2.1 TFAP4 is a potential transcription factor governing TERT mRNA transcription in HCC in TCGA-LIHC cohort

To dissect the transcriptional regulation of TERT in human HCC, we performed dual luciferase reporter assay. We used the wild-type TERT promoter [10] and an overexpression approach, cloning a series of transcription factors suggested to alter TERT promoter activity. We then subjected these factors to reporter assay using Huh7 and PLC/PRF/5 HCC cells. We screened a total of 25 transcription factors and found that eight of them significantly induced the wild-type TERT promoter activity upon overexpression of the transcription factors compared to the empty vector control in both Huh7 and PLC/PRF/5 cells (Figure 1A and 1B). To further analyze the correlation between the transcription factors and TERT promoter activity in the context of HCC, we performed correlation analysis of their mRNA expression using TCGA-LIHC cohort. From this analysis, we found that TFAP4 and MYBL2 were significantly associated with the TERT mRNA expression in TCGA-LIHC cohort (Figure 1C).

Figure 1. Screening of transcription factors governing TERT mRNA expression in human HCC. (A-B) Overexpression approach was used for screening transcription factors that might govern TERT mRNA transcription in Huh 7 (A) and PLC/PRF/5 cells (B) by dual luciferase reporter assay. (C) Correlation analysis between potential transcription factors and TERT mRNA expression in human HCC in TCGA-LIHC cohort (n=371). (D) Correlation analysis between MYBL2, TFAP4, and TERT mRNA expression in the Cancer Cell Line Encyclopedia (CCLE) liver dataset (ns=P>0.05, *P<0.05, **P<0.001, ***P<0.001).

To further assess the correlation between transcription factor and TERT mRNA expression in the context of liver cancer cell lines, we conducted a correlation analysis between TFAP4, MYBL2, and TERT mRNA expression using the Cancer Cell Line Encyclopedia (CCLE). We observed that TFAP4 was significantly and positively associated with the TERT mRNA expression in the liver cancer cell lines in the CCLE dataset, while MYBL2 was negatively associated with the TERT mRNA expression. The findings suggest that TFAP4 is a potential candidate governing TERT mRNA transcription in HCC (Figure 1D).

2.2 TFAP4 is overexpressed in HCCs and associated with TERT mRNA expression in HCCs of all etiologies but not in HBV-associated HCC

We used TIMER (Tumor Immune Estimation Resource) [15] to determine the TFAP4 expression levels in different tumor types from TCGA database. Differential gene expression analysis showed that TFAP4 was upregulated in tumors compared to their corresponding non-tumor tissues in most cancer types, including liver cancer (P<0.001) (Figure 2A). We used TCGA RNA-sequencing database to analyze the expression of the TFAP4 gene in HCC tumors. The boxplots showing the association of gene expression and tumor stage revealed that the TFAP4 mRNA expression levels in HCC tumors (n=371), which include all etiologies, were upregulated compared with the corresponding non-tumor liver samples (Figure 2B). Similarly, TERT mRNA expression levels in tumor tissues (n=371) were upregulated compared with the corresponding non-tumor liver samples (Figure 2C). Additionally, the correlation analysis suggests that the mRNA expression of TFAP4 is significantly associated with the TERT mRNA expression in TCGA-LIHC cohort (Figure 2D). Intriguingly, we found that TFAP4 mRNA expression was not significantly associated with TERT mRNA expression in our HBV-associated HCC cohort consisting of 41 pairs of HCC (P=0.2168) (Figure 2E). Furthermore, in TCGA-LIHC cohort, patients with high mRNA expression of either TFAP4 or both TFAP4 and TERT had significantly reduced overall survival rates. However, such correlation was not observed in our HBV-associated HCC cohort (Figure S1). It is of note that TCGA cohort included HCC of all etiologies, i.e., both non-HBV infection and HBV infection, while our HCC cohort included only HBV-associated HCC. Hence, these discrepancies suggest that the positive association between TFAP4 and TERT mRNA expression may be independent of HBV infection or HBV viral integrations.

Figure 2. TFAP4 is up-regulated in human HCC and significantly correlates with TERT mRNA expression in TCGA-LIHC cohort. (A) TFAP4 expression levels in different tumor types from TCGA database were determined by TIMER. The distributions of gene expression levels are displayed, with statistical significance indicated. TFAP4 is upregulated in tumors compared to their corresponding non-tumor tissues in almost all cancer types, including liver cancer. The red rectangle indicates the expression levels in liver cancer. (B) A tumor stage plot of TFAP4 using TCGA-LIHC database showing an upregulation of TFAP4 mRNA levels in tumors compared to the corresponding non-tumor livers. (RPKM: reads per kilobase million) (C) A tumor stage plot of TERT using TCGA-LIHC database showing an upregulation of TERT mRNA levels in tumors compared to the corresponding non-tumor liver samples. (D) Correlation between TFAP4 and TERT in human HCCs in TCGA-LIHC cohort. (E) Correlation between TFAP4 and TERT in human HCCs in in-house HBV-associated HCC cohort (ns = P>0.05, *P<0.05, **P<0.001, ***P<0.001).

2.3 TFAP4 is a direct transcription factor driving telomerase reactivation in non-HBV-associated HCCs

To further investigate the role of TFAP4 in TERT gene transcription in HCC, we examined both TFAP4 protein expression and telomere length in HCC cell lines. We found that TFAP4 protein was ubiquitously expressed in most of the HCC cell lines tested as well as the immortalized normal liver cell line MIHA (Figure 3A). With qRT-PCR used to measure the telomere length, Huh7 and Hep3B cells had relatively more telomere copies compared to other HCC cell lines and MIHA cells (Figure 3B). Therefore, we selected Huh7 and Hep3B cells for subsequent functional validations. To study the functional role of TFAP4 in human HCC in vitro, we established TFAP4 stable knockdown in these two cell lines, using shRNA targeting lentiviral expression system. We examined the TERT mRNA expression and relative telomere copy numbers. Upon TFAP4 knockdown in Huh7 cells, there was a significant reduction in both TERT mRNA expression and relative telomere copy numbers compared to non-target control (NTC) cells (Figure 3C). In contrast, upon TFAP4 knockdown in Hep3B cells, which are HBV-infected HCC cells [16], both the TERT mRNA expression and relative telomere copy numbers were not significantly altered (Figure 3D).

Figure 3. TFAP4 is a direct transcription factor driving telomerase reactivation in non-HBV-associated HCCs. (A) The protein expression levels of TFAP4 in HCC cell lines. α-Tubulin served as internal control in western blot analysis. (B) The relative telomere length in HCC cell lines measured by qRT-PCR. RPLP0 was used as a normalizer for DNA input in this measurement. (C) Establishment of TFAP4 stable knockdown clones in Huh7 cells. TERT mRNA expression and relative telomere copy numbers were significantly reduced upon TFAP4 knockdown. (D) Establishment of TFAP4 stable knockdown clones in Hep3B cells. TERT mRNA expression and relative telomere copy numbers were not significantly impacted upon TFAP4 knockdown (ns = P>0.05, *P<0.05).

Regarding the cellular functional significance of TFAP4 in HCC cells, we observed that in shTFAP4 Huh7 cells, the cell invasive ability was significantly reduced as compared with NTC (Figure S2A). However, the shTFAP4 Huh7 cells showed a significant reduction of migratory ability only with sequence shTFAP4#5 (Figure S2A). In shTFAP4 Hep3B cells, only sequence shTFAP4#5 but not shTFAP4#2 showed a significant reduction in migratory and invasive abilities, as compared with NTC (Figure S2B). Taking together, the findings suggest that presence of both TFAP4 and TERT mRNA expression may contribute to cell invasiveness in Huh7 cells (in which TERT mRNA was altered), while single knockdown of TFAP4 without TERT reduction as in Hep3B cells (in which TERT mRNA was not significantly altered), such cellular function was diminished.

Using the INSECT (In-silico Search Co-occurring Transcription Factors) 2.0 webserver, we identified the putative binding sites of TFAP4 on the TERT promoter region. We analyzed the score threshold of TFAP4 occurrence on the TERT promoter and chose the four positions (–327, –286, –234, and –111nt) with relatively higher scores (Figure 4A). To assess the binding of TFAP4 on the TERT promoter region, we performed dual luciferase reporter assay. The results showed that upon mutation at positions –327, –234, and –111nt, there was a significant decrease in promoter activity compared to Huh7 cells transfected with the wild-type TERT promoter (Figure 4B). Similarly, in HepG2 cells transfected with the wild-type TERT promoter, any mutation at the putative TFAP4 binding site among the four suggested positions significantly decreased promoter activity (Figure 4C). Notably, when the putative TFAP4 binding site at the –111nt position was mutated, the promoter activities drastically decreased in both Huh7 and HepG2 cells (reduced by 69.4% and 90%, respectively) (Figure 4B and 4C). Finally, with ChIP-qPCR assay using the TFAP4 antibody, we demonstrated that TFAP4 physically interacted with the TERT promoter at the –111nt position, but not at either the –234 or –286nt position in Huh7 cells (Figure 4D). In summary, TFAP4 is a direct transcription factor which physically interacts with the TERT promoter region and drives telomerase reactivation in non-HBV-associated HCC cells.

Figure 4. TFAP4 as a transcriptional regulator of TERT in HCC cell line. (A) A schematic diagram of the wild-type TERT promoter and mutations of the four putative transcription factor binding motifs at –327, –286, –234, and –111nt in the TERT promoter. (B) Dual luciferase reporter assays of TERT promoters of wild type and with the corresponding mutations in Huh7 cells are shown. (C) Results of dual luciferase reporter assays of TERT promoters of wild type and with the corresponding mutations in HepG2 cells. (D) ChIP assays using specific antibodies against TFAP4 in Huh7 cells. The results confirmed that TFAP4 specifically bound to and were enriched on –111nt but not –286 and –234nt of the TERT promoter region (ns = P>0.05, *P<0.05, **P<0.01, ***P<0.001).

2.4 CTCF is crucial for TFAP4 gene transcription in human HCCs

While TFAP4 is upregulated in HCC, its gene transcription regulation control in HCC remains unknown. To investigate the transcription regulator of TFAP4, we searched for putative DNA binding proteins from the Encyclopedia of DNA Elements (ENCODE) data portal [17] in the context of HepG2 cells and liver tissue. According to the ChIP-sequencing database from ENCODE data portal, CTCF, FOXA2, MYC, and MAZ are suggested to physically interact with the TFAP4 promoter region. Using dual luciferase reporter assay, we examined the transcription activity among the four putative transcription factors on the TFAP4 promoter by using an overexpression approach in Huh7 cells. Only CTCF, FOXA2, and MAZ significantly increased TFAP4 promoter activity (Figure 5A). To further explore which transcription factors drove TFAP4 gene transcription in HCC, we conducted a correlation analysis among CTCF, MAZ, and TFAP4 mRNA expression using the Cancer Cell Line Encyclopedia (CCLE). The correlation analysis suggested that both CTCF and MAZ were significantly associated with TFAP4 mRNA expression in liver cancer cell lines in the CCLE dataset, with CTCF having the most significant positive correlation compared to MAZ. This suggests that CTCF is one of the potential candidates governing TFAP4 mRNA transcription in HCC (Figure 5B). Moreover, upon CTCF knockdown in Huh7 cells, both TFAP4 and TERT showed a significant reduction at the mRNA and protein expression level compared with shNTC (Figure 5C). In addition, CTCF knockdown led to significant reductions in relative telomere copy number compared with shNTC Huh7 cells (Figure 5D). Finally, with immunohistochemistry, we found that CTCF protein is overexpressed in human HCC compared to corresponding non-tumor counterparts (Figure 5E), suggesting that CTCF protein is overexpressed in human HCC and facilitates TFAP4 gene transcription.

Figure 5. CTCF is crucial for TFAP4 gene transcription in human HCCs. (A) A schematic diagram of the wild-type TFAP4 promoter (upper panel). Results of dual luciferase reporter assays of wild type TFAP4 promoter with overexpression of CTCF, FOXA2, MAZ, and MYC in Huh7 cells are shown (lower panel). (B) Correlation analysis between CTCF, MAZ, and TFAP4 mRNA expression in the CCLE dataset. (C) TFAP4 and TERT mRNA (left panel) and protein expression (right panel) upon CTCF knockdown in Huh7 cells. (D) The relative telomere copy numbers upon knockdown of CTCF compared with non-target control in Huh7 cells. (E) A representative example of immunohistochemical staining for CTCF protein in human HCCs. ‘T’, tumor; ‘NT’, non-tumor liver. Scale bar, 100 μm (ns = P>0.05, *P<0.05, **P<0.01).

2.5 Ethanol enhances both TFAP4 and TERT mRNA expression in non-HBV-associated human HCC cells

To further dissect the possible mechanism underlying the positive correlation between TFAP4 and TERT mRNA expression in TCGA cohort which includes HCCs of all etiologies, but not in our in-house HBV-associated HCCs, we further stratified the patients in TCGA cohort according to the etiologies for analysis. Interestingly, a significant correlation between TFAP4 and TERT mRNA expression was observed only in patients with chronic alcohol consumption, but not those with either HBV- or HCV-associated HCC (Figure 6A and Figure S3).

Figure 6. Ethanol enhances both TFAP4 and TERT mRNA expression in human non-HBV-associated HCCs and non-HBV-infected HCC cells. (A) Correlation analysis between TRAP4 and TERT mRNA expression levels in human HCCs related to chronic alcoholism (n=72, left panel) and HBV-associated HCC (n=76, right panel) in TCGA-LIHC cohort. (B) TFAP4 and TERT mRNA expression upon ethanol treatment at 50 mM for 24 hours in HepG2 cells (left 2 panels) and Hep3B (right 2 panels). (C) Methylation status at –96nt of the TERT promoter region upon ethanol treatment in HepG2 and Hep3B cells. (D) Dual luciferase reporter assays of wild-type 6x TFAP4 consensus DNA binding motif-containing promoter, with or without 50 mM ethanol treatment in HepG2 and Hep3B cells. Empty vector (EV) with no treatment served as normalization control (ns = P>0.05, *P<0.05, **P<0.01).

Therefore, we conducted in vitro experiments on HCC cells with or without HBV infection. We treated HepG2 and Hep3B cells with 50 mM ethanol for 24 hours. We observed a significant up-regulation of both TFAP4 and TERT mRNA expression in HepG2 cells upon ethanol treatment (Figure 6B), while there was no significant impact on TFAP4 mRNA expression in Hep3B cells. In contrast, the TERT mRNA expression was significantly reduced in Hep3B cells (Figure 6B). Given that it has been reported that ethanol might alter the methylation status using a rat model [18], we further investigated the methylation status of the TERT promoter region upon ethanol treatment. From above finding (Figure 4) suggested that proximity of transcription start site of TERT promoter region crucial for TFAP4 binding (–111nt). To further investigate the methylation status of this region, we performed in-silico analysis of the CpG methylation site within this region and found that -96nt is the putative CpG methylation site, which is in close proximity of the TFAP4 binding site in the TERT promoter. Using methylation-specific qPCR targeting the TERT promoter region, we found there was an induction of DNA methylation status at the TERT promoter region upon ethanol treatment in Hep3B, but not HepG2 cells (Figure 6C). To further assess whether ethanol would alter TFAP4 transcription activity in HCC cells, we employed the six times repeat TFAP4 consensus DNA binding sequence (encode nucleotide sequence “CAGCTG”) driven luciferase reporter constructs to conduct Dual luciferase reporter assay to assess TFAP4 transcription activity upon ethanol treatment. With ethanol treatment, there were no significant alterations of TFAP4 transcription activity in both HepG2 and Hep3B cells (Figure 6D). The findings suggest that ethanol may induce TFAP4 and TERT mRNA expression in HCC cells and alcohol-related HCC.

4.1 Patient Samples

We randomly selected HCC cases from patients who underwent surgical resection of the tumors at Queen Mary Hospital, Hong Kong. Following surgical resection, all specimens were immediately snap frozen in liquid nitrogen and stored at -80°C, with informed consents from the patients. The use of human clinical samples was approved by the Institutional Review Board of the University of Hong Kong and the Hong Kong Hospital Authority Hong Kong West Cluster (UW 17-056).

4.2 Cell lines

Human HCC cells, HepG2, Hep3B and PLC/PRF/5 were obtained from American Type Culture Collection. Huh7 were purchased from the Japanese Collection of Research Bioresources Cell Bank. Human HCC cell line MHCC97L was a gift from Dr. ZY Tang (Fudan University, Shanghai, China) and the STR authentication was conducted. Immortalized liver cell line MIHA was obtained from Dr. J Roy-Chowdhury (Albert Einstein College of Medicine, New York, USA). CLC7 was a gift from Dr. Lijian Hui (Shanghai Institutes for Biological Sciences, CAS, Shanghai, China). Human embryonic kidney cell line 293FT was obtained from Invitrogen (Carlsbad, USA). HepG2, Hep3B and PLC/PRF/5 were cultured in Minimum Essential Medium containing 10% fetal bovine serum and 1mM sodium pyruvate, CLC7 was cultured in RPMI-1640 Medium containing 10% fetal bovine serum, while the others were cultured in Dulbecco’s modified Eagle’s medium high glucose supplemented with 10% fetal bovine serum.

4.3 Plasmids

We amplified full-length forms of transcription factors from HepG2 complementary DNA (cDNA) and subcloned them into the pcDNA3.1+neo vector. We amplified the TERT promoter (chr 5. 52,411,030-52,410,031) and TFAP4 promoter (chr 16. 4,274,618-4,256,186) from HepG2 genomic DNA and subcloned them into the pGL3-Basic vector. We also amplified a series of TERT or TFAP4 promoter DNA fragments with mutations from the wild-type TERT or TFAP4 promoter/pGL3-Basic vector and sub-cloned them into the empty vector of pGL3-Basic. We purchased short-hairpin (sh) RNA interfering sequences targeting CTCF and TFAP4 from Horizon Discovery (Cambridge, United Kingdom) and synthesized them by Integrated DNA Technologies (Coralville, IA, USA), before subcloning them into the pLKO.1-Puro vector. The primers used for cloning are listed in Table S1.

4.4 Dual luciferase reporter assay

Huh7 and PLC/PRF/5 cells underwent transfection with various combinations of plasmids using Lipofectamine 3000 (Invitrogen) as per the manufacturer’s protocol. The plasmids included TERT wild-type promoter/pGL3-Basic, TFAP4 wild-type promoter/pGL3-Basic, different forms of mutated TERT and TFAP4 promoter/pGL3-Basic reporter constructs, and an internal control pRL/PGK. After 24 hours of transfection, we measured luciferase and Renilla luciferase activities using a Dual Luciferase Reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The Renilla luciferase activity was used to normalize transfection efficiency. We conducted these experiments independently at least three times.

4.5 Stable knockdown HCC cell establishment

We employed a lentiviral-mediated approach to construct stable CTCF and TFAP4 knockdown HCC cell lines. We purchased human ON-TARGETplus SMARTpool siRNA duplexes, which target CTCF and TFAP4, along with a non-target control from Horizon Discovery. We stably transduced the pLKO.1-Puro vectors, which contain shRNAs targeting corresponding gene targets and a non-target control, into HCC cell lines. We performed puromycin selection to achieve the stable expression of shRNAs and shNTCs. The sequences of all shRNAs are listed in Table S1.

4.6 Western blot analysis

Cells were lyzed in NET (150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH8.0) buffer with 1%NP-40 and equal amounts of protein were separated in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gel for western blot analysis. Immunodetection was performed using antibodies which are listed in Table S2.

4.7 RNA extraction, cDNA synthesis and qPCR analysis

Total RNA was extracted from HCC cell lines using Trizol reagent (Invitrogen). The cDNA synthesis using GeneAmp PCR Reagent Kit (Applied Biosystems, Foster City, CA, USA) was used to reverse messenger RNA into cDNA and ABI Power SYBR™ Green PCR Master Mix (Applied Biosystems) coupled with primer specific for TERT, TFAP4 and HPRT1 were used for measurement. The primer sequences are listed in Table S3.

4.8 Chromatin immunoprecipitation assay

The detailed protocol has been described [47]. The antibody against TFAP4 protein (HPA001912, Sigma-Aldrich) and normal Mouse IgG antibody (sc-3877, Santa Cruz biotechnology) were used for the chromatin immunoprecipitation (ChIP) assay. The primers (–127nt to +1nt: 5’-CCGCCTCCTCCGCGCGGACC-3’ and 5’-CAGCGCTGCCTGAAACTC-3’; –286nt to –234nt: 5’-GGGAGCGCG AGCGGCGCGCGGG-3’ and 5’-CGACAGCGCAGCTGCTCCGGGC-3’) covering the putative TFAP4 binding site on TERT DNA region were employed for standard qRT-PCR using ABI Sybr green Gene Expression Assay supplements of AmpliTaq Gold 360 master mix (Applied Biosystems) and detected by ABI QuantStudio 5 Real-Time PCR System (Applied Biosystems) in the ChIP assay.

4.9 Cell proliferation assay

We seeded 1,000 cells on each well of a 96-well plate and cultured them for 5 days. Every 24 hours (Day 1 to Day 5), we fixed designated cells using 30 μL of 100% methanol per well for 5 minutes, and then replaced it with 100 μL/well of PBS after cell fixation. After 5 days, we removed the PBS and used diluted DAPI at 1 μg/mL to stain the cell nuclei. We then subjected the plate to fluorescent photo capturing and cell counting by the ImageXpress Pico Automated cell Imaging System (Molecular Devices, San Jose, CA, USA). The cell counting program setting was pre-configured by the manufacturer. We conducted these experiments independently at least three times.

4.10 Transwell migration and invasion assay

100,000 cells were resuspended into 100 μL of serum-free medium and added to the upper chamber of the insert with 8 μm pore size membrane (Merck Millipore, Burlington, MA, USA). For the invasion assay, the transwell was pre-coated with cold 1:4 diluted matrix gel and incubated at 37 C for 1 hour before use. For the bottom chamber, 500 CL of standard culture medium was added to the bottom chamber of cell culture insert in the well of 24-well plate acting as chemoattractant. After incubation, cells on the insert membrane were fixed by 100% methanol for 5 minutes and stained with crystal violet solution for 20 minutes. After washing the insert with water, non-migrated cells on the upper side of the membrane were gently removed by cotton swab. Photos were taken from three randomly selected fields with migrated cells on the lower side of the membrane and cells counted manually. Experiments were done at least three independently.

4.11 Telomere length quantification

We quantified the relative telomere length using quantitative real-time PCR (qRT-PCR), according to the method described by O’Callaghan and Fenech in 2011 [48] with minor modifications. Firstly, cells were centrifuged for 5 minutes at 300 x g. After discarding the supernatant, we re-suspended the cell pellet in 200 μL PBS. Next, we isolated the DNA using the TIANamp Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China), as per the manufacturer’s instructions. We used these samples for standard qRT-PCR using the ABI Sybr Gene Expression Assay (Applied Biosystems) and detected them using the ABI QuantStudio 5 Real-Time PCR System (Applied Biosystems). The 36B4 gene, which encodes an acidic ribosomal protein lateral stalk subunit P0 (RPLP0), served as internal control. The qRT-PCR primers used are listed in Table S4. We performed all reactions in triplicate.

4.12 Methylation-specific quantitative PCR

We isolated genomic DNA from HCC cell lines using the TIANamp Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. To perform methylation-specific quantitative PCR, we subjected 1 μg of genomic DNA to methylation-sensitive restrictions enzyme Hha I digestion at 37 °C for three hours. We then subjected 50 ηg of digested genomic DNA to standard quantitative PCR using the ABI Sybr Gene Expression Assay (Applied Biosystems) and detected them using the ABI QuantStudio 5 Real-Time PCR System (Applied Biosystems). The primers (–127nt to +1nt: 5’-CCGCCTCCTCCGCGC GGACC-3’ and 5’-CAGCGCTGCCTGAAACTC-3’) covering the putative methylation site on TERT DNA region were employed for standard qPCR in this assay. The same amount of undigested genomic DNA served as input DNA control.

4.13 Statistical analysis

We analyzed the data using the Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). Depending on the nature of the study, we subjected the data to either paired or unpaired t-test. We used paired t-test to compare gene expressions in HCC tumor tissues with corresponding non-tumor liver samples, as well as to analyze gene expression changes in independent samples via qRT-PCR. For Dual luciferase reporter assay analyses, we employed paired t-test. In contrast, we used non-linear regression test (curve fit) to analyze cell proliferation assay data. We considered test results significant when the P-value was less than 0.05. For vitro functional assays, we expressed data as mean ± SEM. We defined statistical significance as *P<0.05, **P<0.01, and ***P<0.001.

Ethics Statement

The use of human clinical samples was approved by the Institutional Review Board of the University of Hong Kong and the Hong Kong Hospital Authority Hong Kong West Cluster (UW 17-056).

Consent for Publication

Not applicable.

Availability of Data and Material

The original data presented in the study are openly available in UCSC Xena browser at (https://xenabrowser.net/).

Funding

The study was supported by the Hong Kong Research Grants Council (RGC) Theme-based Research Scheme (T12-704/16-R and T12-716/22-R), Innovation and Technology Commission grant to the State Key Laboratory of Liver Research of the University of Hong Kong (ITC PD/17-9), University Development Fund of The University of Hong Kong. I.O.L. Ng is Loke Yew Professor in Pathology.

Competing Interests

The authors have declared that no conflict of interest exists.

Author Contributions

Conceptualization: K.M.F.S. and I.O.N.; Methodology: H.L., T.K.H.W., K.M.F.S. and I.O.N.; Investigation: H.L., T.K.H.W., K.M.F.S., E.M.S.S., T.C.Y.Y., J.M.F.L., Y.M.T. and D.W.H.H.; Resources: I.O.N; Writing: H.L., T.K.H.W., K.M.F.S. and I.O.N.; Supervision: K.M.F.S. and I.O.N.; Visualization: K.M.F.S. and I.O.N.; Project administration: K.M.F.S. and I.O.N.; Funding acquisition: I.O.N.

Abbreviations

cDNA

complementary DNA