Developmental Stage-Specific Hepatocytes Induce Maturation of HepG2 Cells by Rebuilding the Regulatory Circuit
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On the basis of their characteristics, we presume that developmental stage-specific hepatocytes should have the ability to induce maturation of hepatoma cells. A regulatory circuit formed by hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-6 and the upstream stimulatory factor (USF-1) play a key role in the maturation of embryonic hepatocytes; however, it is unclear whether the regulatory circuit mediates the embryonic induction of hepatoma cell maturation. In this study, 12.5-d to 15.5-d mouse embryonic hepatocytes or their medium were used to coculture or treat HepG2 cells, and the induced maturation was evaluated in vitro and in vivo. In the induced HepG2 cells, the components of the regulatory circuit were detected, their cross-regulation was evaluated and HNF-4α RNA interference was performed. We found that 13.5-d to 14.5-d embryonic hepatocytes could induce HepG2 cell maturation, demonstrated by morphological changes, increased maturation markers and decreased c-Myc and α-fetoprotein (AFP) in vitro. The majority of HepG2 tumors were eliminated by 13.5-d embryonic induction in vivo. All components of the regulatory circuit were upregulated and the binding of HNF-4α, HNF-1α, HNF-6 and USF-1 to their target sites was promoted to rebuild the regulatory circuit in the induced HepG2 cells. Moreover, RNA interference targeting HNF-4α, which is the core of the regulatory circuit, attenuated the induced maturation of HepG2 cells with downregulation of the regulatory circuit. These results revealed that developmental stage-specific hepatocytes could induce the maturation of HepG2 cells by rebuilding the regulatory circuit.
Hepatocellular carcinoma (HCC), which is a major liver malignancy, is the third leading cause of cancer death (1, 2, 3). Although the current treatments are able to effectively kill tumor cells, they fail to completely cure HCC and inhibit its recurrence (4,5). Therefore, the search for a specific therapy remains a struggle in medical research.
HCC cells own common characteristics that are often found to be hepatic stem/progenitor cell biomarkers (2,6, 7, 8, 9, 10), although multiple environmental and genetic risk factors exist. HCC progression and embryonic liver development share many same properties, and a tumor may originate from progenitor/stem cells that receive abnormal proliferation and maturation signals (11, 12, 13). Thus, knowledge about cancer stem cells may reveal the exact biology of tumor cells and will offer new insights on HCC therapy (5,14, 15, 16).
A series of studies focused on the embryo (including embryonic stem cells, embryonary sac and parts of fetus) to induce tumor cell maturation (17,18). However, not all the induction experiments succeed, and the intrinsic explanation is not fully clarified (19,20). On the basis of their characteristics, we presumed that developmental stage-specific hepatocytes, in theory, should have the ability to direct and induce maturation of tumor cells (21). The question of whether developmental stage-specific hepatocytes could induce the maturation of HepG2 cells has not been explored.
In this study, HepG2 cells were cocultured with mouse embryonic hepatocytes at gestation of 12.5-15.5 d, because the mouse hepatocytes differentiated at 12.5 d, and the liver structure became firmly established between 12 and 15 d of gestation (35, 36, 37). The induced maturation of HepG2 cells was evaluated on the basis of their morphological changes, HNF-4α and c-Myc expression and α-fetoprotein (AFP) content. In addition, expressions of cytochrome P450, family 3, subfamily A, polypeptide 4 (CYP3A4), CYP1B1, ornithine carbamoyltransferase (OTC), arginase 1 (ARG1) and alcohol dehydrogenase 1C (class I), γ polypeptide (ADH1C), in 13.5-d embryonic hepatocyte medium-treated HepG2 cells were evaluated. Moreover, the effects of the 13.5-d embryonic hepatocyte medium on the HepG2 tumor cells that were inoculated into nude mice were observed to confirm their induction effects in vivo. All components of the regulatory circuit (HNF-4α, HNF-1α, HNF-6 and USF-1) were detected, and their cross-regulation was measured to investigate whether the regulatory circuit was rebuilt in the induced HepG2 cells. In addition, RNA interference of HNF-4α was performed to confirm the role of the regulatory circuit in the induced maturation of HepG2 cells. In parallel, 10.5-d to 13.5-d embryonic hepatocyte medium was used to treat another human hepatoma cell line SMMC-7721 for 48 h, and its proliferation and maturation markers CYP1B1 and ADH1C were evaluated.
Materials and Methods
Cells and Treatment
HepG2 (human hepatoma cell line) and SMMC-7721 cells were a product of the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Gibco [Thermo Fisher Scientific Inc., Waltham, MA, USA]).
Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction
Total RNA of HepG2 cells was isolated by using Trizol reagent (Takara, Japan) and reverse-transcribed into cDNA by using the RevertAid First-Strand cDNA Synthesis Kit (Fermentas, Canada) followed by real-time polymerase chain reaction (PCR) amplification with specific primers (Supplementary Table S1). Actin was used as a normalization gene.
HNF-4α, HNF-1α, HNF-6, USF-1 and c-Myc protein content was measured by Western blotting by using a previously described protocol (38,39). Anti-HNF-4α, anti-HNF-1α, anti-HNF-6, anti-USF-1, anti-c-Myc and anti-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used, and band intensities were quantified and calculated.
In Vivo Experiment
The 13.5-d embryonic hepatocytes were cultured for 48 h, and then the medium was collected. After having been centrifuged at 8000g, the supernatant was collected, filtered and used to treat HepG2 tumors.
Four-week-old nude mice were a product of Beijing HFK Bioscience (11401300001123; Beijing HFK Biotechnology Co. Ltd., Beijing, P.R. China), and the animal experimental protocol was approved by the institutional animal care committee. Sixteen mice were inoculated subcutaneously in armpit with 1 × 107 HepG2 cells in 1 mL phosphate-buffered saline (PBS) and divided equally into control and treated groups. When tumors became apparent, the mice in the treated group were locally injected with 0.1 mL of the 13.5-d embryonic hepatocyte medium, and the same volume of medium was injected into the control group once daily. The mice were treated for 17 d, and the changes in the tumors were observed for 25 d.
The binding of HNF-4α, HNF-1α, HNF-6 and USF-1 to their target promoters was analyzed by chromatin immunoprecipitation (ChIP), which was performed according to a previously published protocol (39,40). Briefly, the cells were cross-linked, collected and washed. Then, the cell suspension was sonicated, and an aliquot of the sonicated DNA was precipitated with ethanol and analyzed by electrophoresis. The average fragment sizes were 0.5–1 kb. Anti-HNF-4α, anti-HNF-1α, anti-HNF-6, anti-USF-1 (Santa Cruz Biotechnology) or control rabbit IgG (Santa Cruz Biotechnology) was added to an aliquot of 200 µL sonicated lysate, and 20 µL washed protein G-agarose beads (Santa Cruz Biotechnology) was then added. The mixture was rotated and subsequently centrifuged at 500g to wash the beads. The washed beads were resuspended, vortexed and boiled, and the sample and the sonicated lysate were treated with proteinase K. After centrifugation at 12,000g, the digested DNA was used in real-time PCR assays. The primers for the binding sites are listed in Supplementary Table S2. Additionally, controls of the nuclear factor (NF)-κB binding site in inducible nitric oxide synthase (inos) gene promoter and controls with only the antibody or beads were performed.
The protein contents of HNF-4α bound in hnf-1α and usf-1 promoters were measured by DNA pull-down assay. The cells were collected and nuclear proteins were extracted. After protein concentration was determined, DNA affinity precipitation assay was performed. The oligonucleotides containing biotin on the 5′-end of the each strand were used. The sequences of oligonucleotides for the predicted HNF-4α binding sites were in Supplementary Table S3. Each pair of oligonucleotides was annealed following standard protocols. Nuclear protein extracts (200 µg) were precleared with ImmunoPure streptavidin-agarose beads (20 µL/sample, Thermo Fisher Scientific). After centrifugation at 12,000g, the supernatant was incubated with 4 µg biotinylated double-stranded oligonucleotides overnight. A total of 20 µL streptavidin-agarose beads was added and incubated, and the protein-DNA-streptavidin-agarose complex was subjected to Western blotting with anti-HNF-4α antibody.
A small interfering RNA (siRNA) targeting HNF-4α (si-HNF-4α: 5′-CCACA UGUACUCCUGCAGATT-3′ and 5′-UCUGCAGGAGUACAUGUGGTT-3′), a siRNA control (si-control: 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′) and fluorescein-labeled (FAM)-siRNA were products of Integrated Biotech Solutions (Shanghai, China). Transfections were performed by using the Lipo-fectamine Reagent (Invitrogen [Thermo Fisher Scientific]) following the manufacturer’s instructions. First, after transfection with the FAM-siRNA, the cells were observed by using a fluorescence microscope (Olympus IX51, Olympus, Tokyo, Japan) and were then subjected to flow cytometry (Guava EasyCyte Mini, EMD Millipore) to evaluate the transfection efficiency. Then, 24 h after transfection, HepG2 cells were cocultured with 13.5-d embryonic hepatocytes for 48 h, and the HNF-4α mRNA and protein contents were examined to confirm the interfering effect. After the cells were successfully treated, the AFP content was determined by immunofluorescence, and the mRNA and protein contents of HNF-1α, HNF-6, USF-1 and c-Myc were measured.
Albumin in primary embryonic hepatocytes and AFP in the cocultured HepG2 cells or with si-HNF-4α were visualized by immunofluorescence detection. After fixation, the cells were incubated with 1% Triton X-100 for 30 min, blocked with 10% goat serum for 1 h and incubated with primary antibody (albumin or AFP; ZSGB-BIO, Beijing, China) at 37°C for 2 h. After washing with PBS, the cells were incubated with the secondary antibody (fluorescein isothiocyanate [FITC]; ZSGB-BIO). Subsequently, the cells were exposed to 0.5 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at room temperature and sealed with 50% glycerol. Images were taken by using a fluorescence microscope (Olympus IX51). Controls were also performed with PBS in place of the first or second antibody.
Data are presented as the means ± standard deviation (SD), and all statistical tests were two-sided. Student t test was used to assess statistical significance, and a P value <0.05 was considered significant.
All supplementary materials are available online at https://doi.org/www.molmed.org.
Developmental Stage-Specific Hepatocytes Induced Maturation of HepG2 Cells
In addition, this embryonic induction effect was evaluated in vivo. When inoculated HepG2 tumors became apparent, the mice were treated with 13.5-d embryonic hepatocyte medium, and the tumor masses were observed once daily (Figure 4C). Over the course of the study, tumor masses shrunk and gradually disappeared in the treated mice, whereas no changes were observed in the control mice. Importantly, after termination of the treatment, no recurrence of the tumors was observed in the treated mice. At the end of the experiment, in contrast to the control mice, no tumor masses were observed in the majority of treated mice (Figure 4D). The results provided in vivo evidence that HepG2 cells that were inoculated into nude mice could be eliminated by 13.5-d embryonic hepatocyte medium.
Developmental Stage-Specific Hepatocytes Rebuilt the Regulatory Circuit in HepG2 Cells
After HepG2 cells were cocultured with 13.5-d embryonic hepatocytes for 48 h, the cross-regulation of the circuit components was evaluated. The binding of HNF-4α, HNF-1a, HNF-6 and USF-1 to their predicted sites was detected by ChIP (Figure 1C). The 13.5-d embryonic hepatocyte coculture promoted binding of the factors to their sites, which might result in their elevated expression and rebuilding of the regulatory circuit. The NF-κB binding site in inos gene promoter control showed no meaningful signal and was not shown. The binding of HNF-4α to hnf-1α and HNF-4α to usf-1 was promoted, verifying the core position of HNF-4α. Meanwhile, the content of HNF-4α bound in hnf-1α and usf-1 promoters was detected by DNA pulldown assay, and the results showed that 13.5-d embryonic hepatocyte medium could significantly increase the content of bound HNF-4α in HepG2 cells. All the data indicated that the developmental stage-specific hepatocytes could rebuild the regulatory circuit that drives the maturation of HepG2 cells.
Inhibition of the Regulatory Circuit Attenuated the Maturation of HepG2 Cells
In addition, the mRNA and protein contents of other elements of the regulatory circuit as well as c-Myc were measured after HNF-4α interference in the cocultured HepG2 cells (Figures 7A, B). The si-HNF-4α treatment significantly inhibited the induced expression of hnf-1α, hnf-6 and usf-1 in HepG2 cells that were cocultured with 13.5-d embryonic hepatocytes. However, no obvious change in c-myc expression was observed after si-HNF-4α treatment, but HNF-1α and USF-1 protein expression was significantly inhibited (Figure 7C). These data revealed that inhibition of HNF-4α may result in suppression of the circuit components HNF-1α and USF-1.
Although current treatments can effectively kill tumor cells, they fail to completely cure cancer and inhibit its recurrence. The treatment strategy should transit to the induction of tumor cell normalization (41, 42, 43, 44). Because embryonic development process has the ability to coordinate the synchronism of cell maturation, we questioned whether developmental stage-specific hepatocytes induce the maturation of HCC cells. If so, we hypothesized that this method would provide an effective and harmless strategy for HCC therapy.
On the basis of their characteristics, we presumed that only developmental stage-specific hepatocytes should be able to direct and induce their maturation. In this study, HepG2 cells were cocultured with 12.5-d to 15.5-d mouse embryonic hepatocytes because of hepatocyte development (35, 36, 37). Embryonic hepatocytes at different developmental stages had distinct effects on the HepG2 cells, and the 13.5-d to 14.5-d embryonic hepatocytes could completely inhibit the proliferation and apparently induce the maturation of HepG2 cells.
The molecular mechanism underlying the induced maturation of HepG2 cells is not clarified. During normal liver development, the transcription factors HNF-4α, HNF-1α, HNF-6 and USF-1 cross-regulate each other to form a positive feedback regulatory circuit, which drives hepatocyte maturation (24, 25, 26). And we revealed that compared with that in 13.5-d embryonic hepatocytes, the circuit components HNF-4α, HNF-1α, HNF-6 and USF-1 were decreased in HepG2 cells. However, it is unclear whether the developmental stage-specific hepatocytes induce the maturation of HepG2 cells by rebuilding the regulatory circuit.
First, the components of the regulatory circuit were evaluated in the cocultured HepG2 cells. After coculture, HNF-4α, HNF-1α, HNF-6 and USF-1 increased; however, c-Myc expression decreased with time, showing that developmental stage-specific hepatocytes could upregulate the regulatory circuit components and inhibit the oncogene c-Myc. Then, the cross-regulation of HNF-4α, HNF-1α, HNF-6 and USF-1 was measured by ChIP. The 13.5-d embryonic hepatocyte cocultures promoted the binding of the factors to their target sites and rebuilt the regulatory circuit in HepG2 cells. Promotion of the binding of these factors to their promoters might result in their elevated expression, thus forming a positive feedback circuit that drives the maturation of HepG2 cells. The binding of HNF-4α to hnf-1α and HNF-4α to usf-1 was especially promoted and the accumulation of HNF-4α bound in hnf-1α and usf-1 promoters was proved again by DNA pull-down assay, verifying the core position of HNF-4α.
RNA interference of HNF-4α was performed to confirm the role of the regulatory circuit in the induced maturation of HepG2 cells. The results suggested that HNF-4α RNA interference could reverse the inhibited proliferation rate and the near disappearance of AFP protein in HepG2 cells that were cocultured with 13.5-d embryonic hepatocytes, indicating that inhibition of the regulatory circuit may attenuate the induced HepG2 cell maturation. In addition, si-HNF-4α treatment significantly inhibited the induction of hnf-1 α, hnf-6 and usf-1 expression and the increased HNF-1α and USF-1 protein contents in HepG2 cells that were cocultured with 13.5-d embryonic hepatocytes. These data suggested that inhibition of the regulatory circuit might result in the attenuation of the induced maturation of HepG2 cells.
This study revealed that developmental stage-specific hepatocytes could induce the maturation of HepG2 cells in vitro and in vivo, and this induced maturation was mediated by rebuilding the regulatory circuit. It was confirmed that differences in the expression of genes in embryonic stem cells at different developmental stages results in the production of diverse types and intensity of signaling molecules (45,46). And several signals and pathways of regulation mechanism of induced maturation of tumor cells have been identified (20). Therefore, clarification of the molecules expressed in tumor cells and the signaling pathways that mediate their inductive effects may reveal the intrinsic mechanism of induced maturation of tumor cells and then allow for the clinical application of this embryonic-induced maturation therapy.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by the National Natural Science Foundation of China (81470595) and Hebei Natural Science Foundation (H2012206005).
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