Application of E3 ubiquitin ligase RBCK1 in the preparation of drugs for treating liver cancer

Abstract

The present invention discloses an application of an E3 ubiquitin ligase RBCK1 in the preparation of a drug for treating liver cancer. The present invention has found that high expression of RBCK1 is significantly associated with poor tumor survival and distant invasion. Functional experiments have shown that RBCK1 promotes migration and invasion by enhancing GLUT1-mediated aerobic glycolysis, and RBCK1 activates the WNT / β-catenin / GLUT1 pathway to induce HCC cell migration and aerobic glycolysis dependent on the destruction of the PPARγ / PGC1α complex. RBCK1 promotes PPARγ ubiquitination degradation, and RBCK1 overexpression enhances WNT / β-catenin transcriptional activity, thereby upregulating GLUT1-mediated aerobic glycolysis of HCC cells. Both in vivo and in vitro, RBCK1 plays an important role in the occurrence and progression of liver cancer cells, and based on the high expression results of RBCK1 in liver cancer tissues, a powerful means is provided for potential targeted treatment of liver cancer patients in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of E3 ubiquitin ligase RBCK1 in the preparation of a medicine for treating liver cancer. Background Art

[0002] Liver cancer is one of the most common malignancies in the world and the second leading cause of cancer-related death. Despite great advances in diagnostic and therapeutic approaches, metastasis remains the main cause of treatment failure and death. Although modern multidisciplinary care strategies have been applied to the treatment of tumor metastasis, the 5-year overall survival rate of patients with advanced liver cancer is only 25%-39%, and the recurrence rate is approximately 80%. Metastasis of malignant tumors is often the main cause of treatment failure in patients with liver cancer. The molecular mechanism of liver cancer metastasis is still unclear.

[0003] Aerobic glycolysis, also known as the Warburg effect, is one of the most typical metabolic phenotypes of cancer cells, including hepatocellular carcinoma (HCC), in which it plays an important role. Aerobic glycolysis is mainly affected by oncogenic signals, such as PI3K / Akt. Notably, metabolic disorders in tumor cells can inhibit metastasis, indicating that aerobic glycolysis is central to tumor growth and survival. Because the liver is an important organ responsible for multiple unique metabolic functions. Therefore, a better understanding of the mechanisms between cellular metabolism and metastasis in diseases such as liver cancer is crucial for the development of new treatments.

[0004] Glucose transporter 1 (GLUT1) belongs to the facilitatory cell surface glucose transporter family, whose members regulate the transport of glucose across the cell membrane. GLUT1 is overexpressed in several cancers. Accumulating evidence suggests that GLUT1 promotes cell proliferation and metastasis and plays a key role in a variety of cancers, including HCC, breast cancer, and renal cancer. Many studies have shown that the SIRT1 / GLUT1 axis promotes the progression of bladder cancer by regulating glucose uptake. In addition, in ER-positive breast cancer cell lines, downregulation of GLUT1 sensitized cells to tamoxifen treatment and even restored the sensitivity of tamoxifen-resistant cells to the drug. However, the mechanism of action of regulating GLUT1-mediated aerobic glycolysis in HCC metastasis is still unclear.

[0005] The ubiquitin-proteasome system (UPS) plays an important regulatory role in intracellular signaling and protein metabolism. Ubiquitination is one of the most extensive and common post-translational modifications in cells, and it is essential for normal cell function. Post-translational modifications by E3 ubiquitin ligases can regulate the functions of target proteins, as well as intracellular mechanisms. Increasing evidence indicates that E3 ubiquitin ligases are key regulators of cancer. Recent studies have shown that RANBP2-type and C3HC4-type zinc finger-containing 1 (RBCK1), an E3 ubiquitin ligase with an N-terminal ubiquitin-like domain (UBL), an Npl4-type zinc finger (NZF), and a catalytic C-terminal RBR domain, plays an important role in tumor development. However, the exact role of RBCK1 in HCC progression and the underlying signaling cascade remain unclear. Summary of the invention

[0006] The present invention aims to provide an application of E3 ubiquitin ligase RBCK1 in preparing a medicine for treating liver cancer.

[0007] The above object of the present invention is achieved by the following methods:

[0008] The present inventors have found that RBCK1 expression is significantly upregulated in liver cancer tissues, and high expression of RBCK1 is significantly associated with poor tumor survival and distant invasion. Functional experiments have shown that RBCK1 promotes migration and invasion by enhancing GLUT1-mediated aerobic glycolysis. Further, RB0K1 activates the WNT / β-catenin / GLUT1 pathway to induce HCC cell migration and aerobic glycolysis dependent on the destruction of the PPARγ / PGC1α complex. Mechanistically, RBCK1 promotes PPARγ ubiquitination degradation, and RBCK1 overexpression enhances WNT / β-catenin transcriptional activity, thereby upregulating GLUT1-mediated aerobic glycolysis in HCC cells. In summary, the present inventors have determined the mechanism by which liver cancer cells survive in a malnourished tumor microenvironment and provide a role for RBCK1 in HCC cell adaptation to metabolic stress.

[0009] Beneficial Effects

[0010] The present invention analyzes the expression level of RBCK1 in clinical liver cancer tissue samples and finds that high expression levels of RBCK1 are associated with poor prognosis in HCC patients. RBCK1 promotes HCC cell metastasis by enhancing the Warburg effect through GLUT1. The HCC cell migration and aerobic glycolysis mechanism induced by the RBCK1-mediated WNT / β-catenin / GLUT1 pathway depend on the destruction of the PPAR7 / PGC1α complex. RBCK1 plays an important role in the occurrence and progression of liver cancer cells both in vivo and in vitro, and based on the high expression results of RBCK1 in liver cancer tissues, the present invention provides a new biomarker RBCK1 for the diagnosis of liver cancer, and RBCK1 can be used as a new target for the treatment of liver cancer, providing a new direction for the screening of drugs for the diagnosis and treatment of liver cancer and the treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 shows that RBCK1 is overexpressed in metastatic liver cancer and is closely related to the poor prognosis of patients.

[0013] in, Figure 1A The mRNA expression levels of RBCK1 in liver cancer tissues and adjacent tissues in the TCGA database and the expression profile of RBCK1 mRNA in TCGA liver cancer data were analyzed by paired analysis; Figure 1B The relationship between RBCK1 expression and clinicopathological characteristics of HCC patients in the TCGA cohort; Figure 1C To collect clinical data on RBCK1 mRNA levels in liver cancer and paired analysis of RBCK1 mRNA expression profiles in clinical HCC tissues and adjacent normal tissues; Figure 1D Western blot was used to detect the expression level of RBCK1 protein in clinical HCC tissues and adjacent normal tissues, **p<0.01 and the statistical graph of the gray value of RBCK1 protein expression; Figure 1E Immunohistochemical detection of RBCK1 protein expression in HCC tissues and adjacent normal tissues, scale bar, 50 μm; Figure 1F Figure 2 is the Kaplan-Meier curve of overall survival of HCC patients in two groups defined by low and high RBCK1 expression, and the Kaplan-Meier curve of disease-free survival of HCC patients in two groups defined by low and high RBCK1 expression.

[0014] FIG. 2 shows that RBCK1 promotes the migration and invasion of HCC cells in vitro and accelerates the metastasis of HCC cells in vivo.

[0015] in, Figure 2A RBCK1 protein levels in HCC cell lines and normal human hepatocytes (HL-7702), RBCK1 mRNA levels in normal human hepatocytes (HL-7702) and HCC cell lines; Figure 2B Western blot and qRT-PCR were used to detect the expression level of RBCK1 in HCCLM3 and Hep3B cells stably transfected with RBCK1 silencing or overexpression plasmids, *p<0.05, **p<0.01; Figure 2C Transwell migration and Transwell invasion experiments of HCCLM3 cells transfected with RBCK1 knockdown vector, *p<0.05, **p<0.01; Figure 2D Transwell migration and Transwell invasion assays of Hep3B cells transfected with RBCK1 overexpression plasmid; Figure 2E The expression of E-cadherin and vimentin in HCC cells with low RBCK1 expression was analyzed by confocal microscopy; Figure 2F The expression of E-cadherin and vimentin in HCC cells overexpressing RBCK1; Figure 2G To monitor the fluorescence produced by metastases in the orthotopic liver transplantation metastasis model in the RBCK1 group and the control group in HCC cells with stable interference, n = 8; Figure 2H To monitor the fluorescence produced by metastases in the orthotopic liver transplantation metastasis model in the RBCK1 stably overexpressing HCC cells group and the control group, n = 8; Fig.2I To stably interfere with RBCK1 in HCC cells H&E staining of paraffin sections of liver and lung metastatic nodules in the control group; Figure 2J Statistical analysis of intrahepatic and pulmonary metastatic nodules in the orthotopic liver transplantation metastasis model in the group with stable overexpression of RBCK1 in HCC cells and the control group (n=8 / group).

[0016] Figure 3 shows that RBCK1 promotes the migration and invasion of HCC cells by regulating the Warburg effect.

[0017] in, Figure 3A G6P levels, glucose consumption, lactate production and ATP levels in HCCLM3 / shRBCK1 cells, *p<0.05, **p<0.01; Figure 3B ECAR data show the glycolytic rate of RBCK1-silenced HCC cells, *p < 0.05; Figure 3C ECAR data show the capacity of RBCK1-silenced HCC cells, *p<0.05; Figure 3D OCR results show the basal respiration of HCC cells with RBCK1 silenced, *p<0.05, **p<0.01; Figure 3E OCR results show the maximum respiration of HCC cells with RBCK1 silenced, *p<0.05, **p<0.01; Figure 3F Cellular G6P levels, glucose consumption, lactate production, and ATP levels in Hep3B cells overexpressing RBCK1; Figure 3G ECAR data show the glycolytic rate of HCC cells with RBCK1 overexpression, *p<0.05, **p<0.01; Figure 3H ECAR data show the glycolytic capacity of HCC cells with RBCK1 overexpression, *p<0.05, **p<0.01; Fig. 3I OCR results show the basal respiration of HCC cells with RBCK1 overexpression, *p<0.05, **p<0.01; Figure 3J OCR results show the maximum respiration of HCC cells overexpressing RBCK1, *p < 0.05, **p < 0.01; Figure 3K Total lactate production by HCCLM3 / shRB0K1 in the presence of different concentrations of 2-DG *p<0.05, **p<0.01; Figure 3L The total amount of lactate produced by Hep3B / p-RBCK1 in the presence of different concentrations of 2-DG *p < 0.05, **p < 0.01; Figure 3M Effects of 2-DG on migration and invasion of HCCLM3 / shRBCK1 cells, *p<0.05, **p<0.01; Figure 3N Effects of 2-DG on Hep3B / p-RBCK1 cell migration and invasion, *p<0.05, **p<0.01; Fig.3O To culture HepG2 cells in medium containing galactose but not glucose to eliminate the effects of RBCK1 overexpression on cell migration and invasion.

[0018] FIG. 4 shows that the effect of RBCK1 in HCC cells is partially dependent on regulating the expression of GLUT1.

[0019] in, Figure 4A Western blotting and qRT-PCR analysis of GLUT1 expression levels in HCCLM3 / shRBCK1 cells, *p<0.05, **p<0.01; Figure 4B Western blotting and qRT-PCR analysis of GLUT1 expression levels in Hep3B / p-RBCK1 cells, *p < 0.05, **p < 0.01; Figure 4CWestern blotting was used to detect the expression of RBCK1 and GLUT1 in each group, and Tubulin was used as an internal control; Figure 4D The number of cell migration between different treatment groups, *p<0.05, **p<0.01; Figure 4E The number of cell invasions among different treatment groups, *p<0.05, **p<0.01; Figure 4F Representative images (right) and quantification (left) of intrahepatic and lung metastases in different groups of nude mice (n=8); Figure 4G Cell G6P levels, glucose consumption, lactate production, and ATP levels in each group; Figure 4H ECAR data show the glycolytic rate of cells in different treatment groups, *p < 0.05; Fig. 4I OCR results show the basal respiration of cells in different treatment groups, *p < 0.05; Figure 4J ECAR data show the glycolytic capacity of cells in different treatment groups, *p < 0.05; Figure 4K OCR results show the maximum respiration of cells in different treatment groups, *p < 0.05; Figure 4L Western blotting was used to detect the expression of RBCK1 and GLUT1 in each group, and Tubulin was used as an internal control; Figure 4M The number of cell migration between different treatment groups, *p<0.05, **p<0.01; Figure 4N The number of cell invasions among different treatment groups, *p<0.05, **p<0.01; Fig.4O Representative images (right) and quantification (left) of intrahepatic and lung metastases in different groups of nude mice (n=8), P, cellular G6P levels, glucose consumption, lactate production, and ATP levels; Figure 4Q ECAR data show the glycolytic rate of cells in different treatment groups, *p < 0.05; Figure 4R OCR results show the basal respiration of cells in different treatment groups, *p < 0.05; Figure 4S ECAR data show the glycolytic capacity of cells in different treatment groups, *p < 0.05; Figure 4T The OCR results show the maximum respiration of cells in different treatment groups, *p<0.05, *p<0.05.

[0020] Figure 5 shows that RBCK1 promotes glut1-mediated Warburg effect through the WNT / β-catenin signaling pathway.

[0021] in, Figure 5AGene set enrichment analysis (GSEA) showed that RBCK1 induced significant changes in WNT / β-catenin, oxidative phosphorylation, and PPAR signaling; Figure 5B To detect the total and nuclear protein levels of β-catenin in RBCK1-silenced HCCLM3 cells by Western blotting, tubulin and histone 3 were used as loading controls; Figure 5C The relative luciferase activity levels of cells transfected with TOP-flash and FOP-flash vectors in RBCK1-silenced HCCLM3 cells are shown, *p<0.05, **p<0.01; Figure 5D To detect the total and nuclear protein levels of β-catenin in RBCK1-overexpressing Hep3B cells by Western blotting, tubulin and histone 3 were used as loading controls; Figure 5E The relative luciferase activity levels of cells transfected with TOP-flash and FOP-flash vectors in RBCK1-overexpressing Hep3B cells are shown, *p < 0.05, **p < 0.01; Fig. 5F Western blotting was used to detect the expression levels of RBCK1, β-catenin and GLUT1 in each group, and Tubulin was used as an internal reference, *p < 0.05, **p < 0.01; Figure 5G The expression levels of RBCK1, β-catenin and GLUT1 in each group were detected by qRT-PCR, and Tubulin was used as an internal reference, *p < 0.05, **p < 0.01; Figure 5H For quantification of Transwell assays of indicated groups, *p<0.05, **p<0.01; Fig.5I Quantification of intrahepatic and pulmonary metastasis in nude mice of different groups (n=8), J, Western blotting showed the expression levels of RBCK1, β-catenin and GLUT1 in HCCLM3 cells transfected with shRBCK1 and XAV-939, Tubulin was used as an internal reference, *p<0.05; Figure 5K qRT-PCR showing the expression levels of RBCK1, β-catenin, and GLUT1 in HCCLM3 cells transfected with shRBCK1 and XAV-939, with Tubulin as an internal reference, *p < 0.05; Figure 5L Quantification of Transwell assay in HCCLM3 cells transfected with shRBCK1 or XAV-939, *p<0.05, **p<0.01; Figure 5MCellular G6P level, glucose consumption, lactate production, and ATP level in HCCLM3 cells transfected with shRBCK1 or XAV-939, *p < 0.05, **p < 0.01; Figure 5N To detect ECAR in HCCLM3 cells transfected with shRBCK1 or XAV-939; Fig.5O Western blotting was used to detect the expression levels of RBCK1, β-catenin, and GLUT1 in Hep3B cells overexpressing RBCK1 after transfection with XAV-939, with Tubulin as an internal reference, *p < 0.05; Figure 5P qRT-PCR was used to detect the expression levels of RBCK1, β-catenin, and GLUT1 in Hep3B cells overexpressing RBCK1 after transfection with XAV-939, with Tubulin as an internal reference, *p < 0.05; Figure 5Q Quantitative analysis of migration of RBCK1-overexpressing Hep3B cells treated with XAV-939, *p<0.05, **p<0.01; Figure 5R Quantitative analysis of invasion of RBCK1-overexpressing Hep3B cells treated with XAV-939, *p<0.05, **p<0.01; Figure 5S Cellular G6P level, glucose consumption, lactate production, and ATP level in Hep3B cells treated with XAV-939 in RBCK1, *p < 0.05, **p < 0.01; Figure 5T The glycolytic rate was detected in Hep3B cells treated with RBCK1 by XAV-939 for ECAR.

[0022] Figure 6 shows that RBCK1 activates the WNT / β-catenin pathway in HCC cells by disrupting the PPARγ / PGC1α complex and is associated with the Warburg effect.

[0023] Fig. 6A Co-IP assay showed no interaction between RBCK1 and β-catenin; Figure 6B Western blotting shows the expression levels of RBCK1, PPARγ, PGC1α, β-catenin, and GLUT1 in RBCK1-silenced HCCLM3 cells, with Tubulin as an internal control; Figure 6C Co-IP combined with Western blotting was used to detect the expression levels of PPARγ and PGC1α in RBCK1-silenced HCCLM3 cells, with Tubulin as an internal control; Fig.6DWestern blotting was used to show the expression levels of RBCK1, PPARγ, PGC1α, β-catenin, and GLUT1 in RBCK1-overexpressing Hep3B cells, with Tubulin as an internal control; Fig. 6E Co-IP combined with Western blotting was used to detect the expression levels of PPARγ and PGC1α in RBCK1-overexpressing Hep3B cells, with Tubulin as an internal control; Fig. 6F qRT-PCR shows the expression levels of RBCK1 and PPARγ in RBCK1-silenced HCCLM3 cells; Figure 6G Western blotting shows the expression levels of RBCK1, PPARγ, β-catenin, and GLUT1 in RBCK1-silenced HCCLM3 cells transfected with shPPARγ, with Tubulin as an internal control; Figure 6H Quantification of Transwell assay in RBCK1-silenced HCCLM3 cells transfected with shPPARγ, *p<0.05; Fig.6I Cellular G6P level, glucose consumption, lactate production and ATP level in RBCK1-silenced HCCLM3 cells transfected with shPPARγ, *p<0.05, **p<0.01; Figure 6J Western blotting was used to show the expression levels of RBCK1, PPARγ, β-catenin, and GLUT1 in Hep3B cells transfected with p-PPARγRBCK1 overexpression, with Tubulin as an internal control; Figure 6K Quantification of the migration number of RBCK1-overexpressing Hep3B cells transfected with p-PPARγ, *p < 0.05; Figure 6L Quantification of invasion number of RBCK1-overexpressing Hep3B cells transfected with p-PPARγ, *p<0.05; Figure 6M Cellular G6P level, glucose consumption, lactate production and ATP level in RBCK1-silenced HCCLM3 cells transfected with shPPARγ, *p<0.05, **p<0.01; Figure 6N Immunohistochemistry was used to detect the expression of RBCK1, Ki67, PPARγ, PGC1α, β-catenin, GLUT1, and vimentin proteins in HCC tissues and adjacent normal tissues. Scale bar, 50 μm.

[0024] FIG. 7 is an illustration showing that RBCK1 interacts with HCC cells and destabilizes PPARγ by promoting PPARγ ubiquitination and degradation.

[0025] in Fig. 7AGo-IP showed direct binding between endogenous RBCK1 and PPARγ in HCCLM3 cells; Figure 7B Go-lP showed that endogenous RBCK1 and PPARγ did not bind directly; Figure 7C GST pull-down experiments showed that RBCK1 and PPARγ directly bind in HCCLM3 cells; Fig.7D GST pull-down experiments showed that RBCK1 and PPARγ directly bind in Hep3B cells; Fig. 7E To treat HCCLM3 and F cells with MG132 (Z-Leu-Leu-Leu-CHO, 15 μmol / L), PPARγ levels were measured; Figure 7F To measure PPARγ levels, MHCC97H cells were treated with MG132 (Z-Leu-Leu-Leu-CHO, 15 μmol / L); Figure 7G To detect the ubiquitination level of HA-PPARγ in HEK293 cells in the presence of E1, E2 (UBCH5c), FBX09, and Ub; Figure 7H To alter the ubiquitination of PPARγ by downregulation or overexpression of RBCK1 in HCCLM3 cells, cells in each group were treated with the proteasome inhibitor MG132, cell lysates were prepared, and immunoprecipitation was performed using anti-PPARγ antibody, and the level of ubiquitin-attached PPARγ was detected by Western blotting using anti-Ub antibody; Fig.7I To alter the ubiquitination of PPARγ by downregulation or overexpression of RBCK1 in Hep3B cells, cells in each group were treated with the proteasome inhibitor MG132, cell lysates were prepared, and immunoprecipitation was performed using anti-PPARγ antibody, and the level of ubiquitin-attached PPARγ was detected by Western blotting using anti-Ub antibody. Figure 7J Ubiquitination of wild-type PPARγ or K-to-R mutant with all Lys sites mutated in PPARγ gene in HEK293 cells; Figure 7K For the determination of PPARγ ubiquitination type in HEK293 cells; Figure 7L To transfect HCCLM3 cells with plasmids encoding HA-PPARγ and Flag-RBCK1 plasmids, the cells were then treated with cycloheximide (CHX) (20 μmol / L) at specific times, and the degradation of PPAR7 was detected using anti-HA antibodies; Figure 7M To transfect MHCC97H cells with plasmids encoding HA-PPARγ and Flag-RBCK1 plasmids, the cells were then treated with cycloheximide (CHX) (20 μmol / L) at specific times, and the degradation of PPARγ was detected using anti-HA antibodies; Figure 7NWe propose a model in which the E3 ubiquitin ligase RBCK1 promotes GLUT1-mediated Warburg effect by disrupting the PPARγ / PGC1α complex. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0027] Example 1

[0028] Patients and specimens:

[0029] The study subjects were 216 patients with primary liver cancer and adjacent tissues from the Department of General Surgery, The Second Affiliated Hospital of Nanchang University. This study was approved by the Clinical Research Ethics Committee of the Second Affiliated Hospital of Nanchang University, and all subjects agreed to provide samples for research. Table 1 summarizes the clinical characteristics of all patients.

[0030] Table 1 Relationship between RBCK1 expression and clinicopathological features

[0031]

[0032] Cell lines and culture:

[0033] Four HCC cell lines (HCCLM3, Hep3B, SMMC7721, and MHCC97H) and normal human hepatocytes (HL-7702) were obtained from Rockville, U.S.A. Cells were cultured in RPMI 1640 or DMEM (Gibco) supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin at 37°C and 5% CO2.

[0034] Immunoblot analysis:

[0035] The cell lysate was separated using 4-12% Bis-Tris gel (Life Technologies, USA) and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and incubated with the corresponding antibodies. The antibodies used were anti-RBCK1 (1:1000, Abcam, ab219955), anti-GLUT1 (1:1000, Abcam, ab115730), anti-PPARγ (1:1000, Abcam, ab176328), anti-PGC1α (1:1000, Abcam, ab176328), anti-β-catenin (1:1000, Abcam, ab16051), a nti-ub (1:1000, Abcam, ab134953), anti-UBE1 (1:1000, Proteintech, 15912-1-AP), anti-UBCH5C (1:1000, Proteintech, 15912-1-AP), anti-FBX09 (1:1000, Proteintech, 11161-1-AP) and anti-Tubulin (1:1000, Santa Cruz, sc-8035).

[0036] Immunohistochemistry (IHC):

[0037] Fixation, embedding, sectioning, and dewaxing were performed. Some dewaxed sections were stained with hematoxylin and eosin (H&E). TM Immunohistochemical staining was performed using the Agilent Technologies system (Agilent Technologies, USA). Cells were incubated with anti-RBCK1 (1:2000, Abcam, ab219955), anti-GLUT1 (1:2000, Abcam, ab115730), anti-PPARγ (1:2000, Abcam, ab45036), anti-PGC1α (1:2000, Abcam, ab191838), anti-β-catenin (1:2000, Abcam, ab92547), anti-vimentin (1:2000, Abcam, ab92547) and anti-Ki67 (1:2000, Proteintech, 27308-1-AP) for 30 minutes.

[0038] shRNA and plasmid construction:

[0039] Shanghai GeneCare Gene Pharmaceuticals Co., Ltd. synthesized shRNA-mediated double-stranded RNA (shRNA) silencing RBCK1 (shRBCK1), β-catenin (shβ-catenin), or GLUT1 (shGLUT1). Full-length cDNAs of human RBCK1, β-catenin, and GLUT1 were synthesized using gene technology and ligated with pcDNA3.1 vectors to generate p-RBCK1, p-β-catenin, and p-GLUT1, respectively. Blank vectors were used as negative controls. These shRNAs and plasmids were transfected using Lipofectamine 2000 transfection reagent (Invitrogen, USA) according to the manufacturer's instructions.

[0040] Cell migration and invasion assays:

[0041] Transwell assay is routinely used to evaluate the migration and invasion of HCC cell lines. A layer of matrix gel is pre-coated on the top of the polycarbonate membrane for cell invasion assay.

[0042] In vivo transfer assay:

[0043] In the in vivo metastasis experiment, 100 μL of phosphate-buffered saline containing 1×106 cells was injected into the thigh of nude mice. Once the subcutaneous tumor reached 1-2 cm in diameter, it was excised and cut into fragments of approximately 1 mm3 in volume, and then the fragments were implanted into the liver of nude mice (8 per group, BALB / c-nu / nu male, 6-8 weeks old). The mice were killed 6 weeks after tumor implantation. The liver and lungs were then processed and embedded in paraffin. The animal experiments were approved by the Animal Experiment Ethics Committee of the Second Affiliated Hospital of Nanchang University and were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

[0044] Determination of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR):

[0045] The extracellular flux analyzer XF96 (Seahorse Bioscience, Billerica, MA, USA) was used to measure the cellular glycolytic capacity and cellular mitochondrial respiration using the XF CellMito Stress Test Kit and the gly glycolysis Stress Test Kit (Seahorse Bioscience), respectively, according to the manufacturer's instructions.

[0046] Co-immunoprecipitation (Co-IP) and ubiquitination analysis:

[0047] Immunoprecipitation analysis was performed as described previously. For in vivo ubiquitination assays, HA-Ub plasmids were stably transfected into RBCK1 knockdown human hepatoma cells or control cells. Two days after transfection, cells were treated with 10 mmol / L MG132 for 10 h to prevent proteasomal degradation. Lysates were extracted with 2 mg HA tag or IgG antibodies. Eluates were separated by SDS-PAGE and subjected to Western blot experiments using RBCK1, PPARγ, and Tubulin antibodies. The standard reaction mixture (25 μL) for in vitro ubiquitination assays consisted of 20 mM HEPES NaOH (pH 7.5), 50 mM NaCl, 0.02 mg mL-1 BSA, 1 mM DTT, 5 mM MgCl2, 1 mM ATP, his-PPARγ (1 pmol as tetramer), E1 (0.85 pmol), UbcH5c (1.25 pmol), FBX09 (0.8 pmol), and Ub (174 pmol). The reaction mixture was placed on ice and incubated at 30°C for 10 minutes. The reaction was terminated by adding SDS buffer, and Western blot experiments were performed using PPARγ and Tubulin antibodies.

[0048] Statistical analysis:

[0049] All results are expressed as mean ± standard deviation, and at least three independent experiments were analyzed using GraphPad Prism 5 software. We used the Kaplan-Meier method to generate survival curves, and the log-rank test was used to assess statistical significance. The differences between the groups were analyzed using two-tailed t-tests and analysis of variance. When p < 0.05, the results were considered statistically significant.

[0050] result:

[0051] 1. High expression of RBCK1 is associated with poor prognosis in HCC patients

[0052] Analysis of the TCGA group (n=424) showed that RBCK1 was highly expressed in liver cancer tissues, and high expression of RBCK1 was positively correlated with tumor stage and distant metastasis ( Figure 1A To further determine the expression level of RBCK1 in hepatocellular carcinoma, we used qRT-PCR assay, and the results showed that the expression of RBCK1 mRNA in liver cancer tissues was higher than that in paired adjacent tissues ( Figure 1C ). Western blot experiment ( Figure 1D ) and immunohistochemical staining ( Figure 1E ) The results showed that RBCK1 protein was upregulated in HCC tissues compared with adjacent paracancerous tissues. These results indicate that RB0K1 expression is significantly upregulated in HCC tissues.

[0053] Further evaluation of the correlation between RBCK1 overexpression and clinical pathological parameters of HCC revealed that there was no significant correlation between RBCK1 expression and tumor size, age, or histological type, but there was a significant correlation with TNM (p < 0.002), distant metastasis (p = 0.013), and clinical stage (p = 0.006) (Table 1). According to the results of immunohistochemical analysis, 216 HCC patients were divided into 146 cases in the RBCK1 high expression group and 70 cases in the RBCK1 low expression group. Kaplan-Meier survival analysis showed that compared with HCC patients with low RBCK1 levels, HCC patients with high RBCK1 expression had worse overall survival (OS) (p = 0.025) and worse disease-free survival (DFS) (p = 0.0034) ( Figure 1F ), it should be noted that the results of multivariate Cox regression analysis showed that RBCK1 overexpression was one of the independent predictors of poor prognosis in HCC patients (Table 2). Collectively, these data indicate that RBCK1 is upregulated in HCC tissues and is associated with poor prognosis in HCC patients.

[0054] Table 2 Univariate and multivariate analysis of overall survival in patients with liver cancer

[0055]

[0056] 2. RBCK1 promotes the metastasis of liver cancer cells in vivo and in vitro

[0057] To investigate the potential biological function of RBCK1 in HCC progression, we first evaluated the expression of RBCK1 in HCC cell lines. qRT-PCR and Western blot results showed that RBCK1 was significantly upregulated in HCC cells compared with normal HL-7702 cells ( Figure 2A Based on the expression level of RBCK1 in HCC cells, we next established a cell line HCCLM3sh-RBX1 cells that stably knocked down RBCK1, and a cell line Hep3B p-RBX1 cells that stably overexpressed RBCK1 in the Hep3B cell line ( Figure 2B ). Transwell experiments have shown that knocking down RBX1 can significantly reduce the invasion and migration ability of liver cancer cells, while overexpressing RBX1 can significantly increase the invasion and migration ability of liver cancer cells ( Figure 2C and D). Similarly, the RTCA test results also showed that knocking down RBCK1 inhibited the metastatic ability of HCCLM3 cells. Since EMT is significantly correlated with the metastatic ability of cancer cells, we studied the effect of RBCK1 expression on the EMT phenotype of HCC cells. Figure 2EAs shown in Figures 2 and 3, immunofluorescence analysis showed that knockdown of RBCK1 increased epithelial markers and decreased mesenchymal markers in HCCLM3 cells. Therefore, it was concluded that stable knockdown of RBCK1 inhibited the invasion and metastasis of HCC.

[0058] We established an orthotopic liver tumor model in nude mice (including shNC group and shRBCK1 group) to detect the effect of RBCK1 on liver cancer metastasis. Histological analysis showed that 5 cases of intrahepatic metastasis occurred in the shNC group, while only 1 case occurred in the shRBCK1 group ( Figure 2G In addition, HE staining of the lungs showed that the number of hepatocellular carcinoma lung cancer micrometastases in the shRBCK1 group was significantly reduced ( Fig.2I In contrast, overexpression of RBCK1 increased the number of intrahepatic and pulmonary metastatic nodules ( Figure 2J ). Taken together, these results indicate that stable downregulation of RBCK1 can inhibit HCC invasion and metastasis both in vitro and in vivo, demonstrating that RBCK1 is an oncogene for HCC progression and metastasis.

[0059] 3. RBCK1 promotes HCC progression by enhancing the Warburg effect

[0060] E3 ubiquitin ligases have been shown to promote metabolic reprogramming in the progression of several cancers. Since the Warburg effect is a well-characterized metabolic shift that is widely present in tumor cells, including HCC, we explored the role of RBCK1 in HCC glucose metabolism. Knockdown of RBCK1 also significantly reduced glucose-6-phosphate (G6P), glucose consumption, lactate production, and ATP levels in HCCLM3 cells ( Figure 3A ), while overexpression of RBCK1 in Hep3B cells resulted in the opposite trend ( Figure 3F To further verify the effect of RBCK1 on glycolysis in HCC, we measured ECAR, which reflects the total glycolytic flux of cells. RBCK1 knockdown significantly reduced the glycolytic rate and total amount of HCCLM3 cells ( Figure 3B and C), while overexpression of RBCK1 increased the ECAR of Hep3B cells ( Figure 3G and H). The measurement results of mitochondrial respiration index OCR showed that the cell OCR value increased when RBCK1 was interfered in HCCLM3 ( Figure 3D and E), while overexpression of RBCK1 resulted in a decrease in the OCR value of Hep3B cells ( Fig. 3I In addition, the above results were also observed in MHCC97h / shRBCK1 and SMCC7721 / p-RBCK1 cells.

[0061] To investigate whether the Warburg effect is associated with the progression of HCC cells, we treated HCCLM3 / shRBCK1 and Hep3B / p-RBCK1 cells with different concentrations (0, 4, or 8 mM) of 2-DG for 24 h. The results showed that 2-DG significantly inhibited glycolysis in a dose-dependent manner in HCCLM3 / shRBCK1 and Hep3B / p-RBCK1 cells ( Figure 3K and L). The migration and invasion abilities of HCCLM3 / shRBCK1 and Hep3B / p-RBCK1 cells were also reduced in a dose-dependent manner ( Figure 3M and N). Cells were cultured in medium containing galactose instead of glucose, which reduced glycolytic flux and forced the cells to rely on oxidative phosphorylation. We observed that the reduction in glycolytic flux greatly abolished the enhanced migration and invasion ability of Hep3B cells caused by RBCK1 overexpression ( Fig.3O ). These results suggest that RBCK1 inhibits oxidative phosphorylation and promotes aerobic glycolysis (Warburg effect) in HCC cells, but promotes migration and invasion of HCC cell lines in vitro by enhancing the Warburg effect.

[0062] 4. RBCK1 promotes the Warburg effect of liver cancer cells through GLUT1

[0063] Previous studies have shown that GLUT1 plays an important role in glycolysis. Next, we investigated whether RBCK1 regulates GLUT1 expression by preliminarily observing the expression of GLUT1 in HCC cells with RBCK1 knockdown and overexpression. Western blotting results showed that knockdown of RBCK1 significantly reduced the expression of GLUT1 in HCCLM3 cells ( Figure 4A In contrast, overexpression of RBCK1 significantly increased the expression of GLUT1 in Hep3B cells ( Figure 4B ). In addition, in HCCLM3 / shRBCK1 cells, upregulation of GLUT1 attenuated the decrease in GLUT1 expression caused by knockdown of RBCK1 ( Figure 4C In addition, the recovery experiment showed that the restoration of GLUT1 expression eliminated the decreased metastatic ability of liver cancer cells caused by RBCK1 silencing ( Figure 4D and E). In vivo tumor metastasis experiments showed that overexpression of GLUT1 increased the incidence of hepatic and lung metastasis in HCCLM3 cells induced by knockdown of RBCK1 ( Figure 4F ). In addition, studies on whether RBCK1 increases glycolysis through GLUT1 expression showed that upregulating GLUT1 expression rescued the RBCK1-mediated decrease in G6P, glucose consumption, lactate production, and ATP levels in HCC cells ( Figure 4G). Meanwhile, knockdown of RBCK1 reduced ECAR and OCR in HCC cells, while overexpression of GLUT1 attenuated the decline in glycolytic rate and capacity ( Figure 4H -K).

[0064] Next, we evaluated the effect of reducing GLUT1 expression on RBCK1 and GLUT1 protein levels and cell migration and invasion abilities in Hep3B cells overexpressing RBCK1. Western blotting analysis showed that overexpression of RBCK1 significantly increased GLUT1 expression in Hep3B cells, whereas downregulating GLUT1 expression significantly inhibited the RBCK1-induced increase in GLUT1 expression ( Figure 4L In addition, downregulation of GLUT1 significantly reduced RBCK1-mediated enhancement of cell migration and invasion ( Figure 4M and N). In addition, the results of in vivo metastasis experiments showed that in the Hep3B p-RBCK1 group, GLUT1 downregulation reduced the incidence of intrahepatic and lung metastases ( Fig.4O ). In addition, we investigated whether RBCK1 increased glycolysis via GLUT1 expression, and the results showed that downregulating GLUT1 expression rescued the RBCK1-mediated increase in G6P, glucose consumption, lactate production, and ATP levels in HCC cells ( Figure 4P Meanwhile, overexpression of RBCK1 increased ECAR and OCR in HCC cells, while simultaneous knockdown of GLUT1 inhibited the increase in glycolytic rate and capacity ( Figure 4Q Collectively, these findings suggest that GLUT1 is a downstream target of RBCK1 in regulating aerobic glycolysis and is critical in RBCK1-mediated tumor progression.

[0065] 5. RBCK1 activates GLUT1 through the WNT / β-catenin signaling pathway

[0066] To further elucidate the mechanism by which RBCK1 regulates GLUT1 in HCC, we performed gene set enrichment analysis (GSEA) to determine the impact of transcriptome changes on biological functions and pathways. In HCCLM3 cells, the WNT signaling pathway was significantly positively correlated with RBCK1 ( Figure 5A). Since GLUT1 is a target gene of WNT / β-catenin, we speculated that RBCK1 regulates GLUT1 through the WNT / β-catenin signaling pathway. To verify this hypothesis, we detected the expression changes of β-catenin in HCCLM3 cells with RBCK1 knockdown. Western blotting analysis showed that in HCCLM3 cells, the overall β-catenin protein and nuclear β-catenin expression levels decreased with the decrease of RBCK1 expression level ( Figure 5B ). TOP-Flash reporter luciferase assay showed that knockdown of RBCK1 reduced the transcriptional activity of TCF4 in HCCLM3 cells compared with shNC control ( Figure 5C In contrast, overexpression of RBCK1 in Hep3B cells produced the opposite effect ( Figure 5D We further determined that β-catenin upregulation restored the reduction of GLUT1 expression, cell migration, and cell invasion caused by RBCK1 downregulation ( Fig. 5F -I).

[0067] To verify that RBCK1 regulates GLUT1 expression through the WNT / β-catenin signaling pathway, we detected the levels of GLUT1 and β-catenin under the action of the WNT / β-catenin signaling pathway inhibitor XAV-939. XAV-939 consistently inhibited the mRNA and protein levels of GLUT1 in HCCLM3 cells ( Figure 5J and K). Transwell assay analysis showed that XAV-939 significantly reduced cell migration and invasion ( Figure 5L XAV-939 leads to a decrease in G6P, glucose consumption, lactate production, and ATP levels in hepatoma cells ( Figure 5M ). Meanwhile, XAV-939 inhibits ECAR in HCC cells ( Figure 5N In addition, rescue experiments showed that XAV-939 attenuated the enhanced metastatic ability and Warburg effect of liver cancer cells caused by RBCK1 overexpression ( Fig.5O -T). In conclusion, RBCK1 promotes HCC cell metastasis by activating the WNT / β-catenin signaling pathway and activating the GLUT1-mediated Warburg effect.

[0068] 6. RBCK1 activates the WNT / β-catenin pathway and Warburg effect in liver cancer cells by disrupting the PPARγ / PGC1α complex

[0069] To elucidate the mechanism by which RBCK1 regulates the WNT / β-catenin signaling pathway in HCC cells, we first determined whether there is a direct interaction between RBCK1 and β-catenin protein. Co-IP showed that there is no direct interaction between the two ( Fig. 6A ). The PPARγ / PGC1α complex inhibits the WNT / β-catenin pathway and contributes to glucose homeostasis. Therefore, we speculated that RBCK1 regulates WNT / β-catenin by disrupting the PPARγ / PGC1α complex. To verify this hypothesis, we first determined whether RBCK1 affects the activation of the β-catenin / GLUT1 pathway by disrupting the PPARγ / PGC1α complex. Changes in the expression of β-catenin, GLUT1, PGC1α, and PPARγ, as well as changes in the PPARγ / PGC1α complex, were detected in RBCK1-knockdown HCC cells. The results showed that in HCCLM3 cells, knockdown of RBCK1 significantly increased the expression level of PPARγ and the PPARγ / PGC1α complex, and reduced the expression of β-catenin and GLUT1, but there was no change in the PGC1α protein level ( Figure 6B and C). In contrast, overexpression of RBCK1 in Hep3B cells significantly reduced PPARγ protein expression levels and PPARγ / PGC1α complex levels, increased β-catenin and GLUT1 expression, but did not change PGC1α protein levels ( Fig.6D and E). PPARγ mRNA levels in HCC cells remain unaffected by changes in RBCK1 expression ( Fig. 6F ). The PPARγ / PGC1α complex is involved in the regulation of the RBCK1-mediated β-catenin / GLUT1 pathway.

[0070] The results verified that RBCK1 regulates the β-catenin / GLUT1 pathway by disrupting the PPARγ / PGC1α complex. PPARγ was knocked down in RBCK1-knocked-down liver cancer cells. It was found that knocking down PPARγ inhibited the decrease in β-catenin and GLUT1 expression caused by knocking down RBCK1, and cell migration and aerobic glycolysis ( Figure 6G -I). In contrast, upregulation of PPARγ inhibited the increased expression of β-catenin and GLUT1, cell migration, and aerobic glycolysis in Hep3B cells with upregulation of RBCK1 ( Figure 6J -M). Importantly, immunohistochemical staining ( Figure 6N) showed that RBCK1, Ki67, β-catenin, GLUT1, and vimentin proteins were upregulated, while PPARγ and PGC1α were downregulated in HCC tissues compared with adjacent non-tumor tissues. These results suggest that RBCK1-activated β-catenin / GLUT1 pathway promotes HCC cell migration and aerobic glycolysis dependent on the disruption of PPARγ / PGC1α complex.

[0071] 7. RBCK1 disrupts the PPARγ / PGC1α complex by promoting the ubiquitination and degradation of PPARγ

[0072] RBCK1 interacts with different substrates to exert its effects. We further investigated whether RBCK1, PPARγ, and PGC1α directly interact with each other in HCC cells. Co-IP analysis detected endogenous RBCK1 and PPARγ in immunoprecipitates, indicating that there was an interaction between RBCK1 and PPARγ, but no direct interaction between RBCK1 and PGC1α ( Fig. 7A In addition, GST pull-down experiments showed that RBCK1 binds to PPARγ in an in vitro system ( Figure 7C and D). These findings confirm that RBCK1 directly binds to PPARγ in HCC cells and that RBCK1 disrupts the PPARγ / PGC1α complex by regulating PPARγ expression.

[0073] Next, we explored the mechanism by which RBCK1 regulates PGC1α. Consistent with previous findings, PPARγ is degraded through the ubiquitin-proteasome pathway, and treatment with the proteasome inhibitor MG132 led to significant accumulation of endogenous PPARγ protein in HCC cells ( Fig. 7E Moreover, the data indicate that efficient His-PPARγ ubiquitination occurs in E1, E2 (UBCH5c), FBX09 (the E3 ubiquitin ligase of PPARγ), and Ub ( Figure 7G ). PPARγ is also degraded by the UPS in HCC cells. Next, we determined whether RBCK1 could directly mediate PPARγ ubiquitination. Interestingly, PPARγ polyubiquitination was significantly increased upon ectopic RBCK1 expression, but significantly decreased upon RBCK1 knockdown ( Figure 7H and I). The data also showed that mutation of all Lys sites of PPARγ abolished its polyubiquitination ( Figure 7J As expected, mutation of the Lys48 position on ubiquitin (Ub) almost completely abolished RBCK1-mediated PPARγ ubiquitination, whereas the K63R mutation had no effect ( Figure 7K). Consistent with the ubiquitination results, degradation kinetic analysis confirmed that the half-life of exogenously expressed PPARγ in RBCK1-overexpressing HCC cells was significantly reduced compared with that in control cells ( Figure 7L and M). These data suggest that RBCK1 mediates PPARγLys48 polyubiquitination, which leads to PPARγ degradation by the proteasome. Together, these results suggest that RBCK1 disrupts the PPARγ / PGC1α complex by promoting PPARγ ubiquitination and degradation ( Figure 7N ).

[0074] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An application of E3 ubiquitin ligase RBCK1 in the preparation of a drug for treating liver cancer, wherein the application refers to using E3 ubiquitin ligase RBCK1 as an intervention target for the treatment of liver cancer; High expression of the E3 ubiquitin ligase RBCK1 is associated with poor prognosis in patients with liver cancer, and can promote the metastasis of liver cancer cells and the progression of liver cancer.

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