Application of colon cancer marker and colon cancer diagnosis kit

Abstract

The invention belongs to the technical field of biology, particularly relates to application of a colon cancer marker and a colon cancer diagnosis kit, and provides application of a reagent for detecting the expression quantity of JOSD1 in preparation of a colon cancer diagnosis product, application of a reagent for inhibiting the expression quantity of JOSD1 in preparation of a colon cancer treatment product and application of a reagent for inhibiting the expression quantity of JOSD1 in preparation of a colon cancer diagnosis product. According to the present invention, the JOSD1 is determined to influence the growth of the Hippo signal and the colon cancer, and the forward feedback between the Hippo pathway and the JOSD1 is determined so as to drive the progress of the colorectal cancer, the JOSD1 is determined to influence the growth of the Hippo signal and the colon cancer, and the forward feedback between the Hippo pathway and the JOSD1 is determined so as to drive the progress of the colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of a colon cancer marker and a kit for colon cancer diagnosis. Background Art

[0002] Colon cancer is a common malignant tumor. Although local colon cancer can be well treated by surgery, advanced colon cancer is always accompanied by lymph node invasion and metastasis, resulting in a low survival rate. With the application of new target therapies and immunotherapies, a small number of advanced colon cancer patients can benefit from extended survival. However, effective treatment targets in colon cancer are still limited, and cancer biologists are urgently in need of clarifying the driving pathways in colon cancer progression and searching for new treatment targets.

[0003] The Hippo signaling pathway was initially discovered in Drosophila, where it plays an important role in the development of the eye disc and wings. The Hippo signaling pathway involves a series of phosphorylation kinases, such as MST1 / 2, LATS1 / 2, MAP4K, and NF2. When upstream stimuli trigger the Hippo signaling pathway, the phosphorylated kinases MST1 / 2 induce the phosphorylation of LATS1 / 2, which in turn phosphorylates YAP at multiple serine / threonine sites, preventing its nuclear localization. This phosphorylates YAP at multiple serine / threonine sites and prevents its nuclear localization. The phosphorylated YAP protein interacts with certain E3 ubiquitin ligases (such as β-TrCP), leading to protein degradation. Conversely, when the Hippo pathway is inactivated, the unphosphorylated YAP protein migrates to the nucleus, where they interact with transcription factors such as TEADs, inducing the expression of Hippo target genes and promoting carcinogenesis. A large number of studies have revealed abnormalities in the Hippo pathway in human malignancies, including colon cancer. For example, increased expression of YAP protein has been found in human colon cancer samples, and its expression has been observed to be related to local invasion and distant metastasis. Clinical survival statistics indicate that YAP expression is associated with a low survival rate in colon cancer patients. Various mechanism studies have shown that YAP plays a crucial role in the occurrence and development of colon cancer, and the growth and invasion of colon cancer cells can be inhibited by inhibiting YAP. To date, based on human understanding of Hippo signaling in colon cancer, targeting YAP is considered a valuable cancer treatment strategy.

[0004] Uncontrolled activation of the Hippo / YAP axis is a prominent feature of colon cancer, and inhibiting the ubiquitination cascade remains effective. Recent studies have pointed out that the processes of ubiquitination and deubiquitination play a crucial role in regulating the stability and function of YAP protein. The human genome contains approximately 100 deubiquitinating enzymes. It is not yet clear which of these deubiquitinating enzymes can affect Hippo signaling and the growth of colon cancer. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an application of a colon cancer marker and a kit for colon cancer diagnosis, and the colon cancer marker is JOSD1.

[0006] Any one of the following applications:

[0007] Application of a reagent for detecting the expression level of JOSD1 in the preparation of a colon cancer diagnostic product;

[0008] Application of a reagent for inhibiting the expression level of JOSD1 in the preparation of a colon cancer treatment product;

[0009] The amino acid sequence of JOSD1 is: MSCVPWKGDKAKSESLELPQAAPPQIYHEKQRRELCALHALNNVFQDSNAFTRDTLQEIFQRLSPNTMVTPHKKSMLGNGNYDVNVIMAALQTKGYEAVWWDKRRDVGVIALTNVMGFIMNLPSSLCWGPLKLPLKRQHWICVREVGGAYYNLDSKLKMPEWIGGESELRKFLKHHLRGKNCELLLVVPEEVEAHQSWRTDV, denoted as SEQ ID NO.19.

[0010] Preferably, the diagnostic product is a reagent for detecting the expression level of JOSD1.

[0011] Preferably, the reagent for detecting the expression level of JOSD1 includes a reagent for detecting the expression level of JOSD1 protein in a sample by using proteomic sequencing technology.

[0012] Preferably, the reagent for detecting the expression level of JOSD1 includes a reagent for detecting the expression level of JOSD1 gene in a sample by using sequencing technology, probe hybridization technology, gene chip technology or fluorescence quantitative PCR technology.

[0013] Preferably, the treatment product increases the total ubiquitination level of YAP.

[0014] Preferably, the treatment product increases the k48-linked polyubiquitination level.

[0015] Preferably, the treatment product includes a primer sequence for knocking out JOSD1, and the primer sequence is the nucleotide sequence shown in any one of SEQ ID NO.9-10 or SEQ ID NO.11-12.

[0016] A kit for colon cancer diagnosis, the kit includes a reagent for detecting the expression level of JOSD1 as described in claim 1 in a sample.

[0017] Preferably, the sample is from a colon cancer patient, a suspected colon cancer patient, a colon cancer susceptible population, a colon cancer high-risk population or a healthy population.

[0018] Preferably, the sample is tissue.

[0019] The present invention concludes that JOSD1 is related to the genetic characteristics of the Hippo signal and is associated with a low survival rate of colon cancer. The study found that JOSD1 enhances Hippo / YAP activity by regulating the K48-linked deubiquitination of YAP, thereby promoting the progression of colon cancer. In addition, the study also found that YAP upregulates the expression of JOSD1, indicating that JOSD1 and the Hippo signal reinforce each other in a positive feedback loop ( Figure 8 ). These findings highlight JOSD1 as a potential therapeutic target for colorectal cancer treatment and reveal a non-genomic regulatory mechanism of the Hippo signal through the newly discovered positive feedback loop. JOSD1 (Josephin Domain-Containing 1) is a putative deubiquitinase belonging to the MJD family of proteases containing the Machado-Josephin domain. The link between Josephin family DUBs and human diseases was first discovered in the neurodegenerative disease Machado-Joseph disease, in which another MJD member, ATXN3, reduces its expression, promoting Machado-Joseph. Although JOSD1 has a high amino acid sequence similarity to ATXN3, there is currently no evidence of its association with neurodegenerative diseases. However, recent studies have emphasized the role of JOSD1 in human malignancies. For example, JOSD1 gene amplification has been observed in uterine cancer and melanoma. It has been reported that JOSD1 can stabilize MCL1 and inhibit mitochondrial-dependent apoptosis, thereby promoting chemoresistance in leukemia. Further studies have shown that targeting JOSD1 with small molecules may be a promising strategy for treating patients with JAK2-mutated leukemia. In solid tumors, JOSD1 has been found to stabilize Snail and promote lung cancer progression. However, how JOSD1 functions, especially in gastrointestinal malignancies, remains to be elucidated. The present invention reveals a new regulatory mechanism by which JOSD1 regulates the Hippo pathway in colon cancer, proposes key regulatory elements of JOSD1 in the Hippo pathway, and reveals the interaction between the DUB family and the Hippo pathway.

[0020] The connection between the Hippo signal and colon cancer has been established for decades. Targeting YAP function shows promise in colon cancer treatment. Previous studies have shown that inhibiting the interaction between YAP and TEAD is a viable strategy for treating Hippo-driven cancers. However, drugs that inhibit the Hippo / YAP pathway, such as verteporfin and Super-TDU, have not been successful in clinical trials for Hippo-driven cancers. One of the main reasons for this failure is that the inhibitors cannot penetrate the cell membrane and block the interaction of membrane proteins. In addition, the small peptide Super-TDU may be attacked by plasma proteases and neutralizing antibodies. Due to the limitations of YAP-targeted drugs, the focus of this invention has shifted to the development of new drugs that target the stability of YAP protein in colon cancer. Considering the important role of JOSD1 in colon cancer, blocking JOSD1 may be a viable strategy for treating colon cancer.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention determines that JOSD1 can affect the Hippo signal and the growth of colon cancer, determines the positive feedback between the Hippo pathway and JOSD1, thereby driving the progression of colorectal cancer. Blocking JOSD1 and blocking the positive cycle may be a promising strategy for colon cancer treatment, and blocking the YAP / TEAD interaction may be challenging in cancer treatment. Therefore, reactivating the Hippo pathway in colon cancer by targeting the stability of YAP protein may be feasible. Brief Description of the Drawings

[0023] Figure 1JOSD1 is associated with Hippo / YAP gene markers in colon cancer samples; among them, A: 10 DUBs closely related to the Hippo pathway were screened by GSEA and their 8×GTIIC luciferase reporter activity was tested; B: Gene Set Enrichment Analysis (GSEA) data input in TCGA showed a highly significant positive correlation between JOSD1 and YAP target genes; C: Through RNA-seq data analysis, the top 10 KEGG pathways enriched by JOSD1 deletion in HCT116 cells were determined, with a threshold of P < 0.05; D: GSEA analysis showed that after JOSD1 deletion in HCT116 cells, the YAP target gene signal was downregulated; E: The volcano plot showed that JOSD1 deletion inhibited the expression of Hippo signaling genes (highlighted in red) in HCT116 cells, and the threshold standard for this analysis was set at P < 0.05, fold change > 1.5; F: A heat map was generated to show that siJOSD1 in HCT116 cells significantly reduced differentially expressed genes related to the Hippo signaling pathway; G: Kaplan-Meier analysis showed that JOSD1 expression was associated with a lower progression-free survival in colorectal cancer patients; H: Immunohistochemical analysis of colorectal cancer specimens showed a positive correlation between JOSD1 and YAP (P < 0.001), and I was the result of the positive correlation between JOSD1 and YAP;

[0024] Figure 2It is shown that JOSD1 is an important regulator of colorectal cancer cell progression; among them, A: Immunoblot analysis of the expression levels of related proteins with JOSD1 knocked down in HCT116 cells; B: Immunoblot analysis of the expression levels of related proteins with JOSD1 knocked down in SW480 cells; C: qRT-PCR detection of the mRNA expression level with JOSD1 knocked down in HCT116 cells; D: qRT-PCR detection of the expression level of JOSD1 after JOSD1 was knocked down in SW480 cells, with β-Actin as the internal control; E: It is shown that the deletion of JOSD1 inhibits the proliferation of colorectal cancer cell HCT116; F: It is shown that the deletion of JOSD1 inhibits the proliferation of colorectal cancer cell SW480. The experiment was repeated three times, and the cell growth was statistically significant; G: Results of the EdU experiment with JOSD1 knocked down in HCT116; H: Statistical results of the EdU experiment with JOSD1 knocked down in HCT116; I: Results of the transwell experiment with JOSD1 knocked down in SW480; J: Statistical results of the transwell experiment with JOSD1 knocked down in SW480; K: The deletion of endogenous JOSD1 significantly reduces the invasion of cell HCT116; L: Statistical results of the reduction of cell HCT116 invasion by the deletion of endogenous JOSD1; M: The deletion of endogenous JOSD1 significantly reduces the invasion of cell SW480; N: Statistical results of the reduction of cell SW480 invasion by the deletion of endogenous JOSD1; O: The deletion of JOSD1 promotes the apoptosis of HCT116 cells; P: Statistical results of the promotion of HCT116 cell apoptosis by the deletion of JOSD1; Q: The deletion of JOSD1 promotes the apoptosis of SW480 cells; R: Statistical results of the promotion of SW480 cell apoptosis by the deletion of JOSD1; S-V indicate that the deletion of JOSD1 or siccontrol transfection inhibits the growth of colorectal tumors in vivo. S: The knockout of JOSD1 affects the migration ability of HCT116 cells, T: The knockout of JOSD1 affects the migration ability of HCT116 cells, U: The knockout of JOSD1 affects the migration ability of SW480 cells, V: The knockout of JOSD1 affects the migration ability of SW480 cells; W-Y: Inhibition of JOSD1 affects the growth of xenograft tumors. W: Comparison chart of tumor growth, X: Statistical chart of tumor volume, Y: Statistical chart of tumor weight; Z: Analysis of xenograft tumors shows that the deletion of JOSD1 leads to a decrease in the expression of the cell proliferation marker Ki67 in tumors, and the number of Ki67-positive cells in the right figure is significantly reduced; All data are expressed as mean±SEM, one-way ANOVA *P<0.05, **P<0.01, ***P<0.001;

[0025] Figure 3JOSD1 was shown to activate the Hippo / YAP axis in colon cancer cells; among which, A: HCT116 cells were transfected with sicontrol or siJOSD1 targeting sequences. Subsequently, 48 hours after transfection, the cells were collected for western blot analysis. Then, Western blotting was performed to evaluate the protein levels of JOSD1 and YAP. Actin was used as an internal control throughout the experiment. The results of these analyses showed that the protein level of YAP decreased after the deletion of JOSD1 in HCT116 cells; B: It was observed that knockdown of JOSD1 led to a decrease in the protein level of YAP in SW480 cells. To determine the protein level, western blot analysis was performed on SW480 cells transfected with sicontrol or siJOSD1 and harvested 48 hours later. β-Actin was used as the internal reference, and western blotting was used to detect the protein levels of JOSD1 and YAP; C-D: qRT-PCR analysis showed that the deletion of JOSD1 decreased the stability of YAP protein but did not decrease the expression of YAP mRNA; HCT116 and SW480 cells were transfected with 50 nM sicontrol or 50 nM JOSD1. C: qRT-PCR analysis results of HCT116, D: qRT-PCR analysis results of SW480; E: Deletion of JOSD1 could reduce the expression of Hippo target genes in HCT116 cells. To analyze gene expression, HCT116 cells were transfected with sicontrol or siJOSD1, and total RNA was extracted 48 hours later. Each group was detected with 3 replicates; F: Deletion of JOSD1 could reduce the expression of Hippo target genes in SW480 cells. To analyze gene expression, SW480 cells were transfected with sicontrol or siJOSD1, and total RNA was extracted 48 hours later. Each group was detected with 3 replicates; G: By transfecting sicontrol or siJOSD1 and then transfecting the TEAD luciferase reporter plasmid, JOSD1 was depleted, and the TEAD luciferase activity in HCT116 cells decreased; H: After the deletion of JOSD1, the TEAD luciferase activity in SW480 cells decreased; I: Schematic diagram of JOSD1 plasmid and its mutant JOSD1C36A plasmid; J: Overexpression of JOSD1 led to a significant increase in the expression of Hippo target genes, while overexpression of JOSD1C36A did not have the same effect; K: Overexpression of JOSD1 led to an increase in the protein level of YAP, while overexpression of JOSD1C36A did not have the same effect; L: In HEK293T cells, overexpression of JOSD1 increased the activity of TEAD luciferase, while JOSD1C36A did not; All data were expressed as mean±SEM, one-way ANOVA *P<0.05, **P<0.01, ***P<0.001；.

[0026] Figure 4It is shown that JOSD1 promotes the progression of colon cells through the Hippo / YAP axis; among them, A: The deletion of JOSD1 reduces the level of YAP protein, which is then restored by overexpressing YAP protein. HCT116 cells were transfected with sicontrol or siJOSD1, and then another round of transfection was performed with Flag-YAP or Flag vector after 24 hours. Subsequently, cells were harvested after 48 hours for western blot analysis. Actin was used as an internal control to evaluate the expression levels of JOSD1 and YAP proteins; B: Deletion of JOSD1 inhibits the expression of Hippo target genes, and YAP overexpression reverses this effect; SW480 cells were transfected with sicontrol or siJOSD1, and then another round of transfection was performed with Flag-YAP or Flag vector after 24 hours. Subsequently, cells were harvested after 48 hours for western blot analysis. Actin was used as an internal control to evaluate the expression levels of JOSD1 and YAP proteins; C: Deletion of JOSD1 reduces the TEAD luciferase activity of HCT116 cells, and YAP overexpression reverses this effect; D: The growth of HCT116 cells was measured by the CCK-8 method, and the deletion of JOSD1 inhibited their proliferation ability; however, this effect could be reversed by overexpressing YAP; E-F show that the deletion of JOSD1 reduces the number of EdU-positive colorectal cancer cells, and YAP overexpression further rescues this effect. The cell proliferation activity is represented by the absolute cell number. E: The deletion of JOSD1 reduces the number of EdU-positive colorectal cancer cells HCT116, F: The deletion of JOSD1 reduces the number of EdU-positive colorectal cancer cells SW480; G-H: Depletion of JOSD1 reduces the migration ability of colorectal cancer cells, and YAP overexpression reverses this effect. G: The deletion of JOSD1 reduces the number of EdU-positive colorectal cancer cells HCT116, H: Wound healing results of HCT116; I-J: Depletion of JOSD1 reduces the invasion ability of colorectal cancer cells, and YAP overexpression reverses this effect. The average cell number was calculated and the standard deviation was given. I: Results of migrating cells J: Knockout of JOSD1 promotes apoptosis of colon cancer cells, and the transfer of YAP reverses this trend; K-L: FACs was used to detect apoptosis of HCT116 cells. Deletion of JOSD1 promotes apoptosis of HCT116 cells, while YAP overexpression weakens this effect. K: Results of migrating cells, L: Shows the quantitative summary of the apoptosis analysis using FACS; M-O: In vivo, YAP overexpression rescues the growth of xenograft tumors of colon cancer cells transfected with siJOSD1. Figures M, N, and O are the tumor growth curve, weight, and photo respectively; P: Immunohistochemical staining of xenografts shows the levels of JOSD1, YAP, and Ki67. The quantitative results of Ki67-positive cells are shown as follows; All data are expressed as mean ± SEM; One-way ANOVA *P<0.05, **P < 0.01, ***P < 0.001;.

[0027] Figure 5 It is shown that JOSD1 is related to YAP and regulates the stability of YAP protein; among them, A: Immunofluorescence staining was used to analyze the intracellular localization of JOSD1 and YAP in HCT116 cells. The results showed the localization of JOSD1 (green) and YAP (red), and the cell nucleus was stained blue with DAPI; B: In HCT116 cells, JOSD1 and YAP were mainly located in the cell nucleus; C: In HCT116 cells, the Co-IP experiment showed that endogenous JOSD1 and YAP were related, and one antibody was used for Co-IP; D: In the presence of the proteasome inhibitor MG132, knockdown of JOSD1 did not lead to further degradation of YAP; E: When the proteasome inhibitor MG132 was present, the stabilizing effect of JOSD1 on YAP did not cause an additional increase in the YAP protein level; F-G: In HEK293T cells, overexpression of JOSD1 prolonged the half-life of YAP, while overexpression of JOSD1C36A did not. F: In HEK293T cells, overexpression of JOSD1 prolonged the half-life of YAP, while overexpression of JOSD1C36A did not. G: Gray value quantification of the YAP half-life; H-I: Deletion of JOSD1 reduced the half-life of YAP protein in HCT116 cells. H: It was shown that the half-life of YAP protein became shorter with the knockdown of JOSD1. I: Quantitative analysis; J-K: Schematic diagrams showing the wild-type and truncated YAP and JOSD1 structures. J: YAP, K: JOSD1; L-M: Immunoblotting showed that JOSD1 interacted with WT or truncated YAP through JOSD1 immunoprecipitation (anti-Flag), and YAP interacted with WT or truncated JOSD1 through YAP immunoprecipitation (anti-Myc). L: JOSD1, M: YAP; All data are expressed as mean ± SEM; One-way ANOVA *P < 0.05, **P < 0.01, ***P < 0.001;

[0028] Figure 6It is shown that JOSD1 stabilizes YAP by inhibiting K48-linked ubiquitination of YAP; among them, A: JOSD1 reduces YAP ubiquitination; after treating HEK-293T cells with MG132 for 6 hours, transfected with 2 μg YAP plasmid, 0.5 μg HA-Ub plasmid and 0.5 μg Flag-tag or Flag-JOSD1 plasmid, subsequently, immunoblotting the cells with specific antibodies, JOSD1 reduced the ubiquitination of YAP; B-C: JOSD1 reduces YAP ubiquitination, after treating HEK-293T cells with MG132 for 6 hours, transfected with 2 μg YAP plasmid, 0.5 μg HA-K48-Ub or HA-K48R-Ub plasmid and 0.5 μg Flag-tag or Flag-JOSD1 plasmid, subsequently, immunoblotting the cells with specific antibodies, YAP was deubiquitinated by JOSD1 via K48-linked ubiquitination, B: transfected with 2 μg YAP plasmid, 0.5 μg HA-K48-Ub plasmid and 0.5 μg Flag-tag, C: transfected with 2 μg YAP plasmid, HA-K48R-Ub plasmid and Flag-JOSD1 plasmid; D: Reducing the level of JOSD1 leads to increased YAP ubiquitination, after treating HCT116 cells with MG132 for 6 hours, transfected HCT116 cells with 0.5 μg HA-Ub plasmid and 20 μM JOSD1 siRNA, immunoblotting with the indicated antibodies; E-F: Deletion of JOSD1 increases K48-related YAP ubiquitination, while K48R does not increase; after treating HCT116 cells with MG132 for 6 hours, transfected HCT116 cells with 0.5 μg HA-K48-Ub or HA-K48R-Ub plasmid and 20 μM siJOSD1, immunoblotting with the indicated antibodies, E: transfected HCT116 cells with 0.5 μg HA-K48-Ub plasmid and 20 μM siJOSD1, F: transfected HCT116 cells with 0.5 μg HA-K48R-Ub plasmid and 20 μM siJOSD1; G-H: The JOSD1 mutant lacking deubiquitinase activity cannot enhance the accumulation of ubiquitinated YAP, transfected HEK-293T cells with 2 μg YAP plasmid, 0.5 μg HA-Ub / HA-K48 Ub plasmid and 0.5 μg Flag-tag or Flag-JOSD1 or Flag-JOSD1C36A, after treating with MG132 for 6 h, immunoblotting with the indicated antibodies; I: JOSD1 deubiquitinates YAP through its N-terminal region, HEK-293T cells were transfected with 2 μg YAP plasmid, 0.5 μg HA-Ub plasmid and 0.5 μg EGFP-Tag full-length or deletion mutant plasmids, after 6 hours of treatment; subsequently, immunoblotting was performed using the indicated antibodies, K: JOSD1 deubiquitinates YAP through its N-terminal region, HEK-293T cells were transfected with 2 μg YAP plasmid, HA-K48 Ub plasmid and EGFP-JOSD1 full-length or deletion mutant plasmids, after 6 hours of treatment; subsequently, immunoblotting was performed using the indicated antibodies;.

[0029] Figure 7It is shown that YAP regulates the expression of JOSD1, forming a positive regulatory loop in YAP signal transduction; among them, A: Analyze the JOSD1 genomic map to study the binding region of YAP and the JOSD1 promoter; B: ChIP analysis shows that YAP binds to the JOSD1 promoter region. After fixation of HCT116 cells for 30 minutes, the DNA was lysed and purified. Rabbit IgG was used as a negative control. The primer sequences can be found in the method section, and then the enriched DNA fragments were analyzed by DNA gel electrophoresis; C-D: HCT116 and SW480 cells were treated with siRNA to delete YAP, and then fixed, lysed, DNA enriched and ChIP-qPCR analysis were performed. The results showed that the binding of YAP to the JOSD1 gene decreased. C: HCT116, D: SW480; E-F: Decreased YAP levels led to a decrease in JOSD1 protein. In HCT116 or SW480 cells, YAP was silenced with siRNA, and then cell lysis and protein extraction were performed. The cell lysates were immunoblotted with specific antibodies, and β-Actin was used as an internal reference. E: HCT116, F: SW480; G-H: Deletion of YAP in HCT116 and SW480 cells inhibited JOSD1 mRNA. After transfection of HCT116 and SW480 cells with sicontrol or siYAP, total RNA was extracted for gene expression analysis. The relative levels of CYR61 and JOSD1 mRNA in each group were detected by qRT-PCR. Each group was repeated 3 times, and statistical significance was determined by comparing the expression levels of the target genes. G: HCT116, H: SW480; I-J: The expression of JOSD1 protein was inhibited in HCT116 and SW480 cells. HCT116 and SW480 cells were treated with the control group and verteporfin, and proteins were extracted and immunoblotted with the specified antibodies. I: HCT116, J: SW480; K-L: Treatment of HCT116 and SW480 cells with the control group and verteporfin led to the inhibition of JOSD1 mRNA. HCT116 and SW480 cells were treated with the vector or VP respectively, and total RNA was extracted for gene expression analysis after 48 hours. Each group was detected in triplicate. K: HCT116, L: SW480; M-N: Treatment with XMU-MP-1 increased the expression of JOSD1 protein in HCT116 and SW480 cells. HCT116 and SW480 cells were treated with the control group and XMU-MP-1, and proteins were extracted and immunoblotted with the specified antibodies. M: HCT116, N: SW480; O-P: Treatment of HCT116 and SW480 cells with XMU-MP-1 increased the expression of JOSD1 mRNA. HCT116 and SW480 cells were treated with the vehicle or XMU-MP-1 respectively, and total RNA was extracted for gene expression analysis after 48 hours. Each group was detected in triplicate. O: HCT116, P: SW480;Q-R: YAP inhibition decreased the expression of JOSD1 in the cell membrane of HCT116 and SW480 cells. Endogenous JOSD1 was labeled green, and the nucleus was stained with DAPI (blue). Scale bar, 20 mm. Q: HCT116, R: SW480; all data are presented as mean ± SEM; one-way ANOVA *P < 0.05, **P < 0.01, ***P < 0.001;

[0030] Figure 8 It is shown that in colorectal cancer, JOSD1 forms a regulatory loop with the Hippo / YAP axis, confirming that JOSD1 regulates YAP signaling and establishes a positive feedback loop. The activation of JOSD1 is thought to increase YAP activity by regulating YAP k48-related deubiquitination, thereby promoting the progression of colorectal cancer. In addition, YAP has been shown to stimulate the expression of JOSD1, thus reinforcing each other between the two factors;

[0031] Figure 9 is the expression of JOSD1. Detailed implementation manners

[0032] The following describes the detailed implementation manners of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the detailed implementation manners. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0033] Cell lines and cell culture: Human cell lines HCT116, SW480, and HEK-293T were obtained from the American Type Culture Collection (ATCC). To verify these cell lines, short tandem repeat (STR) analysis was performed. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, 21063029) supplemented with 10% fetal bovine serum (FBS, Gibco, 12676-029), 1% penicillin-streptomycin-gentamicin solution, 4.5 g / L glucose, and 4 mM L-glutamine. Additionally, 10% fetal bovine serum (FBS), 4.5 g / L glucose, and 4 mM L-glutamine were added to the medium to obtain optimal cell growth and maintenance.

[0034] RNA isolation and quantitative real-time PCR (qRT-PCR)

[0035] Total RNA was extracted using the RNeasy Plus Mini Kit (Tiangen, DP451) according to the manufacturer's protocol, and then reverse transcription was performed using HiScript II Q RT SuperMix (Vazyme, R223-01). Then, qRT-PCR analysis was carried out using SYB R qRT-PCR MasterMix (Vazyme, Q511-02) on a 7500 Fast Real-Time PCR System (apply dBiosystems, Singapore). To normalize the gene expression levels, 36B4 was used as an internal reference.

[0036] The primers used for qRT-PCR were as follows:

[0037] 36B4

[0038] Forward: GGCGACCTGGAAGTCCAACT, denoted as SEQ ID NO.1;

[0039] Forward: CCATCAGCACACAGCCTTC, denoted as SEQ ID NO.2;

[0040] CYR61

[0041] Forward: GGTCAAAGTTACCGGGCAGT, denoted as SEQ ID NO.3;

[0042] Reverse: GGAGGCATCGAATCCCAGC, denoted as SEQ ID NO.4;

[0043] JOSD1

[0044] Forward: GGGATACGCTGCAAGAGATTT, denoted as SEQ ID NO.5;

[0045] Reverse: CCATGACGTTAGTGAGGGCA, denoted as SEQ ID NO.6;

[0046] YAP

[0047] Forward: CAAGAAAGCAGGCTCACAGAA, denoted as SEQ ID NO.7;

[0048] Reverse: GCTGGGTGTTAGGGCTTCG, denoted as SEQ ID NO.8.

[0049] Plasmids and siRNAs

[0050] The plasmids JOSD1 and YAP were from HANBIO (https: / / www.Han-bio.net) and were further subcloned from the full-length plasmid DNA to create deletion constructs. In addition, the established HA-K48 and HA-Ub plasmids were integrated in this study. Plasmid transfection was performed using Lipofectamine 2000 (1,662,298, Invitrogen). Subsequently, specific genes were targeted for knockout by small interfering RNA (siRNA) technology.

[0051] For JOSD1, the siRNA sequences used in the knockout process were as follows:

[0052] (1) CAAGGCCAAAUCUGAAUC, denoted as SEQ ID NO.9,

[0053] and UGAUUCAGAUUUGGCCUU, denoted as SEQ ID NO.10;

[0054] (2) CUAACGUCAUGGGCUUCA, denoted as SEQ ID NO.11,

[0055] and AUGAAGCCCAUGACGUUA, denoted as SEQ ID NO.12.

[0056] The siRNA sequences used for YAP were:

[0057] (1) GUCAGAGAUACUUCUUAA, denoted as SEQ ID NO.13,

[0058] and UUUAAGAAGUAUCUCUGA, denoted as SEQ ID NO.14;

[0059] (2) GUCUCAGGAAUUGAGAAC, denoted as SEQ ID NO.15,

[0060] and UGUUCUCAAUUCCUGAGA, denoted as SEQ ID NO.16.

[0061] The corresponding negative control siRNA sequences were used:

[0062] UUCUCCGAACGUGUCACG, denoted as SEQ ID NO.17,

[0063] and ACGUGACACGUUCGGAGA, denoted as SEQ ID NO.18.

[0064] By inserting shJOSD1 into pLKO, a lentivirus expressing JOSD1 shRNA was obtained. 1 vector was co-transfected with pMD2.1 into HEK293T cells. G envelope plasmid and psPAX2 packaging plasmid. After 48 hours, colon cancer cells were cultured in an antibiotic-free medium with the lentivirus suspension for subsequent experiments.

[0065] Reagents

[0066] The reagents used included XMU-MP-1 (MCE, Cat; NO. HY-100526), CELLSAVING (New Cell and Molecular Biotechnology, C40100 / C40050), MG132 (MCE, HY-13259), cycloheximide (MCE, Cat. NO. HY-12320), and verteporfin (MCE, Cat. NO. HY-B0146).

[0067] The experimental methods involved in the present invention were all carried out according to the following steps:

[0068] Western blot

[0069] After harvesting the cells, they were lysed with RIPA buffer (Beyotime, China) containing protease and phosphatase inhibitors. After lysis, equal amounts of protein were quantified and then separated by 10% SDS-PAGE and then transferred to a PVDF membrane (Millipore, USA). Subsequently, the membrane was blocked with 5% skim milk for 1 hour and then incubated with the primary antibody overnight at 4°C. After washing three times with TBST, the membrane was detected using the ECL system (Bio-rad ChemiDoc), and the image was captured using the Tanon 5200 system (China). The internal control used in this analysis was β-actin. The primary antibodies used were flag antibody (dilution 1:1000, Sigma, #F1804), JOSD1 antibody (dilution 1:1000, Abcam, ab118221), tubulin (11,24-1-ap, Proteintech, 1:1000), anti-histone H3 (17168-1-ap, Proteintech, 1:1000), Myc antibody (1:1000, Abmart, #20002), YAP antibody (dilution 1:5000, #SC101199), anti-β-actin antibody (3700, Cell Signaling Technology, 1:1000). Subsequently, peroxidase-conjugated AffiniPure goat anti-mouse IgG (Beyotime, #A0216) or goat anti-rabbit IgG (Beyotime, #A0208) was used for secondary antibody detection. Color development was achieved using ECL (Meilunbio, #MA0186).

[0070] Luciferase reporter assay

[0071] After culturing the HCT116 or SW480 cell line, the luciferase reporter plasmid, Renila expression plasmid, and specific plasmid were co-transfected using Lipo 2000. Subsequently, after a 48-hour incubation period, the cells were lysed to evaluate Hippo signaling activity.

[0072] Wound healing and invasion assays

[0073] Wound healing assays included seeding HCT116 and SW480 cells transfected with siJOSD1 or sicontrol into 6-well plates. Once the cells were fully grown, a wound was created by scratching the cell monolayer with a sterile tip. After wounding, the cells were imaged at predetermined time points. Subsequently, the distance between the scratched wound edges was quantified using ImageJ software for analysis. To evaluate cell invasion, a Transwell system with an 8-μm pore size from Corning was used. In the invasion assay, the upper chamber membrane was coated with Matrigel from USABD Biocoat. After 24 hours of incubation, after fixation and staining with crystal violet, the number of colorectal cancer cells on the lower surface of the invasion membrane was determined under a 20× objective. Notably, to ensure the reliability of the results, all experiments were performed in triplicate.

[0074] Cycloheximide assay

[0075] After 24 h of transfection with siJOSD1 or sicontrol, HCT-116 cells were treated with 100 μmol / L cycloheximide. HEK293T cells were transfected with 2 μg of Flag-JOSD1 or Flag vector using the same experimental protocol. Subsequently, cell lysates were obtained at specific time points (0, 3, 6, and 9 h after treatment) to evaluate the effect of the intervention on the two cell lines.

[0076] Immunofluorescence (IF) staining

[0077] After inoculating colon cancer cells into 12-well plates, they were treated with small molecule reagents. After 24 hours of incubation, the cells were transferred to small dialysis slides. The cells were fixed by dropping 4% paraformaldehyde solution. Then they were stained with primary antibodies against JOSD1 (1:100, Sigma-Aldrich, HPA001168) and YAP (14074, Santa Cruz) (Sc-271134, 1:2000) for two hours at room temperature. Subsequently, the cells were rinsed with PBS and incubated with fluorophore-conjugated secondary antibodies from Invitrogen (Carlsbad, CA), and then rinsed again with PBS. The cell nuclei were stained with DAPI (Life Technology), and an anti-quenching reagent was added after washing. Images of cell-specific proteins were captured using a laser scanning confocal microscope (Leica TCS SP8 STED) and then processed using ImageJ software for further analysis.

[0078] Co-IP assay

[0079] To obtain a protein sample, cells are first lysed to produce a cell lysate. Then, the bait protein is introduced into the cell lysate. Subsequently, magnetic bead-conjugated antibodies are added to the sample, specifically binding to the bait protein. By using a magnet, the magnetic beads along with the precipitated bait protein are separated. The next step is to separate the protein complex by SDS-PAGE and then perform Western blotting (WB) to identify the target protein within the sample.

[0080] In vitro ubiquitination assay

[0081] HEK293T cells were transfected with Ub, K48 Ubi plasmid, K48R Ubi plasmid, Flag-JOSD1 plasmid and Myc-YAP or vector, and incubated for 6 hours. Subsequently, 20 μM MG132 (MCE, HY-13259) was introduced into the cells. Subsequently, total proteins were extracted, and IgG and 30 μL of protein A+G agarose (Beyotime, P2055) were added and incubated for 2 hours. Then, anti-flag antibody was introduced for immunoprecipitation. The ubiquitination of YAP was analyzed by Western blotting using an anti-HA antibody.

[0082] In vivo tumorigenesis experiment

[0083] For the in vivo tumorigenesis experiment, 5-week-old female BALB / c nude mice were obtained from SPF (Beijing) Biotechnology Company. HCT116 cells were infected with shControl or shJOSD1 lentivirus, and after 48 h of infection, they were treated with 1 μg / ml puromycin for 3 d. Subsequently, HCT116 cells (2×10 6 ) were injected into the right back of 5-week-old BALB / c female nude mice. The tumor formation in the nude mice was monitored over a period of 4 weeks, and the tumor volume was calculated using the formula Tumor volume = 1 / 2 × length × width2. After 5 weeks, the mice were sacrificed, and the tumors were weighed and photographed.

[0084] Cell proliferation assay

[0085] HCT116 and SW480 cells were transfected with siJOSD1 or siccontrol and counted in a 24-well plate. 24 hours after transfection, 4000 cells were seeded in a 96-well plate. The relative cell viability was measured at the designated time points using the CCK8 cell proliferation reagent, and the cell number was determined by the absorbance reading at 450 nm. The cell proliferation was further analyzed by the EdU incorporation method. The colorectal cancer cell count was detected using a 5-ethynyl-2'-deoxyuridine (EdU) detection kit (RiboBio, Guangzhou, China). Then, images were captured using a fluorescence microscope.

[0086] Cell apoptosis experiment

[0087] Cells transfected with siRNA or plasmid were incubated for 24 or 48 hours, then stained with propidium iodide (PI) and Annexin-v, and subsequently the fluorescence intensity was measured using a CytoFLEX flow cytometer.

[0088] Immunohistochemistry

[0089] Tissues were initially fixed with 4% paraformaldehyde and then embedded in paraffin. Next, the tissues were sectioned using a semi-automatic paraffin slicer S700 (RWD, USA). After sectioning, immunohistochemistry (IHC) staining was performed using an IHC kit (Zsbio, Beijing, China). The staining process included incubation with primary and secondary antibodies, and visualization of the immune complexes using DAB. At the same time, the cell nuclei were counterstained with DAPI. After staining, images were captured using a NanoZoomer digital pathology scanner (NanoZoomer S60, HAMAMATSU, Japan). Then the obtained images were evaluated using ImageJ software based on the integrated optical density (IOD).

[0090] RNA sequencing and data analysis

[0091] Three biological replicates were prepared for the study, each containing an equal amount of RNA. Then the samples were sent to Novogene in Beijing, China, for the construction and analysis of RNA-seq libraries. After analysis, a gene was considered to have significantly differential expression if it met two criteria, namely that the log2(fold change) of the gene was greater than 1 and the false discovery rate (FDR) was less than 0.05. Subsequently, KEGG pathway enrichment analysis was performed using the clusterProfiler software package in R. The obtained RNA sequence data were then archived in the Gene Expression Omnibus (GEO) database with the accession number GSE256276. In addition, gene set enrichment analysis (GSEA) was performed using the GSEA software http: / / www.broadinstitute.org / gsea to further explore the biological significance of the results.

[0092] Chromatin immunoprecipitation (ChIP) assay

[0093] After fixation for 30 minutes, add glycine solution to end. Subsequently, wash the cells with pre-chilled PBS containing PMSF, scrape, and centrifuge. After centrifugation, the obtained cell pellet is treated with SDS lysis buffer and sonicated for 10 minutes with a 30-second on / off cycle to break down chromatin. The ChIP procedure is performed using a ChIP kit (Millipore, 17-295), and rabbit anti-yap antibody (#SC101199) is used in the experiment. ChIP extracts DNA, and quantitative PCR analysis is performed using a DNA extraction kit (Qiagen, order number 28106). It should be noted that the specific primers used are JOSD1 F: GGAACTACAGCTCCCGGT GAT, denoted as SEQ ID NO. 21, and R: ATCACCGGGAGCTGTAGTCC, denoted as SEQ ID NO. 22.

[0094] TCGA data and survival data analysis

[0095] The TCGA database provides gene expression data of colorectal cancer patients. The GEPIA online software is used to detect the correlation between JOSD1 expression and the survival of colorectal cancer patients. The results are generated using the default parameters in the GSEA online software. A volcano plot with a threshold of P < 0.05 and an equivalent change > 1.5 is generated using the "ggplot2" package in R.

[0096] Statistical analysis

[0097] Statistical analysis is performed using GraphPad Prism 8.0.1 (GraphPad, USA). For the comparison of two groups, Student's t-test is used, and for multiple comparisons, one-way analysis of variance (ANOVA) is used. The χ2 test is used for categorical variables. Pearson correlation analysis is used to determine the correlation between measured values. The mean ± standard deviation (SD) of the measured data is given. When P < 0.05, the difference is considered significant.

[0098] Example 1

[0099] JOSD1 is associated with the Hippo / YAP gene signature in colon cancer samples

[0100] First, the whole-genome expression profiles of colon cancer samples in the TCGA database were analyzed. The correlation between the expression of each DUB (deubiquitinase) and the Hippo gene signature was analyzed. GSEA (Gene Set Enrichment Analysis) harvested 10 DUBs with the highest NES (Normalized Enrichment Score). The effects of these 10 genes on TEAD luciferase activity were further verified by siRNA, and JOSD1 deletion showed the lowest TEAD luciferase activity in colon cancer cells, as Figure 1A in. In TCGA colon cancer samples, JOSD1 expression was positively correlated with the YAP target gene signature (NES = 1.75; P < 0.05), as shown in Figure 1 B in. Further, JOSD1 was removed in HCT116 cells and RNA sequence analysis was performed. RNA sequencing data showed that JOSD1 silencing caused a sharp change in the Hippo signature, as shown in Figure 1 C in. While JOSD1 deletion reduced the expression of global YAP target genes from GSEA analysis (NES = 1.65; P < 0.001), as shown in Figure 1 D in. Volcano plots and heatmaps showed that a set of classical YAP target genes were reduced, as shown in Figure 1 E - F in. Further survival analysis of the KMPlot database showed that JOSD1 expression was associated with low survival rates in colon cancer patients, as shown in Figure 1 G in. The expression of JOSD1 in colon cancer samples and clinical characteristics were studied by immunohistochemistry (IHC). The expression of JOSD1 was associated with tumor invasion, lymph node invasion, and distant metastasis, as shown in Figure 9 . Immunohistochemical data showed that YAP expression was positively correlated with JOSD1, as shown in Figure 1 H and I (P < 0.001).

[0101] Example 2

[0102] JOSD1 is an important regulator of colon cancer cell progression

[0103] Two colon cancer cell lines, HCT116 and SW480, were selected to study the phenotype of JOSD1 in these cell lines. Endogenous JOSD1 was removed by transfection with siRNA and knockdown, and the efficiency was verified by western blot and qRT - PCR, as shown in Figure 2 A - D in. CCK8 experiments showed that JOSD1 deletion had a significant inhibitory effect on the growth of colon cancer cells, as shown in Figure 2 E - F in. EdU incorporation experiments showed that JOSD1 deletion had a significant inhibitory effect on the proliferation of colon cancer cells, as shown in Figure 2 G - J in. Transwell experiments showed that after removing JOSD1, the invasion ability of colon cancer cells was significantly reduced, as shown in Figure 2 K - N in. Apoptosis experiments showed that after JOSD1 knockdown, the number of apoptotic cells in HCT116 and SW480 cells increased, as shown in Figure 2 O - R in. In addition, wound healing experiments showed that silencing JOSD1 would hinder the migration of colon cancer cells, as shown in Figure 2 S - V in. The function of JOSD1 was further evaluated by in vivo experiments using xenograft tumor models. The results showed that silencing JOSD1 inhibited the tumorigenic potential of colon cancer cells,Figure 2 W-Y in []. In addition, subsequent immunohistochemical analysis of xenograft tumors showed a decrease in the expression of Ki67, as shown in Figure 2 Z in []. Therefore, JOSD1 plays a crucial role in the progression of colorectal cancer cells.

[0104] Example 3

[0105] JOSD1 activates the Hippo / YAP axis in colon cancer cells

[0106] The YAP protein plays a crucial role in Hippo signaling activity, which was confirmed in the study of the effect of JOSD1 on the YAP protein. It was observed that the level of YAP protein decreased after JOSD1 deletion in HCT116 and SW480 cells, while the level of YAP mRNA was not affected, as shown in Figure 3 A-D in []. These results indicate that JOSD1 plays a crucial role in the post-translational regulation of YAP. The qRT-PCR data strongly support this finding, because silencing JOSD1 led to a significant decrease in the expression of YAP target genes (including CTGF and CYR61) in HCT116 and SW480 cells, as shown in Figure 3 E-F in []. The TEAD response element luciferase assay clearly showed that the activity of YAP decreased in HCT116 and SW480 cells when JOSD1 was depleted, as shown in Figure 3 G-H in []. Since JOSD1 is a putative deubiquitinase, to verify whether its effect on the YAP protein depends on the ubiquitin peptidase activity of JOSD1, a mutant form of JOSD1 (JOSD1)C36A was constructed. Western Blot analysis found that wild-type JOSD1 could enhance the expression of YAP target genes CTGF and CYR61, while JOSD1 C36A could not, as shown in Figure 3 I-J in []. In gene expression experiments, wild-type JOSD1 could increase the level of YAP protein, while JOSD1 C36A could not, as shown in Figure 3 K in []. In luciferase experiments, wild-type JOSD1 could increase TEAD luciferase activity, while JOSD1 C36A could not, as shown in Figure 3 L in [].

[0107] Therefore, JOSD1 can promote YAP function in colon cancer cells, which depends on its deubiquitinase activity.

[0108] Example 4

[0109] JOSD1 promotes the progression of colon cancer cells through the Hippo / YAP axis

[0110] To further explore the relationship between the Hippo pathway in JOSD1 function and colon cancer, multiple rescue experiments were conducted based on the above experiments. Western Blot analysis verified the efficiency of JOSD1 silencing and YAP overexpression, as shown in Figure 4 A in. Subsequently, qRT-PCR analysis showed that the deletion of JOSD1 led to the inhibition of the expression of YAP target genes, and after further overexpression of YAP, the expression of target genes was restored, as shown in Figure 4 B in. Luciferase analysis showed that deleting JOSD1 decreased the activity of TEAD luciferase, but overexpressing YAP reversed this effect, as shown in Figure 4 C in. In the CCK8 experiment, this trend was also observed in the proliferation of colon cancer cells, as shown in Figure 4 D in. In the EdU incorporation experiment, the deletion of JOSD1 led to a decrease in the number of proliferating cells. Notably, this decrease could be partially rescued by the overexpression of YAP, as shown in Figure 4 E-F in. Similarly, the results of the wound healing experiment showed that the migration ability of colon cancer cells was significantly hindered after JOSD1 was depleted. However, this inhibition of cell migration could be partially restored by further overexpressing YAP, as shown in Figure 4 G-H in. In addition, the results of the transwell experiment showed that the invasion ability of colon cancer cells was impaired when JOSD1 was exhausted. Interestingly, the invasion ability of these cells could be partially restored by additional overexpression of YAP, as shown in Figure 4 I-J in. In addition, in FACS analysis, it was found that the deletion of JOSD1 increased the apoptosis of colon cancer cells. Notably, further overexpression of YAP could partially alleviate the increase in apoptosis, as shown in Figure 4 K-L in. The xenograft mouse model further demonstrated that inhibiting JOSD1 could effectively inhibit the growth of colon tumors in vivo. Interestingly, the growth arrest induced by JOSD1 silencing could be partially repaired by additional overexpression of YAP, as shown in Figure 4 M-O in. Finally, immunohistochemical analysis showed that the deletion of JOSD1 in vivo was associated with a decrease in the number of Ki67-positive cells. Notably, this effect could be reversed by further overexpressing YAP, as shown in Figure 4 P-Q in.

[0111] Therefore, JOSD1 controls the progression of colorectal cancer cells through the Hippo / YAP pathway.

[0112] Example 5

[0113] JOSD1 is associated with YAP and regulates the stability of YAP protein

[0114] This study detected the localization of JOSD1 and YAP in colon cancer cells. Immunostaining analysis showed that JOSD1 was mainly present in the cytoplasm, while YAP was present in both the cytoplasm and the nucleus, as shown in Figure 5 A in it. This was also confirmed in the nuclear and cytoplasmic separation experiments, as shown in Figure 5 B in it. In addition, endogenous immunoprecipitation experiments showed that JOSD1 could interact with YAP in colon cancer cells, as shown in Figure 5 C in it. After demonstrating the association between JOSD1 and YAP, its biological effect on YAP protein was studied. The degradation of YAP was inhibited using the proteasome inhibitor MG132, and it was found that removing JOSD1 decreased the YAP protein level in colon cancer cells. MG132 reduced the effect of JOSD1 removal on the YAP protein level, as shown in Figure 5 D - E in it. Overexpression of JOSD1 in HEK293T cells further confirmed this effect. In addition, protein stability assays were performed using the protein synthesis inhibitor cycloheximide. The results showed that wild - type JOSD1 increased the stability of YAP, while catalytically defective JOSD1 did not, as shown in Figure 5 F - G in it. Deletion of JOSD1 in colon cancer cells led to a shortened half - life of YAP protein, as shown in Figure 5 H - L in it. The YAP protein consists of three functional domains: the TEAD - binding domain (TBD), the WW domain, and the trans - activation domain (TA), as shown in Figure 5 J in it. The N - terminus of the JOSD1 protein contains a putative deubiquitinase Josephine in the domain, as shown in Figure 5 K in it. Deletion construct experiments were used to study the interaction between YAP and JOSD1. The above results showed that the WW domain of YAP was crucial for its interaction with JOSD1. In addition, JOSD1 interacted with YAP through its N - terminus, as shown in Figure 5 L - M in it.

[0115] Example 6

[0116] JOSD1 stabilizes YAP by inhibiting K48 - linked ubiquitination of YAP

[0117] The role of JOSD1 in YAP ubiquitination in the HEK - 293T model was studied by immunoprecipitation experiments. These experimental results confirmed that JOSD1 inhibited the total ubiquitination level of YAP, especially the K48 - linked ubiquitination level, as shown in Figure 6A-C therein. Subsequently, the effect of JOSD1 on YAP ubiquitination in HCT116 cells was investigated. The present invention also studied including endogenous Co-IP detection combined with ubiquitin signal transduction immunoblotting, and the results showed that after knockdown of the JOSD1 gene, the total ubiquitination level of YAP and the k48-linked ubiquitination level increased, as shown in Figure 6 D-E therein. Consistent with this, the dominant-negative mutant of ubiquitin (K48R) attenuated the effect of JOSD1 on YAP ubiquitination, as shown in Figure 6 F therein. This result indicates that JOSD1 plays a specific role in inhibiting k48-linked ubiquitination of YAP. To further understand the underlying mechanism, the regulatory effects of wild-type JOSD1 and its enzyme-deficient mutant JOSD1 C36A on YAP ubiquitination were explored. Notably, the results showed that the mutant JOSD1 C36A attenuated the effect of JOSD1 on YAP ubiquitination, as shown in Figure 6 G-H therein. Domain-based ubiquitination analysis indicated that the N-terminal domain of JOSD1 is crucial for the deubiquitination of YAP by JOSD1, as shown in Figure 6 I and K therein.

[0118] Example 7

[0119] YAP regulates the expression of JOSD1, forming a feedback loop between the Hippo signal and JOSD1

[0120] Since YAP plays a crucial role in regulating tumor progression, multiple studies have been conducted to investigate its global genomic binding in various cancer models. Based on the analysis of ChIP-sequencing data of YAP, significant binding peaks were found in the promoter region of JOSD1, indicating a potential regulatory role of YAP in JOSD1 expression, as shown in Figure 7 A therein. To verify this regulatory interaction, a Chip experiment was performed, showing a clear association between YAP protein and the promoter region of the JOSD1 gene, as shown in Figure 7 B therein. In addition, subsequent experiments involving the depletion of YAP in colon cancer cells showed a significant reduction in the binding of YAP to the promoter region of JOSD1, as demonstrated by the ChIP assay results, as shown in Figure 7 C-D therein. Consistently, the reduction of YAP level in colon cancer cells was associated with a significant decrease in JOSD1 mRNA and protein levels. This series of events together highlight the potential regulatory role of YAP in regulating JOSD1 expression in colon cancer cells, as shown in Figure 7 E-H therein. Previous studies have shown that verteporfin inhibits YAP function. After application of verteporfin, the levels of JOSD1 mRNA and protein decreased in HCT116 and SW480 cells, as shown in Figure 7I-L in it. In contrast, after XMU-MP-1 activates YAP, the JOSD1 mRNA and protein levels in HCT116 and SW480 cells increase, as shown in Figure 7 M-P in it. Immunostaining confirmed this, as shown in Figure 7 Q-R in it. This finding reveals a positive feedback pathway connecting JOSD1 and YAP.

[0121] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0122] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0123] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. Any of the following applications: Application of a reagent for detecting the expression level of JOSD1 in the preparation of a colon cancer diagnosis product; Use of a reagent for inhibiting the expression of JOSD1 in the preparation of a product for treating colon cancer.

2. The use according to claim 1, characterized in that: The diagnostic product is a reagent for detecting the expression level of JOSD1.

3. The use according to claim 2, characterized in that: The reagent for detecting the expression amount of JOSD1 includes a reagent for detecting the expression level of JOSD1 protein in a sample using a proteome sequencing technique.

4. The use according to claim 2, characterized in that: The reagent for detecting the expression amount of JOSD1 includes a reagent for detecting the expression level of the JOSD1 gene in a sample by using sequencing technology, probe hybridization technology, gene chip technology or fluorescent quantitative PCR technology.

5. The use according to claim 1, characterized in that: The therapeutic product increases the overall ubiquitination level of YAP.

6. The use according to claim 5, characterized in that: The therapeutic product increases the level of K48-linked polyubiquitination.

7. The use according to claim 1, characterized in that: The therapeutic product includes a primer sequence for knocking out JO SD1, and the primer sequence is a nucleotide sequence shown in any one group of SEQ ID NO.9-10 or SEQ ID NO.11-12.

8. A kit for diagnosing colon cancer, characterized in that: The kit comprises a reagent for detecting the expression level of JOSD1 as claimed in claim 1 in a sample.

9. The kit according to claim 8, characterized in that The samples come from colon cancer patients, suspected colon cancer patients, people susceptible to colon cancer, people at high risk of colon cancer or healthy people.

10. The kit according to claim 8, characterized in that The sample is tissue.

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