Hepatocellular carcinoma prognosis evaluation marker TKFC and application thereof

Abstract

The invention discloses a hepatocellular carcinoma prognosis evaluation marker TKFC and application thereof, and belongs to the technical field of prognosis markers. By comparing tumor tissues of patients with different prognosis, the invention discovers that the HCC subtype with relatively strong invasiveness shows a systematic state imbalance, that is, the oxidative metabolism function represented by mitochondrial oxidative phosphorylation and tricarboxylic acid cycle is weakened, and the intracellular proliferation procedure related to chromatin remodeling and cell cycle progress is abnormally active. The state of low oxidative metabolism-high proliferation output may form the metabolism and epigenetic basis of malignant HCC. The low expression of the TKFC is obviously related to high-risk clinical pathological characteristics such as later tumor staging and higher occurrence rate of vascular invasion, and is an independent poor prognosis factor influencing the total lifetime of a patient, which indicates that the TKFC can be used as a prognosis biomarker of HCC.

Description

Technical Field

[0001] This invention relates to the field of prognostic biomarker technology, and in particular to a prognostic biomarker for hepatocellular carcinoma, TKFC, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Despite significant advancements in prevention, screening, diagnosis, and treatment technologies in recent years, the incidence and mortality rates of HCC remain high. Liver cancer typically develops alongside long-term chronic liver disease or hepatitis virus infection, and its progression takes decades. However, many patients are diagnosed at an advanced stage, and the tumors often have aggressive metastases, making it difficult for current treatments to effectively control disease progression, resulting in a generally poor prognosis for HCC patients.

[0003] Currently, prognostic assessment of HCC mainly relies on imaging examinations (such as CT and MRI) and liver function indicators (such as tumor markers like AFP). However, these methods still have limitations in predicting the accuracy of overall survival, recurrence risk, and metastatic trends. Imaging examinations can usually only evaluate tumor progression after obvious lesions have appeared, and biomarkers such as AFP are often affected by non-cancerous diseases such as hepatitis and cirrhosis, leading to false positive or false negative results. In addition, although some novel biomarkers have been proposed, most have not yet entered clinical application, or their association with HCC progression and prognosis lacks large-scale validation, making it difficult to meet the needs of personalized prognostic assessment and treatment strategy development.

[0004] Given the current situation, there is an urgent need to find new, easily detectable molecular markers that are closely related to the malignancy of tumors and patient prognosis in order to promote personalized diagnosis and treatment of HCC. Summary of the Invention

[0005] The purpose of this invention is to provide a prognostic biomarker for hepatocellular carcinoma, TKFC, and its application, in order to solve the problems existing in the prior art. Low expression of TKFC is significantly associated with high-risk clinicopathological features such as later tumor stage and higher incidence of vascular invasion, and is an independent adverse prognostic factor affecting the overall survival of patients.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an application of TKFC as a prognostic biomarker for hepatocellular carcinoma.

[0008] Optionally, patients with high TKFC expression have a better prognosis.

[0009] This invention also provides the application of reagents for detecting TKFC expression levels in the preparation of products for hepatocellular carcinoma prognostic assessment.

[0010] Optionally, the product may include a chip or a reagent kit.

[0011] Optionally, patients with high TKFC expression have a better prognosis.

[0012] The present invention also provides a kit for prognostic assessment of hepatocellular carcinoma, the kit comprising reagents for detecting TKFC expression levels.

[0013] The present invention discloses the following technical effects:

[0014] By comparing tumor tissues from patients with different prognoses, this invention reveals a systemic imbalance in highly aggressive HCC subtypes: weakened oxidative metabolism, exemplified by mitochondrial oxidative phosphorylation and the tricarboxylic acid cycle, coupled with abnormally active nuclear proliferation processes related to chromatin remodeling and cell cycle progression. This state of low oxidative metabolism and high proliferative output may constitute the metabolic and epigenetic basis of malignant HCC. Notably, TKFC is significantly underexpressed in this subtype, a finding validated in multiple independent public databases and clinical cohorts. Furthermore, low TKFC expression is significantly associated with later tumor stage, higher incidence of vascular invasion, and other high-risk clinicopathological features, and is an independent adverse prognostic factor affecting overall survival, suggesting that TKFC can serve as a prognostic biomarker for HCC.

[0015] Meanwhile, the tissue used for detection and analysis in this invention is paraffin-embedded HCC tissue. The proteins detected are stable, not easily degraded, can be preserved for a long time, are easy to detect, and have broad application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The amount of differentially expressed protein (dep) between HCC tissues with poor and good prognosis;

[0018] Figure 2 KEGG enrichment analysis of differentially expressed proteins between HCC tissues with poor and good prognosis;

[0019] Figure 3The biological process of differentially expressed proteins (upregulated) between HCC tissues with poor and good prognosis.

[0020] Figure 4 The differentially expressed protein biological process (downregulated) between HCC tissues with poor and good prognosis.

[0021] Figure 5 Reactome enrichment analysis for HCC patients with poor and good prognosis (upregulation);

[0022] Figure 6 Reactome enrichment analysis for HCC patients with poor and good prognosis (downregulation);

[0023] Figure 7 KEGG enrichment analysis of HCC with poor prognosis compared to matched neighboring non-tumor tissue (upregulated).

[0024] Figure 8 KEGG enrichment analysis of poorly prognostic HCC compared to matched neighboring non-tumor tissue (downregulated).

[0025] Figure 9 Venn diagram (A) showing the overlap of differentially expressed proteins between poor and good prognosis, top 10 most significantly downregulated proteins (B) and volcano diagram (C) showing the difference between HCC tissues with poor and good prognosis.

[0026] Figure 10 GO and KEGG enrichment analysis was performed on genes associated with TKFC expression in the TCGA-HCC cohort;

[0027] Figure 11 The expression levels of TKFC mRNA and protein in HCC tissues compared with neighboring non-tumor tissues from the TCGA and CPTAC datasets (A) and the relationship between TKFC expression and pathological stage (I-IV) and histological grade (G1-G4) in the TCGA-LIHC cohort (n=373) (B).

[0028] Figure 12 Validation analysis results for datasets from 18 HCCDB databases;

[0029] Figure 13 Kaplan-Meier overall survival curves for patients with high and low TKFC expression in the TCGA-LIHC cohort (n=373).

[0030] Figure 14 To validate TKFC using Western blots in three pairs of fresh HCC and adjacent normal tissues;

[0031] Figure 15 To validate differential TKFC expression between HCC and neighboring tissues using TMT-based proteomics in an independent cohort study;

[0032] Figure 16 Representative immunohistochemical images (A) and quantification of TKFC expression (B) in two independent tissue microarrays, and Kaplan-Meier survival analysis based on TKFC immunohistochemical scores in an independent tissue microarray cohort (n=85) (C). Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] First, an extreme phenotypic cohort consisting of patients with poor and good prognoses was constructed, and differentially expressed proteins were screened using DIA-MS-based quantitative proteomics. Bioinformatics analysis identified the candidate molecule, three-kinase / FMN cyclase (TKFC). Subsequently, public databases such as TCGA-LIHC and CPTAC were used to validate the expression pattern of TKFC in HCC and its correlation with clinicopathological features at the transcriptomic and proteomic levels. Furthermore, immunohistochemical staining was performed using a commercially available tissue microarray containing 85 HCC cases to independently validate the relationship between TKFC protein expression and overall survival and relapse-free survival, and its independent prognostic value was assessed using a Cox proportional hazards model.

[0039] Results: Proteomics screening revealed significantly low expression of TKFC in HCC tissues with poor prognosis. Multi-platform validation consistently confirmed that both TKFC mRNA and protein expression levels in HCC tissues were significantly lower than in adjacent normal tissues, and its low expression was significantly associated with later TNM stage, poorer differentiation, microvascular invasion, and elevated serum AFP levels. Survival analysis showed that low TKFC expression was an independent risk factor for poor overall survival and recurrence-free survival.

[0040] The commercially available human HCC tissue chips used in this invention were purchased from Shanghai Aode Biotechnology Co., Ltd., product numbers: HLivH180Su30, OD-CT-DgLiv04-001.

[0041] Example

[0042] 1. Clinical sample collection

[0043] This study collected 20 pairs of paired hepatocellular carcinoma (HCC) tissue and adjacent normal liver tissue samples from Shanxi Bethune Hospital between January 2014 and August 2023. All samples were obtained from fresh surgically removed tissue, fixed in 10% formalin for 12 hours, and then embedded in paraffin.

[0044] The patient inclusion criteria are as follows: (1) undergoing radical surgical resection; (2) postoperative pathological diagnosis of HCC; (3) age greater than 18 years.

[0045] Exclusion criteria include: (1) having received any chemotherapy, radiotherapy or targeted therapy before surgery; (2) having a history of other cancers; (3) having received a liver transplant; and (4) having incomplete clinical data.

[0046] A total of 20 patients were ultimately included, including 13 males and 7 females, aged 35 to 73 years. Patient age, sex, AJCC tumor stage, tumor size, and other clinicopathological characteristics were collected and analyzed. All procedures in this study followed the principles of the Declaration of Helsinki, and all patients signed informed consent forms. The study protocol was reviewed and approved by the Ethics Committee of Shanxi Bethune Hospital (Approval No.: LYLL-2025-005 / PJ76) and the Ethics Committee of the National Human Genetic Resources Sharing Service Platform (Approval No.: 2005DKA21300).

[0047] 2. Quantitative proteomics analysis

[0048] Twenty pairs of paired paraffin-embedded HCC and adjacent normal tissues were included for LC-MS / MS analysis. Peptides were separated using an EASY-nLC 1200 ultra-high performance liquid chromatography system and subsequently detected on an Orbitrap Exploris 480 mass spectrometer in data-independent acquisition (DIA) mode. Raw data were searched and analyzed using DIA-NN software (version 1.8) in the UniProt Homo_sapiens reference protein database (version 20230103, containing 20,365 sequences). Subsequent analyses included protein quantification, repeatability assessment, functional annotation (GO, KEGG), and screening for differentially expressed proteins. Proteomics experiments and preliminary data analysis were commissioned to Beijing Jingjie Biotechnology Co., Ltd.

[0049] The tissue used in this invention for detection and analysis is paraffin-embedded HCC tissue. The proteins detected are stable, not easily degraded, can be preserved for a long time, are easy to detect, and have broad application prospects.

[0050] 3. Results

[0051] 3.1 Clinical proteomics reveals key molecular features of poor prognosis in liver cancer

[0052] To systematically identify the key molecular processes driving the malignant progression of hepatocellular carcinoma (HCC) and reveal their deep association with clinical prognosis, this study first selected paraffin-embedded tissue samples from HCC patients with significant prognostic differences based on strict clinical follow-up criteria. These samples were then divided into a poor prognosis group (recurrence within 1 year post-surgery and death within 3 years) and a good prognosis group (disease-free survival ≥ 5 years, with no recurrence, metastasis, or death during follow-up) (Table 1). Subsequently, quantitative proteomics was used to perform global protein expression profiling analysis on 10 pairs of matched HCC tissues and their paired adjacent normal tissues. After rigorous quality control to ensure the reliability of protein extraction, enzymatic digestion, and labeling processes, a total of 6086 quantifiable proteins were identified.

[0053] Table 1

[0054]

[0055] Using |Fold Change|>1.5 and P<0.05 as the threshold for difference, 250 significantly differentially expressed proteins were screened between the poor prognosis group and the good prognosis group (49 upregulated and 201 downregulated); 437 upregulated proteins and 786 downregulated proteins were identified between HCC tissues with poor prognosis and paired adjacent normal tissues; while 123 upregulated proteins and 362 downregulated proteins were identified between HCC tissues with good prognosis and paired adjacent normal tissues. Figure 1 ).

[0056] Bioinformatics analysis of differentially expressed proteins revealed a highly coordinated pro-cancer molecular network in hepatocellular carcinoma tissues with poor prognosis, characterized by the following two aspects: (1) Nuclear dysfunction: genomic instability and epigenetic dysregulation. A series of pathways related to the regulation of nuclear structure and function were significantly upregulated in the poor prognosis group, including chromatin and chromosome structural abnormalities and abnormally active epigenetic remodeling. At the same time, the upregulation of serine / threonine protein kinase complex, protein kinase complex and cyclin-dependent kinase activity drove uncontrolled cell cycle progression; while the upregulation of protein phosphatase inhibitory activity indicated that the key tumor suppressor dephosphorylation process was inhibited, further exacerbating the imbalance of proliferation signals. (2) Unique metabolic reprogramming: enhanced glycolysis and weakened oxidative metabolism. In tumors with poor prognosis, we observed extensive activation of glucose metabolism pathways (such as glycolysis / gluconeogenesis, fructose and mannose metabolism and pyruvate metabolism). At the same time, core oxidative and catabolistic pathways, represented by mitochondrial oxidative phosphorylation, the tricarboxylic acid cycle, fatty acid β-oxidation, and peroxisome function, were systematically downregulated. Figures 2-8 Furthermore, peroxisome function and the ferroptosis pathway were also suppressed in the poor prognosis group, suggesting that ferroptosis evasion may be a common event in the occurrence and development of hepatocellular carcinoma, and that HCC cells may rely on the suppression of this pathway to maintain survival regardless of prognosis.

[0057] To precisely identify key pivotal proteins driving malignant phenotypes from the aforementioned complex molecular network, this invention intersected differentially expressed proteins between the "poor prognosis HCC vs. good prognosis HCC" group and the "poor prognosis HCC vs. paired adjacent normal tissue" group, obtaining 166 common differentially expressed proteins. Figure 9 (A). Ranking the Fold Change of intersecting proteins, Triokinase and FMN Cyclase (TKFC) were of particular interest among the top 10 genes. Figure 9(B) Proteomics data showed that TKFC was significantly underexpressed in the poor prognosis group (Fold Change=2.146, P=0.025). Figure 9 (C). To further explore the biological functions of TKFC, GO and KEGG pathway enrichment analyses were performed on genes related to TKFC expression using the TCGA database. The results showed that TKFC is closely related to key metabolic processes such as peroxisome function, fatty acid metabolism, PPAR signaling pathway, glucose metabolism, and lipid oxidation. Figure 10 ).

[0058] 3.2 TKFC was significantly underexpressed in hepatocellular carcinoma tissues and was associated with clinicopathological features and poor prognosis.

[0059] TKFC expression was primarily enriched in organs such as the liver, duodenum, and small intestine. In terms of subcellular localization, TKFC was mainly located in peroxisomes and cytoplasm, with some expression also in the nucleus. Expression profiling analysis showed that in paired TCGA samples and multiple independent public datasets such as CPTAC and HCCDB, the mRNA and protein expression levels of TKFC in hepatocellular carcinoma tissues were significantly lower than those in paired adjacent normal liver tissues. Figure 11 China A, Figure 12 Furthermore, the expression level of TKFC gradually decreased with increasing tumor histological grade (from G1 to G3 / G4) and progression of pathological stage (from Stage I to Stage III / IV). Figure 11 (B)

[0060] Clinical association analysis based on the TCGA database further demonstrated that low TKFC expression was significantly associated with several more aggressive pathological features, including later T stages (T2-T4), a higher incidence of vascular invasion, and higher serum alpha-fetoprotein levels (Table 2). Survival analysis confirmed the clinical prognostic value of TKFC expression, with high expression significantly associated with longer overall survival (hazard ratio HR=0.68). Figure 13 ).

[0061] Table 2

[0062]

[0063] To further verify the above expression differences at the protein level, this invention randomly selected three more pairs of fresh frozen liver cancer and adjacent normal tissue samples (from the Hepatobiliary Surgery Laboratory of Shanxi Bethune Hospital, ethically approved: No.:LYLL-2025-005 / PJ76), and detected TKFC protein expression by Western Blot. The results consistently showed that TKFC expression was downregulated in cancer tissues. Figure 14 ).

[0064] Western Blot: Total protein was extracted using RIPA lysis buffer (Thermo Scientific, 89900) containing a protease inhibitor cocktail (MCE, HY-K0010). After boiling denaturation, the protein was separated by 10% SDS-PAGE electrophoresis and transferred to a PVDF membrane (Millipore, IPVH00010) using a wet transfer method. The membrane was blocked with 5% skim milk at room temperature for 1 hour, followed by incubation overnight at 4°C with the corresponding primary antibodies. The primary antibodies used included: anti-TKFC antibody (Thermo, PA5-58810; or UpingBio, YP-Ab-14730) and anti-β-actin antibody (Cell Signaling Technology, 4970). The following day, the membrane was incubated with HRP-labeled goat anti-rabbit secondary antibody (Imunoway, RS0002) at room temperature for 1 hour, and finally developed on a ChemiDoc imaging system (Sagecreation, MiniChemi500) using ECL chemiluminescent substrate (Vazyme, E412-01).

[0065] Furthermore, this invention obtained a published 10×TMT-labeled quantitative proteomics dataset, and the analysis results also showed that TKFC was significantly lowly expressed in liver cancer tissues. Figure 15 ).

[0066] 4. Verification

[0067] This invention used two independent tissue microarrays (HLivH180Su30 and OD-CT-DgLiv04-001) for immunohistochemical analysis, and the results showed that TKFC protein expression was significantly downregulated in both independent cohorts. Figure 16 China A- Figure 16 (B)

[0068] Immunohistochemical scoring and Kaplan-Meier survival curves were performed based on tissue microarrays (OD-CT-DgLiv04-001) containing complete prognostic information from 85 patients. The results showed that high TKFC expression was significantly associated with better patient prognosis. Figure 16 (C)

[0069] The immunohistochemical staining procedure was as follows: After dewaxing and hydration, the sections were incubated with 3% H2O2 solution at room temperature for 10 minutes to block endogenous peroxidase activity. Antigen retrieval was performed in sodium citrate antigen retrieval solution at 95℃ for 20 minutes. Subsequently, the sections were incubated overnight with the primary antibody at 4℃. The primary antibody used was anti-TKFC antibody (Thermo, PA5-58810). The next day, the sections were incubated with the secondary antibody (Dako, K8002) and DAB staining were performed sequentially. Hematoxylin was used to counterstain the cell nuclei, followed by dehydration, clearing, and mounting with neutral resin. The staining results were independently evaluated by two pathologists unaware of the grouping information. In case of disagreement in scoring, both pathologists reviewed the sections together until a consensus was reached. Staining intensity scores: 0 (no staining), 1 (weak), 2 (moderate), 3 (strong). Positive cell percentage score: 0 (<5%), 1 (5-25%), 2 (26-50%), 3 (51-75%), 4 (76-100%). The final immunohistochemical score (IHC score) is the product of the two scores (range 0-12). Samples were divided into low TKFC expression group and high TKFC expression group based on the median score.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of TKFC as a prognostic biomarker for hepatocellular carcinoma.

2. The application as described in claim 1, characterized in that, Patients with high TKFC expression have a better prognosis.

3. Application of reagents for detecting TKFC expression levels in the preparation of products for prognostic assessment of hepatocellular carcinoma.

4. The application as described in claim 3, characterized in that, The products include chips or reagent kits.

5. The application as described in claim 3, characterized in that, Patients with high TKFC expression have a better prognosis.

6. A kit for prognostic assessment of hepatocellular carcinoma, characterized in that, The kit includes reagents for detecting TKFC expression levels.

Images