WO2022148346A1 - Protéine à doigts de zinc zbtb20 en tant que biomarqueur pour la détection et le diagnostic d'un carcinome hépatocellulaire - Google Patents

Protéine à doigts de zinc zbtb20 en tant que biomarqueur pour la détection et le diagnostic d'un carcinome hépatocellulaire Download PDF

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WO2022148346A1
WO2022148346A1 PCT/CN2022/070120 CN2022070120W WO2022148346A1 WO 2022148346 A1 WO2022148346 A1 WO 2022148346A1 CN 2022070120 W CN2022070120 W CN 2022070120W WO 2022148346 A1 WO2022148346 A1 WO 2022148346A1
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zbtb20
seq
hepatocellular carcinoma
hcc
liver
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龚伟强
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香港理工大学
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present application relates to diagnostic methods and kits for hepatocellular carcinoma.
  • Zinc finger proteins are active in the field of gene regulation, participate in the regulation of cell development and metabolism, and are closely related to the occurrence of many developmental and metabolic diseases and cancer.
  • Zinc finger proteins are mainly divided into three subtypes, Cys 2 His 2 , Cys 4 and Cys 6 , according to the differences in the conserved sequences forming the zinc finger domains.
  • ZBTB20 Zinc finger and BTB domain-containing protein 20
  • POK BB/POZdomain-containing
  • ZBTB20 The expression level of ZBTB20 mRNA is difficult to detect in fetal liver, but the expression gradually increases after birth [1]. Due to different translation initiation sites, ZBTB20 has two isoforms, both containing N-terminal BTB/POZ and C-terminal zinc finger domains [2]. Isoform 1 is mainly expressed in the early postnatal period, while isoform 2 is mainly expressed in the later postnatal period [1].
  • Zbtb20 Gene knockout of Zbtb20 results in developmental delay, metabolic dysfunction, and death [3]. Elevated serum bilirubin and alanine aminotransferase levels were observed in the liver of Zbtb20-mutated mice, suggesting hepatic dysfunction [3]. Zbtb20 is also considered to be a positive regulator of liver replication in mouse liver regeneration [4]. Liver-specific inactivation of Zbtb20 results in marked derepression of the alpha-fetoprotein (Afp) gene in the liver throughout adulthood [5]. While Afp is highly expressed in fetal liver, Zbtb20 expression is only activated in liver after birth. It has been shown that ZBTB20 functions as a transcriptional repressor of Afp by specifically inhibiting the transcriptional activity driven by the Afp promoter [5].
  • Afp alpha-fetoprotein
  • liver cancer is the fifth most common cancer worldwide and the third leading cause of cancer-related death, among which hepatocellular carcinoma (HCC) accounts for more than 90% of primary liver cancers.
  • HCC hepatocellular carcinoma
  • ZBTB20 the role of ZBTB20 in liver cancer and hepatocellular carcinoma has been reported [6-11].
  • the present invention proves for the first time that Zbtb20 is an oncogene of HCC.
  • the inventors also confirmed that the oncogenic mechanism is the result of activation of the WNT/CTNNB1 pathway.
  • the present invention confirmed that ZBTB20 can inhibit PPARG expression, resulting in the upregulation of CTNNB1 and promoting HCC tumorigenesis.
  • Figure 5 of this specification briefly describes the interaction of ZBTB20, PPARG, CTNNB1, WNT and other factors.
  • ZBTB20 can be used as a diagnostic marker for hepatocellular carcinoma such as very early, early stage hepatocellular carcinoma. This has important early diagnostic value in eg AFP-negative HCC patients.
  • Figure 1 In vivo validation of oncogenes of Zbtb20 involved in WNT/CTNNB1 pathway-related HCC tumorigenesis.
  • A Structural differences between the two major isoforms of ZBTB20.
  • BTB Broad complex, tramtrack, bric a brac; POZ: poxvirus and zinc finger ; C2H2: Kruppel - type zinc finger; aa: amino acid.
  • B Gene delivery plasmid for hydrodynamic tail vein injection.
  • C 120-day PHI livers removed from Fah/SB11 animals injected with ZBTB20/shp53 (left) and Empty/shp53 (right). Arrows: liver tumor nodules; scale bar: 0.5 cm.
  • (D) Number of liver tumor nodules in ZBTB20/shp53 and Empty/shp53 groups.
  • (G) Representative immunohistochemical staining of CTNNB1 in ZBTB20/shp53-injected animals (left panel). Right panel, no primary antibody control. Scale bar: 250 ⁇ m.
  • FIG. 2 Immunofluorescence staining of ZBTB20-overexpressing transfected cells. No Flag-tagged signal (yellow) was detected in the wild-type HHL7 cell line, whereas a strong Flag was detected in cells transfected with ZBTB20 isoform 1, isoform 2 and OFP under the same excitation parameters Flag signal. Under the same excitation parameters, strong and concentrated CTNNB1 signal (red) was detected in ZBTB20 isoform 1 and 2 overexpressing transfected cells, whereas only very weak CTNNB1 signal was detected in wild-type HHL7 and OFP control cell lines . Nuclei were stained with DAPI (blue). The merged image is shown in the figure on the far right. Scale bar: 10 ⁇ m.
  • Figure 3 In vitro overexpression of ZBTB20 and its effect on the WNT/CTNNB1 pathway.
  • A Overexpression vectors for stable integration of ZBTB20 Flag-tagged isoforms 1 and 2 into various human hepatocyte cell lines using the piggyBac transposon system. Flag-tagged orange fluorescent protein (OFP) was used as a negative control.
  • B Relative mRNA expression levels of ZBTB20 and AFP in transfected HHL cells. ****, P ⁇ 0.00005; **, P ⁇ 0.005; *, P ⁇ 0.05.
  • C Fluorescence of green fluorescent protein (GFP) and OFP in transfected HHL cells was observed under a fluorescence microscope. BF: Bright field microscope.
  • Figure 4 In vitro disruption of ZBTB20 and its effect on the WNT/CTNNB1 pathway.
  • A Schematic of the knockout vector with gRNA for targeting the BTB and zinc finger domains of ZBTB20.
  • B Detection of knockout regions in genomic DNA using specific primers. The PCR product from the band with the missing indel (dashed box) was extracted and sequenced. PCR results from mixed and single cell cultures are shown.
  • C Knockout regions were detected at the transcriptional level using specific primer pairs for cDNA. PCR results from mixed and single cell cultures are shown.
  • D Relative mRNA expression levels of AFP, CTNNB1, AXIN2, and PPARG in ZBTB20-disrupted cells.
  • Figure 5 ZBTB20 as a transcriptional repressor of PPARG expression.
  • A Schematic representation of the PPARG locus and promoter sequences used to construct pGL3-PPARG.
  • B Luciferase promoter analysis showed that cells overexpressing ZBTB20 isoforms 1 and 2 were able to reduce PPARG promoter activity by 47% and 45%, respectively, compared to cells transfected with OFP control vector. HHL7 wild-type cells (HHL7 WT) were also included for comparison. ***, P ⁇ 0.0005.
  • C Summary graph showing the role of ZBTB20 in repressing PPARG expression and subsequent activation of the WNT/CTNNB1 signaling pathway and its downstream target genes.
  • FIG. 6 Identification of Zbtb20 as a proto-oncogene involved in HCC tumorigenesis.
  • A Schematic illustration of the insertion of a mutant transposon (T2/Onc) into the Zbtb20 gene. Transposon insertion profiling suggests that Zbtb20 functions as an oncogene in HCC tumorigenesis.
  • a schematic diagram of the T2/Onc mutagenic transposon is shown.
  • Grey triangles inverted repeat/direct repeat (IR/DR) transposon sequences flanking cassettes containing gain-of-function and loss-of-function DNA components; SA: splice acceptor; polyA: polyadenylation signal; MSCV : LTR of murine stem cell virus; SD: splice donor; open arrows: sense directional insertion of T2/Onc relative to Zbtb20 gene; arrows: antisense directional insertion of T2/Onc relative to Zbtb20 gene; black and gray arrows indicate, respectively Transposon insertion in male or female animals.
  • B RT-PCR of various genes in livers extracted from male and female liver tumors in which a transposon was inserted in Zbtb20.
  • Afp alpha-fetoprotein
  • Spp1 secreted phosphoprotein 1
  • SB11 Sleeping Beauty transposase
  • Zbtb20 endogenous Zbtb20
  • Actb actin beta
  • normal rat liver normal rat liver.
  • C Expression levels of Zbtb20 relative to Actb were obtained using ImageJ; tumors with forward insertion of the transposon relative to the translation initiation site of Zbtb20 showed non-significantly higher expression compared with the reverse inserted transposon high expression.
  • FIG. 7 Representative liver tumors from ZBTB20/shp53 Fah/SB11 mice 120 days after hydrodynamic tail vein injection.
  • A Liver tumor nodules of Fah/SB11 M1582 at 4X (left) and 20X (right) magnifications. Histologically, this liver tumor nodule can be classified as HCC.
  • B Liver tumor nodules of Fah/SB11 M1584 at 4X (left) and 20X (right) magnifications. Histologically, this liver tumor nodule can be classified as a well-differentiated liver tumor.
  • C Liver tumor nodules from Fah/SB11 M1752 magnified 4X (left) and 20X (right).
  • this liver tumor nodule can be classified as a well-differentiated liver tumor.
  • Figure 8 Cell proliferation and liver tumorigenesis in ZBTB20/shp53 injected Fah/SB11 mice.
  • A Representative HE (left panel) and Ki67 (right panel) staining showing similarly high mitotic index was observed in Fah/SB11 injected livers of ZBTB20/shp53 and Empty/shp53.
  • B HE (top panel) and Ki67 (bottom panel) staining showing different levels of Ki67 immunoreactivity was observed in ZBTB20/shp53 injected Fah/SB11 liver tumors.
  • P parenchymal hepatocytes
  • T tumor nodules.
  • HE hematoxylin-eosin staining
  • Ki67 cellular marker of proliferation; all scale bars: 250 ⁇ m.
  • Figure 9 Liver hypertrophy observed in ZBTB20/shp53 injected animals.
  • A Evidence of hypertrophy observed in ZBTB20/shp53-injected experimental animals, with fewer cell counts per field than Empty/shp53-injected controls. P: unpaired t-test.
  • B Enlarged hepatocytes demonstrated in experimental animals injected with ZBTB20/shp53. P: unpaired t-test.
  • C Representative magnified images of ZBTB20/shp53 and Empty/shp53 injected groups showing fewer but enlarged cells in ZBTB20/shp53 animals. Each bin is a 190 ⁇ m square.
  • Figure 10 In vitro overexpression of ZBTB20 and its effect on the WNT/CTNNB1 pathway.
  • A Western blot and relative protein levels of active CTNNB1, total CTNNB1, PPARG and ACTB in PLC/PRF/5 (PLC5) transfected cells.
  • B Western blot and relative protein levels of active CTNNB1, total CTNNB1, PPARG and ACTB in Hep3B-transfected cells. ***, P ⁇ 0.0005; **, P ⁇ 0.005; *, P ⁇ 0.05.
  • FIG 11 In vitro disruption of endogenous ZBTB20 in human hepatoma cell line PLC/PRF/5 (PLC). Knockout regions in PLC genomic DNA were detected using specific primer pairs. Sequencing results showed that gRNA1 and gRNA2 and gRNA4 and gRNA5 deleted the BTB (upper panel) and zinc finger (lower panel) domains in ZBTB20, respectively.
  • WT wild-type PLC cells; scrambled: PLC cells transfected with a pSpCas9-scrambled vector containing a non-targeting control gRNA sequence; transfected: PLC cells were transfected with a pSpCas9 vector containing the Associated gRNAs targeting the ZBTB20 BTB or DNA-binding zinc finger domains.
  • Figure 12 In vitro disruption of ZBTB20 in C3A human hepatoma cell line and its effect on WNT/CTNNB1 signaling pathway.
  • A Relative mRNA expression levels of ZBTB20, PPARG and CTNNB1 in ZBTB20-disrupted C3A cells.
  • B Relative mRNA expression levels of downstream target genes of the WNT/CTNNB1 signaling pathway in ZBTB20-disrupted C3A cells. ****, P ⁇ 0.00005; ***, P ⁇ 0.0005.
  • FIG. 13 PPARG promoter sequence (chromosome 3p25.2, 12392051 to 12393088) with putative binding motifs for ZBTB20 (12392366 to 12392374) highlighted (UCSC Genome Browser, GRCh97/hg1).
  • Figure 14 Expression levels of ZBTB20, PPARG and CTNNB1 target genes in human HCC samples, compared using the online database Gene Expression Omnibus (GEO).
  • GSE6764 dataset contains gene expression profiles of 75 tissue samples from HCC patients, covering four tumor stages (very early HCC, early HCC, advanced HCC, and very advanced HCC), compared with normal livers as controls.
  • A Higher expression of ZBTB20 was detected in the very early stages of HCC, while no significant changes in AFP were detected.
  • B Activation of the WNT signaling pathway in the very early stages of the disease through the upregulation of CTNNB1 and its target genes.
  • Hepatocellular carcinoma can be classified into very early, early, advanced or very advanced HCC according to the stage of development. Screening tests and surveillance tests for hepatocellular carcinoma are the core of early diagnosis and effective treatment. This application provides the most appropriate markers and uses of markers for this effect.
  • the markers of the present application for the diagnosis of hepatocellular carcinoma comprise one or more of the following markers: ZBTB20, alpha-fetoprotein (Afp) and/or secreted phosphoprotein 1 (Spp1).
  • the marker used in the present application for the diagnosis of hepatocellular carcinoma is ZBTB20.
  • markers for diagnosing hepatocellular carcinoma may further include one or more selected from the group consisting of Golgi protein 73 (GP73), alpha-fetoprotein heterogenous 3 (AFP-L3) , phosphatidylglycerol 3 (GPC-3), osteopontin (OPN), serum ferritin (SF), transforming growth factor ⁇ 1 (TGF ⁇ 1), insulin-like growth factor II (IGF-II), hepatocyte growth factor (HGF), tumor-specific growth factor (TSGF), abnormal prothrombin (DCP), ⁇ -L-fucosidase (AFU), serum GGT, matrix metalloproteinase (MMP), squamous cell carcinoma antigen ( SCCA), circulating tumor cells (CTC) or human cervical oncogene (HCCR).
  • GP73 Golgi protein 73
  • AFP-L3 alpha-fetoprotein heterogenous 3
  • GPC-3 phosphatidylglycerol 3
  • osteopontin osteo
  • the biomarkers of the present application can be used in the form of compositions, kits containing detection substances capable of detecting the one or more biomarkers from tissue or blood, or in the form of using one or more organisms of the present application
  • the marker is used for monitoring or diagnosing hepatocellular carcinoma, especially very early and early hepatocellular carcinoma.
  • the markers of the present application can be quantitatively or qualitatively analyzed to detect the presence or absence of nucleic acids, especially proteins and/or mRNAs, and/or, at the level of the marker's expression itself, changes in expression, differences in expression, etc. detection and/or quantification.
  • detection includes quantitative and/or qualitative analysis, including detection of presence and absence and detection of expression level, these methods are well known in the art, and for the implementation of this application, those skilled in the art can Choose the appropriate method.
  • biomarkers of the present application may be performed by ELISA assays, dipstick rapidkit assays, mass spectrometry, microarrays, nucleic acid amplification, or immunoassays.
  • Detection of the markers of the present application can be performed on the basis of the sequence characteristics, functional characteristics and/or antigenic characteristics of the markers.
  • the markers of the present application can be detected by nucleic acids encoding proteins, especially mRNAs and/or proteins.
  • Qualitative or quantitative detection methods at the protein level can be, for example, using Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, and solution/suspension assays.
  • Qualitative or quantitative detection methods at the nucleic acid level may use nucleic acid transfer and amplification methods, methods using eTag systems, systems based on labeled beads, array systems such as nucleic acid arrays, and the like.
  • the present invention relates to the use of an agent that specifically recognizes ZBTB20 in the manufacture of a kit for diagnosing hepatocellular carcinoma (HCC) in a biological sample from a subject.
  • HCC hepatocellular carcinoma
  • hepatocellular carcinoma is very early stage HCC, early stage HCC, advanced stage HCC or very advanced stage HCC.
  • the biological sample for testing may be liver tissue, whole blood, plasma or serum, preferably liver tissue.
  • the biological sample is from a mammal, such as a human.
  • the agent for detecting ZBTB20 comprises an antibody that specifically binds ZBTB20, a primer that specifically binds ZBTB20, or a DNA probe that specifically binds ZBTB20.
  • the kit further comprises preparations for the detection of other hepatocellular carcinoma markers, which may be any of the markers mentioned herein, preferably alpha-fetoprotein and/or secreted Phosphoprotein 1 (Spp1).
  • Kits that can be used in the present invention may be enzyme-linked immunoassay kits or PCR kits.
  • primers that specifically bind ZBTB20 include: forward 5'-ATGAGATTACTCAGCCGGGCGGATC-3' (SEQ ID NO:21) and reverse 5'-AGCTTGTCTTGGAAGAAGGGGCTGC-3' (SEQ ID NO:22); or Forward 5'-CGAGCGCATTCACAGCATCAACCTT-3' (SEQ ID NO:23) and reverse 5'-TCTCGATGTCGCTGTAGCCAAGCAG-3' (SEQ ID NO:24).
  • the primer sequences for detecting alpha-fetoprotein include: forward 5'-CCTGTGAACTCTGGTATCAG-3' (SEQ ID NO: 11) and reverse 5'-GCTCACACCAAAGCGTCAAC-3' (SEQ ID NO: 12); Primers to detect Sppl include: forward 5'-CTTTCACTCCAATCGTCCCTAC-3' (SEQ ID NO: 13) and reverse 5'-GCTCTCTTTGGAATGCTCAAGT-3' (SEQ ID NO: 14).
  • the present invention relates to a method of diagnosing hepatocellular carcinoma (HCC) in a subject, comprising detecting or quantifying ZBTB20 in a biological sample of the subject, and combining the detection or quantification results Compared with the corresponding results of ZBTB20 in the control sample; when compared with the control sample, there is ZBTB20 or the expression of ZBTB20 in the biological sample of the subject is increased, the subject can be diagnosed as hepatocellular carcinoma patient.
  • HCC hepatocellular carcinoma
  • the hepatocellular carcinoma is very early stage HCC, early stage HCC, advanced stage HCC or very advanced stage HCC.
  • the biological sample for testing may be liver tissue, whole blood, plasma or serum.
  • the biological sample is from a mammal, eg, a human.
  • the agent for detecting ZBTB20 comprises an antibody that specifically binds ZBTB20, a primer that specifically binds ZBTB20, or a DNA probe that specifically binds ZBTB20.
  • primers that specifically bind to ZBTB20 include: forward 5'-ATGAGATTACTCAGCCGGGCGGATC-3' (SEQ ID NO:21) and reverse 5'-AGCTTGTCTTGGAAGAAGGGGCTGC-3' (SEQ ID NO:22); or Forward 5'-CGAGCGCATTCACAGCATCAACCTT-3' (SEQ ID NO:23) and reverse 5'-TCTCGATGTCGCTGTAGCCAAGCAG-3' (SEQ ID NO:24).
  • the primer sequences for detecting alpha-fetoprotein include: forward 5'-CCTGTGAACTCTGGTATCAG-3' (SEQ ID NO: 11) and reverse 5'-GCTCACACCAAAGCGTCAAC-3' (SEQ ID NO: 12);
  • the primers for detecting Sppl were: forward 5'-CTTTCACTCCAATCGTCCCTAC-3' (SEQ ID NO: 13) and reverse 5'-GCTCTCTTTGGAATGCTCAAGT-3' (SEQ ID NO: 14).
  • the present invention provides a kit comprising primers, antibodies or probes that specifically recognize ZBTB20, and instructions for using the kit.
  • the kit further comprises preparations for the detection of other hepatocellular carcinoma markers, which may be any of the markers mentioned herein, preferably alpha-fetoprotein and/or secreted phosphoprotein 1 (Spp1).
  • the ZBTB20 of the present application can be used in a method for monitoring the prognosis of hepatocellular carcinoma in a subject, comprising the steps of: performing the detection of the above-mentioned markers or marker combinations disclosed in the present application on a sample of the subject or quantification, and the detection or quantification results are compared with the corresponding results of the corresponding markers in the control sample; and when compared with the control sample, the protein or nucleic acid of the subject's sample is present Or when changes occur, the prognosis of the subject can be determined.
  • the present invention relates to the use of an agent that specifically recognizes ZBTB20 in the manufacture of a kit for predicting the prognosis of hepatocellular carcinoma (HCC) in a subject.
  • HCC hepatocellular carcinoma
  • the present invention provides a primer set for the detection of ZBTB20, the primer set comprising the following primers: forward 5'-ATGAGATTACTCAGCCGGGCGGATC-3' (SEQ ID NO: 21) and reverse 5'-AGCTTGTCTTGGAAGAAGGGGCTGC-3 ' (SEQ ID NO:22); or forward 5'-CGAGCGCATTCACAGCATCAACCTT-3' (SEQ ID NO:23) and reverse 5'-TCTCGATGTCGCTGTAGCCAAGCAG-3' (SEQ ID NO:24).
  • the present invention provides a primer set for detecting alpha-fetoprotein and Sppl, wherein the primer sequences for detecting alpha-fetoprotein include forward 5'-CCTGTGAACTCTGGTATCAG-3' (SEQ ID NO: 11) and reverse 5' '-GCTCACACCAAAGCGTCAAC-3' (SEQ ID NO: 12); primers to detect Spp1 include: forward 5'-CTTTCACTCCAATCGTCCCTAC-3' (SEQ ID NO: 13) and reverse 5'-GCTCTCTTTGGAATGCTCAAGT-3' (SEQ ID NO :14).
  • the pT2/GD-IRES-GFP vector was prepared, which contains the destination cassette (DEST) for the clonase reaction (Life Technologies) [28].
  • ZBTB20 transcript variant 2 (isoform 2) in pCMV6-XL5 vector was obtained from OriGene (NM_015642.2).
  • the ZBTB20 insert was removed from pCMV6-XL5 by standard restriction enzyme digestion and cloned into the pENTR vector (Life Technologies) to obtain pENTR-ZBTB20.
  • the ZBTB20 component from pENTR-ZBTB20 was cloned into pT2/GD-IRES-GFP using the LR clonase reaction to obtain pT2/GD-IRES-GFP-ZBTB20.
  • mice were usually fed 7.5 ⁇ g/ml 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC, Swedish Orphan Biovitrum, Sweden) in drinking water, but Replace with normal drinking water after hydrodynamic injection of the transposon vector.
  • NTBC 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione
  • 10 injected experimental animals should have sufficient statistical power (>95%) with a 95% confidence level assuming that the control vehicle will not acquire tumors (standard deviation ⁇ 3 background tumors). All animals received human care, and the study protocol was approved by the Ethics Subcommittee of Animal Science, The Hong Kong Polytechnic University, Hong Kong SAR.
  • the expression transposon vector also co-expresses the Fah gene, which allows for the selective proliferation of hepatocytes co-expressing the transgene of interest and Fah.
  • the phenotype of the liver following overexpression of the stably incorporated transgene was observed.
  • the body weight change and luciferase activity of the injected experimental animals were observed, see [25-28] for the method.
  • RNA extraction and histopathological analysis Tissue samples of RNA were stored in RNAlater (Sigma) at -80°C to prevent RNase contamination and degradation. Histological sections were only performed on larger tumor nodules (>2 mm in diameter).
  • Formalin-fixed paraffin-embedded sections from various tissues were sectioned at 5 microns using a standard microtome (Leica) and heat-fixed on glass slides. Tissue section slides were processed and stained with hematoxylin-eosin (HE) using standard protocols or used for immunohistochemical (IHC) analysis. Histopathological analysis was performed by a qualified professional.
  • cell numbers in representative fixed areas from experimental and control animals of both sexes were counted. Five representative areas were counted for each animal and expressed as cell number.
  • cell size determination the shorter diameter of hepatocytes from experimental and control animals of both sexes was measured using ImageJ 1.40J software (NIH, Maryland, USA). Fourteen representative hepatocytes were randomly measured and shown as cell size.
  • sections were washed thoroughly in PBS and then incubated with a secondary horseradish peroxidase antibody against the primary antibody used. After thorough washing with PBS, sections were treated with freshly prepared DAB substrate (Vector Laboratories), which should generate sufficient signal before stopping the reaction in water. Finally, sections were lightly counterstained with hematoxylin, dehydrated by gradually increasing concentrations of ethanol, sequined in xylene and mounted in Permount (Thermo Fisher Scientific).
  • RT-PCR reverse transcription polymerase chain reaction
  • the primer sequences for alpha-fetoprotein are forward 5'-CCTGTGAACTCTGGTATCAG-3' (SEQ ID NO: 11) and reverse 5'-GCTCACACCAAAGCGTCAAC-3' (SEQ ID NO: 12) (amplicon 410bp); secretion Type phosphoprotein 1 (Spp1) forward 5'-CTTTCACTCCAATCGTCCCTAC-3' (SEQ ID NO: 13) and reverse 5'-GCTCTCTTTGGAATGCTCAAGT-3' (SEQ ID NO: 14) (amplicon 305bp); actin ⁇ (Actb) forward 5'-GTGACGAGGCCCAGAGCAAGAG-3' (SEQ ID NO: 15) and reverse 5'-AGGGGCCGGACTCATCGTACTC-3' (SEQ ID NO: 16) (amplicon 938bp); fumaric acetoacetate hydrolase (Fah) Forward 5'-ATGAGCTTTTATTCCAGTGGCC-3' (SEQ ID NO: 17) and reverse
  • PCR conditions were 25 to 30 cycles to avoid amplicon saturation.
  • Semi-quantitative analysis of unsaturated RT-PCR amplicons was performed using ImageJ software. Briefly, the intensities of unsaturated RT-PCR amplicon bands were detected as values relative to Actb expression levels using ImageJ software.
  • axin 2 forward 5'-ATGAGTAGCGCCGTGTTAGTG-3' (SEQ ID NO:27) and reverse 5'-GGGCATAGGTTTGGTGGACT-3' (SEQ ID NO:28) (amplicon 150bp);
  • Cyclin D1 forward 5'-CAGAAGTGCGAAGAGGAGGTC-3' (SEQ ID NO:29) and reverse 5'-TCATCTTAGAGGCCACGAACAT-3' (SEQ ID NO:30) (amplicon 130bp);
  • Ctnnb1 forward 5'-ATGGAGCCCGGACAGAAAAGC-3' (SEQ ID NO:31) and reverse 5'-CTTGCCACTCAGGGAAGGA-3' (SEQ ID NO:32) (amplicon 108bp);
  • Lymphoid enhancer binding factor 1 forward 5'-TGTTTATCCCATCACGGGTGG-3' (SEQ ID NO:33) and reverse 5'-CATGGAAGTGTCGCCTGACAG-3' (SEQ ID NO:34) (amplicon 67bp);
  • Myc Myeloma oncogene forward 5'-ATGCCCCTCAACGTGAACTTC-3' (SEQ ID NO:35) and reverse 5'-CGGAGTCGTAGTCGAGGTCATA-3' (SEQ ID NO:36) (amplicon 55bp);
  • T cell specific forward 5'-AGCTTTCTCCACTCTACGAACA-3' (SEQ ID NO:37) and reverse 5'-AATCCAGAGAGATCGGGGGTC-3' (SEQ ID NO:38) (amplicon 115bp) ;
  • Glyceraldehyde-3-phosphate dehydrogenase forward 5'-GTGTTCCTACCCCCAATGTGT-3' (SEQ ID NO:39) and reverse 5'-GAGACAACCTGGTCCCTCAGTGT-3' (SEQ ID NO:40) (amplicon 148bp) .
  • Transfected cell cDNA was synthesized using 500 ng of total RNA extracted from transfected cells using PrimeScript RT Master Mix (Takara). The cDNA was diluted 1:10 in nuclease-free water, and 4 ⁇ L of the diluted cDNA was used to perform qPCR.
  • the master mix (GoTaq qPCR Master Mix, Promega) contained SYBR Green I, and the final concentration of each primer was 0.2 ⁇ M. Reactions were run on a QuantStudio7 Flex real-time PCR system (Thermo Fisher).
  • the primer sequences are as follows: ACTB forward 5'-GCCGTCTTCCCCTCCATCGT-3' (SEQ ID NO:41) and reverse 5'-TGCTCTGGGCCTCGTCGC-3' (SEQ ID NO:42);
  • AXIN2 forward 5'-CTCCCCACCTTGAATGAAGA-3' (SEQ ID NO:43) and reverse 5'-TGGCTGGTGCAAAGACATAG-3' (SEQ ID NO:44);
  • PPARG forward 5'-ACCAAAGTGCAATCAAAGTGGA-3' (SEQ ID NO:47) and reverse 5'-ATGAGGGAGTTGGAAGGCTCT-3' (SEQ ID NO:48);
  • CTNNB1 forward 5'-AAAGCGGCTGTTAGTCACTGG-3' (SEQ ID NO:49) and reverse 5'-CGAGTCATTGCATACTGTCCAT-3' (SEQ ID NO:50);
  • ZBTB20 forward 5'-CGAGCGCATTCACAGCATCAACCTT-3' (SEQ ID NO:51) and reverse 5'-TCTCGATGTCGCTGTAGCCAAGCAG-3' (SEQ ID NO:52);
  • CCND1 forward 5'-GCTGCGAAGTGGAAACCATC-3' (SEQ ID NO:53) and reverse 5'-CCTCCTTCTGCACACATTTGAA-3' (SEQ ID NO:54);
  • Glycogen synthase kinase 3 beta forward 5'-TCGAGAGCCTCCAGATCATGAGAA-3' (SEQ ID NO:55) and reverse 5'-CGGAACATAGTCCAGCACCAGA-3' (SEQ ID NO:56);
  • APC regulators of the WNT signaling pathway forward 5'-AAAACGAGCACAGCGAAGAATAGC-3' (SEQ ID NO:57) and reverse 5'-TCGTGTAGTTGAACCCTGACCAT-3' (SEQ ID NO:58);
  • Flag-tagged ZBTB20 variants were inserted into the pENTR vector using restriction enzymes to generate pENTR-Flag-ZBTB20 (Isoform 1) or (Isoform 2).
  • the expression vector was obtained by introducing the Flag-tagged ZBTB20 variant from the previous clone into the destination vector pPB/SB-DEST-GFP [30] to generate pPB/SB-Flag-ZBTB20 (isoform 1) or (isoform 2) -GFP.
  • a control expression vector containing orange fluorescent protein (OFP) was generated using a similar method to make pPB/SB-Flag-OFP.
  • Cells were co-transfected with 1 microgram of expression vector or control vector and 1 microgram of PB transposase vector using ViaFect Transfection Reagent (Promega) [30]. Transfect 3:1 ViaFect Transfection Reagent to DNA into 5x10 cells in a 24 -well plate as recommended by the manufacturer's instructions. Transfected cells were selected at 1 mg/mL puromycin in medium. Stably transfected cell lines were obtained approximately 4 weeks after transfection. Transfected cells were then collected for DNA, RNA and protein extraction; and for immunofluorescence (IF) staining.
  • IF immunofluorescence
  • the CRISPR/Cas9 system was used to disrupt ZBTB20.
  • Five different guide RNAs (designated gRNA1 to gRNA5) were designed to target the BTB and DNA-binding zinc finger domains of ZBTB20.
  • the gRNA sequences are shown in Table 1. These gRNAs were ligated into Cas9 (pSpCas9(BB)-2A-Puro(PX459) V2.0 vector, kindly provided by Dr. Feng Zhang) (Addgene plasmid #62988; http://n2t.net/addgene:62988; RRID:Addgene_62988 ), the knockout vector carrying gRNA and Cas9 was prepared according to the protocol provided by it [31].
  • each knockout vector was mixed and transfected into 15x104 PLC/PRF/5 and C3A (ATCC) cells in 6-well plates by ViaFect Transfection Reagent (Promega).
  • 1 microgram of pSpCas9-scrambled vector containing the non-targeting control gRNA sequence 5'-CATTTCTCAGTGCTATAGA-3' (SEQ ID NO: 62) was transfected as a negative control.
  • Transfected cells were selected for 5 days in culture medium at 0.4 ⁇ g/mL puromycin. Genomic DNA and RNA were extracted from transfected cells for further analysis. To obtain single cell cultures, 50 cells were diluted in 10 mL of medium and seeded into 96-well plates at 100 ⁇ L/well.
  • Table 1 gRNAs designed to target ZBTB20 domains.
  • primer pairs 1 and 2 were used to amplify the BTB domain of interest—515 bp and 446 bp for the wild-type (WT) and deletion alleles, respectively.
  • primer pairs 3 and 4 were used to amplify the zinc finger domains of interest - 275 bp and 203 bp for the WT and deletion alleles, respectively.
  • primer pairs 1 and 2 were used to amplify the BTB domain of interest - 380 bp and 309 bp for the WT and deletion alleles, respectively.
  • Primer pairs 4 and 5 were used to amplify the zinc finger domains of interest - 501 bp and 441 bp for the WT and deletion alleles, respectively.
  • Stably transfected cells were seeded overnight on round coverslips in 24-well plates. Media was discarded and cells were washed twice with cold PBS (1X). Next, cells were fixed by incubation with 4% formaldehyde for 10 min at room temperature and blocked with 5% BSA for 1 h at room temperature. Cells were then incubated with primary antibodies overnight at 4°C. The primary antibodies used were Flag antibody (Sigma-Aldrich), active CTNNB1 (CST) or total CTNNB1 (CST) and were then diluted according to the dilutions provided by the manufacturer. Cells were then washed twice with cold PBS and incubated with specific secondary antibodies for 1 hour at room temperature.
  • nuclei were stained by incubating with DAPI at a 1:5000 dilution for 5 min. Cells were then mounted on glass slides using Anti-Faded mounting medium. IF was observed using a Leica TCS SP8 MP multiphoton microscope.
  • Proteins were extracted from transfected cells using SDS protein lysis buffer. Protein concentrations were determined using standard protein assays (Bio-Rad) and 30 ⁇ g of protein were loaded into SDS-PAGE and transferred to PVDF membranes. Primary antibodies against active CTNNB1 (CST), total CTNNB1 (CST), PPARG (CST), ACTB (TransGen Biotech) and Flag (Sigma-Aldrich) were diluted 1:1000 in 5% BSA. Secondary antibodies (anti-mouse or anti-rabbit) were diluted 1:2000 in 5% BSA. Membranes were blocked with 5% nonfat milk and then incubated with primary antibodies overnight at 4°C followed by secondary antibodies for 1 hour at room temperature.
  • Membranes were washed 3 times with 1X TBST at 10 min intervals. Finally, the membrane was developed using Immobilon Western chemiluminescent HRP substrate (Millipore). Semi-quantitative analysis of protein bands was performed using ImageJ software. The level of ACTB expression was used as a reference to measure the intensity of the protein bands.
  • the pGL3-PPARG reporter plasmid was constructed to study the transcriptional effect of ZBTB20 on the PPARG promoter.
  • the putative binding motif of ZBTB20 was previously identified as the following sequence - GATGTATA [32]. Interestingly, this binding motif is also present in the promoter region of PPARG.
  • the 1038 bp promoter region of PPARG containing the putative binding site for ZBTB20 [33] was PCR amplified from HHL7 genomic DNA by high-fidelity Taq polymerase using the following primer pair: Forward 5'-GGTACCCACTCATGTGACAAGACCTGCTCC-3' (SEQ ID NO: 63) and reverse 5'-GCTAGCAGCATGGAATAGGGGTTTGCTGTAATTC-3' (SEQ ID NO: 64). Then the 1038bp promoter fragment was directionally cloned into the KpnI and NheI sites of the pGL3 basic vector (Promega) to obtain the expression vector pGL3-PPARG. DNA sequencing to ensure that no mutations were introduced during PCR amplification.
  • HHL7 cells (6X10 cells) previously transfected with ZBTB20 isotype or OFP control were seeded on 12-well plates overnight with 1 ⁇ g pGL3-PPARG and 0.1 ⁇ g pRL-TK (Promega) as endogenous transfection control Transfect cells. Twenty-four hours after transfection, luciferase activity was measured using a Glomax 20/20 Luminometer (Promega).
  • CTNNB1 reporter plasmid by M50Super 8x TOP-Flash (Addgene plasmid #12456; http://n2t.net/addgene:12456; RRID:Addgene_12456) and its mutation control, M51 Super 8x FOP-Flash (Addgene plasmid #12457; http://n2t.net/addgene:12457; RRID:Addgene_12457) composed of a kind gift from Dr. Randall Moon [35].
  • TOP/FOP-Flash reporter, pRL-TK (Promega) and pPB/SB-Flag-ZBTB20 were co-transfected into PLC/PRF/5 and Hep3B cells in 12-well plates at a density of 6X104 cells. 24 to 48 hours after transfection, cells were harvested for luciferase measurements using a dual luciferase reporter assay system (Promega), and luciferase activity was measured by a Glomax 20/20 Luminometer (Promega). Relative luciferase activity was expressed as the ratio of TOP-flash/FOP-flash luciferase activity and normalized to pRL-TK luciferase activity.
  • the GSE6764 dataset includes 75 tissue samples from 38 HCV-infected patients, including 13 samples from cirrhotic tissue, 17 from dysplastic nodules, and 35 from HCC. Normal tissue samples were obtained from healthy livers of 10 patients [Wurmbach, E., et al., Genome ⁇ wide molecular profiles of HCV ⁇ induced dysplasia and hepatocellular carcinoma. Hepatology, 2007.45(4):p.938 ⁇ 47.].
  • HCC pathological stages were defined in 35 HCC samples according to the following criteria: (i) very early HCC (8 cases), including well-differentiated tumors ⁇ 2 cm in diameter without vascular invasion/satellite nodules (size range: 8 -20 mm); (ii) early-stage HCC (10 cases), including tumors smaller than 2 cm with microvascular invasion/satellite nodules; 2-5 cm tall to moderately differentiated tumors without vascular invasion/satellite nodules; or 2-3 well-differentiated nodules smaller than 3 cm (size range: 3-45 mm); (iii) advanced HCC (7 cases), including poorly differentiated tumors >2 cm with microvascular invasion/satellite nodules, or Tumors >5 cm; (iv) very advanced HCC (10 cases), including macrovascular invasion or diffuse liver disease (Wurmbach, E., et al., supra).
  • Tissue specimens were collected for RNA extraction and microarray hybridization was performed using a human U133 plus 2.0 array (Affymetrix). Following analysis of the microarray data, gene expression profiles were obtained and published in the GSE6764 dataset (Wurmbach, E., et al., supra). The inventors extracted the expression values of ZBTB20 and AFP, and compared their expressions in normal liver tissue samples and HCC tissue samples at different tumor stages.
  • liver tumors all expressed Afp and/or Spp1, two known liver cancer markers, and were positive for SB transposase (SB11), suggesting that transposition events are likely responsible for liver tumorigenesis (Fig. 6B).
  • Liver tumors with forward transposon insertion relative to the endogenous Zbtb20 gene tended to show higher expression levels compared to liver tumors with reverse insertion ( Figure 6B and Figure 6C).
  • ZBTB20 is involved in liver tumorigenesis as a novel proto-oncogene
  • livers of Fah/SB11 animals injected with ZBTB20/shp53 were significantly larger relative to overall body weight (6.625% ⁇ 0.306, percent w/w ⁇ standard deviation) compared to controls injected with Empty/shp53 (5.233% ⁇ 0.367) ( Figure 1E).
  • ZBTB20 overexpression together with Trp53 inactivation, can induce hepatomegaly.
  • the liver weight percentage of the injection group is shown in Table 3.
  • the percentages of liver and body weight in the null-only and ZBTB20-only groups were 5.27% ⁇ 0.26 and 7.03% ⁇ 1.45, respectively (Table 3). No tumors were observed in Empty control animals, but only 1 animal showed a single liver nodule in the ZBTB20 only group (Table 3).
  • Table 3 Tumor number and liver weight percentage in injection group.
  • ZBTB20/shp53 PHI Liver weight (%) number of nodules Fah/SB11 M1582 120 7.2 5 Fah/SB11 M1584 120 6.4 7 Fah/SB11 M1751 120 6.5 5 Fah/SB11 M1752 120 7 5 Fah/SB11 M1753 120 6.4 4 Fah/SB11 M1761 120 6.4 3 Fah/SB11 M1762 120 6.6 5 Fah/SB11 M1764 120 6.5 2 ZBTB20 PHI Liver weight (%) number of nodules Fah/SB11 M1971 122 8.7 0 Fah/SB11 M1982 143 6.3 0 Fah/SB11 M1983 143 6.1 1 Empty/shp53 PHI Liver weight (%) number of nodules Fah/SB11 M982 130 5.2 0 Fah/SB11 M983 130 5.5 0 Fah/SB11 M984 131 4.7 0
  • liver weight (%) percentage of liver weight and total mouse weight
  • number of nodules total liver tumor nodules counted in the whole liver.
  • liver tumor from Fah/SB11 M1582 had a well-defined nodule consisting of sheets of pleomorphic hepatocytes with loss of histology, some showing large irregular singular nuclei.
  • varying degrees of micro and macrovesicular fat droplets (steatosis) were observed in most cells. Morphologically, this tumor was most compatible with moderately to poorly differentiated HCC ( Figure 7A).
  • Liver tumors from Fah/SB11 M1584 and M1752 both had prominent nodules composed of hepatocytes, exhibited very mild atypia, and had no apparent steatosis. Morphologically, these tumors were best described as well-differentiated liver tumors (Figure 7B and Figure 7C).
  • the PB transposon system was used to stably transfect each of the two isoforms into various human hepatocyte cell lines (Fig. 3A), with successful overexpression determined by qPCR (Fig. 3B) and detection of GFP signal in culture (Fig. 3C).
  • Fig. 3A human hepatocyte cell lines
  • Fig. 3B successful overexpression determined by qPCR
  • Fig. 3C detection of GFP signal in culture
  • mRNA expression level of AFP the negative target of ZBTB20
  • Reduced expression of AFP was detected in HHL cells transfected with isoform 1 and isoform 2 ZBTB20 overexpression compared to OFP control vector ( Figure 3B). This confirmed the efficiency and function of ZBTB20 overexpression in transfected HHL cell lines.
  • CTNNB1 CTNNB1 mRNA expression levels were not significantly different in ZBTB20-overexpressing-transfected cell lines, but significantly higher AXIN2 and CCND1 expression (FIG. 3D).
  • Components of the canonical WNT/CTNNB1 disruption complex such as glycogen synthase kinase 3 ⁇ (GSK3B) and the APC regulator of WNT signaling (APC), were also significantly reduced in ZBTB20-overexpressing HHL cells compared to the OFP control vector (Fig. 3E)).
  • GSK3B glycogen synthase kinase 3 ⁇
  • APC APC regulator of WNT signaling
  • HHL cells transfected with ZBTB20 overexpression resulted in significantly higher levels of active CTNNB1 (Fig. 3F), which was consistent with immunofluorescence staining (Fig. 2).
  • PPARG is considered to be an intermediate protein that interacts with ZBTB20 and CTNNB1 [15].
  • HHL cells transfected with ZBTB20 overexpression did reflect this interaction, as significantly lower PPARGs were detected at both mRNA (Fig. 3D) and protein levels (Fig. 3F), consistent with the CTNNB1 results.
  • Similar results were reproduced in PLC/PRF/5 and Hep3B cell lines stably transfected with both isoforms of ZBTB20.
  • ZBTB20 overexpression transfected PLC/PRF/5 (FIG. 10A) and Hep3B (FIG. 10B) cell lines resulted in higher CTNNB1 levels and decreased PPARG levels as determined by western blot analysis.
  • TOP/FOP-Flash luciferase reporter gene assay was also performed on Hep3B and PLC/PRF/5 cell lines to confirm the activation of the WNT/CTNNB1 signaling pathway.
  • Cell lines transfected with both ZBTB20 isoforms showed a dose-dependent increase in TOP/FOP luciferase activity (FIG. 10C).
  • ZBTB20-disrupted cells exhibited significantly increased AFP levels compared to scrambled controls (Fig. 4D).
  • AXIN2 levels were reduced in cells lacking ZBTB20 (Fig. 4D).
  • significantly lower levels of active CTNNB1 were detected in ZBTB20-disrupted cells, while total CTNNB1 levels were unchanged ( Figure 4E).
  • ZBTB20-disrupted cells showed significantly increased PPARG levels compared to scrambled controls (FIG. 4D).
  • Similar gene disruption experiments were also performed on the human hepatoma cell line C3A ( Figure 12).
  • the pGL3-PPARG reporter plasmid contained a 1038 bp PPARG promoter sequence and was used to determine whether ZBTB20 acts as a transcriptional repressor of PPARG expression (Figure 5A).
  • the 1038 bp PPARG promoter sequence is located on chromosome 3p25.212392051 to 12393088, while the putative binding motif for ZBTB20 is located at 12392366 to 12392374 (UCSC Genome Browser, GRCh37/hg19) ( Figure 13).
  • ZBTB20 isoforms 1 and 2 overexpressing cells were able to activate the PPARG promoter compared to OFP control transfected or HHL7 wild-type cells, respectively Activity was decreased by 47% and 45% (Fig. 5B).
  • the present invention confirms that ZBTB20 can inhibit the expression of PPARG and activate the WNT/CTNNB1 signaling pathway, which leads to the tumorigenesis of HCC.
  • ZBTB20 as an early detection marker for hepatocellular carcinoma
  • the inventors used the online database Gene Expression Omnibus (GEO) for comparison.
  • GSE6764 dataset contains gene expression profiles of 75 tissue samples from HCC patients, covering four tumor stages (very early HCC, early HCC, advanced HCC, and very advanced HCC), compared with normal livers as controls. Higher expression of ZBTB20 was detected at very early stages of HCC, while no significant changes in AFP were detected ( Figure 14A).
  • PPARG expression was also downregulated in very early stages of disease, although not statistically significant, whereas activation of the WNT signaling pathway was evident in very early stages of disease by upregulation of CTNNB1 and its target genes such as LEF1, TCF7, and CCND1 (Fig. 14B).
  • ZBTB20 is an oncogenic proto-oncogene of HCC, and confirmed its carcinogenic mechanism of action.
  • this gene is clearly shown in the results above and in Fig. 14, for example, that ZBTB20 can be used as a detection marker for hepatocellular carcinoma such as very early hepatocellular carcinoma, especially in the detection of hepatocellular carcinoma in AFP-deficient tissue samples.
  • Zbtb20 is critical for liver development, and mice lacking this gene exhibit liver dysfunction [3]. AFP is currently a well-established tumor marker for HCC, and its levels are often elevated during the disease stage [16]. Zbtb20 has been shown to be a transcriptional repressor of Afp using gene targeting studies [3,5]. It has also been recently reported that another member of the POK transcription factor family, Zbtb7a, can act as a proto-oncogene in some cases, but also has proto-onco-suppressive activity in others [17].
  • livers of Fah/SB11 mice injected with ZBTB20/shp53 also showed hepatomegaly compared to Empty/shp53 controls (Fig. 1E). While ZBTB20 overexpression alone induces hepatomegaly, Trp53 deficiency appears to act synergistically with ZBTB20 overexpression in HCC tumorigenesis.
  • transgenic hepatocytes may remain constitutively hypertrophic even after they have begun to proliferate. Therefore, a combination of increased hypertrophic and proliferative effects may promote liver tumorigenesis.
  • activation of the WNT/CTNNB1 pathway was observed in all experimental animals injected with ZBTB20/shp53 (Table 4).
  • ZBTB20/shp53 activated downstream genes of the WNT/CTNNB1 signaling pathway, such as Axin2, Lef1, Myc, and Tcf7 (Fig. 1H).
  • Table 4 CTNNB1 immunohistochemical staining of experimental and control injection groups.
  • a positive correlation between ZBTB20 and CTNNB1 could be confirmed by overexpression and disruption of ZBTB20 in human hepatocyte cell lines.
  • the novel results of the present invention indicate that the activity of CTNNB1 can be regulated by ZBTB20, further confirming that ZBTB20 acts on the WNT/CTNNB1 signaling pathway and is involved in the carcinogenesis of HCC. Furthermore, this interaction was further confirmed by immunofluorescence staining for the co-localization and localization of ZBTB20 and CTNNB1 in the nucleus ( Figure 2). Studies between PPARG and ZBTB20 have demonstrated the interaction [15].
  • ZBTB20 in liver tumorigenesis appears to involve enhanced hepatic hypertrophy and hepatomegaly due to TP53 inactivation.
  • the role of ZBTB20 as a novel oncogene and its mechanism in relation to HCC tumorigenesis have been demonstrated.
  • Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. P Natl Acad Sci USA 2008; 105: 10859 ⁇ 10864.
  • Zinc finger protein Zbtb20 is essential for postnatal survival and glucose homeostasis. Mol Cell Biol 2009; 29:2804 ⁇ 2815.
  • Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. Proceedings of the National Academy of Sciences of the United States of America 2008;105:10859 ⁇ 10864.

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Abstract

La présente demande concerne une étude de mécanisme de ZBTB20 entraînant un carcinome hépatocellulaire. De plus, la présente invention prouve que ZBTB20 est un oncogène du carcinome hépatocellulaire, et peut être utilisé pour le diagnostic d'un carcinome hépatocellulaire très précoce et précoce. La présente invention concerne également un ensemble d'amorces et un kit de reconnaissance spécifique de ZBTB20.
PCT/CN2022/070120 2021-01-07 2022-01-04 Protéine à doigts de zinc zbtb20 en tant que biomarqueur pour la détection et le diagnostic d'un carcinome hépatocellulaire WO2022148346A1 (fr)

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CN102234687A (zh) * 2010-04-29 2011-11-09 中国科学院上海生命科学研究院 抑制锌指蛋白ZBTB20的miRNA或其前体的应用

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KAN HEPING, HUANG YUQI, LI XIANGHONG, LIU DINGLI, CHEN JIANJIA, SHU MIAOJIANG: "Zinc finger protein ZBTB20 is an independent prognostic marker and promotes tumor growth of human hepatocellular carcinoma by repressing FoxO1", ONCOTARGET, vol. 7, no. 12, 22 March 2016 (2016-03-22), pages 14336 - 14349, XP055949713, DOI: 10.18632/oncotarget.7425 *
LIU GAN, ET AL.: "Biological functions of zinc finger protein ZBTB2c recent advance", ACADEMIC JOURNAL OF SECOND MILITARY MEDICAL UNIVERSITY, DI-ER JUN-YI DAXUE, SHANGHA, CN, vol. 36, no. 5, 31 May 2015 (2015-05-31), CN , pages 525 - 529, XP055949711, ISSN: 0258-879X, DOI: 10.3724/SP.J.1008.2015.00525 *
QING WANG;YE-XIONG TAN;YI-BIN REN;LI-WEI DONG;ZHI-FANG XIE;LIANG TANG;DAN CAO;WEI-PING ZHANG;HE-PING HU;HONG-YANG WANG: "Zinc finger protein ZBTB20 expression is increased in hepatocellular carcinoma and associated with poor prognosis", BMC CANCER, BIOMED CENTRAL, LONDON, GB, vol. 11, no. 1, 25 June 2011 (2011-06-25), LONDON, GB , pages 271, XP021102382, ISSN: 1471-2407, DOI: 10.1186/1471-2407-11-271 *
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