A marker HNF1B for identifying HCC subtypes and construction of a DPHCC in vitro model

Abstract

The application discloses a marker HNF1B for identifying HCC subtypes and construction of a DPHCC in-vitro model, and belongs to the technical field of biological medicines. The application can identify DPHCC subtypes and common HCC by detecting the expression of HNF1B in situ liver cancer, can more effectively predict the survival time of patients, and provides more powerful support for effective diagnosis and treatment, so as to help doctors select more suitable patient groups and methods for treatment and perform more accurate personalized treatment. Meanwhile, the application provides a method for preparing a DPHCC in-vitro cell model, and is beneficial to researches in aspects of drug screening and mechanism exploration for DPHCC.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a marker HNF1B for identifying HCC subtypes and construction of a DPHCC in vitro model. BACKGROUND

[0002] Primary liver cancer is the sixth most commonly diagnosed cancer in the world and the third leading cause of cancer death, and the incidence is rising. According to the pathological type of primary liver cancer, WHO divides primary liver cancer into hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and hepatocellular-cholangiocarcinoma composite carcinoma (CHC), and HCC and ICC are the most common malignant types of liver cancer (90%). Since liver cancer patients are mostly in the middle and advanced stages when diagnosed, and there is no ideal treatment method, the prognosis is usually poor, and the 5-year survival rate is less than 5%. Therefore, effectively predicting the survival period of patients after surgery will help to select the appropriate treatment method and provide strong support for timely and effective diagnosis and treatment.

[0003] At present, there are various methods for diagnosing liver cancer, including imaging examination, liver function examination, liver cancer marker examination, histological examination, etc., and the diagnosis efficiency is getting higher and higher. However, for the same type of diagnosis, different patients may have different prognosis conditions. For example, patients diagnosed as HCC, patients with TP53 tumor suppressor gene mutation often have poor prognosis. Among the subtypes of HCC, there is a dual-phenotype hepatocellular carcinoma (DPHCC), which was first reported in China in 2011, with an incidence of about 10% of HCC, belonging to a newly defined subtype. DPHCC is similar to ordinary HCC in histopathological morphology, but this subtype can express the immunophenotype of HCC and ICC at the same time. Clinically, it is observed that DPHCC has higher microvascular invasion, intrahepatic and extrahepatic metastasis and postoperative recurrence rate, and the prognosis of patients with this subtype is significantly poorer than that of ordinary HCC. For different prognosis conditions, the preferred diagnosis and treatment method is also different. In the cases of ICC and DPHCC, if feasible, surgical resection is the main treatment choice, often accompanied by lymph node dissection due to the high frequency of lymph node metastasis. The treatment options for HCC include resection, ablation, transplantation, transarterial chemotherapy and radiotherapy, and systemic therapy. The resection of ordinary HCC does not involve lymph node dissection. Therefore, clear diagnosis of DPHCC and ordinary HCC is of great significance for guiding clinical diagnosis and treatment.

[0004] Currently, for ICC, bile duct marker detection (C-reactive protein, CK7, CK19) is the main diagnostic method. However, there is no clear and effective differential diagnostic index for DPHCC. Therefore, the diagnostic method of ICC is usually also used to identify the bile duct type in HCC, but studies have shown that the sensitivity of C-reactive protein for ICC diagnosis is only 75%, and the diagnostic efficiency for DPHCC is not clear. In addition, the current research on DPHCC is mostly in the identification of clinical phenotypes, and lacks in-depth mechanism exploration.

[0005] HNF1B is located on chromosome 17q12 and is a member of the POU cis-acting transcription factor family, which is involved in the regulation of systemic lipid, carbohydrate and protein metabolism, and plays an important role in regulating liver development and hepatocyte differentiation. In recent years, studies have shown that HNF1B is significantly related to the occurrence and development of ovarian clear cell carcinoma, prostate cancer, pancreatic cancer and other tumors. However, the expression significance of HNF1B in liver malignant tumors and its clinical application have not been fully studied. SUMMARY

[0006] To solve the above problems, the present application finds that HNF1B has an important relationship with the prognosis of HCC patients through bioinformatics analysis, and explores the regulation of the gene on the proliferation, migration and invasion ability of hepatocellular carcinoma cells through basic experiments, finds that HNF1B can promote the expression of ICC immunophenotype of HCC cells, thereby proposing the value of HNF1B in the diagnosis of DPHCC subtype, and providing a method for preparing a DPHCC cell model.

[0007] The first object of the present application is to provide a biomarker for identifying a subtype of hepatocellular carcinoma, wherein the biomarker is hepatocyte nuclear factor 1 beta (HNF1B).

[0008] Further, the subtype of hepatocellular carcinoma is common hepatocellular carcinoma or double phenotype hepatocellular carcinoma (DPHCC).

[0009] Further, the biomarker has a low expression level in common hepatocellular carcinoma and a high expression level in double phenotype hepatocellular carcinoma.

[0010] The second object of the present application is to provide the use of the above biomarker in preparing a detection reagent for a subtype of hepatocellular carcinoma.

[0011] Further, the detection sample is liver cancer tissue or liver cancer paracancerous tissue.

[0012] Further, the detection reagent is used for detecting the expression level of HNF1B in the to-be-detected tissue.

[0013] Further, the detection reagent can be an immunohistochemical detection reagent.

[0014] A third object of the present application is to provide the use of the biomarker in the preparation of a detection reagent for the prognosis of a hepatocellular carcinoma patient.

[0015] Further, the prognosis prediction of liver cancer is the survival prediction of a patient after liver cancer resection.

[0016] A fourth object of the present application is to provide a kit for detecting the prognosis of a hepatocellular carcinoma patient, the kit comprising a reagent for detecting the expression of HNF1B.

[0017] A fifth object of the present application is to provide a dual phenotype hepatocellular carcinoma in vitro cell model, the cell model being obtained by introducing an HNF1B-encoding gene into a liver cancer cell.

[0018] Further, the nucleotide sequence of the HNF1B-encoding gene is NM_000458.4.

[0019] Further, the liver cancer cell can be from a liver cancer tissue, a liver cancer paracancerous tissue, or a liver cancer cell line, wherein the liver cancer cell line includes but is not limited to SMMC-7721, Bel-7402, MHCC97, HepG2, Hep3B, Huh-7, PLC / PRF / 5, etc.

[0020] A sixth object of the present application is to provide the use of the dual phenotype hepatocellular carcinoma in vitro cell model in the screening of a dual phenotype hepatocellular carcinoma treatment drug.

[0021] Further, the drug to be screened is evaluated according to the difference in the tumor biological behavior of the dual phenotype hepatocellular carcinoma in vitro cell model after administration of different drugs.

[0022] The beneficial effects of the present application are:

[0023] (1) Currently, the diagnosis of DPHCC requires the detection of multiple immunophenotype markers of HCC and ICC, which increases the clinical workload and the burden on patients. The present application can distinguish the DPHCC subtype and the common type in HCC patients through HNF1B only, which is more economical and efficient than the existing multiple marker detection method, and can also timely detect the high-risk subtype in HCC patients, facilitating doctors to take active treatment measures and improving the survival period of patients.

[0024] (2) Currently, most of the research on DPHCC is in the identification of clinical phenotypes, and lacks in-depth mechanism exploration, partly due to the lack of a DPHCC in vitro cell model. The present application finds that the up-regulation of the HNF1B gene in an HCC cell line can express the ICC immunophenotype. At the same time, the proliferation, migration, and invasion ability of the cell are also significantly improved, which is consistent with the clinical characteristics of DPHCC. A cell model for conducting related research on DPHCC in vitro is obtained.

[0025] (3) Lack of cell model for in vitro research of DPHCC limits researches on drug screening, mechanism exploration, etc. of DPHCC. The application provides a cell model for in vitro DPHCC, which facilitates targeted research experiments. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 For bioinformatics analysis of differentially expressed gene results;

[0027] Figure 2 For HNF1B immunohistochemistry of liver cancer tissue and paracancerous tissue, the left graph is paracancerous tissue, the middle graph is DPHCC with low expression of HNF1B, and the right graph is DPHCC with high expression of HNF1B;

[0028] Figure 3 For Kaplan-Meier analysis of OS, DSS and PFI according to the expression level of HNF1B in HCC cases;

[0029] Figure 4 For green fluorescence expression of HepG2 cells transfected with HNF1B plasmid;

[0030] Figure 5 For CCK8 method to detect the proliferation ability of recombinant HepG2 cells;

[0031] Figure 6 For scratch test to detect the migration ability of recombinant HepG2 cells;

[0032] Figure 7 For Transwell experiment to detect the invasion ability of recombinant HepG2 cells;

[0033] Figure 8 For recombinant HepG2 cells to express cholangiocarcinoma markers CK7 and CK19;

[0034] Figure 9 For HNF1B to participate in the regulation of various signal pathways in HCC. DETAILED DESCRIPTION

[0035] The application will be further described below in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement it, but the embodiments are not limiting to the application.

[0036] The application provides a diagnostic marker for identifying DPHCC and common HCC, HCC patients with high expression of HNF1B can be prompted as DPHCC subtype, so that differential diagnosis can be made according to the expression level of the marker, and since the prognosis of DPHCC is poorer than that of common HCC patients, the marker has both auxiliary diagnosis and evaluation of prognosis.

[0037] The application provides a method for preparing an in vitro DPHCC cell model, which provides a cell model for researchers to carry out researches such as drug screening and mechanism exploration for DPHCC, and promotes the research progress of DPHCC.

[0038] The HNF1B has a nucleotide sequence with a NCBI accession number of NM_000458.4.

[0039] Example 1: Bioinformatics screening of markers

[0040] 1. Data acquisition

[0041] A large amount of transcriptome and clinical information data of liver cancer samples were obtained from the TCGA database using R (version 4.2.1) and R package TCGAbiolinks (version 2.24.3). A total of 371 hepatocellular carcinoma samples (referred to as LIHC in TCGA), 35 cholangiocarcinoma samples (referred to as CHOL in TCGA) and 50 normal samples were included. Single-cell sequencing data files of HCC and ICC were downloaded from the GEO database (GSE125449, GSE138709).

[0042] 2. Bioinformatics analysis

[0043] The CPM value of the expression matrix was calculated using the R software package edgeR (version 3.38.4), and genes with CPM>1 were screened for differential analysis. The R package limma (version 3.52.2) was used to analyze hepatocellular carcinoma samples and cholangiocarcinoma samples. The adjusted P value <0.01 and |logFC|>0.5 were used as the cutoff points for significant differential expression of genes. The results are shown in Figure 1 There was no significant difference in the expression of HNF1B between the normal (Normal) and tumor (Tumor) samples in hepatocellular carcinoma patients Figure 1 a). However, a significant HNF1B high and low expression population was observed in hepatocellular carcinoma samples Figure 1 b, c). Then, the tumor samples were divided into two groups with high and low expression of HNF1B. Receiver operating characteristic (ROC) analysis confirmed the diagnostic value of HNF1B for DPHCC, and the diagnostic value in the first year was the highest (AUC=0.582)Figure 1 d).

[0044] Pathological detection and prognosis of HCC tissue samples in Example 2

[0045] I. Implementation steps:

[0046] Material preparation: tumor tissue samples obtained by surgery, HNF1B detection kit (first antibody is rabbit anti-human HNF1B, second antibody is horseradish peroxidase labeled goat anti-rabbit antibody)

[0047] 1. Specimen detection

[0048] (1) Select a liver cancer paraffin section containing a tumor area, and ensure that there is no large necrosis;

[0049] (2) Obtain 8 paraffin sections of 4 μm, after baking at 60°C for 2 hours, deparaffinize with pure xylene for 2 times, 10 minutes each time;

[0050] (3) The deparaffinized sections were sequentially placed in 100%, 95%, and 90% series alcohol for 2 minutes each time for step-by-step hydration, and then rinsed with tap water until the glass slide was clean and transparent;

[0051] (4) Block endogenous peroxidase activity with 0.3% H2O2 for 10 minutes at room temperature, and wash 4 times in PBS for 5 minutes each time;

[0052] (5) Antigen repair: 10 mM citric acid buffer (0.01 M PBS, pH 6.0), high pressure heat repair for 15 minutes, room temperature natural cooling for 30 minutes, TBS buffer containing 0.1% Tween-20 wash 3 times, 3 minutes each time;

[0053] (6) Block with 5% BSA, 0.1% Triton-100 in PBS solution at room temperature for 30 minutes;

[0054] (7) Add the first antibody HNF1B (purchased from abcam, product number ab187744) to the tissue section, incubate at room temperature for 1 hour, wash 3 times with TBS buffer containing 0.1% Tween-20, 5 minutes each time;

[0055] (8) Add the second antibody labeled with horseradish peroxidase (purchased from Beyotime Company, product number A0208), incubate at room temperature for 20 minutes; wash 3 times with TBS buffer containing 0.1% Tween-20, 5 minutes each time;

[0056] (9)DAB solution (purchased from Beyotime Company, item number P0203) was added dropwise, and color development was controlled under a microscope for 3-10 minutes. Color development was terminated by washing with distilled water, and the slices were washed with TBS buffer containing 0.1% Tween-20 for 3 times, each for 5 minutes;

[0057] (10) The slices were placed in an antigen repair solution and placed in a microwave oven for 5 minutes at high heat and 15 minutes at low heat. The slices were naturally cooled at room temperature for 30 minutes, and then washed with TBS buffer containing 0.1% Tween-20 for 3 times, each for 3 minutes;

[0058] (11) Hematoxylin re-staining, bluing with distilled water, dehydration, transparency, and mounting with a quenching agent. Each batch of staining was performed with a known positive slice as a positive control, and PBS instead of the primary antibody was used as a negative control.

[0059] 2. Judgment of immunohistochemical staining results

[0060] The diagnosis results were interpreted by two or more senior pathologists in a double-blind manner. Five fields of view were randomly selected for each slice. (-): no color or <10% of the cells showed light brown granular staining of the cell membrane and cytoplasm; (+): 10%-60% of the cells showed moderate intensity brown granular staining of the cell membrane and cytoplasm; (++) >60% of the cells showed high intensity brown granular staining of the cell membrane and cytoplasm. The negative reference range was “-”. Negative patients were ordinary HCC, and positive patients could be diagnosed as DPHCC (see Figure 2 ). According to the expression level, it could be suggested that the higher the positive degree, the worse the prognosis of the patient. A more targeted diagnosis and treatment plan could be developed for HCC patients positive for HNF1B.

[0061] II. Predicting the survival period of HCC patients through the expression level of HNF1B

[0062] The R software packages survive and survminer were used to perform survival analysis of the patients in the data set related to the target gene. The OS, DSS and PFI were calculated using the Kaplan-Meier method, and P<0.05 was considered significant. The results are shown in Figure 3 It can be seen that HNF1B can be used to identify the DPHCC subtype in HCC patients. Since the prognosis of DPHCC is worse, the survival period of HCC patients can be predicted through the expression level of HNF1B. Clinicians can be guided to take more targeted diagnosis and treatment measures for HCC patients with high expression of HNF1B, and improve the prognosis of patients.

[0063] Example 3: Preparation of an in vitro DPHCC cell model and its application

[0064] 1. HCC cell line culture

[0065] HCC cell line HepG2 was cultured in high glucose DMEM medium (GIBCO) containing 10% fetal bovine serum (GIBCO). Cells were evenly inoculated in a six-well culture plate and cultured at 37°C, 5% CO2. The medium was replaced every 2 days, and when the cell confluence reached 90%, the cells were passaged or cryopreserved.

[0066] 2. Construction of DPHCC cell model

[0067] The plasmid with the HNF1B target gene was transfected into HepG2 cells by liposome transfection to construct a recombinant hepatocellular carcinoma cell with high expression of HNF1B. The cell model can express ICC immunophenotype, and the proliferation, migration and invasion ability is significantly improved compared with untransfected cells, which is a DPHCC cell model.

[0068] (1) HepG2 cells were cultured in a 6-well plate to 90% confluence, passaged into a 12-well plate at 2x10 5 cells per well, and cultured to 70%-90% confluence.

[0069] (2) Transfection solution preparation: A. Dilute 1.6 μg HNF1B plasmid DNA (containing GFP) with 100 μl Opti-MEM low serum medium (or other serum-free medium) and mix gently; B. Shake gently before use Lipofectamine2000, then dilute 4.0 μl Lipofectamine2000 in 100 μl Opti-MEM medium, and incubate at room temperature for 5 minutes. Note: please perform the next step within 25 minutes; C. Mix the diluted DNA and Lipofectamine2000 (total volume 200 μl) and mix gently, and incubate at room temperature for 20 minutes.

[0070] (3) Add 200 μl transfection solution to each well of cells and shake gently.

[0071] (4) Incubate at 37°C, remove the transfection solution after 4-6 hours of transfection, and add complete culture medium (10% fetal bovine serum + 90% high glucose DMEM), and detect green fluorescence expression after 48 hours to verify successful transfection. Figure 4 ).

[0072] (5) Detection of the proliferation ability of recombinant HepG2 cells by CCK8 method:

[0073] The cells are inoculated in a six-hole plate one day before the experiment, so that the desired confluence can be reached the next day; the culture medium in the culture is aspirated, and the cells are washed once with PBS; the cells are trypsinized, and when the trypsinization reaches an appropriate degree, the trypsin is discarded, and the culture medium is added to terminate the trypsinization, and the cells are blown down and mixed evenly; a certain amount of cells are counted using a hemocytometer to calculate the cell concentration, and a cell suspension of a corresponding concentration is prepared according to the counting result and the required number of cells, and is inoculated into a 96-hole plate, with 4-5 replicate holes; the 96-hole plate is placed in an incubator for incubation, and the incubation time is detected according to the experimental design of 24h, 48h, 72h, and 96h time nodes; when detecting, the culture medium in the culture plate is discarded, and CCK8 reagent is added to the 96-hole plate according to a CCK8: culture medium ratio of 1:9; the incubator is incubated, and generally 1h or 2h of incubation is sufficient; the absorbance at 450nm is measured by an enzyme-labeled instrument, and data calculation is performed. Figure 5 ).

[0074] (6) The migration ability of the recombinant HepG2 cells is detected by a scratch test.

[0075] A. Scratch: First, use a marker pen to draw horizontal lines on the back of a 6-hole plate, evenly spaced at about 0.5-1cm, crossing the holes. At least 5 lines cross each hole.

[0076] B. Plating: Cells in the logarithmic growth phase are trypsinized into a single-cell suspension and inoculated into a 6-hole culture plate; 6x105 cells are plated per hole to ensure that the cells will be full the next day, and the total volume of the culture medium in each hole is 2mL.

[0077] C. Cell culture: The cells are cultured in a 37℃, 5% CO2 incubator for 24h.

[0078] D. Scratch: The next day, use a gun head to draw a straight line perpendicular to the horizontal line on the back, and the gun head should be perpendicular and cannot be inclined (the same gun head is preferably used between different holes).

[0079] E. Washing: The cells are washed with PBS 3 times to remove the scratched cells, and serum-free medium is added.

[0080] F. Photography: The scratch is photographed under a microscope to ensure that it is centered and perpendicular, and the background is consistent. Samples can be taken at 0, 6, 12, 24, and 48 hours, and photographs are taken. Figure 6 ).

[0081] (7) The invasion ability of the recombinant HepG2 cells is detected by a Transwell test.

[0082] 8.0 μm PET membrane (Corning) was installed on a 24-well plate. Using Matrigel (1:8 Opti dilution), 200 ul was added to each well of the upper chamber, and incubated at 37°C for 30 minutes. 15 μl of serum-free cell suspension (2 x 10 5 ) was plated in each well of the upper chamber, and 500 μl of DMEM containing 10% fetal bovine serum was added to the lower chamber as a chemoattractant. After 36 hours of regular culture, the cells on the upper surface were wiped off with a cotton swab. The cells that penetrated to the lower surface of the filter were fixed with 4% formaldehyde for 10 minutes, and then stained with 0.1% crystal violet for 30 minutes. The stained cells were observed under a microscope Figure 7 ).

[0083] (8) Detection of cholangiocarcinoma marker expression in recombinant HepG2 cells by immunofluorescence

[0084] A. The day before the experiment, sterilized small round glass pieces were placed in a 12-well plate. Cells were transferred to the 12-well plate at an appropriate density and cultured overnight.

[0085] B. The cells were fixed with 4% buffered paraformaldehyde (PFA) for 1 hour, and after removing the fixative, the cells were washed with PBS.

[0086] C. Incubate with 0.5% Triton X100 at room temperature for 15 min, and then wash with PBS for 3 times, 5 min each time.

[0087] D. Seal the sample in PBS containing 1% bovine serum albumin (BSA) for 60 minutes.

[0088] E. Dilute CK7 and CK19 to 1:100 in 1% BSA, and treat overnight at 4°C. After removing the primary antibody, wash the cells with PBS twice.

[0089] F. Incubate the cells with secondary antibody (Alexa 488 AffiniPure Goat Anti-Rabbit IgG (H+L)) for 2 hours, and carefully wash the cells with PBS 3 times.

[0090] G. Cover the stained glass pieces with a glass slide with 50 μl of DAPI added, and mount the slide.

[0091] H. Take pictures using a fluorescence microscope, and analyze using ImageJ Figure 8 ).

[0092] (9) Research direction based on the results of HNF1B-related KEGG analysis in the HCC dataset

[0093] The differentially expressed genes between the two groups of samples with high and low expression of HNF1B were clustered for analysis, and the top 50 most different genes were screened out for visual analysis with the screening conditions of |logFC|>1 and fdr<0.05. The KEGG pathway enrichment results showed that the up-regulation of HNF1B expression was mainly related to signal pathways such as neuroactive ligand-receptor interaction, bile secretion, ECM-receptor interaction, and regulation of stem cell pluripotency, providing support for subsequent mechanism research. The results are shown in Figure 9 .

[0094] Obviously, the above embodiments are only examples for clearly illustrating, and are not limitation to the embodiments. For ordinary skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. The application of reagents for detecting biomarkers in the preparation of products for detecting biphenotype hepatocellular carcinoma subtypes, characterized in that: The biomarker is hepatocyte nuclear factor 1β.

2. The application according to claim 1, characterized in that: Hepatocellular carcinoma nuclear factor 1β (HCC) expression levels are low in conventional hepatocellular carcinoma but high in biphenotype hepatocellular carcinoma.

3. The application according to claim 1, characterized in that: The test sample is liver cancer tissue or adjacent tissue of liver cancer.

4. The application of reagents for detecting biomarkers in the preparation of diagnostic products for the prognosis of patients with biphenotype hepatocellular carcinoma, characterized in that: The biomarker is hepatocyte nuclear factor 1β.

5. A kit for detecting the prognosis of patients with biphenotype hepatocellular carcinoma, characterized in that: The kit includes reagents for detecting hepatocyte nuclear factor 1β.

6. The application of a biphenotype hepatocellular carcinoma in vitro cell model in screening therapeutic drugs for biphenotype hepatocellular carcinoma, characterized in that: The cell model was obtained by introducing the gene encoding hepatocyte nuclear factor 1β into liver cancer cells.

7. The application according to claim 6, characterized in that: The nucleotide sequence of the gene encoding hepatocyte nuclear factor 1β is NM_000458.4.

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