screening method. Background Technology

[0002] Colorectal cancer (CRC) is a prevalent malignant tumor worldwide and one of the leading causes of cancer-related morbidity and mortality. While current clinical treatments for CRC, such as surgery, chemotherapy, targeted therapy, and immunotherapy, can control tumor progression to some extent, significant clinical challenges remain, including high rates of recurrence and metastasis, widespread treatment resistance, and poor overall patient prognosis. Therefore, in-depth exploration of the core molecular mechanisms underlying the occurrence, development, and malignant progression of CRC, screening and identifying novel targets for precision diagnosis and treatment of CRC, and developing new intervention strategies are of significant theoretical and clinical value in improving the clinical prognosis of CRC patients and overcoming existing treatment bottlenecks.

[0003] Current clinical studies have shown that HMGB1 is abnormally highly expressed in colorectal cancer tumor tissues, and its expression level is significantly negatively correlated with overall survival in colorectal cancer patients, making it an important prognostic factor. Furthermore, the subcellular localization of HMGB1 is closely related to tumor malignancy; cytoplasmic enrichment of HMGB1 corresponds to higher tumor grades and poorer prognosis, while nuclear HMGB1 is associated with better prognosis, exhibiting significant functional differences. Currently, the regulation of HMGB1 in colorectal cancer is mainly focused on the transcriptional level, with limited understanding of its protein stability and post-translational modification mechanisms. This significantly restricts the application and development of HMGB1 as a therapeutic target for colorectal cancer, presenting considerable limitations. Summary of the Invention

[0004] The inventors of this invention, aiming to elucidate the regulatory mechanisms of HMGB1 stability and post-translational modifications in colorectal cancer cells, have creatively discovered, through extensive and in-depth research and numerous experiments, that ubiquitin-specific protease 5 (USP5) is an important stabilizer of the HMGB1 protein. USP5 can protect HMGB1 from proteasome degradation by reducing K48-linked ubiquitination, thereby enhancing HMGB1 stability in colorectal cancer cells. Further research revealed that the USP5 / HMGB1 signaling axis is highly correlated with processes such as cell viability, division, cell cycle progression, migration and invasion, as well as thermal necrosis and autophagy. This axis has significant potential as a predictive marker for colorectal cancer, including assessing its malignancy and progression, and evaluating the efficacy of anti-colorectal cancer drug treatments. This discovery is of great significance for the application and development of HMGB1 as a therapeutic target for colorectal cancer.

[0005] The primary objective of this invention is to address the gap in existing research on the regulatory mechanisms of HMGB1 protein stability and post-translational modification. It provides the application of USP5 as a target in the preparation of formulations that regulate HMGB1 protein degradation. By targeting USP5, the degradation process of HMGB1 can be precisely intervened to regulate the level of HMGB1 in colorectal cancer cells, which has excellent application prospects in the development of drugs for the treatment of colorectal cancer.

[0006] Furthermore, the applications specifically include: preparing formulations that regulate USP5-mediated HMGB1 de-K48-linked ubiquitination; and / or preparing formulations that regulate the level of HMGB1 protein in colorectal cancer cells.

[0007] A second objective of this invention is to provide the application of a USP5 inhibitor in the preparation of a formulation that promotes the degradation of HMGB1 protein. Specifically, the USP5 inhibitor depletes USP5 in colorectal cancer cells, thereby increasing the level of HMGB1 de-K48-linked ubiquitination, promoting HMGB1 degradation, and reducing HMGB1 levels in colorectal cells. This induces colorectal cancer cells to enter a low-ROS, metabolically dormant state, impairing autophagy and malignant transformation, ultimately achieving improved therapeutic effects in colorectal cancer.

[0008] Furthermore, the USP5 inhibitor includes: substances that knock down or silence USP5 gene expression; and / or substances that reduce or inhibit USP5 protease activity.

[0009] Furthermore, the substance for knocking down or silencing USP5 gene expression includes: siRNA, shRNA, or sgRNA targeting the USP5 gene; and / or, a vector carrying the siRNA, shRNA, and / or sgRNA targeting the USP5 gene.

[0010] A third objective of this invention is to provide the application of the USP5 / HMGB1 signaling axis as a target in the preparation of drugs for the treatment of colorectal cancer. Specifically, the application includes: preparing drugs that regulate USP5-mediated HMGB1 de-K48-linked ubiquitination; preparing drugs that regulate the level of HMGB1 protein in colorectal cancer cells; and / or preparing drugs that regulate the proliferation, migration, invasion, pyroptosis, or autophagy of colorectal cancer cells mediated by the USP5 / HMGB1 / JAK2 / STAT3 signaling axis.

[0011] A fourth objective of this invention is to provide a predictive marker associated with colorectal cancer, the predictive marker comprising: the USP5 gene and its mRNA and expressed protein and its antibody; the HMGB1 gene and its mRNA and expressed protein and its antibody; and / or, K48-linked ubiquitinated HMGB1 protein and its antibody.

[0012] A fifth objective of the present invention is to provide a colorectal cancer-related detection kit, the detection kit comprising the above-mentioned predictive markers and / or substances for detecting the predictive markers.

[0013] The sixth objective of this invention is to provide a method for screening drugs for the treatment of colorectal cancer, characterized in that the screening method includes: screening based on the regulatory effect of candidate molecules on the USP5 / HMGB1 / JAK2 / STAT3 signaling axis.

[0014] Furthermore, the screening method includes: treating colorectal cancer cells with the candidate molecules, detecting the degree of change in USP5 expression level, HMGB1 expression level and / or HMGB1 ubiquitination modification level in colorectal cancer cells, and evaluating the effect of the candidate molecules on the activity, proliferation, invasion, spread, autophagy and / or pyroptosis of colorectal cancer cells based on the degree of change. Attached Figure Description

[0015] Figure 1 The figure shows the qPCR experimental results of the inhibition effect of USP5 gene expression in colorectal cancer cells infected with shUSP5-1 / 2 / 3 virus provided in Example 1 of this invention. Figure 2 The image shows the Western blot results of the test on the inhibition effect of USP gene expression in colorectal cancer cells infected with shUSP5-1 / 2 / 3 virus provided in Example 1 of this invention. Figure 3 Immunohistochemical results of cancerous tissue and adjacent normal tissue USP5 / HMGB1 in one of the colorectal cancer tissue microarrays provided in Example 2 of the present invention; Figure 4 This is a statistical chart of the average optical density score of USP5 in the colorectal cancer tissue chip provided in Example 2 of the present invention (unpaired). Figure 5 This is a statistical chart of the average optical density score of HMGB1 in the colorectal cancer tissue chip provided in Example 2 of the present invention (unpaired). Figure 6 This is a paired statistical chart of the average optical density score of USP5 in the colorectal cancer tissue chip provided in Example 2 of the present invention. Figure 7 This is a statistical chart (paired) of the average optical density score of HMGB1 in the colorectal cancer tissue chip provided in Example 2 of the present invention. Figure 8 The Spearman correlation analysis results of the average optical density scores of USP5 and HMGB1 in the colorectal cancer tissue chip provided in Example 2 of this invention are shown in Figure 2. Figure 9This is a paired statistical chart of the relative levels of USP5 mRNA in a colorectal cancer tissue microarray provided in Example 2 of the present invention. Figure 10 This is a paired statistical chart of the relative levels of HMGB1 mRNA in a colorectal cancer tissue microarray provided in Example 2 of this invention. Figure 11 The image shows the results of Western blot experiments on USP5 and HMGB1 in the colorectal cancer tissue chip provided in Example 2 of this invention. Figure 12 This is a paired statistical chart of the relative levels of USP5 protein in a colorectal cancer tissue microarray provided in Example 2 of the present invention. Figure 13 This is a statistical chart (paired) of the relative levels of HMGB1 protein in the colorectal cancer tissue microarray provided in Example 2 of the present invention. Figure 14 The figure shows the experimental results of relative luciferase activity assay after co-transfection of HEK293T cells with USP overexpression plasmid and HMGB1-Nanoluc plasmid in Example 2 of this invention. Figure 15 This is one of the experimental results of testing the relative level of HMGB1 protein in HEK293T cells after co-transfection with USP overexpression plasmid in Example 2 of the present invention; Figure 16 The second figure shows the experimental results of the relative level of HMGB1 protein in HEK293T cells after co-transfection with USP overexpression plasmid in Example 2 of this invention. Figure 17 This is a graph showing the experimental results of the relative levels of USP5 mRNA in normal human colonic epithelial cell lines and colorectal cancer cell lines CCD-841CoN, SW480, SW620, HT-29, CW2, and HCT-116 provided in Example 2 of this invention. Figure 18 This is a graph showing the experimental results of the relative levels of HMGB1 mRNA in normal human colonic epithelial cell lines and colorectal cancer cell lines CCD-841CoN, SW480, SW620, HT-29, CW2, and HCT-116 provided in Example 2 of the present invention. Figure 19 This is one of the experimental results provided in Example 2 of the present invention, showing the relative levels of USP5 and HMGB1 proteins in normal human colonic epithelial cell lines and colorectal cancer cell lines CCD-841CoN, SW480, SW620, HT-29, CW2, and HCT-116. Figure 20Figure 2 shows the experimental results of the relative levels of USP5 and HMGB1 proteins in normal human colonic epithelial cell lines and colorectal cancer cell lines CCD-841CoN, SW480, SW620, HT-29, CW2, and HCT-116 provided in Example 2 of this invention. Figure 21 Figure 3 shows the experimental results of the relative levels of USP5 and HMGB1 proteins in normal human colonic epithelial cell lines and colorectal cancer cell lines CCD-841CoN, SW480, SW620, HT-29, CW2, and HCT-116 provided in Example 2 of this invention. Figure 22 This is one of the experimental results provided in Example 3 of the present invention, showing the relative level of HMGB1 protein in colorectal cancer cells HCT-116 after treatment with actinomycin and / or the proteasome inhibitor MG132. Figure 23 Figure 2 shows the experimental results of the relative level of HMGB1 protein in colorectal cancer cells HCT-116 after treatment with actinomycin and / or the proteasome inhibitor MG132, as provided in Example 3 of this invention. Figure 24 This is one of the experimental results provided in Example 3 of the present invention, showing the relative level of HMGB1 protein in colorectal cancer cells SW620 after treatment with actinomycin and / or the proteasome inhibitor MG132. Figure 25 Figure 2 shows the experimental results of the relative level of HMGB1 protein in colorectal cancer cells after treatment with actinomycin and / or the proteasome inhibitor MG132 in Example 3 of this invention. Figure 26 This is a diagram showing the experimental results of Western blot analysis of the supernatant of cell lysis after treating HA-USP5 colorectal cancer cells or their corresponding colorectal cancer cells with HMGB1 OE plasmid in Example 3 of the present invention. Figure 27 This is a graph showing the GST pull-down experimental results of HMGB1 and USP5 proteins provided in Example 3 of this invention; Figure 28 This is one of the experimental results of Western blot analysis of the supernatant of cell lysis after treating shUSP5 colorectal cancer cells with HMGB1 OE plasmid and / or His-Ub plasmid in Example 3 of the present invention. Figure 29This is the second figure showing the results of Western blot analysis of the supernatant of shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid and / or His-Ub plasmid in Example 3 of this invention. Figure 30 This is one of the experimental results of Western blot detection of the cell lysis supernatant after treating HA-USP5 colorectal cancer cells or corresponding colorectal cancer cells with HMGB1 OE plasmid, His-Ub (WT) plasmid and / or His-Ub (mutant) plasmid provided in Example 3 of the present invention. Figure 31 Figure 2 shows the experimental results of Western blot detection of the cell lysate supernatant after treating HA-USP5 colorectal cancer cells or corresponding colorectal cancer cells with HMGB1 OE plasmid, His-Ub (WT) plasmid and / or His-Ub (mutant) plasmid provided in Example 2 of this invention. Figure 32 This is one of the experimental results of Western blot detection of cell lysis supernatant after treating shUSP5 colorectal cancer cells or corresponding colorectal cancer cells with HMGB1 OE plasmid in Example 3 of the present invention. Figure 33 This is the second figure of the experimental results of Western blot detection of cell lysis supernatant after treating shUSP5 colorectal cancer cells or corresponding colorectal cancer cells with HMGB1 OE plasmid in Example 3 of the present invention. Figure 34 Figure 3 shows the experimental results of Western blot detection of cell lysis supernatant after treating shUSP5 colorectal cancer cells or corresponding colorectal cancer cells with HMGB1 OE plasmid in Example 3 of this invention. Figure 35 This is one of the experimental results of Western blot detection of cell lysate supernatant after treating USP5 OE colorectal cancer cells or corresponding colorectal cancer cells with HMGB1 OE plasmid in Example 3 of the present invention. Figure 36 Figure 2 shows the experimental results of Western blot analysis of the cell lysis supernatant after treating USP5 OE colorectal cancer cells or corresponding colorectal cancer cells with HMGB1 OE plasmid in Example 3 of this invention. Figure 37 Figure 3 shows the experimental results of Western blot analysis of the cell lysis supernatant after treating USP5 OE colorectal cancer cells or corresponding colorectal cancer cells with HMGB1 OE plasmid in Example 3 of this invention. Figure 38 This is one of the experimental results of Western blot analysis of the cell lysis supernatant during the process of culturing shUSP5 colorectal cancer cells or the corresponding colorectal cancer cells SW620 after treatment with HMGB1 OE plasmid until USP5 was exhausted, as provided in Example 3 of the present invention. Figure 39 This is the second figure of the experimental results of Western blot detection of the cell lysis supernatant during the process of culturing shUSP5 colorectal cancer cells or the corresponding colorectal cancer cells SW620 after treatment with HMGB1 OE plasmid until USP5 was exhausted, as provided in Example 3 of the present invention. Figure 40 This is a graph showing the experimental results of MTT assay in the culture medium after treating shUSP5 colorectal cancer cells HCT-116 with HMGB1 OE plasmid in Example 4 of the present invention. Figure 41 The experimental results of MTT assay of the culture medium obtained from culturing USP5 OE colorectal cancer cells HCT-116 provided in Example 4 of this invention are shown in the figure. Figure 42 This is a graph showing the MTT assay results of the culture medium during the culture process of shUSP5 colorectal cancer cells SW620 after treatment with HMGB1 OE plasmid provided in Example 4 of this invention. Figure 43 The figure shows the experimental results of MTT assay of the culture medium obtained from culturing USP5 OE colorectal cancer cells SW620 provided in Example 4 of this invention. Figure 44 The image shows the experimental results of cloning USP5 OE colorectal cancer cells provided in Example 4 of this invention, or shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid. Figure 45 This is the result of the clone formation experiment of shUSP5 colorectal cancer cells HCT-116 after treatment with HMGB1 OE plasmid provided in Example 4 of the present invention; Figure 46 The results of the clonogenic experiment of USP5 OE colorectal cancer cells HCT-116 provided in Example 4 of this invention; Figure 47 This is a diagram showing the results of the clone formation experiment after treating shUSP5 colorectal cancer cells SW620 with HMGB1 OE plasmid in Example 4 of this invention. Figure 48 The results of the clone formation experiment of USP5 OE colorectal cancer cells SW620 provided in Example 4 of this invention; Figure 49 This is one of the cell cycle detection results provided in Example 4 of the present invention after treating shUSP5 colorectal cancer cells HCT-116 with HMGB1 OE plasmid. Figure 50 This is the second figure showing the cell cycle detection results after treating shUSP5 colorectal cancer cells HCT-116 with HMGB1 OE plasmid in Example 4 of this invention. Figure 51 This is one of the cell cycle detection results after treating shUSP5 colorectal cancer cells SW620 with the HMGB1 OE plasmid provided in Example 4 of this invention. Figure 52 This is the second image showing the cell cycle detection results after treating shUSP5 colorectal cancer cells SW620 with HMGB1 OE plasmid in Example 4 of this invention. Figure 53 The image shows the cell migration results of USP5 OE colorectal cancer cells provided in Example 4 of this invention, or the cell migration results of shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid. Figure 54 This is a diagram showing the cell migration results of shUSP5 colorectal cancer cells HCT-116 after treatment with HMGB1 OE plasmid, as provided in Example 4 of this invention. Figure 55 This is a diagram showing the cell migration results of USP5 OE colorectal cancer cells HCT-116 provided in Example 4 of this invention; Figure 56 This is a diagram showing the cell migration results of shUSP5 colorectal cancer cells SW620 after treatment with HMGB1 OE plasmid, as provided in Example 4 of this invention. Figure 57 This is a diagram showing the cell migration results of USP5 OE colorectal cancer cells SW620 provided in Example 4 of this invention; Figure 58 The image shows the cell invasion results of USP5 OE colorectal cancer cells provided in Example 4 of this invention, or the results of cell invasion experiments after treatment of shUSP5 colorectal cancer cells with HMGB1 OE plasmid. Figure 59 This is a diagram showing the cell invasion results of shUSP5 colorectal cancer cells HCT-116 after treatment with HMGB1 OE plasmid in Example 4 of this invention. Figure 60 This is a diagram showing the cell invasion results of USP5 OE colorectal cancer cells HCT-116 provided in Example 4 of this invention; Figure 61 This is the third figure showing the cell invasion results after treating SW620 colorectal cancer cells with HMGB1 OE plasmid in Example 4 of this invention. Figure 62 This is a diagram showing the cell invasion results of USP5 OE colorectal cancer cells SW620 provided in Example 4 of this invention; Figure 63 This is a TEM image of shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid, as provided in Example 4 of this invention. Figure 64 This is one of the experimental results of ROS level detection in shUSP5 colorectal cancer cells HCT-116 after treatment with HMGB1 OE plasmid and Nigerian cytosine in Example 4 of the present invention, using DCF as a probe. Figure 65 The second figure shows the experimental results of ROS level detection in shUSP5 colorectal cancer cells HCT-116 after treatment with HMGB1 OE plasmid and Nigerian styracin in Example 4 of this invention, using DCF as a probe. Figure 66 This is one of the experimental results of ROS level detection in shUSP5 colorectal cancer cells SW620 after treatment with HMGB1 OE plasmid and Nigerian cytosine in Example 4 of the present invention, using DCF as a probe. Figure 67 The second figure shows the experimental results of ROS level detection in shUSP5 colorectal cancer cells SW620 after treatment with HMGB1 OE plasmid and Nigerian cytosine in Example 4 of this invention, using DCF as a probe. Figure 68 This is one of the experimental results of ROS level detection in shUSP5 colorectal cancer cells HCT-116 after treatment with HMGB1 OE plasmid and Nigerian styraxin in Example 4 of the present invention, using MitoSOX Red as a probe. Figure 69 The second figure shows the experimental results of ROS level detection in shUSP5 colorectal cancer cells HCT-116 after treatment with HMGB1 OE plasmid and Nigerian styraxin in Example 4 of this invention, using MitoSOX Red as a probe. Figure 70 This is one of the experimental results of ROS level detection in shUSP5 colorectal cancer cells SW620 after treatment with HMGB1 OE plasmid and Nigerian styraxin in Example 4 of the present invention, using MitoSOX Red as a probe. Figure 71 Figure 2 shows the experimental results of ROS level detection in shUSP5 colorectal cancer cells SW620 after treatment with HMGB1 OE plasmid and Nigerian styraxin in Example 4 of this invention, using MitoSOX Red as a probe. Figure 72 The figure shows the experimental results of testing the LDH level in the culture supernatant after treating shUSP5 colorectal cancer cells HCT-116 with HMGB1 OE plasmid and Nigerian styracin in Example 4 of this invention. Figure 73 The figure shows the experimental results of testing the LDH level in the culture supernatant after treating shUSP5 colorectal cancer cells SW620 with HMGB1 OE plasmid and Nigerian cytosine in Example 4 of this invention. Figure 74 This is a graph showing the experimental results of detecting the relative mRNA levels of cytokines IL-18, IL-1β, and TNF-α in shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid and Nigerian cytosine, as provided in Example 4 of this invention. Figure 75 This is a graph showing the experimental results of detecting the relative mRNA levels of cytokine TNF-α in shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid and Nigerian cytokine in Example 4 of this invention. Figure 76 This is a graph showing the experimental results of detecting the relative mRNA level of cytokine IL-18 in shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid and Nigerian cytokine in Example 4 of this invention. Figure 77 This is a graph showing the experimental results of detecting the relative mRNA level of cytokine IL-1β in shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid and Nigerian cytokine in Example 4 of this invention. Figure 78 This is a graph showing the experimental results of detecting the relative protein levels of pyroptosis-related proteins in shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid and Nigerian cytosine, as provided in Example 4 of this invention. Figure 79 This is a diagram showing the experimental results of detecting the relative protein levels of autophagy markers in shUSP5 colorectal cancer cells after treatment with HMGB1 OE plasmid and Nigerian cytosine, as provided in Example 4 of this invention. Figure 80This is a photograph of a tumor obtained by treating mice with shUSP5 cells HCT-116, as provided in Example 4 of this invention. Figure 81 This is a diagram showing the experimental results of tumor volume obtained after treating mice with shUSP5 cells HCT-116, as provided in Example 4 of this invention. Figure 82 This is a graph showing the experimental results of tumor weight obtained after treating mice with shUSP5 cells HCT-116, as provided in Example 4 of this invention. Figure 83 This is an H&E staining image of a tumor obtained by treating mice with shUSP5 cells HCT-116, as provided in Example 4 of this invention. Figure 84 This is one of the results of the tissue immunochemical detection experiment of Ki-67 tumors obtained by treating mice with shUSP5 cells HCT-116 provided in Example 4 of the present invention; Figure 85 This is the second image of the immunochemical detection results of Ki-67 tumors obtained by treating mice with shUSP5 cells HCT-116 in Example 4 of the present invention. Figure 86 This is one of the experimental results of ROS level detection in tumors obtained after treating mice with shUSP5 cells HCT-116, as provided in Example 4 of this invention. Figure 87 This is the second figure showing the experimental results of ROS level detection of tumors obtained after treating mice with shUSP5 cells HCT-116 in Example 4 of this invention. Figure 88 This is a diagram showing the experimental results of detecting the cytokine expression level of tumors obtained after treating mice with shUSP5 cells HCT-116, as provided in Example 4 of this invention. Figure 89 This is one of the experimental results provided in Example 4 of this invention, showing the expression levels of pyroptosis-related proteins and autophagy markers in the tumors obtained after treating mice with shUSP5 cells HCT-116. Figure 90 This is the second figure showing the experimental results of detecting the expression levels of pyroptosis-related proteins and autophagy markers in tumors obtained after treating mice with shUSP5 cells HCT-116 in Example 4 of this invention. Figure 91 This is a bright-field image of the organoid obtained after processing primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus, as provided in Example 4 of the present invention. Figure 92 This is a graph showing the change rate of organoid diameter after treating primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus, as provided in Example 4 of this invention. Figure 93 This is a graph showing the results of ATP level detection in primary colorectal cancer organoids after treatment with shUSP5 or shNC lentiviruses, as provided in Example 4 of this invention. Figure 94 This is one of the results of histoimmunochemical detection of Ki-67 organoids obtained after processing primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus in Example 4 of the present invention. Figure 95 This is the second image showing the results of histoimmunochemical detection of Ki-67 organoids obtained after processing primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus in Example 4 of this invention. Figure 96 This is one of the results of histoimmunochemical detection of CD45 in organoids obtained after processing primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus, as provided in Example 4 of this invention. Figure 97 This is the second image showing the results of histoimmunochemical detection of CD45 in organoids obtained after processing primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus, as provided in Example 4 of this invention. Figure 98 This is a graph showing the results of detecting the expression level of the organoid cytokine TNF-α after treating primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus in Example 4 of the present invention. Figure 99 This is a graph showing the results of detecting the expression level of organoid cytokine IL-18 after treating primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus in Example 4 of the present invention. Figure 100 This is a graph showing the results of detecting the expression level of organoid cytokine IL-1β after treating primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus in Example 4 of this invention. Figure 101 This is a graph showing the results of detecting the expression levels of HMGB1 and pyroptosis-related proteins in primary colorectal cancer organoids obtained by treating them with shUSP5 or shNC lentiviruses, as provided in Example 4 of this invention. Figure 102This is a graph showing the results of detecting the expression level of autophagy markers obtained after processing primary colorectal cancer organoids with shUSP5 lentivirus or shNC lentivirus in Example 4 of the present invention. Detailed Implementation

[0016] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0017] The plasmids involved in the following embodiments specifically include: (1) USP overexpression plasmids: purchased from Xiamen Anhela Biotechnology Co., Ltd., specifically including USPL1 overexpression plasmid, USP1-8 overexpression plasmid, USP10-11 overexpression plasmid, USP13-15 overexpression plasmid, USP18-22 overexpression plasmid, USP25-26 overexpression plasmid, USP27X overexpression plasmid, USP29-30 overexpression plasmid, USP32-33 overexpression plasmid, USP35-36 overexpression plasmid, USP38-40 overexpression plasmid, USP44-46 overexpression plasmid, USP48-49 overexpression plasmid and USP51-54 overexpression plasmid.

[0018] (2) The preparation of HMGB1-Nanoluc plasmid includes: cloning the HMGB1 gene (Gene ID: 3146) into the pmirGLO vector to obtain HMGB1-Nanoluc plasmid.

[0019] (3) The preparation of HMGB1 OE plasmid includes: cloning the HMGB1 gene (Gene ID: 3146) into the pcDNA3.1 vector to obtain HMGB1 OE plasmid.

[0020] (4) Preparation of USP5 OE plasmid includes: cloning the USP5 gene (Gene ID: 8078) with Flag tag into the pLV-CMV vector to obtain USP5 OE plasmid.

[0021] (5) The preparation of pLKO.1-shUSP5-1 plasmid to pLKO.1-shUSP5-3 plasmid includes: based on the USP5 gene, three shRNAs targeting USP5 were designed, a sticky end of the restriction enzyme site was added to the 5' end of the shRNA, and a termination sequence TTTTT and a sticky end of the restriction enzyme site were added to the 3' end. The primers obtained are shown in Table 1. The primers were cloned into the pLKO.1 vector to obtain pLKO.1-shUSP5-1 plasmid to pLKO.1-shUSP5-3 plasmid (pLKO.1-shNC was prepared in the same way).

[0022] Table 1.

[0023] (6) His-Ub (WT) plasmid: The ubiquitin chain encoding gene after human codon optimization was cloned into the pcDNA3.1 vector to obtain the His-Ub (WT) plasmid. The specific amino acid sequence of the ubiquitin chain is shown in Table 2.

[0024] (7) His-Ub (mutant) plasmids: Specifically, His-Ub (K48) plasmid, His-Ub (K63) plasmid, His-Ub (K48R) plasmid and His-Ub (K63R) plasmid are used. The ubiquitin chain mutant coding gene after human codon optimization is cloned into the pcDNA3.1 vector to obtain the corresponding plasmid. The specific amino acid sequence of the ubiquitin chain mutant is shown in Table 2.

[0025] Table 2.

[0026] (8) Virus packaging plasmid pMD2.G (preserved in this laboratory); (9) Viral envelope plasmid psPAX2 (preserved in this laboratory); The reagents and their sources involved in the following examples specifically include: HCT-116 colorectal cancer cells (Xiamen Aimeng Cell Technology Co., Ltd., catalog number IM-H098); Colorectal cancer cells SW620 (Xiamen Aimeng Cell Technology Co., Ltd., catalog number IM-H112); Colorectal cancer cells CCD-841CoN (Xiamen Aimeng Cell Technology Co., Ltd., catalog number IM-H477); Colorectal cancer cells SW480 (Xiamen Aimeng Cell Technology Co., Ltd., catalog number IM-H111); Colorectal cancer cells HT-29 (Xiamen Aimeng Cell Technology Co., Ltd., catalog number IM-H102); CW2 colorectal cancer cells (Xiamen Aimeng Cell Technology Co., Ltd., catalog number IM-H097); HEK293T cells (cryopreserved in our laboratory); Fetal bovine serum (purchased from Xiamen Aimeng Cell Technology Co., Ltd., catalog number IMC-101); Penicillin-streptomycin (purchased from Gbico, catalog number 15140122); RPMI 1640 medium (Gbico, catalog number 11875119); DMEM medium (purchased from Gbico, catalog number 11966025); polybrene (Beyotime, product number C0351); ExFect Transfection Reagent (Novozymes, product number T101-01); G418 (Beyotime, item number ST081); RNA isolater Total RNA Extraction Reagent (Novizan, catalog number R401-01); HiScript II 1st Strand cDNA Synthesis Kit (NovaZan, catalog number R211-01); ChamQ SYBR Color qPCR Master Mix (Novazan, product number Q411-02); RIPA lysis buffer (purchased from Beyotime); 75 colorectal cancer tissue microarrays (containing 75 cancer tissues and paired adjacent normal tissues, purchased from Shanghai Chipchao Biotechnology Co., Ltd., catalog number HClA150CS02); USP5 primary antibody (Proteintech, catalog number 10473-1-AP). HMGB1 primary antibody (Proteintech, catalog number 10829-1-AP). IgG(H+L) HRP (Jackson ImmunoResearch, catalog number 111-035-003); Pierce TM BCA Protein Quantitative Kit (Invitrogen, catalog number #23227); Sample loading buffer (Yaxin, catalog number LT101); Duo-Lite Dual Fluorescence Detection Kit (Novizan, catalog number DD1205); His-HMGB1 (constructed by Xiamen Anhera Biotechnology Co., Ltd.); GST-USP5 (constructed by Xiamen Anhera Biotechnology Co., Ltd.); GST control protein (constructed by Xiamen Anhera Biotechnology Co., Ltd.); Glutathione agarose 4B magnetic beads (Sigma-Aldrich, catalog number G0924). Lysis buffer (Proteintech, catalog number PR20037); Anti-Flag-tag (Proteintech, catalog number 20543-1-AP); Anti-HA-tag (Proteintech, catalog number 51064-2-AP); Protein A / G magnetic beads (Millipore (Sigma), catalog number Cat#16-663); MTT kit (Yisheng Biotechnology, catalog number 40201ES72); 4% paraformaldehyde fixative (purchased from Beyotime); 0.1% crystal violet stain (purchased from Antikythera); RNase A (Soleb, product number R8020); 7-AAD staining solution (Thermo Fisher Scientific, catalog number A1310) Transwell cell (purchased from Corning); DCFDA / H2DCFDA Cell ROS Detection Kit (Abcam, catalog number ab113851). Digestive fluid (IMMOCELL, catalog number IMV-A006); Matrigel matrix adhesive (BD, product number 356234); Complete culture medium for colorectal cancer organoids (IMMOCELL, catalog number IMV-T12). DMEM / F12 basal medium (Gbico, catalog number 11320033). ATP assay kit (Beyotime, catalog number S0026).

[0027] Example 1 This embodiment illustrates the construction of shUSP5 colorectal cancer cells and the inhibitory effect of the USP5 gene expression in them. The specific tests include: 1. Preparation of shUSP5 colorectal cancer cells: (1) According to 5×10 5HEK293T cells were seeded at a seeding rate of 10% fetal bovine serum and 1× penicillin-streptomycin in 6 cm culture dishes containing RPMI 1640 or DMEM medium. Lentiviral knockdown plasmids (pLKO.1-shUSP5-1, pLKO.1-shUSP5-2, or pLKO.1-shUSP5-3), viral packaging plasmid pMD2.G, and viral envelope plasmid psPAX2 were added at 6 μg / well, 3 μg / well, and 3 μg / well, respectively. Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions. After 12 h, the medium was changed and cultured for another 60 h. The culture supernatant was then filtered through a 0.45 μm filter to obtain the viral supernatant.

[0028] (2) According to 5×10 5 Inoculate HCT-116 and SW620 colorectal cancer cells at a seeding rate of 1 cell per well. After the cells have fully adhered, add 2 mL of viral supernatant and 2 mL of the corresponding culture medium to the cells. Add polybrene to the cells at a final concentration of 8 μg / mL. After 12 h of infection, replace the culture medium with fresh medium. After 48 h, add G418 at a final concentration of 800 μg / mL for stable cell selection for 7-10 days (passage the cells after they reach confluence, changing the culture medium and adding G418 every two days) to obtain shUSP5 colorectal cancer cells.

[0029] The lentivirus knockdown plasmid, viral packaging plasmid pMD2.G, and viral envelope plasmid psPAX2 were added in the form of a virus-culture medium mixture. The method for preparing the virus-culture medium mixture was a conventional technique in the field, and the conditions used in each group were kept consistent.

[0030] 2. USP5 mRNA level in colorectal cancer cells: (1) Total RNA was extracted from shUSP5 colorectal cancer cells using RNA isolater Total RNA Extraction Reagent in accordance with the instructions.

[0031] (2) The total RNA obtained after (1) treatment was reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit according to the instructions.

[0032] (3) The cDNA obtained in (2) was qPCR performed on an ABI 7300 Plus real-time PCR instrument (Applied Biosystems, USA) using ChamQ SYBR Color qPCR Master Mix according to the instructions. The relative mRNA level of USP5 was calculated using the 2-ΔΔCt method in the dark. The results are as follows. Figure 1 As shown.

[0033] 3. USP5 protein level in colorectal cancer cells: (1) shUSP5 colorectal cancer cells were lysed and homogenized using RIPA lysis buffer according to the instructions, and the supernatant was collected by centrifugation.

[0034] (2) The supernatant obtained after treatment (1) was subjected to Western blot analysis, and the results are as follows: Figure 2 As shown.

[0035] Depend on Figure 1 and 2 The results show that pLKO.1-shUSP5-1, pLKO.1-shUSP5-2, and pLKO.1-shUSP5-3 all have good knockdown efficiency for USP5, and the pLKO.1-shUSP5-3 plasmid has a better inhibitory effect on the USP5 gene at the protein level.

[0036] In the following examples, the shUSP5, HA-USP5, and USP5 OE colorectal cancer cells involved were all constructed using the method provided in Example 1. Lentiviral knockdown plasmid (pLKO.1-shUSP5-3 plasmid), USP5 overexpression plasmid, or USP5 OE plasmid, along with viral packaging plasmid pMD2.G and viral envelope plasmid psPAX2, were used in the form of a virus-culture medium mixture to treat and screen colorectal cancer cells HCT-116 or SW620, thereby constructing stable transfected cell lines.

[0037] Example 2 This embodiment illustrates the differential expression of USP5 and HMGB1 in colorectal cancer tissues and cancer cells, specifically including: I. USP5 and HMGB1 are highly expressed in colorectal cancer tissues 1. Immunochemical detection of tissues: (1) 75 colorectal cancer tissue chips were dewaxed and hydrated using an automated staining machine in accordance with the instructions; the colorectal cancer tissue chips were treated with 3% hydrogen peroxide to block endogenous peroxidase; then the colorectal cancer tissue chips were repaired with antigens using an antigen repair instrument in accordance with the instructions, and then the colorectal cancer tissue chips were blocked with 5% goat serum.

[0038] (2) The colorectal cancer tissue microarray obtained by (1) was incubated overnight at 4°C with USP5 primary antibody and HMGB1 primary antibody; then IgG(H+L) HRP was used to incubate at room temperature for 1 h; the colorectal cancer tissue microarray was washed with PBS solution.

[0039] (3) The colorectal cancer tissue microarray obtained by (2) was incubated with DAB staining solution at room temperature for 2 min; then hematoxylin staining was performed for 1 min, followed by immersion in 0.25% hydrochloric acid alcohol (prepared by mixing 400 mL of 70% alcohol and 1 mL of 12 mol / L concentrated hydrochloric acid) for 10 s, and rinsed with tap water for 15 min. The colorectal cancer tissue microarray was then sectioned and dehydrated using a fully automated staining machine. The microarray images were acquired and ImageJ quantification was performed using an Aperio scanner (Aperio XT, Leica), and paired two-sided Student's t test and Spearman correlation analysis were performed. The results are as follows: Figures 3-8 As shown.

[0040] Depend on Figures 3-8 The results show that, compared with adjacent non-tumor tissue samples, the protein expression levels of USP5 and HMGB1 are increased in tumor samples, and the protein expression levels of USP5 and HMGB1 in cancer tissue are positively correlated.

[0041] 2. qPCR detection: Following the detection method provided in "2. USP5 mRNA level in colorectal cancer cells" in Example 1, the relative mRNA levels of USP5 and HMGB1 genes in 20 out of 75 colorectal cancer tissue microarray samples were detected, and a paired two-sided Student's t-test was performed. The results are as follows: Figure 9 and 10 As shown.

[0042] Depend on Figure 9 and 10 The results show that, compared to adjacent non-tumor tissue samples, the mRNA levels of USP5 and HMGB1 were elevated in tumor samples.

[0043] 3. Protein blotting: (1) Using RIPA lysis buffer, 20 of the above 75 colorectal cancer tissue microarrays were lysed and homogenized according to the instructions, and the supernatant was collected by centrifugation.

[0044] (2) Pierce TM The protein concentration of the protein solution obtained by (1) was tested according to the instructions of the BCA protein quantitative kit.

[0045] (3) The protein concentration of the protein solution obtained after (1) treatment was diluted to 2 μg / mL using loading buffer. After denaturation at 100℃ for 10 min, the diluted protein solution was added at a rate of 15 μg / well for SDS-PAGE electrophoresis. The resulting gel image was transferred to a PVDF membrane, blocked with 5% skim milk, and then incubated overnight at 4℃ with USP5 primary antibody and HMGB1 primary antibody. Then, IgG(H+L) HRP was added and incubated at room temperature for 2 h. ECL chemiluminescence imaging and X-ray exposure were performed. The band intensity was quantified using ImageJ and normalized with actin. The experiment was repeated in triplicate, and the results are as follows: Figures 11-13 As shown.

[0046] Depend on Figures 11-13 The results show that the levels of USP5 and HMGB1 proteins were elevated compared to adjacent non-tumor tissue samples.

[0047] II. Overexpression of USP5 significantly increased HMGB1 protein levels. 1. Effect of USP overexpression on HMGB1 expression in HEK293T cells: 1. Construction of USP5 overexpressing cells: according to 3×10 4 HEK293T cells were seeded into 96-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin in DMEM medium at a seeding rate of 0.6 μg / well and 0.3 μg / well respectively. The cells were then transfected using ExFect Transfection Reagent for 48 h according to the manufacturer's instructions to obtain the corresponding USP-overexpressing cells.

[0048] 2. Luciferase experiment: (1) Use RIPA lysis buffer to lyse USP overexpressing cells according to the instructions, collect the lysate and centrifuge.

[0049] (2) The activities of firefly luciferase and kidney luciferase in the supernatant obtained after treatment in (1) were detected using the Duo-Lite dual fluorescence detection kit and the Orion II microplate reader (Berthold, Germany, according to the instruction manual). The kidney luciferase activity was normalized to the firefly luciferase activity. The experiment was repeated in triplicate, and the results are as follows: Figure 14 As shown.

[0050] Depend on Figure 14 The results show that USP39, USP14, USP5, and USP27X have a stronger upregulation effect on HMGB1 expression.

[0051] 3. Western blotting: Following the method described in "I. High Expression of USP5 and HMGB1 in Colorectal Cancer Tissues" under "3. Western Blotting," the HMGB1 protein level in USP-overexpressing cells was detected. The results are as follows: Figure 15 and 16 As shown.

[0052] Depend on Figure 15 and 16 The results show that overexpression of USP39, USP14, USP5 and USP27X significantly increased the expression of HMGB1 in HEK293T cells, with USP5 showing the strongest upregulation effect, indicating that overexpression of USP5 significantly increased the protein level of HMGB1.

[0053] III. Differential expression of USP5 and HMGB1 genes in colorectal cancer cell lines 1. qPCR detection: Following the detection method provided in "2. USP5 mRNA level in colorectal cancer cells" in Example 1, the relative mRNA levels of USP5 and HMGB1 genes in normal human colonic epithelial cell lines and colorectal cancer cell lines CCD-841CoN, SW480, SW620, HT-29, CW2, and HCT-116 were detected. The results are as follows: Figure 17 and 18 As shown.

[0054] Depend on Figure 17 and 18 The results show that, compared with normal human colonic epithelial cell lines, USP5 was upregulated in colorectal cancer cell lines SW620, CW2 and HCT-116, and downregulated in SW480 and HT-29 cells; HMGB1 was upregulated in colorectal cancer cell lines SW620 and HCT-116, and downregulated in SW480.

[0055] 2. Western Blotting: Following the method provided in "I. High Expression of USP5 and HMGB1 in Colorectal Cancer Tissues" and "3. Western Blotting," the protein levels of USP5 and HMGB1 in normal human colonic epithelial cell lines and colorectal cancer cell lines CCD-841CoN, SW480, SW620, HT-29, CW2, and HCT-116 were detected. The results are as follows: Figures 19-21 As shown.

[0056] Depend on Figures 19-21 The results show that, compared with normal human colonic epithelial cell lines, the levels of USP5 and HMGB1 proteins are elevated in colorectal cancer cell lines SW620 and HCT-116.

[0057] comprehensive Figures 14-21The results show that the expression level of USP5 is positively correlated with the expression level of HMGB1, that is, overexpression of USP5 promotes the upregulation of HMGB1 expression.

[0058] Example 3 This embodiment illustrates the stabilizing effect of USP5 on HMGB1 in colorectal cancer cells, specifically including: I. Ubiquitin-Proteasome System Mediates HMGB1 Degradation 1. Colorectal cancer cell culture: according to 5×10 5 Inoculation rate was 1 cell / well. Colorectal cancer cells HCT-116 and SW620 were inoculated into 6 cm culture dishes of RPMI 1640 or DMEM medium containing 10% fetal bovine serum and 1× penicillin-streptomycin antibiotics. Then, a certain concentration of actinomycin (CHX, protein synthesis inhibitor) and / or proteasome inhibitor MG132 were added, and the dishes were cultured at 37°C and 5% CO2 saturated humidity for 3 h.

[0059] 2. Western blotting: Referring to the method provided in "3. Western blotting" under "I. High expression of USP5 and HMGB1 in colorectal cancer tissue" in Example 2, the protein level of HMGB1 in colorectal cancer cells obtained after "1. Colorectal cancer cell culture" was detected. The results are as follows: Figures 22-25 As shown.

[0060] Depend on Figures 22-25 The results show that in colorectal cancer cells HCT-116 and SW620, the proteasome inhibitor MG132 prevented the reduction of HMGB1 in a dose-dependent manner, indicating that the ubiquitin-proteasome system mediates the degradation of HMGB1.

[0061] II. There is a strong interaction between USP5 and HMGB1. 1. Immunoprecipitation test: (1) According to 1×10 6 The seeding rate was 10 cm per well. HA-USP5 colorectal cancer cells or their corresponding colorectal cancer cells were seeded into 10 cm culture dishes containing 10% fetal bovine serum and 1× penicillin-streptomycin antibiotics in RPMI 1640 or DMEM medium. HMGB1 OE plasmid was added at a rate of 5 μg / well. Transfection was performed using ExFectTransfection Reagen according to the manufacturer's instructions for 48 h. Colorectal cancer cells transfected with the corresponding pLKO.1-shNC plasmid were used as a control.

[0062] (2) The cells obtained after (1) were lysed using lysis buffer according to the instructions, and the lysate was collected and centrifuged.

[0063] (3) Referring to the method provided in "I. High expression of USP5 and HMGB1 in colorectal cancer tissue" in Example 2, "3. Western blotting", the supernatant obtained after (1) treatment was subjected to Co-IP experiment, and the results are as follows: Figure 26 As shown.

[0064] Depend on Figure 26 The results show that there is a strong interaction between USP5 and HMGB1.

[0065] 2. Glutathione thiol transferase (GST) pull-down experiment: (1) Take 20 mg of His-HMGB1 and 20 mg of GST-USP5 or GST control protein and mix them thoroughly.

[0066] (2) Take a portion of the mixture obtained by (1) and denature it by centrifugation as an input control. The remaining mixture is gently incubated with 20 μL of glutathione agarose 4B magnetic beads at 4°C for 6 h by inversion. The magnetic beads are collected by centrifugation and washed to remove unbound proteins.

[0067] (3) Take 80 μL of the elution buffer for glutathione agarose 4B magnetic beads and mix it with the magnetic beads obtained after (2) treatment. Incubate at 100℃ for 10 min to elute the bound protein. Centrifuge and collect the supernatant for Western blot detection. The results are as follows: Figure 27 As shown.

[0068] Depend on Figure 27 The results show that the GST-coupled USP5 can effectively bind to the His-tagged HMGB1, indicating that there is a direct binding between USP5 and HMGB1.

[0069] III. USP5 inhibits K48-linked ubiquitination of HMGB1 1. USP5 knockdown increases HMGB1 ubiquitination level: (1) according to 5×10 5 Seedling density was set at 3 μg / well for shUSP5 colorectal cancer cells or corresponding colorectal cancer cells in 6 cm culture dishes containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. HMGB1 OE plasmid and / or His-Ub (WT) plasmid were added at 3 μg / well and 1.5 μg / well respectively. Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h, with corresponding colorectal cancer cells transfected with pLKO.1-shNC plasmid as a control.

[0070] (2) The cells obtained after (1) were lysed using lysis buffer according to the instructions, and the lysate was collected and centrifuged.

[0071] (3) Anti-Flag-tag and / or Anti-HA-tag were added to the lysis supernatant obtained after (2) and incubated overnight to enrich Flag-HMGB1 protein. Then, protein A / G magnetic beads were incubated for 3 hours. The magnetic beads and protein complex were washed 5 times with lysis buffer, the supernatant was removed by centrifugation, the precipitate was resuspended and denatured at 100°C for 10 minutes. The results were detected according to the method provided in "I. High expression of USP5 and HMGB1 in colorectal cancer tissue" "3. Western blotting" in Example 2. The results are as follows. Figure 28 and 29 As shown.

[0072] Depend on Figure 28 and 29 The results show that USP5 knockdown significantly increased the ubiquitination level of HMGB1, while USP5 overexpression significantly inhibited this process.

[0073] 2. USP5 removes K48-linked ubiquitination of HMGB1: (1) according to 5×10 5 Seedling density was 1 cell / well. HA-USP5 colorectal cancer cells or corresponding colorectal cancer cells were seeded into 6 cm culture dishes containing 10% fetal bovine serum and 1× penicillin-streptomycin in DMEM medium. HMGB1 OE plasmid, His-Ub (WT) plasmid and / or His-Ub (mutant) plasmid were added at 3 μg / well, 1.5 μg / well and 1.5 μg / well, and transfected for 48 h using ExFect Transfection Reagen according to the manufacturer's instructions.

[0074] (2) The cells obtained after treatment (1) were tested according to the method provided in “1. USP5 knockdown increases HMGB1 ubiquitination level”. The results are as follows: Figure 30 and 31 As shown.

[0075] Depend on Figure 30 and 31 The results show that USP5 can effectively reduce HMGB1 ubiquitination only in cells in which His-Ub (K48) plasmid or His-Ub (K63R) plasmid has been introduced. This means that USP5 achieves deubiquitination of HMGB1 by removing the K48-linked polyubiquitin chain on HMGB1.

[0076] 3. USP5 knockdown accelerates HMGB1 degradation: (1) According to 5×10 5Seeding density was 3 μg / well. shUSP5 colorectal cancer cells, USP5 OE colorectal cancer cells, or corresponding colorectal cancer cells were seeded into 6 cm culture dishes containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. HMGB1 OE plasmid was added at a rate of 3 μg / well, and transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h. Colorectal cancer cells transfected with pLKO.1-shNC plasmid were used as a control.

[0077] (2) Add actinomycin (CHX, protein synthesis inhibitor) to the cells obtained after treatment (1) according to the final concentration of 100 μg / mL, and incubate them in a 37℃, 5% CO2 saturated humidity incubator for 0, 6h, 9h or 18h respectively.

[0078] (3) The cells obtained after treatment (2) were tested according to the method provided in “1. USP5 knockdown increases HMGB1 ubiquitination level”. The results are as follows: Figures 32-39 As shown.

[0079] Depend on Figures 32-39 The results show that the lack of USP5 accelerates the degradation of HMGB1, while the overexpression of USP5 improves the stability of HMGB1 in colorectal cancer cells.

[0080] comprehensive Figures 28-39 The results show that in colorectal cancer cells, USP5 enhances the stability of HMGB1 by reducing K48-linked ubiquitination of HMGB1, thus protecting it from proteasome degradation.

[0081] Example 4 This embodiment illustrates the role of the USP5 / HMGB1 signaling axis in colorectal cancer progression and its sensitivity to Nigerian serotonin, specifically including: I. Correlation between the USP5 / HMGB1 signaling axis and malignant phenotypes of colorectal cancer cells 1. Correlation between the USP5 / HMGB1 signaling axis and colorectal cancer cell viability: (1) According to 3×10 4Seeding density was set at 0.6 μg / well. shUSP5 colorectal cancer cells, USP5 OE colorectal cancer cells, and their corresponding unmodified colorectal cancer cells were seeded into 96-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. HMGB1 OE plasmid or pLV-CMV plasmid (vector) was added at a rate of 0.6 μg / well. Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h. The corresponding colorectal cancer cells (shNC) obtained by treatment with pLKO.1-shNC plasmid were used as a control.

[0082] (2) During the culture process, the culture medium was sampled regularly, and the MTT assay kit was used to test the culture medium according to the instructions. The average value of three parallel experiments was taken. The results are as follows: Figures 40-43 As shown.

[0083] Depend on Figures 40-43 The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, the survival rate of shUSP5 colorectal cancer cells was significantly reduced, while the survival rate of USP5 OE colorectal cancer cells was increased. HMGB1 overexpression completely rescued the decreased cell viability of shUSP5 colorectal cancer cells.

[0084] 2. Correlation between the USP5 / HMGB1 signal axis and the clonogenic ability of colorectal cancer cells: (1) According to 3×10 4 The seeding rate was 0.6 μg / well. shUSP5 colorectal cancer cells were seeded into 96-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. HMGB1 OE plasmid was added at a rate of 0.6 μg / well, and transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h. Colorectal cancer cells transfected with pLKO.1-shNC plasmid were used as a control.

[0085] (2) At an inoculation rate of 3000 cells / well, cells obtained from (1) were seeded into 6-well plates containing RPMI 1640 medium or DMEM medium containing 10% fetal bovine serum and 1× penicillin-streptomycin antibiotics. The plates were then placed in a 37°C, 5% CO2 saturated humidity incubator for 14 days, and the medium was changed every 3 days during the culture process.

[0086] (3) After discarding the culture medium in the culture solution obtained after (2) treatment, the solution was washed, then fixed with 4% paraformaldehyde fixative for 15 min, and then stained with 0.1% crystal violet at room temperature for 30 min. After washing and air drying, the solution was photographed and quantified using ImageJ software. The results are as follows: Figures 44-48 As shown.

[0087] Depend on Figures 44-48 The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, the clonogenic ability of shUSP5 colorectal cancer cells was significantly reduced, the clonogenic ability of USP5 OE colorectal cancer cells was enhanced, and HMGB1 overexpression completely rescued the clonogenic ability of shUSP5 colorectal cancer cells.

[0088] 3. Correlation between the USP5 / HMGB1 signal axis and the cell cycle of colorectal cancer cells: (1) According to 3×10 4 The seeding rate was 1 cell / well. shUSP5 colorectal cancer cells were seeded into 96-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. HMGB1 OE plasmid or pLV-CMV plasmid (vector) was added at a rate of 0.6 μg / well. Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h. Colorectal cancer cells transfected with pLKO.1-shNC plasmid were used as a control.

[0089] (2) Collect the cells obtained after treatment (1), fix them with ethanol at -20℃ overnight and wash them, add 20 μL of RNase A and incubate at 37℃ for 30 min, add 5 μL of 7-AAD staining solution and incubate at 4℃ in the dark for 30 min, then resuspend the cells in PBS buffer (0.02M, pH=7.4) to 1×10⁻⁶. 6 Cells / 100 μL were analyzed by flow cytometry, and the results are as follows: Figures 49-52 As shown.

[0090] Depend on Figures 49-52 The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, the proportion of cells in the G0 / G1 phase in shUSP5 colorectal cancer cells increased, indicating that USP5 depletion can lead to G1 / S cell cycle arrest; while HMGB1 overexpression rescued the cell cycle arrest of shUSP5 colorectal cancer cells.

[0091] 4. Correlation between the USP5 / HMGB1 signal axis and the migration and invasion ability of colorectal cancer cells: (1) According to 3×10 4Seeding density was determined by seeding shUSP5 colorectal cancer cells, USP5 OE colorectal cancer cells, and their corresponding unmodified colorectal cancer cells into 96-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin antibiotics in RPMI 1640 or DMEM medium. HMGB1 OE plasmid or pLV-CMV plasmid (vector) was added at a rate of 0.6 μg / well. Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h, with the corresponding colorectal cancer cells transfected with pLKO.1-shNC plasmid serving as a control.

[0092] (2) Take 6×10 4 The cells obtained after treatment (1) were resuspended in 100 μL of RPMI 1640 medium or DMEM medium, and then seeded into the upper chamber of a Transwell chamber with an 8 μm pore size. 500 μL of complete medium was added to the lower chamber, and the cells were cultured in a 37℃, 5% CO2 saturated humidity incubator for 48 h.

[0093] (3) The upper chamber membrane obtained after treatment (2) was washed with PBS buffer (0.02M, pH=7.4), fixed with 4% paraformaldehyde fixative for 30 min, stained with 0.1% crystal violet at room temperature for 20 min, washed, air-dried, and then photographed and quantified using ImageJ software. The results are as follows: Figures 53-62 As shown.

[0094] Depend on Figures 53-62 The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, the migration and invasion abilities of shUSP5 colorectal cancer cells were significantly impaired, while the migration and invasion abilities of USP5 OE colorectal cancer cells were enhanced, and HMGB1 overexpression restored the motility of shUSP5 colorectal cancer cells.

[0095] II. Correlation between the USP5 / HMGB1 signaling axis and pyroptosis and autophagy in Nigeria-induced colorectal cancer cells 1. Correlation between the USP5 / HMGB1 signaling axis and mitochondrial structural damage in colorectal cancer cells: (1) According to 1×10 5At a seeding rate of cells / well, shUSP5 colorectal cancer cells were seeded into 6-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. HMGB1 OE plasmid or pcDNA3.1 vector was added at a rate of 0.6 μg / well. Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h, with corresponding colorectal cancer cells transfected with pLKO.1-shNC plasmid as a control.

[0096] (2) The cells obtained after treatment (1) were photographed using a transmission electron microscope, and the results are as follows: Figure 63 As shown.

[0097] Depend on Figure 63 The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, shUSP5 colorectal cancer cells have clusters of damaged mitochondria, indicating that USP5 knockdown causes mitochondrial damage in colorectal cancer cells; while HMGB1 overexpression improves the integrity of mitochondrial structure in shUSP5 colorectal cancer cells.

[0098] 2. Correlation between the USP5 / HMGB1 signaling axis and mitochondrial function in colorectal cancer cells: (1) According to 3×10 4 At a seeding rate of cells / well, shUSP5 colorectal cancer cells and their corresponding unmodified colorectal cancer cells were seeded into 96-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. Then, 2 μg of nigra was added per well, followed by 0.6 μg of HMGB1 OE plasmid or pLV-CMV plasmid (vector). Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h, with colorectal cancer cells transfected with pLKO.1-shNC plasmid serving as a control.

[0099] (2) The cells obtained after treatment (1) were tested using the DCFDA / H2DCFDA cell ROS detection kit according to the instructions. The results are as follows: Figures 64-71 As shown.

[0100] Depend on Figures 64-71The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, the overall and mitochondrial ROS levels in shUSP5 colorectal cancer cells were reduced, indicating that USP5 knockdown can induce mitochondrial dysfunction in colorectal cancer cells; while HMGB1 overexpression significantly upregulated ROS levels in shUSP5 colorectal cancer cells and improved mitochondrial dysfunction.

[0101] 3. Correlation between the USP5 / HMGB1 signal axis and the integrity of colorectal cancer cell membranes: (1) According to 3×10 4 At a seeding rate of cells / well, shUSP5 colorectal cancer cells and their corresponding unmodified colorectal cancer cells were seeded into 96-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. Then, 2 μg of nigra was added per well, followed by 0.6 μg of HMGB1 OE plasmid or pLV-CMV plasmid (vector). Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h, with colorectal cancer cells transfected with pLKO.1-shNC plasmid serving as a control.

[0102] (2) The culture supernatant obtained after treatment (1) was used to detect the level of lactate dehydrogenase (LDH). The results are as follows: Figure 72 and 73 As shown.

[0103] Depend on Figure 72 and 73 The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, the LDH level in the culture supernatant of shUSP5 colorectal cancer cells was reduced, indicating that knockdown of USP5 inhibited LDH release and enhanced the membrane integrity of colorectal cancer cells; while HMGB1 overexpression restored LDH release in shUSP5 colorectal cancer cells.

[0104] 4. Correlation between the USP5 / HMGB1 signaling axis and the expression levels of pro-inflammatory cytokines in colorectal cancer cells: (1) According to 3×10 4At a seeding rate of cells / well, shUSP5 colorectal cancer cells were seeded into 96-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. Then, 2 μg of nigra was added per well, followed by 0.6 μg of HMGB1 OE plasmid or pLV-CMV plasmid (vector) per well. Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h, with colorectal cancer cells transfected with pLKO.1-shNC plasmid serving as a control.

[0105] (2) Total RNA was extracted from the cells obtained after (1) treatment using the RNA isolater Total RNA Extraction Reagent according to the instructions.

[0106] (3) The total RNA obtained after (2) was reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit according to the instructions.

[0107] (4) The cDNA obtained in (3) was qPCR performed on an ABI 7300 Plus real-time PCR instrument using ChamQ SYBR Color qPCR Master Mix according to the instructions. The relative mRNA levels of cytokines IL-18, IL-1β and TNF-α were calculated using the 2-ΔΔCt method in the dark. The results are as follows. Figures 74-77 As shown.

[0108] Depend on Figures 74-77 The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, the expression levels of IL-18, IL-1β and TNF-α in shUSP5 colorectal cancer cells were reduced; while HMGB1 overexpression increased the expression levels of IL-18, IL-1β and TNF-α in shUSP5 colorectal cancer cells.

[0109] 5. Correlation between the USP5 / HMGB1 signaling axis and the expression levels of pyroptosis-related proteins in colorectal cancer cells: (1) According to 5×10 5At a seeding rate of 1 cell / well, shUSP5 colorectal cancer cells and their corresponding unmodified colorectal cancer cells were seeded into 6cm culture plates containing 10% fetal bovine serum and 1× penicillin-streptomycin antibiotics in RPMI 1640 or DMEM medium. Then, 2 μg of nigra was added per well, followed by 0.6 μg of HMGB1 OE plasmid or pLV-CMV plasmid (vector) per well. Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h, with colorectal cancer cells transfected with pLKO.1-shNC plasmid serving as a control.

[0110] (2) The cells obtained after (1) were lysed and homogenized using RIPA lysis buffer according to the instructions, and the supernatant was collected by centrifugation.

[0111] (3) Pierce TM The protein concentration of the protein solution obtained by treatment (2) was tested according to the instructions of the BCA protein quantitative kit.

[0112] (4) Referring to the method provided in "I. High Expression of USP5 and HMGB1 in Colorectal Cancer Tissues" in Example 1, "3. Western Blotting", the levels of USP5, HMGB1, and pyroptosis-related proteins in USP-overexpressing cells were detected, and the results are as follows: Figure 78 As shown.

[0113] Depend on Figure 78 The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, the expression of pyroptosis-related proteins such as NLRP3, ASC, cleaved caspase 1, and GSDMD-N was downregulated in shUSP5 colorectal cancer cells; while HMGB1 overexpression could restore the expression of the above-mentioned pyroptosis-related proteins in shUSP5 colorectal cancer cells, which means that USP5 can indirectly increase Beclin 1 levels by stabilizing HMGB1.

[0114] 6. Correlation between the USP5 / HMGB1 signaling axis and the expression levels of autophagy markers in colorectal cancer cells: (1) According to 5×10 5At a seeding rate of cells / well, shUSP5 colorectal cancer cells and their corresponding unmodified colorectal cancer cells were seeded into 96-well plates containing 10% fetal bovine serum and 1× penicillin-streptomycin in RPMI 1640 or DMEM medium. Then, 2 μg of nigra was added per well, followed by 3 μg of HMGB1 OE plasmid or pLV-CMV plasmid (vector) per well. Transfection was performed using ExFect Transfection Reagen according to the manufacturer's instructions for 48 h, with the corresponding colorectal cancer cells transfected with pLKO.1-shNC plasmid as a control.

[0115] (2) The cells obtained after (1) were lysed and homogenized using RIPA lysis buffer according to the instructions, and the supernatant was collected by centrifugation.

[0116] (3) Pierce TM The protein concentration of the protein solution obtained by treatment (2) was tested according to the instructions of the BCA protein quantitative kit.

[0117] (4) Referring to the method provided in "I. High Expression of USP5 and HMGB1 in Colorectal Cancer Tissue" in Example 1, "3. Western Blotting", the levels of USP5, HMGB1, and autophagy markers in USP-overexpressing cells were detected, and the results are as follows: Figure 79 As shown.

[0118] Depend on Figure 79 The results show that, compared with shNC colorectal cancer cells introduced with pLKO.1-shNC plasmid, the activity level of the JAK2 / STAT3 pathway, which is closely related to autophagy, is decreased in shUSP5 colorectal cancer cells; while HMGB1 overexpression can restore the activity level of the JAK2 / STAT3 pathway, which is closely related to autophagy, in shUSP5 colorectal cancer cells.

[0119] comprehensive Figures 63-79 The results show that USP5 knockdown can alleviate pyroptosis and autophagy induced by nigrain in colorectal cancer cells; it is speculated that low expression or absence of USP5 induces colorectal cancer cells to enter a low ROS, metabolic dormant state, while overexpression of HMGB1 can restore pyroptosis and autophagy induced by nigrain in USP5-deficient colorectal cancer (CRC) cells.

[0120] III. Improvement and therapeutic effects of USP5 expression inhibitors in colorectal cancer 1. Animal experiment: (1) Twenty 6-week-old female nude mice weighing about 20g were randomly divided into two groups. After acclimatization under 12h light / dark cycle and free access to food and water, the mice were anesthetized with 2% isoflurane and the following operations were performed: (i) shNC group (n=10): according to 1×10 6 The dosage was 1 / mouse. shNC colorectal cancer cells HCT-116 were subcutaneously injected into mice, which were then fed under 12-hour light / dark cycles and free access to food and water. (ii) shUSP5 group (n=10): according to 1×10 6 The dosage was shUSP5 colorectal cancer HCT-116 was administered subcutaneously to mice, which were then fed under 12-hour light / dark cycles and free access to food and water. The day of cell injection was designated as day 0. Tumor size was monitored periodically during the feeding process. Mice were sacrificed at week 7, the tumors were dissected, and images were taken. The results are as follows: Figures 80-82 As shown.

[0121] Depend on Figures 80-82 The results show that, compared with the shNC group mice, the tumors grown in mice that underwent xenografting of shUSP5 colorectal cancer cells HCT-116 were significantly smaller in size and weight.

[0122] (3) Half of the tumors obtained after treatment (2) were fixed with 4% PFA, then dewaxed and hydrated using a fully automated staining machine. Next, the tumors were treated with 3% hydrogen peroxide to block endogenous peroxidase. Then, the tumors were antigen-repaired using an antigen retrieval instrument according to the instructions. Finally, the tumors were blocked with 5% goat serum, followed by hematoxylin-eosin staining and Ki-67 immunochemical detection. The results are as follows: Figures 83-85 As shown.

[0123] Depend on Figures 83-85 The results show that, compared to the shNC group mice, mice that underwent xenografting of shUSP5 colorectal cancer cells HCT-116 showed a reduced proportion of tumor cells and an increased proportion of stromal cells in their tumors, and Ki-67... + The reduced cell percentage indicates decreased cell division, suggesting that inhibiting the USP5 / HMGB1 signaling axis can effectively suppress the growth and division of tumor cells, thereby inhibiting the growth of colorectal cancer tissue.

[0124] (4) The ROS level in the tumors obtained after (2) treatment was detected using the DCFDA / H2DCFDA cell ROS detection kit according to the instructions. The results are as follows: Figure 86 and 87 As shown.

[0125] Depend on Figure 86 and 87The results show that, compared with the shNC group mice, the tumors grown in mice that underwent xenografting of shUSP5 colorectal cancer cells HCT-116 had lower ROS levels.

[0126] (5) Referring to the method provided in “4. Correlation between USP5 / HMGB1 signal axis and expression level of pro-inflammatory cytokines in colorectal cancer cells”, the expression level of cytokines in the tumor obtained after (2) treatment was detected, and the results are as follows: Figure 88 As shown.

[0127] Depend on Figure 88 The results show that, compared with the shNC group mice, the expression levels of pro-inflammatory cytokines TNF-α, IL-18, and IL-1β in the tumors grown in mice that underwent xenografting of shUSP5 colorectal cancer cells HCT-116 were significantly reduced. In other words, by inhibiting the USP5 / HMGB1 signaling axis, the inflammation level of tumor tissue can be reduced.

[0128] (6) Referring to the methods provided in “5. Correlation between the USP5 / HMGB1 signaling axis and the expression level of pyroptosis-related proteins in colorectal cancer cells” and “6. Correlation between the USP5 / HMGB1 signaling axis and the expression level of autophagy markers in colorectal cancer cells”, the expression levels of pyroptosis-related proteins and autophagy markers in the tumors treated in (2) were detected, and the results are as follows: Figure 89 and 90 As shown.

[0129] Depend on Figure 89 and 90 The results show that, compared with the shNC group mice, the tumors grown in mice that underwent xenografting of shUSP5 colorectal cancer cells HCT-116 showed decreased expression levels of pyroptosis markers such as NLRP3, lysed caspase 1, ASC, and GSDMD-N, as well as autophagy markers Beclin 1 and LC3 II / I, while the expression level of p62 was increased. In other words, by inhibiting the USP5 / HMGB1 signaling axis, the pyroptosis and autophagy of colorectal cancer cells can be suppressed.

[0130] comprehensive Figures 40-90The results show that the in vivo experimental data are consistent with the in vitro experimental data, further confirming the correlation between the USP5 / HMGB1 signaling axis and colorectal cancer growth, pyroptosis, and autophagy. USP5 increases the level of HMGB1 protein in cells by reducing K48-linked ubiquitination of HMGB1 protein, thereby exerting its pro-cancer effect in colorectal cancer by promoting tumor growth, pyroptosis, and autophagy. Targeting and inhibiting the USP5 / HMGB1 signaling axis has anti-cancer effects by inhibiting tumor growth, pyroptosis, and autophagy, which is a highly promising direction for the development of drugs for the treatment of colorectal cancer.

[0131] 2. Organoid experiment: (1) Three human colorectal cancer tissues (approximately 0.25 cm³ to 1 cm³) were surgically collected, washed with 1% streptomycin-bromide PBS buffer (0.2 M, pH=7.4), and then cut into pieces approximately 1 mm in size. 3 The fragments were incubated with a preheated digestion solution at 37°C, and repeatedly pipetted during incubation until most of the tissue was dissociated. The fragments were then filtered through a 70μm filter and digested and filtered repeatedly until the cell clusters were 5-15μm in diameter.

[0132] (2) The cells obtained by (1) were resuspended in liquid Matrigel and then seeded into 24-well plates at a rate of 35 μL / well. The plates were allowed to stand at 37°C for 20 min to solidify. Then, 500 μL of complete colorectal cancer organoid culture medium containing 10 μM Y27632 was added and cultured at 37°C and 5% CO2 saturated humidity. The medium was changed every 2 days for a total of 10 days.

[0133] (3) Take the stable and proliferating primary colorectal cancer organoids obtained by (2) treatment, discard the culture medium, add PBS buffer (0.02M, pH=7.4) at 1mL / well to dissolve Matrigel, then transfer to a 1.5mL centrifuge tube, centrifuge at 200×g for 3min, and collect the organoid pellet; take 1mL of digestion solution to resuspend the organoid pellet, gently pipette 5-8 times to loosen the clumps, incubate at 37℃ for 5min, then gently pipette 10 times again, centrifuge at 200×g for 3min, and collect the cell pellet; resuspend the cell pellet in 1mL of DMEM / F12 basal culture medium, gently pipette to obtain small cell pellets, centrifuge at 200×g for 3min, collect the cell pellet, and resuspend it in 500μL of complete colorectal cancer organoid culture medium.

[0134] (4) Take 50 μL of shUSP5 lentivirus (prepared by Xiamen Anhera Biotechnology Co., Ltd., with PLKO.1 backbone; the primers used to construct this lentivirus are shown in Table 3, and the titer is 1×10⁻⁶). 7After mixing the cell suspension obtained by (3) with the cell cluster suspension obtained by (3), the mixture was added to a 24-well low-adsorption plate and incubated for 24 h. After centrifugation, the precipitate was resuspended in Matrigel and then seeded into a 24-well plate. After solidification at 37 °C for 20 min, 1 μg / mL of puromycin solution was added at a rate of 0.5 μL / well. The medium was changed every 2 days for 10 days. The shNC lentivirus (prepared by Xiamen Anhela Biotechnology Co., Ltd., with a backbone of PLKO.1, and the primers used to construct the lentivirus are shown in Table 3, with a titer of 1×10⁻⁶) was used. 7 ( ) as a comparison.

[0135] Table 3.

[0136] (5) The organoids obtained after (4) were photographed using an electron microscope, and the results are as follows: Figure 91 and 92 As shown.

[0137] Depend on Figure 91 and 92 The results show that, compared with organoids treated with shNC lentivirus, USP5-deficient organoids treated with shUSP5 lentivirus showed less size variation, indicating that USP5 expression inhibitors can inhibit the growth of human colorectal cancer organoids by suppressing the USP5 / HMGB1 signaling axis.

[0138] (6) The ATP levels of the organoids obtained after treatment (4) were detected using an ATP assay kit according to the instructions. The results are as follows: Figure 93 As shown.

[0139] Depend on Figure 93 The results show that, compared with organoids treated with shNC lentivirus, the ATP level in USP5-deficient organoids treated with shUSP5 lentivirus is reduced, which means that the cell activity of USP5-deficient organoids is reduced.

[0140] (7) Organoids obtained from (4) were fixed with 4% PFA, dewaxed and hydrated using an automated staining machine, and then treated with 3% hydrogen peroxide to block endogenous peroxidase. Following antigen retrieval using an antigen retrieval instrument and according to the instructions, the colorectal cancer tissue was blocked with 5% goat serum. Ki-67 and CD45 were then detected using histochemical methods. The results are as follows: Figures 94-97 As shown.

[0141] Depend on Figures 96-97The results show that, compared with organoids treated with shNC lentivirus, the expression levels of the proliferation marker Ki-67 and the oncogene CD45 were decreased in USP5-deficient organoids treated with shUSP5 lentivirus, indicating that the proliferation capacity of USP5-deficient organoids was significantly reduced.

[0142] (8) The expression levels of cytokines in the organoids obtained after treatment (4) were detected using the method provided in “4. Correlation between the USP5 / HMGB1 signaling axis and the expression levels of pro-inflammatory cytokines in colorectal cancer cells”. The results are as follows: Figures 98-100 As shown.

[0143] Depend on Figures 99-100 The results show that, compared with organoids treated with shNC lentivirus, the expression of cytokines IL-18, IL-1β and TNF-α was reduced in USP5-deficient organoids obtained after treatment with shUSP5 lentivirus.

[0144] (9) Add 1 μmol / L of nigra to the organoids obtained after treatment (4). Then, refer to the method provided in "5. Correlation between the USP5 / HMGB1 signal axis and the expression level of pyroptosis-related proteins in colorectal cancer cells" to detect the expression levels of HMGB1 and pyroptosis-related proteins in the organoids. The results are as follows. Figure 101 As shown.

[0145] Depend on Figure 101 The results show that, compared with organoids treated with shNC lentivirus, the expression of HMGB1 protein and pyroptosis-related proteins was downregulated in USP5-deficient organoids obtained after treatment with shUSP5 lentivirus.

[0146] (10) Add 1 μmol / L of nigra to the organoids obtained after (4) treatment and then treat them. Then, according to the method provided in "6. Correlation between USP5 / HMGB1 signal axis and autophagy marker expression level in colorectal cancer cells", the expression level of autophagy markers in the organoids was detected. The results are as follows: Figure 102 As shown.

[0147] Depend on Figure 102 The results show that, compared with the organoids treated with shNC lentivirus, the expression of NLRP3, Caspase 1, ASC, GSDMD-N, Beclin 1, and LC3 II / I was reduced, while the expression of p62 was upregulated in the USP5-deficient organoids obtained after treatment with shUSP5 lentivirus.

[0148] comprehensive Figures 91-102The results show that the USP5 / HMGB1 signaling axis plays an important role in promoting tumorigenesis in human CRC organoids and regulating pyroptosis and autophagy induced by nigericin. USP5 expression inhibitors also showed good improvement and treatment effects in human CRC organoids.

[0149] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. Application of USP5 as a target in the preparation of formulations that regulate the degradation of HMGB1 protein.

2. The application according to claim 1, characterized in that, The specific applications include: Preparation of formulations that regulate USP5-mediated HMGB1 de-K48-linked ubiquitination; And / or, to prepare formulations that regulate the level of HMGB1 protein in colorectal cancer cells.

3. Application of USP5 inhibitors in the preparation of formulations that promote the degradation of HMGB1 protein.

4. The application according to claim 3, characterized in that, The USP5 inhibitors include: Substances that knock down or silence the expression of the USP5 gene; And / or substances that reduce or inhibit the activity of the USP5 protease.

5. The application of the USP5 inhibitor according to claim 4 in the preparation of an HMGB1 protein ubiquitination degradation promoter, characterized in that, The substances that knock down or silence USP5 gene expression include: siRNA, shRNA, or sgRNA targeting the USP5 gene; And / or, a vector carrying the siRNA, shRNA, and / or sgRNA targeting the USP5 gene.

6. The application of the USP5 / HMGB1 signal axis as a target in the preparation of drugs for the treatment of colorectal cancer, characterized in that, The applications include: Prepare drugs that regulate USP5-mediated HMGB1 de-K48-linked ubiquitination; To prepare a drug that regulates the level of HMGB1 protein in colorectal cancer cells; And / or, to prepare drugs that regulate the proliferation, migration, invasion, pyroptosis, or autophagy of colorectal cancer cells mediated by the USP5 / HMGB1 / JAK2 / STAT3 signaling axis.

7. A predictive marker for colorectal cancer, characterized in that, The prediction markers include: USP5 gene and its mRNA and expressed protein, and its antibody; HMGB1 gene and its mRNA and expressed protein, and its antibody; And / or, K48-linked ubiquitinated HMGB1 protein and its antibody.

8. A colorectal cancer-related diagnostic kit, characterized in that, The detection kit includes the predictive marker as described in claim 7 and / or a substance for detecting the predictive marker.

9. A method for screening drugs for the treatment of colorectal cancer, characterized in that, The screening method includes screening based on the regulatory effects of candidate molecules on the USP5 / HMGB1 / JAK2 / STAT3 signaling axis.

10. The method for screening drugs for the treatment of colorectal cancer according to claim 9, characterized in that, The screening method includes: treating colorectal cancer cells with the candidate molecules, detecting the changes in USP5 expression level, HMGB1 expression level and / or HMGB1 ubiquitination modification level in colorectal cancer cells, and evaluating the effects of the candidate molecules on the activity, proliferation, invasion, spread, autophagy and / or pyroptosis of colorectal cancer cells based on the changes.

Images