Liver cancer prognosis marker and application thereof

Abstract

The invention discloses a liver cancer prognostic marker and application thereof, relates to the technical field of molecular biomedicine, and discovers that significant correlation exists between mRNA expression quantity increase of a KDM4B gene and overall survival rate reduction of LIHC patients. GO analysis and KEGG analysis are respectively carried out on the co-expression gene of the KDM4B to obtain the correlation between the co-expression gene and signal channels such as cell cycle and AMPK, and the expression of the KDM4B is positively correlated with the immune cell infiltration level. Westernblot shows that the expression of the KDM4B in the liver cancer cell line is higher than that in normal cells. The invention provides a new marker for prognosis of liver cancer, and provides an important basis for selection of an LIHC precise treatment strategy.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biomedicine, and in particular to a liver cancer prognosis marker and application thereof. Background Art

[0002] Hepatocellular carcinoma (LIHC) is one of the most common malignant tumors worldwide, ranking third among the causes of cancer-related deaths, with approximately 830,000 related deaths in 2020. Smoking, alcoholism, hepatitis, and metabolic syndrome are all high-risk factors for LIHC. The prognosis of patients has gradually improved with the improvement of LIHC treatment level. However, postoperative metastasis is an important reason for the high recurrence of LIHC, and the overall prognosis of patients with advanced LIHC is still poor. Therefore, in-depth analysis and research on the molecular mechanisms related to LIHC metastasis and recurrence, and exploration of LIHC prognostic markers and drug treatment targets are of great significance for improving long-term efficacy. Summary of the invention

[0003] In view of this, the main purpose of the present invention is to provide a liver cancer prognosis diagnostic marker, wherein the diagnostic marker is KDM4B, namely histone demethylase KDM4B, whose NCBI Gene ID is 23030.

[0004] The present invention preliminarily explores the expression of KDM4B in liver cancer and its possible regulatory mechanism, and clarifies its influence on the occurrence and development of LIHC and its test value as a prognostic marker.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] A liver cancer prognosis diagnostic marker, wherein the diagnostic marker is KDM4B.

[0007] A second object of the present invention is to provide an application of the above-mentioned diagnostic marker or its detection reagent in the preparation of a product for liver cancer prognosis assessment.

[0008] Furthermore, the product is a reagent or a kit for detecting the expression level of the marker KDM4B gene.

[0009] Specifically, the reagents include reagents for detecting the expression level of diagnostic markers in samples by protein immunoassay technology, dye technology, nucleic acid sequencing technology, nucleic acid hybridization technology, chromatography technology, and mass spectrometry technology;

[0010] Preferably, the sample comprises a peripheral blood sample, a serum sample, a plasma sample, a urine sample, a saliva sample or a tissue sample.

[0011] Furthermore, the expression level of the marker is negatively correlated with the prognosis of liver cancer.

[0012] The third object of the present invention is to provide a product for predicting the prognosis of liver cancer, wherein the product comprises a reagent or a kit or a chip for detecting the above-mentioned markers.

[0013] Preferably, the reagent includes a probe that recognizes the marker gene; or a primer that amplifies the marker gene; or a binding agent that binds to the marker protein.

[0014] Preferably, the chip comprises a gene chip and a protein chip, the gene chip comprises an oligonucleotide probe for the marker gene for detecting the transcription level of the marker gene, and the protein chip comprises a specific binding agent for the marker protein; the kit comprises a gene detection kit and a protein detection kit, the gene detection kit comprises a reagent or a chip for detecting the transcription level of the marker gene, and the protein detection kit comprises a reagent or a chip for detecting the expression level of the marker protein;

[0015] Preferably, the kit comprises a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit and an electrochemiluminescence detection kit.

[0016] The present invention also aims to provide the use of KDM4B gene and / or its encoded protein as a target in the preparation of a drug for treating liver cancer.

[0017] Furthermore, the application is to inhibit the expression of KDM4B gene or inhibit the function of the protein encoded by KDM4B gene.

[0018] Furthermore, the expression of the KDM4B gene or the function of the protein encoded by the KDM4B gene is inhibited by RNA technology or gene editing technology; or the expression of the KDM4B gene or the function of the protein encoded by the KDM4B gene is inhibited by antisense nucleotide drugs or antibody drugs.

[0019] The present invention also aims to provide the use of an expression inhibitor or a knockout agent or a silencing agent of the KDM4B gene or an agent for inhibiting the protein encoded by the KDM4B gene in the preparation of a drug for treating liver cancer.

[0020] The present invention also aims to provide a drug for treating liver cancer, wherein the drug is at least one of the following:

[0021] 1) siRNA that inhibits KDM4B gene expression;

[0022] 2) DNA or RNA that inhibits the expression or transcription of the protein encoded by the KDM4B gene;

[0023] 3) Inhibitors against KDM4B encoded protein, including small molecule compounds, antibody drugs, proteins, nucleic acid molecules, polypeptides, lipids, carbohydrates or combinations thereof.

[0024] The beneficial effects of the present invention include at least:

[0025] (1) This invention opens up a new research direction and theoretical basis for the targeted treatment of LIHC;

[0026] (2) This invention provides a new marker for the prognosis of liver cancer and an important basis for the selection of precise treatment strategies for LIHC;

[0027] (3) The present invention provides a new target for preparing drugs for treating liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The differential expression results of KDM4B gene in different tumors and adjacent tissues, *P<0.05, **P<0.01, ***P<0.001 vs normal group. TPM: transcripts per million; LIHC: hepatocellular carcinoma.

[0029] Figure 2 The differential expression results of KDM4B in LIHC. The expression level of KDM4B gene in LIHC tissue samples showed a significant upregulation trend compared with healthy tissues (Ⅰ), and its expression was significantly correlated with (Ⅱ) race, (Ⅲ) gender, (Ⅳ) age of onset, (Ⅴ) body weight, and (Ⅵ) TP53 attenuation status. TCGA: The Cancer Genome Atlas.

[0030] Figure 3 Kaplan-Meier plot (GEPIA) of KDM4B mRNA expression in LIHC patients. Ⅰ: High KDM4B expression was significantly associated with poor prognosis of OS (P=0.0029); Ⅱ: High KDM4B expression was not significantly associated with DFS (P=0.098). Red indicates high expression. HR: hazard ratio; LIHC: hepatocellular carcinoma cell; mRNA: messenger RNA; GEPIA: gene expression profile interaction analysis; OS: overall survival; DFS: disease-free survival.

[0031] Figure 4 Analysis of the sites where KDM4B is prone to mutation in liver cancer patients. Ⅰ: TNR site mutation of KDM4B; Ⅱ: CLTC site mutation of KDM4B. TNR: trinucleotide repeat; CLTC: clathrin heavy chain.

[0032] Figure 5Construction and visualization of molecular network for the PPI network of KDM4B co-expressed genes. Ⅰ: Construction diagram of the PPI network of KDM4B co-expressed genes; Ⅱ: Visualization diagram of molecular network. PPI: protein-protein interaction.

[0033] Figure 6 GO functional annotation enrichment analysis of KDM4B co-expressed genes.

[0034] Figure 7 KEGG pathway enrichment analysis of KDM4B co-expressed genes.

[0035] Figure 8 Figure 1 shows the relationship between KDM4B expression and LIHC immune microenvironment. Ⅰ: Correlation diagram between KDM4B expression and LIHC immune microenvironment. Ⅱ: There was no statistically significant difference between KDM4B expression and immune cells (P>0.05). TCGA: The Cancer Genome Atlas; LIHC: Hepatocellular carcinoma.

[0036] Fig. 9 is the expression level of KDM4B in liver cancer cells and normal liver cells.

[0037] Fig.10 Identification of KDM4B gene knockdown efficiency. Ⅰ: Identification of KDM4B gene knockdown efficiency, detection of KDM4B mRNA in HepG2 cells by RT-qPCR. Ⅱ: Western blot detection of KDM4B protein level in HepG2 cells.

[0038] Fig.11 RNA interference with KDM4B expression inhibits the proliferation of liver cancer cells. Ⅰ: Knockdown of KDM4B in HepG2 cells, and observation of cell proliferation by CCK8 assay. Ⅱ: Knockdown of KDM4B in HepG2 cells, and detection of cell proliferation by clone formation assay.

[0039] Fig.12 After knocking down KDM4B, transcriptome sequencing was performed on HepG2 cells. A: Heat map of differential clustering analysis; B: Go analysis of its function at the molecular level. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] The solution proposed by the present invention is specifically described below through specific embodiments:

[0043] The materials and methods used in the process of the present invention are as follows:

[0044] 1. TIMER2.0 analysis

[0045] Tumor Immune Estimation Resource 2 (TIMER2) (http: / / timer.cistrome.org / ) is an interactive web analysis tool. Its main purpose is to comprehensively and flexibly study the situation of tumor-infiltrating immune cells (TIICs) and enable these data to be visualized. By using this tool, it is hoped that the expression differences of the KDM4B gene in various cancer types can be further explored. The present invention uses an independent sample t-test to comprehensively evaluate the mRNA expression of the KDM4B gene in clinical tumor samples, and compares the corresponding expression levels with those of normal tissues in the control group. The statistical significance level was set to P<0.001. When the P value was lower than 0.001, the difference between the two groups was considered to be highly statistically significant, thereby accepting the hypothesis that the mRNA expression level of the KDM4B gene in tumor tissues was significantly different from that in normal tissues.

[0046] 2. UALCAN analysis

[0047] In order to more comprehensively evaluate the changes in the transcriptional level of the KDM4B gene in cancer and its relationship with clinical characteristics, the rich resources of The Cancer Genome Atlas (TCGA) were further utilized and combined with the UALCAN platform for in-depth analysis. The UALCAN (http: / / ualcan.path.uab.edu) database not only allows the retrieval of the relative expression profiles of genes within a specific cancer type, but also allows grouping queries according to the different clinical pathological characteristics of cancer, such as different stages, grades, and patient races, thus providing a more comprehensive and detailed analysis perspective.

[0048] 3. Co-expressed genes and PPI network analysis

[0049] The top 100 genes co-expressed with KDM4B were screened from the Coexpedia tumor dataset based on the screening criteria of P ≤ 0.01 and fold difference ≥ 1.5. In order to further study the interaction between these genes, the bioinformatics resource library STRING (Search Tool for Retrieval of Interacting Genes) was used. Its website is https: / / string-db.org, which is dedicated to the prediction and analysis of protein-protein interactions (PPI). This database combines known and expected PPI networks with the aim of deeply exploring the complex functional relationships between proteins. In this study, PPIs with a score greater than 0.4 were selected as the criteria for statistically significant interaction effects. In addition, the Cytoscape (version 3.7.2) network analysis tool and visualization platform were used to more deeply analyze and understand the network data.

[0050] 4. GO analysis of Gene Ontology and enrichment study of KEGG pathway

[0051] The gene ontology (GO) resource platform is a comprehensive analysis tool that is specifically used to provide detailed annotations of gene functions and perform gene enrichment analysis. This study mainly focuses on exploring the similarities of genes in cellular components (Cellular Component CC), molecular functions (Molecular Function MF) and biological processes (Biological Process Biological Process BP). By comparing the distribution of the top 100 genes co-expressed with KDM4B in specific GO entries, it can be determined whether these genes are enriched in certain biological functions or processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a comprehensive bioinformatics database whose main purpose is to promote in-depth analysis and research of large molecular data sets. By using the KEGG database, the functions and regulatory modes of the top 100 genes co-expressed with KDM4B in known metabolic pathways were analyzed. R software was used for statistical analysis and interpretation of the results, and these results were visualized.

[0052] 5. Sangerbox3.0 immune infiltration analysis and immune cell analysis

[0053] Sangerbox3.0 is a platform that provides biotechnology and gene sequencing analysis. Its immune cell analysis webpage analyzes the correlation between single genes and multiple immune cells in the tumor microenvironment, and the immune infiltration analysis webpage performs immune infiltration analysis of single genes in pan-cancer.

[0054] 6. Kaplan-Meier survival curve

[0055] To better evaluate the prognostic significance of KDM4B expression in LIHC, the Kaplan-Meier website (https: / / www.kmplot.com / ) was used to discover and validate gene expression as a prognostic biomarker. In this study, Kaplan-Meier survival curves of mRNAs that met specific conditions were obtained. The relationship between these mRNAs and the overall survival (OS) of patients with hepatocellular carcinoma (LIHC) was further explored by calculating the risk score, and the specific effects of these molecular markers on the survival prognosis of patients were quantified by calculating the hazard ratio (HR).

[0056] 7. Western blot method

[0057] After lysing the cells with RIPA lysis buffer, the protein concentration was quantified by BCA method. The samples were mixed with 3x (x is the concentration multiple) sample buffer solution and boiled for 5 minutes. The protein samples were loaded on a 10% SDS gel and separated by 100V voltage for 90 minutes. The separated proteins were transferred to a nitrocellulose membrane. After blocking with 5% skim milk for 1 hour at room temperature, the primary antibody of KDM4B was incubated overnight at 4°C.

[0058] 8. Statistical processing

[0059] The data analysis process was processed using the SPSS19.0 statistical software package. For continuous measurement data, the format of mean plus minus standard deviation (x±s) was used for description. When comparing the data differences between different groups, the t-test method was applied. In terms of graphical presentation, GraphPad Prism 6.0 software was used to ensure the visual presentation of the results. The statistical significance level was set at P<0.05, that is, when the P value was less than 0.05, the difference was considered statistically significant.

[0060] result:

[0061] 1. Expression of KDM4B in LIHC patients

[0062] In this study, we first compared the mRNA expression of the KDM4B gene in the TIMER2 database in LIHC samples and normal samples ( Figure 1 ). By using samples from the TCGA database for UALCAN analysis, it was observed that the expression level of the KDM4B gene in LIHC tissue samples showed a significant upregulation trend compared with healthy tissues; at the same time, tumor samples were grouped based on clinical data such as age of onset, race, gender, weight, and TP53 status, and these grouped tumor samples were compared with normal samples. Compared with normal samples, the difference in tumor samples was statistically significant ( Figure 2 ).

[0063] 2. Relationship between KDM4B expression and prognosis of LIHC patients

[0064] Using Kaplan-Meier survival analysis combined with the gene expression profile data interactive platform, we explored the correlation between the expression level of KDM4B in LIHC patients and their overall survival (OS) and disease-free survival (DFS). The results showed that the mRNA expression level of KDM4B significantly affected the overall survival (OS) of patients, showing a strong statistical correlation (P = 0.0029, see Figure 3 Ⅰ), but its effect on disease-free survival (DFS) was relatively weak and did not reach statistical significance (P = 0.098, see Figure 3 II). Taken together, these findings highlight that decreased mRNA levels of KDM4B are significantly associated with decreased OS rates in LIHC patients.

[0065] 3. Analysis of KDM4B mutation sites in patients with liver cancer

[0066] The trinucleotide repeat (TNR) and clathrin heavy chain (CLTC) sites of KDM4B are prone to mutation in LIHC, such as Figure 4 As shown, the expression of KDM4B was decreased after both mutations (P=7.76e-03, P=9.87e-03).

[0067] 4. KDM4B co-expressed genes and enrichment analysis of functions and biological processes

[0068] In order to analyze the gene functions and enrichment of biological processes in more depth, the top 100 genes co-expressed with KDM4B gene in LIHC were screened from the Coexpedia database (see Table 1). The STRING database was used to create the PPI map, and the intersection targets were uploaded to the Multipleprotein option. After submitting these data, the PPI network map of KDM4B was obtained ( Figure 5 Ⅰ). In order to conduct more in-depth visualization analysis, it was imported into the network visualization software platform Cytoscape (version 3.7.1) ( Figure 5 II). In the samples of the top 100 genes co-expressed with the KDM4B gene, functional annotation enrichment analysis was performed using GO, which confirmed the role of GO in biosynthetic processes such as positive regulation of cells and mononuclear cell differentiation. Figure 6 In addition, it has molecular functions such as modification-dependent protein binding and histone binding, and is significantly enriched in cells with secretory granule membrane, apical part of cell, and neuronal cell body. Through KEGG pathway enrichment analysis, it was proved that its enrichment pathway in Alzheimer's disease is significant ( Figure 7 ).

[0069] Table 1 Top 100 genes co-expressed with KDM4B

[0070]

[0071]

[0072] 5. Relationship between KDM4B expression and LIHC immune environment

[0073] Through the immune infiltration analysis performed on the Sangerbox3.0 platform, it was found that there was no significant correlation between the immune infiltration environment of LIHC and the expression of KDM4B, and the P values ​​were all greater than 0.05. Through the analysis of immune cells using Sangerbox3.0, it was found that the changes in the infiltration levels of CD4+T cells, CD8+T cells, B cells, neutrophils, macrophages, and dendritic cells were related to the expression of KDM4B, and this change was positively correlated ( Figure 8 ).

[0074] 6. Western blot results

[0075] The protein level of KDM4B Fig. 9As shown, β-actin was used as an internal reference. Compared with the control group, the expression level of KDM4B in the liver cancer cell line was higher than that in the normal group, further verifying the above bioinformatics analysis results.

[0076] 7. Identification of KDM4B gene knockdown efficiency

[0077] Using the construction experiment of different RNA interference vectors, two RNA interference vectors KDM4B#1 and KDM4B#2 of KDM4B were constructed. The sequences of RNA were GUCCCGAGCUGGUCAAUGA(dT)(dT) and GCGACAACCUGUACCCUGA(dT)(dT), respectively. Lentivirus was packaged in 293T cells, and different liver cancer cell lines were infected. The cells were suspended and screened in the culture medium with puromycin. After obtaining the stable cell line of KDM4B with RNA interference, the cells were collected for real-time fluorescence quantitative PCR experiments and immunoblotting experiments to verify the RNA interference efficiency of KDM4B. The results showed that the two constructed RNA interference fragments could significantly downregulate the mRNA level and protein level of KDM4B in HepG (such as Fig.10 shown).

[0078] 8. RNA interference with KDM4B expression inhibits the proliferation of liver cancer cells

[0079] First, after RNA interference with KDM4B in HepG cells, the cell status was observed under white light. Compared with the control group, the number of cells was significantly reduced, and the cells underwent phenotypic changes. The most prominent feature was that the cells became larger, which may have inhibited the ability of tumor cells to divide. Further observation and detection by the incucyte living cell system found that the proliferation rate of cells in the KDM4B knockdown group was significantly reduced within 7 days (e.g. Fig.11 Ⅰ). Then, through the plate cloning experiment, it was found that the cloning ability of single cells in the KDM4B knockdown group was also significantly reduced (as shown in Fig.11 Ⅱ).

[0080] 9. Molecular mechanism of KDM4B regulating liver cancer progression

[0081] Afterwards, transcriptome sequencing was used to further explore the mechanism of KDM4B cell migration and invasion. The results showed that after RNA interference with KDM4B, the transcriptome sequencing results were significantly different from those of the control group. GO analysis showed that after knocking down KDM4B, the molecular functions of the cells changed significantly, including the function of nucleic acid binding, suggesting that KDM4B may play a role in the cell through its epigenetic modification enzyme function ( Fig.12 ).

[0082] 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.

[0083] 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.

[0084] 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. A liver cancer prognosis diagnostic marker, characterized in that: The diagnostic marker is KDM4B.

2. Use of the diagnostic marker or detection reagent thereof according to claim 1 in the preparation of a product for liver cancer prognosis assessment.

3. The use according to claim 2, characterized in that: The product is a reagent or a kit for detecting the expression level of the marker KDM4B gene.

4. The use according to claim 2, characterized in that: The expression level of the marker is negatively correlated with the prognosis of liver cancer.

5. A product for predicting the prognosis of liver cancer, characterized in that: The product comprises a reagent or a kit for detecting the marker according to claim 1.

6. Use of KDM4B gene and / or its encoded protein as a target in the preparation of drugs for treating liver cancer.

7. The use according to claim 6, characterized in that: The application is to inhibit the expression of KDM4B gene or inhibit the function of protein encoded by KDM4B gene.

8. The use according to claim 7, characterized in that: The expression of the KDM4B gene or the function of the protein encoded by the KDM4B gene is inhibited by RNA technology or gene editing technology; or the expression of the KDM4B gene or the function of the protein encoded by the KDM4B gene is inhibited by antisense nucleotide drugs or antibody drugs.

9. Use of an expression inhibitor or knockout agent or silencing agent of the KDM4B gene or an agent that inhibits the protein encoded by the KDM4B gene in the preparation of a drug for treating liver cancer.

10. A drug for treating liver cancer, characterized in that: The drug is at least one of the following: 1) siRNA that inhibits KDM4B gene expression; 2) DNA or RNA that inhibits the expression or transcription of the protein encoded by the KDM4B gene; 3) Inhibitors targeting KDM4B encoded protein, including small molecule compounds, antibody drugs, proteins, nucleic acid molecules, polypeptides, lipids, carbohydrates or combinations thereof.

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