Background
Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide [
1,
2]. According to data published by the International Agency for Research on Cancer in 2012, over 78 million new cases of HCC and more than 70 million deaths due to HCC are recorded per year. As HCC is a highly heterogeneous disease, several genes and proteins are known to contribute to its tumorigenesis and progression [
3].
Forkhead box A1 (FOXA1), also called HNF3A [
4], is a member of the forkhead family of DNA-binding proteins, which are known for their role in regulating metabolism. FOXA proteins include three members, FOXA1, FOXA2, and FOXA3, each encoded by an individual gene [
5]. Increasing evidence indicates that FOXA factors are involved in the development and progression of several tumors [
6].
In the present study, we focused on FOXA1 as a transcriptional regulator of HCC. Function gain and loss analysis was performed to determine the role of FOXA1 in cancer cells derived from male HCC patients.
Methods
Cell culture
Human liver carcinoma cell lines HepG2 and Hep3B, both derived from male hepatocellular carcinoma patients according to ATCC, were purchased from the Cell Bank at the Chinese Academy of Sciences (Shanghai, China). According to the manufacturer’s instructions, the cells were cultured in Minimum Essential Medium (MEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere containing 5% CO2.
Cell transfection
Cells were seeded in 6-well plates (1.5 × 10
5 or 3 × 10
5 cells/well) and maintained in complete medium for 12 h prior to transfection with siRNA (GenePharma, Shanghai, China) or plasmid DNA (Vigene Bioscience Inc., Shandong, China). siRNA sequences are available in Additional file
1: Table S1. siRNA and the FOXA1 plasmid were transfected into HepG2 and Hep3B cells at working concentrations of 100 nM using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Scrambled-sequence siRNA and the pEnter plasmid were transfected as negative controls. Cells were harvested after 48–72 h and used for further experiments.
Lentivirus infection
HepG2 and Hep3B cells were infected with FOXA1-overexpressing lentivirus (GeneChem, Shanghai, China), after which FOXA1 expression was confirmed by quantitative reverse transcription polymerase chain reaction (qPCR) and western blotting.
Quantitative reverse transcription PCR
Trizol reagent (Takara Bio Inc., Shiga, Japan) was used to extract RNA from the cells and total RNA was then reverse transcribed according to the manufacturer’s protocol. PCR cycling conditions were 95 °C for 30 s to denature the cDNA template, followed by 40 cycles at 95 °C for 5 s and 60 °C for 20 s. The specificity of amplification products was confirmed by melting curve analysis. Data were analyzed using the 2
−ΔΔCt method. The primers used for qPCR are available in Additional file
1: Table S2. Independent experiments were performed in triplicate.
Western blotting
Cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride and phosphatase inhibitors (Roche, Basel, Switzerland). After the protein concentration was determined with the BCA kit (Beyotime), cell lysates were subjected to SDS polyacrylamide gel electrophoresis and the protein bands were transferred to polyvinylidene fluoride membranes. The membranes were then probed with the following primary antibodies: anti-FOXA1 (ab170933; Abcam, Cambridge, UK), PI3Kp85 (ab86714; Abcam), anti-Akt (2920; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Akt (Ser473, 12,694; Cell Signaling Technology), anti-glyceraldehyde 3-phosphate dehydrogenase (RM2002; Beijing Ray Antibody Biotech, Beijing, China), and anti-Flag (F1804; Sigma, St. Louis, MO, USA). Bands were visualized using horseradish peroxidase-conjugated secondary antibodies and electrochemiluminescence detection kit. All western blot images were processed by ImageJ software (
https://imagej.nih.gov/ij/).
Cell proliferation assays
For the Cell Counting Kit-8 (CCK-8) assay, transfected HepG2 and Hep3B cells were seeded in 96-well plates at a density of 103 cells/well. For transient transfection with siFOXA1 and siPIK3R1, cells were cultured for 1, 2, 3, 4, or 5 days. For lentivirus-mediated FOXA1 overexpression, cells were incubated for 1, 2, 3, 4, 5, 6, or 7 days. Subsequently, 100 μL of complete medium supplemented with 10 μL of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well, the plates were incubated for 2 h, and the absorbance was measured at 450 nm.
EdU assays: Transfected HepG2 and Hep3B cells were seeded in 96-well plates at a density of 4 × 103 cells/well. After incubation with 10 mM EdU (RiboBio, Guangzhou, China) for 2 h, cells were fixed and stained according to the manufacturer’s protocol. EdU-positive cells were counted under a fluorescence microscope in five random fields.
Colony formation assays: Lentivirus-infected HepG2 and Hep3B cells were seeded in 6-well plates at a density of 300 cells/well and cultured for 14 days. Subsequently, colonies were fixed with 100% methanol and stained with 1% crystal violet. Colonies composed of more than 50 cells in a well were counted under a microscope. All experiments were performed three times.
Migration and invasion assay
For the transwell migration or invasion assay (8.0 μm, #3442; Corning, Corning, NY, USA), 1 × 105 treated cells were seeded into the upper chamber in the presence of an uncoated or a Matrigel-precoated membrane (356,234; Corning) containing 200 μL of serum-free MEM. Complete medium (600 μL) containing 10% FBS was added to the bottom chamber. Following incubation for 24–30 h, the chambers were washed twice with phosphate-buffered saline, fixed with 100% methanol, and stained with 1% crystal violet at room temperature. Cells were counted under a microscope in five random fields.
Chromatin immunoprecipitation (ChIP) assay
To examine whether FOXA1 bound to the promoter sequence of PIK3R1, a ChIP assay (17–371; Millipore, Merck, Darmstadt, Germany) was performed following the manufacturer’s protocol. Briefly, untreated Hep3B cells were fixed using 1% formaldehyde for 10 min to crosslink proteins to DNA, and then soluble chromatin was sheared into 200–1000 bp fragments using sonication. The fragmented chromatin samples were incubated with anti-FOXA1 antibody (ab23738; Abcam) to precipitate the putative binding sequences. Finally, PCR was used to detect enrichment of PIK3R1 promoter fragments on the putative FOXA1 binding sites. Primers used in ChIP are available in Additional file
1: Table S3.
Dual luciferase reporter assay
Hep3B cells were plated at 2 × 105 cells/well in 24-well tissue culture plates. PIK3R1-promoter-pGL3-basic plasmid (Kidan Bio Co. Ltd., Guangzhou, China), pRL-TK plasmid (Kidan Bio Co. Ltd), and FOXA1-pEnter plasmid (Vigene Bioscience Inc.) or pEnter (Vigene Bioscience Inc.) vector were co-transfected into Hep3B cells. After culturing for 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA).
Immunohistochemistry assay
Immunohistochemistry assays were employed to detect expression of FOXA1 and PI3Kp85 proteins in paraffin-embedded human tissue microarrays (Shanghai Outdo Biotech, Shanghai, China), using a standard immunoperoxidase staining procedure and anti-FOXA1 (1:100, ab170933; Abcam) and anti-PI3Kp85 (1:50, ab86714; Abcam) antibodies. Stained tissue sections were examined separately by two pathologists. Protein expression was evaluated in terms of the proportion and intensity of stained cells. Thus, positive cells were scored based on their staining proportions as 1 (< 25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%); and in terms of intensity as negative (0), weak (1), medium (2), or strong (3).
Computational analysis of putative target genes regulated by FOXA1
Putative target genes controlled by FOXA1 were identified in silico analysis using the Cistrome Dataset Browser (
http://cistrome.org/db/#/) [
7] and ChIPBase v2.0 (
http://rna.sysu.edu.cn/chipbase/) [
8] databases on ChIP-seq data from HepG2 cells. Genes that scored higher than 2.5 points or encoded proteins were selected for further analysis using the Cistrome Dataset Browser and ChIPBase v2.0, respectively. Gene Ontology (GO) enrichment analysis was employed to cluster predicted genes from the datasets.
Statistical analysis
SPSS 20.0 software (SPSS Inc. Chicago, IL, USA) was used for statistical analyses. Values represent the mean ± standard error of the mean of at least three independent experiments. Comparisons between two groups were performed using Student’s t-test. Multi-way classification analysis of variance was performed for the results of the CCK-8 assays [
9]. Associations between FOXA1 and PI3Kp85 were analyzed using Spearman’s correlation coefficient. Survival analysis was performed using the Kaplan-Meier method. All statistical tests were two-sided, with statistical significance defined as *
P < 0.05, **
P < 0.01, and ***
P < 0.001.
Discussion
FOXA1 has been linked to various types of tumors [
10,
11]. Intriguingly, it exhibits a dual role even in the same pathological condition [
12], partly owing to FOXA1 acting both as a pioneer factor and as a transcription factor [
4,
13]. In the latter case, FOXA1 acts as a critical regulator of metabolism, tissue function, and tumor development. In this study, we investigated the role and molecular mechanism of FOXA1 in HCC. Our findings revealed that FOXA1 protein directly regulates transcription of PIK3R1, which encodes PI3Kp85, and blocks HCC proliferation, migration, and invasion. Consistently, in HCC patients, FOXA1 expression was negatively correlated with PI3Kp85 expression in male subjects, and low expression of PI3Kp85 was a favorable factor in stage II male patients with HCC. These results suggest that FOXA1 functions as a potential HCC suppressor.
According to previous studies, FOXA1 is a polytropic gene, often associated with sex hormones [
4]. In estrogen receptor-positive breast cancer, prostate cancer [
14], acute myeloid leukemia, and thyroid carcinoma [
6], FOXA1 exhibits a potential cancer-promoting effect; however, it causes tumor inhibition in estrogen receptor-negative breast cancer [
15], advanced prostate cancer, and pancreatic cancer [
10]. Further molecular mechanistic studies have revealed that FOXA1 promotes tumor progression by recruiting other transcription factors, while acting as a transcription factor for suppressing tumor development by directly regulating target gene expression [
12,
16]. Until now, studies on the role of FOXA1 in carcinogenesis have focused mainly on breast and prostate cancers. Although FOXA1 was first detected in the liver, its role in HCC remains unclear. Recently, Li et al. [
17] demonstrated that the sexual dimorphism of HCC was reversed in Foxa1/Foxa2-deficient mice. Mostly, though, it is believed that FOXA1 and FOXA2 do not interact with each other [
18]. The exact mechanism by which FOXA1 regulates HCC progression remains poorly understood.
The PI3K/Akt signaling pathway is well known for mediating fundamental carcinogenic processes, including cancer cell survival, differentiation, proliferation, and motility [
18]. The PI3K/Akt signaling pathway is constitutively activated in nearly all cancer types, probably through activation of upstream signaling molecules [
19] or mutation of pathway components [
20]. PI3K is composed of a regulatory subunit, PI3Kp85, and a catalytic subunit, PI3Kp110 [
21], whose combination determines the biological activity of PI3K [
22]. Recent studies have suggested that PI3Kp85 may act as an oncogene in several tumor types [
23,
24], but the role of PI3Kp85 in HCC remains unclear.
In this study, function loss and gain experiments showed that FOXA1 knockdown induced proliferation, migration, and invasion of HepG2 and Hep3B cells, whereas FOXA1 overexpression decreased cell viability and motility. Moreover, the biological functions of FOXA1 identified in this study provide a mechanistic explanation for its role in carcinogenesis. We obtained the FOXA1 putative target gene, PIK3R1, by retrieving information from combined ChIP databases and GO enrichment analysis. Based on the FOXA1 conserved motif, we predicted its binding sites on the PIK3R1 promoter, designed eight pairs of primers to validate the possibility of a successful combination, and observed at least three binding sites. Simultaneously, the luciferase reporter assay indicated that FOXA1 functions as a transcriptional inhibitor of PIK3R1. The reduction in phospho-Akt activity by FOXA1 was mediated by PI3Kp85 and was involved in the PI3K/Akt signaling pathway, as determined by western blotting.
Finally, we confirmed a negative correlation between FOXA1 and PI3Kp85 in stage II male HCC patients, but not in female patients. Moreover, stage II male patients with HCC with low PI3Kp85 were predicted to have a long survival time. This might be linked to the origin of our clinical samples, which were collected from patients with HCC, who underwent surgery, and were in the early stage of the disease. Additionally, it should be noted that both HepG2 and Hep3B cell lines were constructed using samples from Caucasian male patients. These limitations emphasize the importance of investigating the regulatory effects of FOXA1 on PIK3R1 in female patients and in those diagnosed with HCC at different stages. For example, the Mahlavu cell line could be used to investigate how FOXA1 functions in female patients in vitro and more specimens from female patients should be collected to verify the role of FOXA1 in female patients.