Platelet marker for detecting liver cancer and detection kit

Abstract

The invention belongs to the field of liver cancer diagnosis, and particularly relates to a platelet marker for detecting liver cancer and a detection kit. According to the invention, platelets of a healthy (HC) individual and a hepatoma individual are collected for transcriptome sequencing, genes with high specific expression in the platelets of a hepatoma patient are identified for the first time through differential expression analysis, and FCGR2A, CPQ, KLRF1 and CDC5L are screened out. And in the subject working characteristic curve analysis of the training set, the area under the curve is up to 0.99. The mRNA expression levels of the FCGR2A, CPQ, KLRF1 and CDC5L genes identified by the invention can be used for diagnosing the liver cancer, and the discovery not only has important clinical significance, but also predicts wide popularization potential, and provides a new way and method for detecting the liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of liver cancer diagnosis, and in particular relates to a platelet marker and a detection kit for detecting liver cancer. Background Art

[0002] Hepatocellular carcinoma (HCC), as a highly prevalent and lethal malignant tumor, is the sixth most common cancer worldwide and the third leading cause of cancer-related death. Although surgical resection can provide patients with the opportunity for curative treatment in the early stages of the disease, the disease is located deep in the abdominal cavity, the early symptoms are not obvious, the liver itself has no pain nerves, high-risk groups (such as those infected with hepatitis B virus or hepatitis C virus) fail to undergo regular physical examinations, and most cancer patients suffer from chronic liver disease, resulting in the diagnosis of the disease in the middle and late stages. Although some progress has been made in new diagnostic methods and effective treatments in the past decade, research progress has been relatively slow. To date, alpha-fetoprotein (AFP), AFP isoform-L3 (AFP-L3), and des-gamma-carboxy prothrombin (DCP) are biomarkers approved for the diagnosis of liver cancer and preoperative prognostic assessment. However, these three markers have obvious limitations, including suboptimal sensitivity and specificity, and a high false positive rate, which makes it difficult to meet clinical needs.

[0003] Therefore, finding new biomarkers, especially non-invasive early diagnosis methods with high sensitivity and specificity, is crucial to improve the curative surgical resection rate and reduce the mortality rate of HCC patients. Platelets, as non-nuclear cell fragments shed by mature megakaryocytes, have high concentrations in peripheral blood and are easy to obtain. Platelets can absorb RNA and proteins through factors secreted by tumor cells (such as exosomes or microvesicles), become carriers of tumor-related molecules, and can also participate in cancer-related processes such as epithelial-mesenchymal transition, metastasis, angiogenesis, and immune evasion. In liquid biopsy technology, platelets have unique advantages due to their good stability, abundant number, and strong sensitivity. Platelets frequently interact with the tumor microenvironment in the circulation, and the molecules they carry may reflect the dynamic changes of the tumor. This information is of great value for early diagnosis, real-time monitoring of disease progression, and prediction of tumor site. These characteristics of platelets make them a promising tool in the field of liquid biopsy, which will help promote the development of personalized medicine and provide patients with more accurate and efficient cancer management strategies.

[0004] It is also based on this that the technical solution of the present invention is proposed. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a platelet marker for detecting liver cancer, wherein the platelet marker includes FCGR2A, CPQ, CDC5L and KLRF1.

[0006] Based on the same technical concept, another embodiment of the present invention is to provide a use of a platelet marker in preparing a kit for detecting liver cancer, wherein the platelet marker includes FCGR2A, CPQ, KLRF1 and CDC5L.

[0007] Based on the same technical concept, another embodiment of the present invention is to provide a detection kit for liver cancer, wherein the detection kit comprises detection reagents for detecting platelet markers, and the platelet markers include FCGR2A, CPQ, KLRF1 and CDC5L.

[0008] Preferably, the detection reagent is a primer, and the primer is used to detect the mRNA expression level of FCGR2A, CPQ, KLRF1 and CDC5L.

[0009] Preferably, the detection kit also includes the sample cDNA to be tested, DNA polymerase, four deoxynucleotide triphosphates, SYBR Green I, Mg 2+ , buffer, enzyme-free water, EDTA reagent, Trizol reagent and primers of internal reference genes GAPDH and β-actin.

[0010] Preferably, the sample to be tested is a human blood sample.

[0011] The beneficial effects of the present invention are:

[0012] In the present invention, by analyzing the platelet transcriptome of HC individuals and hepatoma individuals, it was found that FCGR2A and CPQ were significantly highly expressed in hepatoma individuals, KLRF1 and CDC5L genes were significantly lowly expressed in hepatoma individuals, and the diagnostic model constructed by the above four genes showed excellent performance. This model not only showed high sensitivity and specificity in distinguishing hepatoma individuals from HC individuals, but also achieved an accuracy of more than 95% in the independent training set, showing its great potential in clinical applications. At present, there is still a blank in the research on platelet FCGR2A, CPQ, KLRF1 and CDC5L in liver cancer, and the molecular mechanism of these genes in liver cancer occurrence has not been fully revealed. In addition, platelet FCGR2A, CPQ, KLRF1 and CDC5L have not yet been used in the development of liver cancer diagnostic reagents. However, with the deepening of research, the combination of these genes is expected to provide new strategies and targets for the accurate diagnosis of liver cancer.

[0013] In summary, the present invention provides a new biological marker for differential diagnosis of liver cancer. The mRNA expression levels of platelet genes FCGR2A, CPQ, KLRF1 and CDC5L are used as highly specific markers for diagnosing liver cancer. The present invention not only reduces the trauma of patients and reduces the cost of detection, but also makes the method easier to promote in clinical practice, especially in underdeveloped areas with limited resources, because RT-qPCR technology has strong anti-interference ability and easy-to-standardize operating procedures. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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.

[0015] Figure 1 This is the result diagram of genes that are significantly differentially expressed in platelets of HC individuals and hepatoma patients, screened out through differential expression analysis.

[0016] Figure 2 This is the result of univariate logistic regression analysis of differentially expressed genes.

[0017] Figure 3 This is the analysis result of the univariate Lasso regression screening model of the 4 genes with the largest odds ratio among the differentially expressed genes.

[0018] Figure 4 This is the result diagram of genes that are significantly differentially expressed in platelets of HC individuals and hepatoma individuals, screened out through differential expression analysis.

[0019] Figure 5 This is a graph showing the differential expression of FCGR2A in platelets of HC individuals (n=55) and hepatoma individuals (n=14) obtained by transcriptome sequencing (Wilcoxon rank sum test was used for statistics, ****P<0.0001).

[0020] Figure 6 This is a graph showing the differential expression of CPQ in platelets of HC individuals (n=55) and hepatoma individuals (n=14) obtained by transcriptome sequencing (Wilcoxon rank sum test was used for statistics, ****P<0.0001).

[0021] Figure 7This is a graph showing the differential expression of KLRF1 in platelets of HC individuals (n=55) and hepatoma individuals (n=14) obtained by transcriptome sequencing (Wilcoxon rank sum test was used for statistics, ****P<0.0001).

[0022] Figure 8 This is a graph showing the differential expression of CDC5L in platelets of HC individuals (n=55) and hepatoma individuals (n=14) obtained by transcriptome sequencing (Wilcoxon rank sum test was used for statistics, ****P<0.0001).

[0023] Fig. 9 The expression level of FCGR2A in platelets of HC individuals (n=15) and hepatoma individuals (n=14) obtained by RT-qPCR (Wilcoxon rank sum test was used for statistics; ****P<0.0001).

[0024] Fig.10 The expression level of CPQ in platelets of HC individuals (n=15) and hepatoma individuals (n=14) obtained by RT-qPCR (Wilcoxon rank sum test was used for statistics; ****P<0.0001).

[0025] Fig.11 The expression level of KLRF1 in platelets of HC individuals (n=15) and hepatoma individuals (n=14) obtained by RT-qPCR (Wilcoxon rank sum test was used for statistics; ****P<0.0001).

[0026] Fig.12 The expression level of CDC5L in platelets of HC individuals (n=15) and hepatoma individuals (n=14) obtained by RT-qPCR (Wilcoxon rank sum test was used for statistics; ****P<0.0001).

[0027] Fig.13 It is the ROC curve diagram of the multivariate logistic regression model constructed by integrating the expression levels of the two genes FCGR2A and KLRF1 in the training set (the X-axis represents the true negative rate, that is, specificity %; the Y-axis represents the true positive rate, that is, sensitivity %).

[0028] Fig.14 It is the ROC curve diagram of the multivariate logistic regression model constructed by integrating the expression levels of the two genes CPQ and CDC5L in the training set (the X-axis represents the true negative rate, that is, specificity %; the Y-axis represents the true positive rate, that is, sensitivity %). DETAILED DESCRIPTION

[0029] In order to fully illustrate the purpose, technical solutions and advantages of the present invention, the technical solutions will be described in detail below, and supplemented by specific embodiments and drawings. It should be noted that the embodiments shown are only a part of the many embodiments of the present invention, and their purpose is to illustrate rather than define the scope of the present invention. All other implementation methods derived from these embodiments by those skilled in the art without creative thinking are within the scope of protection of the present invention. In addition, the professional vocabulary and terms used in this article, unless otherwise defined, should be understood in accordance with the common understanding of technical experts in the relevant fields of the invention.

[0030] Example 1: Identification of key platelet genes for diagnosis of liver cancer

[0031] 1.1 Clinical sample platelet transcriptome sequencing

[0032] 1. Collection of Clinical Samples

[0033] In order to screen new highly specific biomarkers that can diagnose liver cancer, the present study collected peripheral whole blood from 69 patients recruited from West China Hospital of Sichuan University by venipuncture. Among these 69 patients, 55 were judged to be HC individuals, while the remaining 14 were hepatoma individuals. The collected whole blood was stored in 10 ml EDTA-coated BD vacuum blood collection tubes containing the anticoagulant EDTA. EDTA inhibits the immediate activation and aggregation of platelets through calcium chelation, thereby blocking the coagulation cascade reaction. In order to minimize the degradation of platelet RNA, whole blood stored in vacuum tubes needs to be separated within 48 hours at room temperature or 4°C.

[0034] 2. Platelet Separation

[0035] In order to ensure the quality and integrity of platelet RNA, the present invention adopts a set of optimized platelet separation steps. First, the whole blood stored in the EDTA-coated vacuum blood collection tube is gently inverted and mixed at room temperature or 4°C to ensure that the anticoagulant is evenly distributed. Subsequently, the platelets are separated according to the following steps: First, the blood sample is centrifuged at room temperature at a centrifugal force of 200g for 15 minutes to separate the platelet-rich plasma from the red blood cells and white blood cells. Then, the plasma is transferred to a 15 ml BD tube. During the operation, be careful to avoid inhaling the lower cell sediment, and some more plasma can be appropriately retained. Subsequently, the plasma is centrifuged again at room temperature at a centrifugal force of 200g for 5 minutes, and the supernatant is carefully transferred to a new BD tube, and the sediment is discarded. Immediately afterwards, it is centrifuged again at room temperature for 3 minutes at a centrifugal force of 300g, the supernatant is transferred to a new tube, and the sediment is discarded. Finally, centrifuge at 1500g for 10 minutes at room temperature, pour the supernatant directly into the waste bottle, and use a pipette to suck up and discard the remaining liquid, then invert the tube for a moment to ensure that as little plasma as possible is left. Use 1mL of Trizol reagent to resuspend the platelets in the pellet and transfer the resuspended liquid to the seed tube.

[0036] Under the protection of RNA stabilizing solution, platelet samples can be stored for a long time at a low temperature of -80°C while maintaining the integrity and quality of their RNA. Through this sophisticated platelet separation and preservation process, the present invention ensures the purity of the sample and the long-term stability of RNA, providing a solid foundation for subsequent biomarker screening and diagnostic analysis.

[0037] 3. Isolation, library construction and sequencing of platelet RNA

[0038] For the extraction of platelet RNA, first, the frozen platelet samples were placed on ice for slow thawing to minimize RNA degradation, and then total RNA was isolated from the platelets. The isolated RNA samples were subjected to strict quality assessment on the Bioanalyzer 2100 system using RNA6000Picochip. The present invention only selects platelet RNA samples with an RNA integrity number (RIN) greater than 7 and / or showing an obvious rRNA characteristic curve for inclusion in subsequent experiments, including reverse transcription to generate cDNA, synthesis of double-stranded DNA, library construction, and final sequencing.

[0039] (II) Identification of platelet genes as novel markers for liver cancer

[0040] 1. Processing of Raw Platelet Transcriptome Data

[0041] First, the raw data images obtained by sequencing were converted into standard Fastq format sequence information with double-end 150 base pair pairing using Illumina's bcl2fastq software. Then, the raw RNA sequencing reads data were strictly quality controlled using TrimGalore (v0.6.7) software, including the removal of low-quality data and trimming and shearing of adapter sequences to improve data quality. Subsequently, the cleaned reads were accurately aligned to the human reference genome (GRCh38) using HISAT2 (v2.2.1). Finally, featureCounts (v2.0.0) software was used in combination with the NCBI gene annotation gtf file GRCh38.84 version to calculate the number of reads mapped on genomic features (such as genes, exons, promoters, etc.).

[0042] 2. Screening of differentially expressed genes

[0043] In order to find differentially expressed genes in liver cancer platelets, the present invention used edgeR (v3.40.2) to perform statistical tests between HC individual platelets and hepatoma individual platelets. The present invention sets the threshold of logFC (log-fold change) to 1 to ensure the significance of gene expression changes; at the same time, the threshold of logCPM (log-counts per million) is set to 3 to exclude the interference of low-expression genes. In addition, the present invention uses FDR (false discovery rate) correction and sets the threshold to 0.05 to control the false positive rate caused by multiple comparisons. The present invention also excludes genes located on the Y chromosome and limits the analysis objects to protein-coding genes to ensure the biological significance and clinical relevance of the research results. After this series of screening conditions, we finally identified a series of genes that were significantly upregulated or downregulated in liver cancer platelets (such as Figure 1 ), and a univariate regression analysis was performed (as shown in Figure 2 These genes may serve as potential biomarkers and provide new insights into the diagnosis of liver cancer.

[0044] 3. Screening for genes with liver cancer diagnostic specificity

[0045] In order to evaluate the relationship between the mRNA expression level of a single gene and the binary dependent variable (HC individuals and hepatoma individuals), the present invention performed a univariate Lasso regression screening analysis on the above genes that were significantly upregulated in liver cancer (FCGR2A and CPQ) and significantly downregulated (CDC5L and KLRF1). Among these genes, FCGR2A, CPQ, CDC5L and KLRF1 genes showed significantly better odds ratios (OR, OR>100; such as Figure 3 This indicates that the expression levels of FCGR2A and CPQ genes in HC individuals are significantly increased compared with those in hepatoma individuals, and the expression levels of KLRF1 and CDC5L genes in HC individuals are significantly decreased compared with those in hepatoma individuals, and their expression changes have a strong predictive ability for the occurrence of HC individuals. The differential expression of the four genes in different individuals can also be intuitively observed through the heat map (as shown in Figure 2). Figure 4 Example 2: Verification of FCGR2A, CPQ, KLRF1 and CDC5L expression levels in platelets of clinical samples

[0046] First, the present invention uses the platelet transcriptome data in Example 1, takes the platelets of HC individuals as the control, normalizes the gene expression values, and calculates the expression levels of FCGR2A, CPQ, KLRF1, and CDC5L in HC individuals and hepatoma individuals. Figures 5 to 8 As shown, FCGR2A and CPQ were significantly upregulated in platelets of hepatoma individuals, and KLRF1 and CDC5L were significantly upregulated in platelets of hepatoma individuals.

[0047] At the same time, in the present invention, according to the above-mentioned platelet separation method, additional platelet samples from 15 HC individuals and 14 hepatoma individuals were collected. Further, in this example, RT-qPCR technology was used to detect the mRNA expression levels of FCGR2A, CPQ, KLRF1 and CDC5L in these samples. Table 1 below shows the primers used.

[0048] Table 1

[0049]

[0050]

[0051] The results are as follows Figures 9 to 12 As shown, the mRNA expression levels of FCGR2A and CPQ in platelets of hepatoma individuals were significantly increased, while the mRNA expression levels of CDC5L and KLRF1 were significantly decreased compared with those of HC individuals.

[0052] Example 3: Diagnostic value of platelet FCGR2A, CPQ, KLRF1 and CDC5L for liver cancer

[0053] By using the receiver operating curve (ROC) and its area under the curve (AUC) for verification, the present invention evaluated the differences in the mRNA expression levels of FCGR2A, CPQ, KLRF1 and CDC5L in platelets between HC individuals and hepatoma individuals to determine their potential as diagnostic markers for liver cancer, and further evaluated their test efficacy.

[0054] First, the platelet transcriptome data in Example 2 was used as a training set, and a univariate logistic regression model was constructed based on the expression levels of FCGR2A and KLRF1 to predict the occurrence of liver cancer. On the training set, these univariate regression models all performed well, with AUC values ​​reaching above 0.99 (e.g. Fig.13 In addition, a univariate logistic regression model was constructed based on the expression levels of CPQ and CDC5L to predict the occurrence of liver cancer. In the training set, these univariate regression models performed well, with AUC values ​​exceeding 0.99 (as shown in Figure 2). Fig.14 These results fully confirmed the strong ability and stability of platelet genes FCGR2A, CPQ, KLRF1, and CDC5L in liver cancer prediction.

[0055] Conclusion:

[0056] The present invention collects platelets from HC individuals and hepatoma individuals for transcriptome sequencing, and uses bioinformatics technology to analyze the differentially expressed genes of platelets at the population sequencing level. The results show that the mRNA levels of FCGR2A and CPQ genes are significantly upregulated in HC individuals, and the mRNA levels of CDC5L and KLRF1 genes are significantly downregulated in hepatoma individuals. Based on this, a logistic regression model is constructed according to the expression levels of FCGR2A, CPQ, KLRF1 and CDC5L genes. The model performs ROC analysis in the training set, and the results show that the AUC values ​​are all higher than 0.99. Further, the mRNA expression levels of FCGR2A, CPQ, KLRF1 and CDC5L are detected in HC individuals and hepatoma individuals using real-time quantitative polymerase chain (RT-qPCR) technology. The results show that their differences are statistically significant (P<0.05).

[0057] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which 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. A platelet marker for detecting liver cancer, characterized in that: The platelet markers include FCGR2A, CPQ, KLRF1 and CDC5L.

2. Use of a platelet marker in the preparation of a kit for detecting liver cancer, characterized in that: The platelet markers include FCGR2A, CPQ, KLRF1 and CDC5L.

3. A detection kit for liver cancer, characterized in that: The detection kit comprises detection reagents for detecting platelet markers, and the platelet markers comprise FCGR2A, CPQ, KLRF1 and CDC5L.

4. The detection kit according to claim 3, characterized in that The detection reagent is a primer, and the primer is used to detect the mRNA expression level of FCGR2A, CPQ, KLRF1 and CDC5L.

5. The detection kit according to claim 3, characterized in that The detection kit also includes a sample cDNA to be tested, DNA polymerase, four deoxynucleotide triphosphates, SYBR Green I, Mg 2+ , buffer, enzyme-free water, EDTA reagent, Trizol reagent and primers of internal reference genes GAPDH and β-actin.

6. The detection kit according to claim 5, characterized in that The sample to be tested is a human blood sample.

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