Application of RPL6 gene and encoded protein thereof as marker and therapeutic target of liver cancer

Abstract

The invention discloses an application of an RPL6 gene and an encoded protein thereof as a marker and a therapeutic target of liver cancer, and relates to the field of biological medicines. The invention provides the key regulatory factor RPL6 of the liver cancer, and the RPL6 shows that the RPL6 is highly related to the prognosis of the liver cancer; and a new marker is provided for preparing products for diagnosis and prognosis of liver cancer. Researches find that the expression of the overexpressed RPL6 gene or the encoded protein thereof can effectively reduce the proliferation of liver cancer cells, and a new target is provided for preparing products for treating liver cancer.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of ribosomal protein L6 in preparing liver cancer diagnosis kits and medicines. Background Art

[0002] Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide. Although treatment advances have improved the prognosis of patients with advanced HCC, only about 30% of patients achieve an objective response. In addition, the three-year overall survival (OS) rate remains significantly below 50%. Despite significant progress in research, HCC remains one of the few cancers that continues to show an increase in morbidity and mortality year by year. Understanding the molecular mechanisms that contribute to the occurrence and progression of HCC remains a difficult task, and it is also difficult to find new effective therapeutic targets. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide an application of an RPL6 gene and its encoded protein (ribosomal protein L6) as a marker and therapeutic target for liver cancer. The present invention found that the expression of RPL6 in HCC tissue was significantly reduced. The reduction in RPL6 expression was closely related to tumor size, the presence of vascular invasion and poor prognosis. Functional experiments showed that the expression of RPL6 inhibited the proliferation of HCC cells and the progression of xenograft tumors. Mechanistically, the inventors found that RPL6 bound to and was degraded by the E3 ubiquitin ligase FBXO22, thereby inhibiting the polyubiquitination and subsequent degradation of p53. The enhancement of p53 activity further promoted the inhibition of cell growth. In contrast, p53 levels decreased significantly after RPL6 loss, indicating that RPL6 is essential for the stability of p53. In summary, RPL6 inhibits HCC cell proliferation through the FBXO22 / p53 signaling pathway, indicating its potential as a biomarker and therapeutic target for HCC.

[0004] Among them, protein p53, as a tumor suppressor, has become one of the most intensively studied proteins. Since the discovery of p53 protein in 1979, its important function in tumor development has been well documented. As the "guardian of the genome", p53 triggers various responses, such as cell cycle arrest, programmed cell death, and cell senescence, in the face of DNA damage or abnormal activation of oncogenes. Increasing evidence suggests that the loss of p53 function is due to post-transcriptional modifications, including phosphorylation, ubiquitination, acetylation, methylation, and neoligandization, which play an important role in the occurrence and progression of HCC.

[0005] Exploring new mechanisms that affect p53 protein function is crucial for developing targeted therapeutic strategies and promoting the development of effective HCC treatments.

[0006] The present invention found that the interaction between RPL6 and FBXO22 reduced the ubiquitination and degradation of p53 protein, a process mediated by FBXO22. RPL6 was identified as a target that can be cleared through FBXO22-mediated ubiquitination and proteasomal degradation. Taken together, the present invention's research results reveal RPL6 as a novel and important regulator of the FBXO22-p53 signaling pathway and illustrate how RPL6 contributes to the stability of p53 protein.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] Use of the RPL6 gene and / or its encoded protein as a target in the preparation of a drug for treating liver cancer.

[0009] The sequence of the RPL6 gene is as shown in SEQ ID NO.1; or the nucleotide sequence shown in SEQ ID NO: 1 is replaced, deleted and / or one or more nucleotides are added and the nucleotide sequence expressing the same functional protein is expressed.

[0010] The amino acid sequence of the encoded protein is shown in SEQ ID NO.2, or the amino acid sequence of the amino acid sequence shown in SEQ ID NO.2 is obtained by replacing and / or deleting and / or adding one or more amino acid residues to obtain the amino acid sequence of the protein with the same biological function.

[0011] Furthermore, the application is to enhance the expression of the RPL6 gene or enhance the function of the protein encoded by the RPL6 gene.

[0012] Furthermore, the expression of RPL6 gene or the function of the protein encoded by RPL6 gene is enhanced by RNA technology or gene editing technology; or the expression of RPL6 gene or the function of the protein encoded by RPL6 gene is enhanced by antisense nucleotide drugs or antibody drugs.

[0013] Furthermore, the application is to enhance the expression of RPL6 gene or enhance the function of protein encoded by RPL6 gene, so that the protein encoded by RPL6 gene binds to E3 ubiquitin ligase FBXO22, thereby inhibiting the polyubiquitination and / or degradation of p53 protein.

[0014] The second object of the present invention is to provide the use of the RPL6 gene and / or its encoded protein as a marker in the preparation of products for diagnosing liver cancer.

[0015] Furthermore, the product includes a reagent, a kit or a chip for detecting the activity of the RPL6 gene or the protein encoded by the RPL6 gene.

[0016] Furthermore, the expression level of the marker is negatively correlated with liver cancer.

[0017] The present invention also aims to provide an agent for enhancing the expression of the RPL6 gene and / or enhancing the protein encoded by the RPL6 gene for use in the preparation of a drug for treating liver cancer.

[0018] The present invention also aims to provide a drug for treating liver cancer, characterized in that the drug is:

[0019] 1) siRNA that enhances RPL6 gene expression;

[0020] 2) DNA or RNA that increases the expression or transcription of RPL6 encoding protein;

[0021] 3) An activator targeting the protein encoded by RPL6, wherein the activator includes a small molecule compound, an antibody drug, a protein, a nucleic acid molecule, a polypeptide, a lipid, a carbohydrate or a combination thereof.

[0022] The beneficial effects of the present invention include at least:

[0023] (1) The present invention provides a new marker for preparing products for the prognosis and diagnosis of liver cancer;

[0024] (2) The present invention provides a new target for preparing products for treating liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The expression level of RPL6 in HCC. A) The expression level of RPL6 in HCC samples; B) The expression of RPL6 protein in HCC in the GEO dataset.

[0026] Figure 2 Figure 1 shows the changes after RPL6 knockdown. A) Schematic diagram of the expression level of RPL6 in 6 HCC cells; B) Schematic diagram of the expression level of RPL6 after RPL6 knockdown experiment in SK-Hep1 and HepG2 cells; C) Schematic diagram of cell proliferation after RPL6 inhibition in HepG2 and SK-Hep1 cell lines; D) Schematic diagram of EdU-positive cells after RPL6 expression inhibition in HepG2 and SK-Hep1 cell lines; E) Schematic diagram of cell cloning experiment after RPL6 expression inhibition in HepG2 and SK-Hep1 cell lines; F) Graph of tumor size in the RPL6 normal group and knockdown group; G) Schematic diagram of Ki-67 level after RPL6 expression reduction.

[0027] Figure 3The diagram shows the changes after RPL6 overexpression. A) Schematic diagram of RPL6 protein level after RPL6 overexpression in SK-Hep1 and HepG2 cells; B) Schematic diagram of cell proliferation after RPL6 overexpression in SK-Hep1 and HepG2 cells; C) Schematic diagram of cell cloning experiment after RPL6 overexpression in SK-Hep1 and HepG2 cells; D) Schematic diagram of EdU positive cells after RPL6 overexpression in HepG2 and SK-Hep1 cell lines; E) Schematic diagram of tumor size in RPL6 normal group and overexpression group; F) Schematic diagram of Ki-67 level after RPL6 overexpression.

[0028] Figure 4 Figures for the study of the mechanism of action of RPL6 and p53. A) Correlation diagram of RPL6 and cell proliferation in TCGA-LIHC; B) Schematic diagram of p53 protein level when SK-Hep1 and HepG2 cells overexpress RPL6; C) Localization diagram of RPL6 and p53 in HepG2 cells; D) Interaction diagram of RPL6 and p53 in HepG2 cells; E) Schematic diagram of p53 mRNA level in HepG2 and SK-Hep1 cells; F) Schematic diagram of RPL6 protein expression in HepG2 cells after treatment with the proteasome inhibitor MG132; G) Schematic diagram of p53 protein expression level in RPL6-inhibited HepG2 cells after cycloheximide induction; H) Schematic diagram of p53 ubiquitination and degradation in HepG2 cells overexpressing RPL6.

[0029] Figure 5 A) Mass spectrometry analysis of FBXO22 binding to RPL6; B) Schematic diagram of p53 expression level after FBXO22 knockdown in HepG2 cells; C) Schematic diagram of p53 protein expression level after FBXO22 and RPL6 knockdown in HepG2 cells at the same time; D) Polyubiquitination level of p53 after FBXO22 overexpression in HepG2 cells; E) Schematic diagram of cell cloning experiment results after FBXO22 expression inhibition in HepG2 cells.

[0030] Figure 6This is a diagram related to the action mechanism of RPL6 and FBXO22. A) Schematic diagram of the expression levels of RPL6 and FBXO22 in HepG2 cells; B) Schematic diagram of the localization of RPL6 and FBXO22 in HepG2 cells; C) Schematic diagram of the interaction of RPL6 specifically with the N-terminal domain of FBXO22; D) Schematic diagram of the study of the interaction mechanism between RPL6 and FBXO22; E) The mRNA expression level of RPL6 after overexpression and knockdown of FBXO22 in HepG2 and SK-Hep1 cells; F) The expression level of RPL6 after overexpression of FBXO22 in SK-Hep1 cells; G) The expression level of RPL6 protein after overexpression or knockdown of FBXO22 in HepG2 and SK-Hep1 cells and treatment with the proteasome inhibitor MG132; H) Schematic diagram of the half-life of RPL6 after overexpression of FBXO22 in HepG2 and SK-Hep1 cells; I) Schematic diagram of the ubiquitination of RPL6 after overexpression or knockdown of FBXO22 in HepG2 and SK-Hep1 cells.

[0031] Figure 7 Figure 2 shows the correlation between RPL6 level, p53 level and tumor size in samples. A) p53 and RPL6 expression levels in samples from cohorts 1 and 2; B) RPL6 and p53 immunohistochemical scores in HCC tissues; C) Schematic diagram of the correlation between RPL6 level, p53 and tumor size in tumor cells. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0034] The solution proposed by the present invention is specifically described below through specific embodiments:

[0035] The materials and methods used in the present invention are as follows:

[0036] 1. HCC samples and patient data

[0037] Immunohistochemistry and prognostic analysis used a tissue microarray containing 120 pairs of primary HCC tissues (cohort I), all samples had similar clinicopathological characteristics and follow-up data. The second cohort (cohort II) contained 40 pairs of fresh tumor and non-tumor tissue samples for Western Blot analysis. Samples from both cohorts were obtained from the clinical specimen bank of liver surgery, Tongji Hospital, Huazhong University of Science and Technology. Before the start of the study, ethical approval was obtained from the Ethics Committee of Tongji Hospital and compliance with established ethical guidelines was ensured. Before participation, all participants signed an informed consent form to ensure that they understood the nature and significance of the study.

[0038] 2. Cell lines and culture conditions

[0039] MHCC97H and HLF cells were from the laboratory, and HEK293, SK-Hep1, Huh7, HCCLM3 and HepG2 cells were from the Cell Bank of the Chinese Academy of Sciences (CCTCC) in Wuhan. The cells were cultured in high-glucose DMEM medium (HyClone, USA) with 10% fetal bovine serum (Bovogen, Argentina) added to the medium. The cells were cultured in a humidified environment at 37°C and 5% carbon dioxide.

[0040] 3. Cell proliferation assay

[0041] Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) from Dojindo Corp, Japan. 1000 cells were seeded in a 96-well plate with 100 μL of culture medium per well and incubated overnight at 37°C to ensure adequate adhesion to the plate surface. After the initial incubation, 10 μL of CCK-8 reagent was added to each well every 24 hours for a maximum of 120 hours in the laboratory. After each addition of the reagent, the sample was incubated at 37°C for 1.5 hours to promote the reaction. The optical density (OD) of each well was measured at 450 nm using a Bio-Tek Instruments microplate reader from the United States. All experiments were repeated three times to ensure the reliability and consistency of the results.

[0042] 4. Immunofluorescence staining

[0043] Cells were fixed with 4% paraformaldehyde for 15 minutes. After fixation, cells were permeabilized with 0.5% Triton X-100 for 15 minutes at room temperature. Samples were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature before immunostaining. Primary antibodies targeting specific intracellular antigens were then used at 0.2 μg / ml. For detection, goat anti-mouse IgG secondary antibodies conjugated with FITC or DyLight 549, and goat anti-rabbit IgG secondary antibodies conjugated with DyLight 549 were used. In addition, to visualize the cell nucleus, DAPI, a fluorescent dye that strongly binds to DNA, was used for counterstaining. Images of stained samples were taken using a Nikon ECLIPSE C1 laser confocal microscope.

[0044] 5. EdU incorporation experiment

[0045] EdU incorporation experiments used a cell proliferation kit from Servicebio, following the manufacturer's instructions. Briefly, cells were cultured in 96-well plates. Once the cells were attached, medium containing EdU storage solution (G1603-1) was added and incubated for 2 hours. Afterwards, the cells were fixed with 4% paraformaldehyde solution for 15 minutes. The cells were subjected to a 30-minute click reaction and stained with Hoechst 33342 for 10 minutes to visualize the nuclei. Cells were observed using a confocal microscope with laser scanning technology, and cell proliferation was assessed by the proportion of EdU-positive cells.

[0046] 6. Clone formation experiment

[0047] Cells were cultured in 6-well plates at a density of 1000 cells per well for 14 days. After this culture period, cells were fixed with 4% paraformaldehyde solution. After fixation, cells were stained with crystal violet dye from Shanghai Beidatong. Clonogenicity was assessed by observing the emergence of cell clones.

[0048] 7. Co-immunoprecipitation (Co-IP)

[0049] Cells were lysed with 500 μL of immunoprecipitation (IP) buffer. Subsequently, the cell lysate was incubated with specific antibodies overnight at 4°C using a vertical rotating shaker. After overnight incubation, protein A / G magnetic beads (HY-K0202, MedChemExpress) were incubated at 4°C for 2 to 4 hours using a vertical rotating shaker to effectively capture the antibody-antigen complex. After the incubation was completed, the supernatant was carefully discarded and the magnetic beads were washed with TBST to remove unbound components.

[0050] 8. Plasmid construction and transfection

[0051] The present invention uses cDNA templates from wild-type HCC cells to amplify the full-length coding sequence (CDS) of RPL6 by polymerase chain reaction (PCR), and the primer sequence table is shown in Table 1. These sequences were inserted into expression vectors pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA (all purchased from Jikai Company). Plasmids GV115, GV118 and GV365 (purchased from Jikai Company) containing human RPL6 were constructed and integrated into the lentiviral expression vector Helper 1.0 (purchased from Jikai Company). In addition, short hairpin RNA (shRNA) for RPL6 gene knockdown was designed, and these sequences are in the lentiviral vector Helper 1.0, and the sequences are shown in Table 2. Cells were transfected using these expression vectors, and transfection was performed at a cell density of 50-60% to evaluate protein expression. Plasmids were transfected into HEK293T cells using polyethyleneimine (PEI, Invitrogen, USA) at a plasmid to PEI ratio of 1:4. HCC cell lines were transfected using Lipofectamine 3000 and P3000 (Invitrogen, USA). The lentiviral vector packaging mixture was transfected into 293T cells with 20 μg of GV vector plasmid, 15 μg of Helper 1.0 plasmid, and 10 μg of Helper 2.0 plasmid together with PEI and incubated in a 6 cm culture dish at a ratio of 4:1. The density of 293T cells reached about 80%. After 12 hours of incubation, the culture medium containing lentivirus was replaced with complete DMEM medium. Cell supernatants were collected after 48 hours. Subsequently, RNA and protein were extracted from HCC cell lines with specific gene knockdown and overexpression.

[0052] Table 1 Primer sequence list

[0053]

[0054] Table 2 shRNA sequence list

[0055]

[0056] 9. Animal Models

[0057] To establish an orthotopic liver tumor model, four-week-old male BALB / c nude mice were anesthetized and 1 million HCC cell suspensions (30 μL culture medium) were injected directly into the left liver lobe of the mice using a microinjector. The injection was performed under sterile conditions to reduce contamination. After injection, the mice were regularly observed for stress responses and tumor growth. 28 days after injection, the mice were euthanized and tumors were removed. The excised tumors were then fixed with 4% paraformaldehyde, weighed, photographed, and stored at -80°C for further analysis.

[0058] 10. Statistical analysis

[0059] When evaluating statistical significance between two groups, the Student t test for normally distributed data or the Wilcoxon signed rank test for paired samples was used. For multi-group analysis, one-way or two-way analysis of variance (ANOVA) was used. The chi-square test was used for immunohistochemistry scoring. Survival curves were drawn using the Kaplan-Meier method, and the Log-rank test was used to assess statistical significance. The results are expressed as mean ± standard deviation (SD). Each experiment was performed at least three times independently. The Pearson correlation method was used to determine the correlation. In the statistical analysis of the charts, a p value less than 0.05 was considered statistically significant (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, indicating no significant difference). All statistical calculations were performed using GraphPadPrism8.0 software.

[0060] result:

[0061] 1. Low expression of RPL6 in HCC

[0062] According to the Human Protein Atlas (HPA) database, it was observed that the expression of RPL6 protein in HCC samples was generally low ( Figure 1 A). To further investigate the expression of RPL6 in HCC, the GEO dataset (GSE25097) was analyzed and confirmed to be downregulated in HCC ( Figure 1 B). The expression level of RPL6 mRNA was evaluated in 110 pairs of HCC tissue samples and their corresponding adjacent normal tissues (cohort I). The results showed that the level of RPL6 mRNA in HCC tissues was significantly reduced compared with the surrounding normal tissues.

[0063] 2. Decreased expression of RPL6 promotes the growth of HCC cells in vitro and promotes tumor development in vivo

[0064] The present invention used Western Blot and quantitative real-time polymerase chain reaction (qRT-PCR) to analyze the expression levels of RPL6 in six HCC cell lines ( Figure 2 A). SK-Hep1 and HepG2 cells were selected for RPL6 silencing experiment ( Figure 2 B). Subsequently, the results of CCK-8 experiments in vitro showed that inhibition of RPL6 significantly increased the proliferation of HepG2 and SK-Hep1 cell lines ( Figure 2 C). In addition, cell proliferation was assessed by EdU assay and quantitatively analyzed by calculating the proportion of EdU positive (EdU+) cells. The results showed that reducing RPL6 levels resulted in an increase in the proportion of EdU+ cells ( Figure 2 D). The clone formation experiment showed that the reduction of RPL6 level enhanced the clone formation ability of HCC cells ( Figure 2 E). To investigate the effect of RPL6 on tumor growth, HepG2 cells with stable knockdown of RPL6 and their control group were first injected orthotopically into BALB / c nude mice. Tumor size was monitored every three days. Compared with the control group, the tumor volume of the RPL6 knockdown group was significantly increased ( Figure 2 F). Immunohistochemical evaluation showed that decreased RPL6 expression resulted in increased Ki-67 levels in RPL6 knockdown xenograft tumor tissues ( Figure 2 G).

[0065] 3. Increased RPL6 expression inhibits HCC cell growth in vitro and reduces tumor development in vivo

[0066] RPL6 overexpression experiments were performed using SK-Hep1 and HepG2 cells ( Figure 3 A). Subsequently, several in vitro experiments were performed to evaluate the effect of RPL6 on HCC cell growth. CCK-8 experiments showed that RPL6 inhibited the growth of SK-Hep1 and HepG2 cells ( Figure 3 B). In addition, clone formation assays showed that increased RPL6 expression led to a decrease in the number of clones formed by SK-Hep1 and HepG2 cells ( Figure 3 C). EdU experiments showed that overexpression of RPL6 led to a decrease in the proportion of EdU-positive cells in SK-Hep1 and HepG2 cells ( Figure 3 D). In vivo experiments, HepG2 cells were injected into nude mice, and the results confirmed that RPL6 inhibited tumor growth ( Figure 3 E). Immunohistochemical evaluation showed that increased RPL6 expression was associated with decreased Ki-67 levels in xenograft tumor tissues ( Figure 3 F).

[0067] 4. RPL6 inhibits the ubiquitination and degradation of p53 in HCC

[0068] The study showed that RPL6 was significantly associated with cell proliferation in TCGA-LIHC ( Figure 4 A). Western Blot (WB) experiments confirmed that knockdown of RPL6 led to a decrease in p53 expression in HCC cell lines, while overexpression of RPL6 was associated with an increase in p53 expression ( Figure 4 B). The present invention further explores the direct interaction between RPL6 and p53 proteins. Immunofluorescence analysis showed that RPL6 and p53 were co-localized in the nuclei of HCC cells ( Figure 4C). In addition, co-immunoprecipitation (Co-IP) experiments confirmed the endogenous interaction between RPL6 and p53 in HepG2 cells ( Figure 4 D). Since the change of RPL6 did not significantly affect the mRNA level of p53 ( Figure 4 E), so RPL6 regulates p53 expression through a post-transcriptional mechanism. HCC cells with RPL6 knockdown were treated with the proteasome inhibitor MG132, and the results showed that MG132 significantly reduced the stimulatory effect of RPL6 on p53 expression ( Figure 4 F). In addition, RPL6 inhibition resulted in increased p53 degradation induced by cycloheximide ( Figure 4 G). At the same time, it was also observed that in cells overexpressing RPL6, the ubiquitination of p53 was reduced ( Figure 4 H). Data indicate that RPL6 reduces the ubiquitination and subsequent degradation of p53 in HCC cells.

[0069] 5. RPL6 inhibits cell proliferation by attenuating FBXO22-mediated p53 polyubiquitination and degradation, enhancing p53 stability

[0070] FBXO22-mediated p53 polyubiquitination is essential for regulating p53 stability. The present invention conducted an immunoprecipitation experiment in HEK293T cells and identified RPL6 as a protein that interacts with FBXO22 ( Figure 5 A). In addition, given that RPL6 inhibits the ubiquitination and degradation of p53, its potential mechanism was further explored. Knockdown of FBXO22 significantly reversed the decrease in p53 levels caused by RPL6 knockdown ( Figure 5 B). Knockdown of FBXO22 stabilized the decreased p53 level caused by RPL6 knockdown ( Figure 5 C). In addition, overexpression of FBXO22 promoted the polyubiquitination of p53; however, RPL6 significantly attenuated this effect in a concentration-dependent manner ( Figure 5 D). We further examined whether the RPL6 / FBXO22 / p53 pathway plays a role in the invasive characteristics of HCC cells. Using CCK-8 and clone formation assays, our results showed that reducing FBXO22 expression reduced the proliferation and clone formation abilities of tumor cells enhanced by RPL6 knockdown ( Figure 5 E). The above results indicate that RPL6 promotes the invasive behavior of HCC cells through the FBXO22 / p53 signaling pathway.

[0071] 6. RPL6 as a substrate for FBXO22

[0072] To further investigate the interaction between RPL6 and FBXO22, we investigated whether RPL6 serves as a substrate for FBXO22. Figure 6 A), and both proteins are mainly localized in the nucleus ( Figure 6 B) RPL6 specifically interacts with the N-terminal domain of FBXO22 ( Figure 6 C). Notably, p53 also interacts with the N-terminal domain of FBXO22. When the interaction between RPL6 and FBXO22 was investigated using domain deletions (ΔM1 and ΔM2), it was found that the interaction was significantly weakened after deleting the M1 domain of RPL6. This suggests that the interaction between RPL6 and FBXO22 depends on the functional domain M1 of RPL6 ( Figure 6 D). To further explore this interaction, WB analysis was performed. The experimental results showed that the expression level of FBXO22 protein did not change significantly after RPL6 overexpression or knockdown ( Figure 6 E). In addition, as FBXO22 expression increased, RPL6 protein levels decreased in a dose-dependent manner ( Figure 6 F), suggesting a post-transcriptional modification pathway. Initially, changes in RPL6 protein levels caused by FBXO22 overexpression or knockdown were reversed by treatment with the proteasome inhibitor MG132 ( Figure 6 G). In addition, cycloheximide (CHX) treatment showed that the half-life of RPL6 was prolonged after FBXO22 knockdown, while FBXO22 overexpression significantly shortened the half-life of RPL6 ( Figure 6 H). Co-immunoprecipitation analysis showed that FBXO22 knockdown resulted in reduced ubiquitination of RPL6, whereas increased FBXO22 levels enhanced the ubiquitination of RPL6 ( Figure 6 I).

[0073] 7. Relationship between RPL6 and p53 in clinical samples

[0074] To explore the pathological characteristics of RPL6 in HCC tissues and its relationship with p53, WB and immunohistochemical staining analysis were performed on HCC tissue samples. In cohort 2 consisting of 40 pairs of tissue samples, RPL6 expression in HCC tissues was low and significantly correlated with p53 levels ( Figure 7 A). Similarly, immunohistochemical scoring revealed that the expression of RPL6 was positively correlated with that of p53 in HCC tissues ( Figure 7 B) In the experimental cohort, the vast majority of liver cancer tissue samples had low p53 expression, while adjacent normal tissues showed higher p53 expression, and only a few liver cancer samples showed high p53 levels. Figure 7As shown in C, the increased level of RPL6 in tumor cells promoted its interaction with FBXO22, thereby attenuating FBXO22-mediated polyubiquitination and degradation of p53.

[0075] In summary, the present invention explores the role of RPL6 as a tumor suppressor in the development of HCC and recognizes that RPL6 is an important new regulator of p53. Figure 7 As shown in C, the increase in RPL6 levels in tumor cells promotes its interaction with FBXO22, thereby attenuating FBXO22-mediated p53 polyubiquitination and degradation. The increase in p53 levels subsequently inhibits cell growth. Conversely, when RPL6 levels decrease, especially when the inhibitory effect of RPL6 on FBXO22-mediated ubiquitination and subsequent p53 degradation is reduced, p53 levels decrease, thereby promoting tumor development.

[0076] That is, both in vitro and in vivo experiments of the present invention show that RPL6 can inhibit the ubiquitination and degradation of p53 through FBXO22. This action stabilizes p53 and further inhibits tumor proliferation.

[0077] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0078] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Use of the RPL6 gene and / or its encoded protein as a target in the preparation of drugs for the treatment of liver cancer.

2. The use according to claim 1, characterized in that: The application is to enhance the expression of the RPL6 gene or enhance the function of the protein encoded by the RPL6 gene.

3. The use according to claim 2, characterized in that: Enhance the expression of RPL6 gene or enhance the function of the protein encoded by RPL6 gene through RNA technology or gene editing technology; or enhance the expression of RPL6 gene or enhance the function of the protein encoded by RPL6 gene through antisense nucleotide drugs or antibody drugs.

4. The use according to claim 2, characterized in that: The application is to enhance the expression of the RPL6 gene or enhance the function of the protein encoded by the RPL6 gene, so that the protein encoded by the RPL6 gene binds to the E3 ubiquitin ligase FBXO22, thereby inhibiting the polyubiquitination and / or degradation of the p53 protein.

5. Use of the RPL6 gene and / or its encoded protein as a marker in the preparation of products for diagnosing liver cancer.

6. The use according to claim 5, characterized in that: The product includes a reagent, a kit or a chip for detecting the activity of the RPL6 gene or the protein encoded by the RPL6 gene.

7. The use according to claim 5, characterized in that: The expression level of the marker is negatively correlated with liver cancer.

8. Use of an agent for enhancing the expression of the RPL6 gene and / or enhancing the protein encoded by the RPL6 gene in the preparation of a drug for treating liver cancer.

9. A drug for treating liver cancer, characterized in that: The drugs are: 1) siRNA that enhances RPL6 gene expression; 2) DNA or RNA that increases the expression or transcription of RPL6 encoding protein; 3) An activator for the protein encoded by RPL6, wherein the activator includes a small molecule compound, an antibody drug, a protein, a nucleic acid molecule, a polypeptide, a lipid, a carbohydrate or a combination thereof.

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