Novel blood biomarker SH3PXD2b for diagnosing hepatitis b virus-associated hepatocellular carcinoma and use thereof

Abstract

The present invention relates to a novel blood biomarker SH3PXD2B for diagnosing hepatitis B virus (HBV)-associated hepatocellular carcinoma and a use thereof. In order to identify a novel serum biomarker, transcriptome profiling including buffy coat WTS data was used to distinguish blood biomarkers specific to the caused of hepatocellular carcinoma Confirmation was made through validation using publicly available large-scale RNA-seq data, and qRT-PCR and AUC analyses were performed. Through this, it was confirmed that SH3PXD2B was upregulated exclusively in hepatitis B virus hepatocellular carcinoma in buffy coat transcripts, and qRT-PCR data demonstrated significant upregulation of SH3PXD2B in HBV-HCC. Therefore, SH3PXD2B is expected to be advantageously utilized as a blood biomarker for hepatitis B virus-associated hepatocellular carcinoma.

Description

SH3PXD2B, a novel blood biomarker for the diagnosis of hepatitis B virus-induced hepatocellular carcinoma and its uses

[0001] The present invention relates to a novel blood biomarker SH3PXD2B for diagnosing hepatitis B-related hepatocellular carcinoma (HBV-HCC) and its use.

[0002] Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, accounting for 70-85% of all cases. Despite recent advances in targeted and immunotherapies, HCC-related mortality has increased over the past 30 years, necessitating continued efforts to improve survival rates.

[0003] Although several biomarkers for the diagnosis of hepatocellular carcinoma (HCC) have been identified, early detection is limited by challenges such as low sensitivity and intratumoral heterogeneity. These limitations highlight the need for blood-based biomarkers to improve the early detection of HCC.

[0004] Fifty-four percent of hepatocellular carcinomas (HCCs) are caused by the hepatitis B virus (HBV), and HBV-associated HCC generally carries a high mortality rate. If HBV-induced HCC progresses, liver cancer often presents no symptoms in its early stages, making early detection difficult. Therefore, there is a critical need for novel blood biomarkers that can complement and improve the diagnostic power of existing markers.

[0005] Meanwhile, PBMCs and plasma samples have been widely used to identify blood-based biomarkers through transcriptome profiling, whereas buffy coat profiling for HCC blood-based markers has not yet been performed.

[0006] The purpose of the present invention is to provide a biomarker composition for diagnosing hepatitis B-related hepatocellular carcinoma (HBV-HCC), which comprises SH3PXD2B protein or a gene encoding the same as an active ingredient.

[0007] In addition, another object of the present invention is to provide a composition for diagnosing HBV-HCC, which includes as an active ingredient an agent capable of measuring the expression level of SH3PXD2B in blood.

[0008] In addition, another object of the present invention is to provide a kit for diagnosing HBV-HCC comprising the composition.

[0009] In addition, another object of the present invention is to provide a method for providing information necessary for diagnosing HBV-HCC, including a step of measuring the expression level of SH3PXD2B in blood.

[0010] To achieve the above purpose, the present invention provides a biomarker composition for diagnosing HBV-HCC, which comprises SH3PXD2B protein or a gene encoding the same as an active ingredient.

[0011] In addition, the present invention provides a composition for diagnosing HBV-HCC, which comprises as an active ingredient a preparation capable of measuring the expression level of SH3PXD2B in blood.

[0012] In addition, the present invention provides a kit for diagnosing HBV-HCC comprising the composition.

[0013] In addition, the present invention provides a method for providing information necessary for diagnosing HBV-HCC, comprising the steps of (1) measuring the expression level of SH3PXD2B from a blood sample isolated from a patient; (2) comparing the measured expression level of SH3PXD2B with a control sample; and (3) determining HBV-HCC if the measured expression level of SH3PXD2B is higher than that of the control sample.

[0014] The present invention relates to a novel blood biomarker, SH3PXD2B, for the diagnosis of hepatitis B virus-associated hepatocellular carcinoma and its use. In order to discover a novel serum biomarker, transcriptome profiling including buffy coat WTS data was used to identify a blood biomarker specific to the cause of hepatocellular carcinoma. The blood biomarker was confirmed through validation using publicly available large-scale RNA-seq data, and qRT-PCR and AUC analyses were performed. Through this, it was confirmed that SH3PXD2B was upregulated only in hepatitis B virus-associated hepatocellular carcinoma in the buffy coat transcriptome, and that qRT-PCR data showed a significant upregulation in HBV-HCC. Therefore, SH3PXD2B is expected to be useful as a blood biomarker for hepatitis B virus-associated hepatocellular carcinoma.

[0015] Figure 1 shows a schematic diagram of buffy coat separation from patient blood.

[0016] Figure 2 shows the validation data of PBMCs.

[0017] Figure 3 shows the results of Gene Ontology analysis.

[0018] Figure 4 shows the significant DEG results of HBV-HCC vs HBV Non-HCC verified by each VD.

[0019] Figure 5 shows the results of qRT-PCR and AUC analysis of SH3PXD2B.

[0020] The present invention provides a biomarker composition for diagnosing HBV-HCC, comprising SH3PXD2B protein or a gene encoding the same as an active ingredient.

[0021]

[0022] Meanwhile, the NCBI accession No. of SH3PXD2B used in the present invention may be NM_001308175.2, but is not limited thereto.

[0023]

[0024] The term “diagnosis” as used herein includes determining the susceptibility of a subject to a particular disease or condition, determining whether a subject currently has a particular disease or condition, determining the prognosis of a subject having a particular disease or condition, or therametrics (e.g., monitoring the condition of a subject to provide information about the efficacy of a treatment).

[0025]

[0026] In addition, the present invention provides a composition for diagnosing HBV-HCC, which comprises as an active ingredient a preparation capable of measuring the expression level of SH3PXD2B in blood.

[0027] Preferably, the blood may be buffy coat, but is not limited thereto.

[0028] Preferably, the agent capable of measuring the expression level may be, but is not limited to, a primer or probe that specifically binds to the SH3PXD2B gene, an antibody, peptide, aptamer or compound that specifically binds to the SH3PXD2B protein.

[0029] In addition, the present invention provides a kit for diagnosing HBV-HCC comprising the composition.

[0030] As used herein, the term "primer" refers to a short nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

[0031] As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several hundred bases in length, capable of specifically binding to mRNA, and is labeled so that the presence or absence of a specific mRNA and its expression level can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.

[0032] As used herein, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the biomarker of the present invention, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes specialized antibodies such as humanized antibodies.

[0033] In addition, the kit of the present invention may include an antibody that specifically binds to a marker component, a secondary antibody conjugate to which a label that develops color by reaction with a substrate is conjugated, a chromogenic substrate solution that reacts with the label, a washing solution, an enzyme reaction stop solution, etc., and may be manufactured into a plurality of separate packagings or compartments containing the reagent components used.

[0034] The term "peptide" used herein has the advantage of high binding affinity to target substances and resists denaturation even during heat and chemical treatments. Furthermore, due to its small molecular size, it can be attached to other proteins to form fusion proteins. Specifically, it can be attached to polymer protein chains, making it suitable for use as a diagnostic kit and drug delivery material.

[0035] As used herein, the term "aptamer" refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristics of being able to bind to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to an antigenic substance in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easy to synthesize, and therefore can be used as a substitute for antibodies.

[0036]

[0037] In addition, the present invention provides a method for providing information necessary for diagnosing HBV-HCC, comprising the steps of (1) measuring the expression level of SH3PXD2B from a blood sample isolated from a patient; (2) comparing the measured expression level of SH3PXD2B with a control sample; and (3) determining HBV-HCC if the measured expression level of SH3PXD2B is higher than that of the control sample.

[0038] Preferably, the blood may be buffy coat, but is not limited thereto.

[0039]

[0040] In the present invention, the method for measuring the expression level of SH3PXD2B may use, but is not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, or protein chip.

[0041] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0042]

[0043] <Experimental Example>

[0044] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0045]

[0046] 1. Prepare blood samples

[0047] Blood samples and data were obtained from the Ajou University Hospital Human Resources Bank with IRB approval from 6 normal control subjects, 46 patients with chronic hepatitis and cirrhosis (LC), and 129 patients with hepatocellular carcinoma (HCC).

[0048] Patient cohort

[0049] NV: Normal → Non-alcoholic steatohepatitis → Non-alcoholic / alcoholic cirrhosis → Liver cancer

[0050] Viral: Normal → Chronic viral hepatitis → Cirrhosis → Liver cancer

[0051]

[0052] 2. Extraction of buffy coat from patient blood

[0053] Blood was centrifuged at 2000 × g, 4°C, for 5 minutes to separate plasma or buffy coat. Total RNA was isolated from the separated buffy coat using TRIzol-LS reagent, and then whole transcriptome sequencing was performed (Fig. 1).

[0054]

[0055] 3. Whole transcriptome sequencing of buffy coat samples

[0056] Total RNA concentration was calculated using Quant-IT RiboGreen, and samples were run on a TapeStation RNA sreentape to assess the integrity of total RNA, and only high-quality RNA with an RIN ≥7.0 was used for library construction.

[0057] For each sample, libraries were independently prepared using 0.5 μg of total RNA using the Illumina TruSeq Stranded Total RNA Library Prep Globin Kit (Illumina) (Cat#20020613), and the first step was to remove rRNA from total RNA using the Ribo-Zero rRNA Removal Kit (Human / Mouse / Rat Globin) (Cat#20037135).

[0058] After this step, the remaining mRNA was fragmented into smaller fragments at high temperature using the total amount of ions. The fragmented RNA fragments were then subjected to cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen, Cat#18064014) and random primers. These cDNA fragments then underwent an end repair process, adding a single 'A' base, and then ligating adapters. The products were then purified and enriched by PCR to generate the final cDNA library.

[0059] Libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms and verified according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS, #KK4854). Indexed libraries were subsequently submitted to Illumina NovaSeq (Illumina, Inc., San Diego, CA, USA) and paired-end (2 × 100 bp) sequencing was performed at Macrogen Incorporated.

[0060]

[0061] 4. Analysis of differences in WTS data

[0062] Raw data (FASTQ) files were processed using AltAnalyze software (v.2.1.4), which used Kallisto and Ensemble72 annotation.

[0063] Raw count values ​​were obtained and used for differential expression analysis using the RDESeq2 package (v.1.34.0), and genes with total read counts less than 10 were excluded from further analysis.

[0064] Significant DEGs with p.adj < 0.05 and logIFCI > 0.5 were identified through comparative analysis using DESeq2 (v1.34.0), and volcano plots showing the DEGs were drawn using the R EnhancedVolcano package (v1.12.0). To evaluate the biological process enrichment of the significant DEGs, Gene Ontology analysis was performed using the compareCluster function of the R clusterProfiler package (v. 4.2.2), and the top 10 terms of up-DEGs or down-DEGs were displayed through dotplots.

[0065]

[0066] 5. DE Analysis of the Validation Dataset

[0067] The bulk RNA-seq datasets of hepatocellular carcinoma PBMCs were downloaded as raw data (FASTQ) files from NCBI BioProject with accession codes RPJNA739257, RPJNA909469, and RPJNA717231, respectively. Raw count data were obtained from the downloaded files using AltAnalyze software (v. 2.1.4) (Fig. 2).

[0068] RPJNA739257 was detected in 17 patients with hepatocellular carcinoma and 17 normal controls, RPJNA909469 was detected in 3 patients with hepatocellular carcinoma and 3 normal controls, and RPJNA717231 was detected in 8 patients with hepatocellular carcinoma and 4 normal controls.

[0069] Raw count values ​​were obtained and used for differential expression analysis using the R DESeq2 package (v.1.34.0), and genes with a total read count of less than 1 were excluded from further analysis.

[0070] Significant expression of buffy coat DEGs was verified using p.adj <0.05, and count values ​​were obtained using the plotCounts function of the R DESeq2 package (v.1.34.0) and visualized as haploid plots using the ggplot and geom_boxjitter functions of the R ggplot2 (v3.4.2) and ggpol (v.0.0.7) packages.

[0071]

[0072] 6. Redundancy measurement between rank-rank sets

[0073] Log2 fold values ​​were obtained from the DESeq2 results, and all genes included in the buffy coat WTS data and validation datasets were ranked based on their log2 fold values. These ranked lists were processed to include only genes common between the buffy coat and each validation dataset.

[0074] The web-based file-based RRHO (https: / systems.crump.ucla.edu / rankrank / rankranksimple.php) was loaded, and in all cases the step size was set to 100 to generate the Benjamini-Yekutieli corrected hypergeometric matrix and RRHO heatmap.

[0075]

[0076] 7. Measurement of mRNA expression in buffy coat using qRT-PCR analysis

[0077] Buffy coat RNA was synthesized into cDNA using SuperScript™IV VILO™ Master Mix (Invitrogen, Cat#11756050), and qRT-PCR was performed using amfiSure qGreen Q-PCR Master Mix (GenDEPOT, Cat#Q5602).

[0078] It was monitored in real time on a CFX Connect Real-Time PCR System (Bio-Rad Laboratories).

[0079]

[0080] qRT-PCR conditions were set as follows.

[0081] A. Stage 1: 95℃, 2 minutes

[0082] B. Stage 2: 95℃, 15 seconds

[0083] C. : 58℃, 34 seconds

[0084] D. : 72℃, 30 seconds

[0085] E. Repeat Stage 2 40 cycles

[0086]

[0087] qRT-PCR was performed using primers with the sequences below.

[0088] GeneAccession No.Forward sequenceReverse sequenceSH3PXD2BNM_001308175.25'-TCAGGTTGGTGGTTCGTCAG-3'5'-TCTCCTCTTCTTCAGGCTGC-3'GAPDHNM_001357943.25'-AGTATGACAACAGCCTCAAG-3'5'-TCATGAGTCCTTCCACGATA-3'

[0089]

[0090] 8. AUC (area under the curve) analysis

[0091] ROC analysis was performed using Graphpaid Prism software (version 10.0). This analytical approach involves generating ROC curves to assess the diagnostic accuracy of the biomarker under investigation. These curves were used to assess the specificity and sensitivity of the biomarker at various thresholds.

[0092] The area under the ROC curve (AUROC) was calculated, which provides a quantitative measure of the overall diagnostic effectiveness.

[0093] This analysis was performed with 95% confidence intervals (CIs) for the AUROC, which provide a statistical range of where the true value of the area under the curve would be expected to be, indicating the precision of the assessment.

[0094]

[0095] <Example 1> Gene Ontology Analysis of Biological Processes of Up-regulated and Down-regulated DEGs

[0096] Forty differentially expressed genes (DEGs) were identified in the hepatitis B virus-induced liver cancer group, and 987 differentially expressed genes (DEGs) were identified in the hepatitis B virus-induced non-liver cancer group.

[0097] Gene ontology (GO) analysis revealed that in the hepatitis B virus-induced liver cancer group, genes related to regulation of innate immune response and activation of immune cells were upregulated, while genes related to metabolism were downregulated (Fig. 3).

[0098]

[0099] <Example 2> Verification of Buffy Coat DEG

[0100] We analyzed DEGs in each validation dataset (VD) and compared each DE result with the buffy coat DE result. Among the 40 significant DEGs between the hepatitis B virus-induced liver cancer group and the non-liver cancer group in the buffy coat transcriptome analysis, a total of 27 genes showed significant differential expression in at least one validation dataset.

[0101] In VD1-3, 20, 16, and 9 DEGs overlapped with buffy coat DEGs, respectively.

[0102] Excluding HBA2, HBA1, and HBG2, which are considered to be derived from red blood cell contamination, a total of 24 genes overlapped with the buffy coat transcriptome, indicating that the buffy coat dataset of the present invention can be reliably used in HCC blood transcriptome analysis and that these genes have the potential to be used as blood biomarkers for HBV-associated HCC.

[0103] Combined with a validation dataset from buffy coat WTS analysis, we identified genes co-upregulated in hepatitis B virus-induced liver cancer.

[0104] Among them, IFITM3, SH3PXD2B, and BAMBI were reported to be upregulated in HCC tissues and have diagnostic and prognostic values. In particular, SH3PXD2B upregulation was repeatedly confirmed in VD1-3 in hepatitis B virus-induced liver cancer (Fig. 4).

[0105]

[0106] qRT-PCR and AUC (area under the curve) analysis for SH3PXD2B identified this gene as a significant DEG in buffy coat WTS data and all VDs (Fig. 5).

[0107] SH3PXD2B was upregulated only in hepatitis B virus-induced liver cancer in the buffy coat transcriptome, and showed significant upregulation in HBV-HCC in qRT-PCR data (AUC: 0.73, 95% confidence interval: 0.63–0.84, P < 0.001). The cut-off value was 0.88, based on the delta delta Ct value normalized to the mean of the normal group for the entire sample, and a value above this value is considered positive. This indicates that this gene has diagnostic potential as a blood biomarker for HBV-HCC.

[0108]

[0109] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for diagnosing hepatitis B-related hepatocellular carcinoma (HBV-HCC), comprising SH3PXD2B protein or a gene encoding the same as an active ingredient.

2. A composition for diagnosing HBV-HCC, comprising as an active ingredient a preparation capable of measuring the expression level of SH3PXD2B in blood.

3. A composition for diagnosing HBV-HCC, characterized in that the blood in the second paragraph is a buffy coat.

4. A composition for diagnosing HBV-HCC, characterized in that in the second paragraph, the agent capable of measuring the expression level is a primer or probe that specifically binds to the SH3PXD2B gene, or an antibody, peptide, aptamer, or compound that specifically binds to the SH3PXD2B protein.

5. A kit for diagnosing HBV-HCC comprising a composition according to any one of claims 2 to 4. 6.(1) A step of measuring the expression level of SH3PXD2B from a blood sample isolated from a patient; (2) a step of comparing the measured SH3PXD2B expression level with a control sample; and (3) A method for providing information necessary for diagnosing HBV-HCC, including a step of determining that the sample is HBV-HCC if the measured SH3PXD2B expression level is higher than that of the control sample.

7. A method according to claim 6, characterized in that the blood sample is a buffy coat.

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