SIRT1 regulates YAP2-mediated cell proliferation and chemoresistance in hepatocellular carcinoma

Oncogene volume 33, pages1468–1474(2014)Cite this article

Subjects

Abstract

The MST/YAP (mammalian Ste20-like kinase/Yes-associated protein 2) pathway plays an important role in hepatocellular carcinoma (HCC). Although post-translational modification—especially MST/Lats (large tumor suppressor)-mediated phosphorylation and PP1 (protein phosphatase-1)-mediated dephosphorylation—has been found to regulate the activity of YAP2, very little is known about its acetylation. In our experiments, we observed that the expression of SIRT1 is significantly upregulated in the tumor samples of the hepatocarcinoma patients, and SIRT1 mRNA level positively correlates with connective tissue growth factor (CTGF) mRNA level. We then found that SIRT1 deacetylates YAP2 protein in HCC cells and SIRT1-mediated deacetylation increases the YAP2/TEAD4 association, leading to YAP2/TEAD4 transcriptional activation and upregulated cell growth in HCC cells. Moreover, knockdown of SIRT1 blocks the cisplatin (CDDP)-induced nuclear translocation of YAP2 and enhances the chemosensitivity of HCC cells to CDDP treatment. Together, our findings reveal a new regulatory mechanism of YAP2 by the SIRT1-mediated deacetylation that may be involved in HCC tumorigenesis and drug resistance.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most frequent neoplasm1 and ranks third among the most lethal cancers worldwide.2 HCC has a poor prognosis with only ∼5% of patients surviving more than 5 years,3 reflecting the need for a better understanding of the molecular pathogenesis of HCC.

The Yes-associated protein (YAP) is a downstream effector of the Hippo signaling pathway that controls cell growth and organ size.4 Both YAP and its homolog TAZ act as transcriptional co-activators of TEAD and regulate transcription of target genes involved in cell growth, proliferation and survival.5, 6, 7 Using a conditional YAP1 transgenic mouse model, it was shown that the overexpression of YAP1 in mice leads to HCC development, suggesting a direct link between deregulation of the Hippo size-control pathway and liver tumorigenesis.8, 9 Moreover, recent work highlighted the role of Mst1/2 (mammalian Ste20-like 1/2) kinases as tumor suppressors by showing that combined deficiency of Mst1/2 kinases led to the loss of inactivating phosphorylation of YAP, massive liver overgrowth and development of HCC.10 Consistently, connective tissue growth factor (CTGF), which is the target gene of YAP/TEAD, has been also found over-expressed in HCCs.11

SIRT1, a NAD+-dependent class III deacetylase, is a key modulator of cell proliferation, apoptosis and cell metabolism. Many transcriptional factors have been identified as the targets of SIRT1, including p53, E2F1, FOXO, NF-κB and c-Myc.12, 13, 14, 15, 16 The interaction of SIRT1 with tumor-suppressor proteins and oncoproteins implicates its role in cancer development. However, the biological function of SIRT1 in tumorigenesis continues to be debated, and may depend on cancer types.

In this study, we identified that SIRT1 deacetylates YAP2 protein, and the deacetylation of YAP2 by SIRT1 upregulates the YAP2/TEAD4 association, leading to YAP2/TEAD4 transcriptional activation and cell growth in HCC cells. Knockdown of SIRT1 blocks the CDDP-induced nuclear translocation of YAP2 and enhances the chemosensitivity of HCC cells to CDDP treatment. We also observed that SIRT1 is significantly upregulated in the tumor samples of the hepatocarcinoma patients. Our findings reveal a new regulatory mechanism of YAP2 by the SIRT1-mediated deacetylation that may be involved in liver tumorigenesis.

Results and Discussion

The expression of SIRT1 and YAP2 in human HCC tissues

To better understand the role of SIRT1 in HCCs, we first measured the expression and subcellular localization of SIRT1 in human liver tumor tissues. We found that SIRT1 protein levels in 31 HCC tumor tissues were significantly higher than that of the self-paired adjacent nontumor tissues (Figure 1a). However, YAP2 expression is similar between tumor and nontumor tissues (Figure 1a). Moreover, immunohistochemical staining showed that SIRT1 is mostly located in the nucleus of HCC cells, whereas SIRT1 showed a strong cytoplasmic staining in the self-paired nontumor tissues (Figure 1b). We also examined the mRNA levels of SIRT1 and CTGF in liver tumor tissues. Interestingly, we observed that mRNA levels of CTGF were significantly increased in the group with higher SIRT1 mRNA levels compared with that of lower SIRT1 expression (Figure 1c). Together, these results indicate that both SIRT1 and CTGF expression are involved in liver carcinogenesis and raise the possibility that SIRT1 might regulate YAP2-mediated gene transcription in the hepatocarcinoma development.

Figure 1
figure1

SIRT1 and CTGF are highly expressed in clinical HCC specimens. (a) Left panel shows the representative western blotting of the YAP2 and SIRT1 expression in 31 HCC tumor tissues (T) compared with the self-paired adjacent nontumor tissue (N). HD, healthy donors. Right panel shows the quantitation of SIRT1 and YAP2 protein levels from the 31 self-paired tumors by using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression as the internal control. SIRT1 is highly expressed in the HCC tumors (P<0.001, t-test). (b) SIRT1 immunohistochemical staining is mostly located in the nuclear. Case 1 represents high immunoreactivity of SIRT1 in T tissues whereas low expression in the self-paired N tissues. Case 2 represents both low immunoreactivity of SIRT1 in the T and N tissues. (c) Real-time PCR results of CTGF from 10 liver cancer samples with higher SIRT1 expression and the lower SIRT1 expression, respectively. The quantitation of CTGF mRNA was calculated by using liver samples from five healthy donors as a reference (horizontal lines represent the median; the bottom and top of the boxes represent the 25th and 75th percentiles, respectively; the vertical bars represent the range of data, Mann–Whitney test).

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SIRT1 deacetylates YAP2 in vitro and in vivo

As CTGF is a target of YAP2, and YAP2 has been found to be critical for HCC tumorigenesis, we decided to examine whether SIRT1 alters the protein level of YAP2. However, there is no difference in YAP2 expression between the control and SIRT1-overexpressed or -deficient HepG2 cells (Figure 2a).

Figure 2
figure2

SIRT1 deacetylases YAP2 in vitro and in vivo. (a) HepG2 cells infected with adenovirus expressing SIRT1 or short hairpin RNA (shRNA) for SIRT1 were analyzed for the expression of YAP2. The protein levels of YAP2 are not altered by SIRT1 expression or knockdown. (b) 293T cells were co-transfected with Myc-YAP2 and p300, CBP or PCAF. Cell extracts were immunoprecipitated (IP) with anti-Myc antibody followed by western blotting with pan-anti-acetylation antibody. p300 and CBP are responsible for YAP2 acetylation. (c) Cell lysates from 293T cells that were co-transfected with Myc-YAP2 and SIRT1 were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting (IB) with anti-SIRT1 antibody. (d) 293T cells were co-transfected with Myc-YAP2 and Flag-SIRT1. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed by western blotting. (e) The recombinant GST-YAP2 was acetylated by p300, and incubated with recombinant SIRT1 protein in the presence of NAD+ (the right panel shows the working flowchart). Right panel: reactions were subjected to electrophoresis and immunoblotted with anti-pan-Ac antibody. SIRT1 deacetylates YAP2 in vitro. (f) 293T cells were transfected with the plasmids encoding Myc-YAP2, p300 and wild-type SIRT1 or dominant-negative SIRT1 (H363Y) as indicated. At 24 h after transfection, cells were treated with trichostatin A and nicotinamide for 16 h before harvest. Cell lysates were immunoprecipitated with anti-Myc antibody followed by immunoblotting with anti-pan-Ac antibody. SIRT1 deacetylates YAP2 in cells. (g) 293T cells were transfected with Myc-YAP2 and treated with nicotinamide at different concentrations for 16 h. Cell lysates were immunoprecipitated with anti-Myc antibody and analyzed as in (f). SIRT1 inhibition increases YAP2 acetylation.

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SIRT1 is a class III deacetylase.12 It is possible that YAP2 is a substrate of SIRT1. To test this possibility, we first examined whether YAP2 could be acetylated. 293T cells were cotransfected with Myc-YAP2 and different acetyltransferases. As shown in Figure 2b, we observed the YAP2 acetylation in cells that were co-transfected with p300 and CBP (CREB-binding protein), but not in cells co-transfected with PCAF (p300/CBP-associated factor), suggesting that YAP2 is an acetylated protein and p300/CBP are responsible for the YAP2 acetylation.

Next, we examined the protein interaction of YAP2 and SIRT1. In 293T cells, we found that YAP2 was co-precipitated with SIRT1 and vice versa (Figures 2c and d). We then examined whether SIRT1 could deacetylate YAP2 proteins. By incubating the in vitro p300-acetylated GST-YAP2, we performed the in vitro deacetylation assay and we observed that SIRT1 could deacetylate YAP2 (Figure 2e). Then, we expressed YAP2 and SIRT1 (wild-type or dominant-negative form H363Y) in 293T cells. As shown in Figure 2f, the expression of wild-type SIRT1, but not H363Y SIRT1, reduced the acetylation levels of YAP2 proteins. It has been shown that cell density regulated YAP2 localization via the YAP2 phosphorylation at serine 127.17 Here we also found higher S127 phosphorylation of YAP2 in dense culture condition (Supplementary Figure S1A). However, we observed similar SIRT1-mediated deacetylation of WT-YAP2 and S127A-YAP2, indicating that the deacetylation of YAP2 by SIRT1 is independent of YAP2 phosphorylation at serine 127 (Supplementary Figure S1B). Furthermore, NAM, the inhibitor of SIRT1, significantly reduced the SIRT1-mediated deacetylation of YAP2 (Figure 2g). In conjunction, these results strongly indicate that SIRT1 is a deacetylase for YAP2 proteins.

SIRT1 enhances the YAP2/TEAD4 transcriptional activation and promotes HCC cell growth

Reversible lysine acetylation of target proteins plays an important role in the regulation of protein localization, stability, and protein–protein or protein–DNA interactions.18, 19, 20, 21 As SIRT1 did not alter the protein level of YAP2, we wondered whether SIRT1-mediated deacetylation of YAP2 could affect the association between YAP2 and TEAD proteins. As shown in Figure 3a and Supplementary Figure S2A, the expression of wild-type SIRT1 dramatically increased the interaction of YAP2 with TEAD4. Consistently, knockdown of SIRT1 decreased the endogenous association between YAP2 and TEAD4 proteins in HepG2 cells (Figure 3b).

Figure 3
figure3

SIRT1 promotes the transcriptional activity of YAP2/TEAD4 and YAP2-dependent HCC cell growth. (a) 293T cells were transfected with Myc-YAP2 and HA-TEAD4 together with wild-type SIRT1. Cell lysates were immunoprecipitated (IP) with anti-Myc antibody followed by immunoblotting with anti-HA (TEAD4) antibody. SIRT1 expression increases the interaction of YAP2 and TEAD4. (b) HepG2 cells were infected with adenovirus-directed plasmid encoding SIRT1 short hairpin RNA (shRNA). Lysates were immunoprecipitated with anti-YAP2 and immunoblotted with anti-TEAD4 antibody. SIRT1 knockdown impaired the YAP2/TEAD4 association. (c) 293T cells were transfected with 3*SD reporter plasmids and expression vectors for YAP2 and SIRT1 as indicated. Luciferase activity was normalized to co-transfected pRL-CMV. All the relative luciferase activities are presented as mean±s.d. of triplicate samples and are representative of three independent experiments. SIRT1 expression increases YAP2-mediated reporter activity. (d) HepG2 cells were infected with adenovirus expressing shRNA for YAP2 or SIRT1. At 24 h after infection, HepG2 cells were transfected with 3*SD reporter plasmids and luciferase activity was analyzed as in (c). (e) Total RNA isolated from HepG2 cells were infected with adenovirus expressing shRNA for YAP2 or SIRT1 were analyzed for the mRNA levels of CTGF and p21 by real-time PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The middle panel shows the knockdown efficiency of SIRT1 RNA interference (RNAi). SIRT1 knockdown decreases CTGF activation (left panel) and p21 repression (right panel). (f) HepG2 cells infected with adenovirus expressing shRNA for SIRT1 or retrovirus expressing shRNA for YAP2 or the control vectors. At 72 h after infection, cells were stained with propidium iodide (PI) and analyzed by fluorescence-activated cell sorting (FACS) analysis. (g) HepG2 cells stably expressing shRNA for YAP2 were infected with adenovirus expressing shRNA for SIRT1. Colony formation assay was performed and colonies were visualized with crystal violet staining and counted. The right panel shows knockdown of either YAP2 or SIRT1 reduces HCC colony formation (P<0.01, t-test, n=3). Depletion of YAP2 attenuated the effect of SIRT1 deficiency-induced reduction of colony formation.

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As YAP2/TEAD binding is important for the transcriptional activation of YAP2, we took advantage of the 3*SD-luciferase reporter activity to examine the effect of SIRT1 expression on the YAP2 co-transcriptional activation. Coexpression of SIRT1 increased the transactivity of YAP2 on 3*SD reporter approximately threefold (Figure 3c). Consistently, SIRT1 expression increased the mRNA levels of CTGF (Supplementary Figure S2B). Moreover, both YAP2 and SIRT1 knockdown caused decrease of the 3*SD-luciferase activity in HepG2 cells (Figure 3d), suggesting that SIRT1 positively regulates the transcriptional activity of YAP2.

To further estimate the effect of SIRT1 on YAP2 transcriptional activity, we determined the role of SIRT1 in the regulation of YAP2-mediated gene transcription. As shown in Figure 3e and Supplementary Figure S2C, knockdown of SIRT1 in HepG2 cells resulted in a dramatic decrease in CTGF mRNA levels. We also found that the knockdown of either YAP2 or SIRT1 increased the expression of p21, which controls the G1/S transition (Figure 3e).

We next examined the effect of SIRT1 on YAP2-mediated cell growth. As shown in Figure 3f, knockdown of YAP2 induced slightly but reproducible G1/S arrest and a similar G1/S arrest was observed in SIRT1-deficient HepG2 cells. Moreover, knockdown of YAP2 or SIRT1 also led to the reduced colony formation in HepG2 cells. In addition, we also found that depletion of YAP2 attenuated the effect of SIRT1 deficiency-induced reduction of colony formation in HepG2 cells (Figure 3g), suggesting that YAP2 is a mediator of SIRT1 in promoting hepatocarcinoma cell growth. We also examined the effect of SIRT1/YAP2 in other hepatocarcinoma cells and we found that knockdown of either YAP2 or SIRT1 significantly decreased colony formation in SMMC-7221 cells (Supplementary Figure S3A). In Huh7 cells, we observed that knockdown of SIRT1 significantly decreased the mRNA level of CTGF (Supplementary Figure S3B) and knockdown of SIRT1 decreased the activity of 3*SD luciferase (Supplementary Figure S3C). Moreover, knockdown of either SIRT1 or YAP2 in Bel-7402 cells increased the level of cleaved PARP-1 (poly (ADP-ribose) polymerase-1), which means SIRT1 and YAP2 had antiapoptotic roles in this cell line (Supplementary Figure S3D). Taken together, SIRT1 and YAP2 play important roles in proliferation in hepatocarcinoma cell lines.

SIRT1 promotes CDDP-induced YAP2 nuclear accumulation and inhibits CDDP-induced apoptosis

The findings that SIRT1-mediated YAP2 deacetylation promotes cell growth led us to next address the question of whether SIRT1/YAP2 signaling mediated drug resistance in HCCs. First, we treated the cells with different stimulations as indicated in Figure 4a. Notably, we observed that in our tested reagents, only CDDP dramatically increased the interaction between SIRT1 and YAP2, suggesting that SIRT1 and YAP2 may functionally participate in the response to CDDP of HCC. In another line of experiments, we found that CDDP treatment enhanced the enrichment of YAP2 in the CTGF promoter region (Supplementary Figure S4A) and increased CTGF transcription level (Supplementary Figure S4B). Consistently, the interaction between YAP2 and TEAD4 was also increased on CDDP treatment (Supplementary Figure S4C). Together, these findings suggest that CDDP treatment affects the interaction of SIRT1 and YAP2 as well as the expression of YAP2 target genes.

Figure 4
figure4

SIRT1 promotes the nuclear accumulation of YAP2 and HCC cell death in response to CDDP treatment. (a) 293T cells co-transfected with Myc-YAP2 and Flag-SIRT1 expression plasmids were treated with different stimuli as indicated. Cell lysates were immunoprecipitated (IP) with anti-Flag antibody and analyzed by immunoblotting with anti-Myc (YAP2) antibody. (b) Immunofluorescent staining of YAP2 in HepG2 cells transfected with SIRT1 shRNA or control cells treated with or without CDDP. The upper panel shows the quantification of the YAP2 subcellular localization. The lower panel shows SIRT1 knockdown significantly decreased YAP2 nuclear accumulation (P<0.001, t-test, n=3). (c) HepG2 cells infected with adenovirus expressing YAP2 or SIRT1 were treated with 30 μM CDDP for 16 h. (d) HepG2 cells infected with adenovirus expressing shRNA for YAP2 or SIRT1 were treated with 30 μM CDDP for 16 h. Cells in (c, d) were lysed and analyzed by western blotting with anti-cleaved Caspase3 and PARP-1 antibodies.

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We next found that YAP2 translocated from the cytoplasm to the nucleus in response to CDDP in HepG2 cells, which was consistent with the result that CDDP could enhance the binding of YAP2 and SIRT1. Consistently, the nuclear accumulation of YAP2 was impaired in HepG2 cells transfected with SIRT1 RNA interference (Figure 4b). Moreover, overexpression H363Y/SIRT1, which lacks acetyltransferase activity, failed to increase YAP2 nuclear accumulation on CDDP treatment (Supplementary Figure S5A) or enhance the 3*SD-luciferase activity (Supplementary Figure S5B).

To further define the acetylation sites on YAP2, we made the lysine to arginine mutations: 4KR, K76R/K90R/K97R/K102R (lysines are mutated to arginines) in TBD (TEAD binding domain), and 2KR, K494R/K497R (C-terminus,22). To our surprise, we found similar acetylation of wild type (WT), 4KR and 2KR YAP2, indicating there might be other acetylated lysines on YAP2 (Supplementary Figure S5C). We also found that SIRT1 expression could deacetylate WT, 4KR and 2KR YAP2 (Supplementary Figure S5C). YAP2 4KR, similar to WT or 2KR, still associated with TEAD4. However, the interaction between YAP2 4KR and TEAD4 was not affected by the overexpressed SIRT1 when compared with YAP2 WT or 2KR (Supplementary Figure S5D). Consistently, SIRT1 increased the transcriptional activity of WT and 2KR, but not 4KR YAP2 (Supplementary Figure S5E), indicating the four lysines in the TBD are important for the SIRT1-mediated regulation of YAP2. 4KR confers a higher co-transcriptional activity in comparison with YAP2 WT in the presence of CBP expression (Supplementary Figure S5F). Taken together, these data suggest that SIRT1 enhances the activity of YAP2 through deacetylating YAP2 proteins in response to CDDP treatment and protects cells from CDDP-induced apoptosis.

As expected, both YAP2 and SIRT1 expression conferred resistance of HepG2 cells to CDDP as evidenced by the detection of cleaved PARP-1 and caspase3 (Figure 4c). Knockdown of either YAP2 or SIRT1 increased the cleavage of PARP-1 and caspase3, suggesting that both YAP2 and SIRT1 protect HCC cells from anticancer drug CDDP-induced cell apoptosis (Figure 4d and Supplementary Figure S3D).

In addition to Lats (large tumor suppressor)-mediated phosphorylation and PP1A-mediated dephosphorylation,23, 24 our study found a new modification of YAP2 acetylation and deacetylation. SIRT1 is responsible for the deacetylation of YAP2. Through deacetylation, SIRT1 upregulates the binding between YAP2 and TEAD4 and detains the nuclear localization of YAP2 in response to CDDP, therefore stimulating its activity and promoting the HCC cell growth and survival. It has been reported that SIRT1 interacts with p73 and suppresses p73-dependent transcriptional activity.25, 26 However, in our hands, we found that SIRT1 expression does not alter the interaction between YAP2 and p73α in HepG2 cells with or without CDDP treatment (Supplementary Figures S6A and B), indicating that SIRT1-mediated YAP2 deacetylation might function in parallel to p73 pathway in the development of HCC tumorigenesis and drug resistance, which needs to be further investigated.

Acetylation and deacetylation are counteracting post-translational modifications that affect a large number of histone and nonhistone proteins, especially the transcription factors. Many nonhistone proteins targeted by acetylation are products of oncogenes or tumor-suppressor genes, including p53,27 E2F1,28 c-Myc16 and E1A.29 Our finding adds a new target—YAP2—to this growing list. As a transcriptional cofactor, the TEAD family is required for YAP2 to coordinate gene expression, cell growth and epithelial–mesenchymal transition.30 Recently, Hata et al.22 reported that YAP2 could be acetylated and deacetylated at Lysine 494/497 of C-terminal YAP2, which supports our finding that SIRT1 regulates YAP2 activation through deacetylation. However, we found that the four lysines in the TBD of YAP2 (not the two lysines in the C-terminal YAP2) are critical for SIRT1-mediated regulation (Supplementary Figures S5C–E). In our study, we also reported that YAP2 deacetylation by SIRT1 led to enhanced YAP2/TEAD4 association without affecting its stability. This is in agreement with the finding that SIRT1 promotes the transactivity of the YAP2/TEAD complex. A similar mechanism was suggested for SIRT1 and FXR. It has been demonstrated that FXR deacetylation by SIRT1 increases FXR and RXR heterodimerization.31 Meanwhile, CDDP induces the cytoplasmic YAP2 to relocate into nucleus, subsequently enhancing the YAP2/SIRT1 interaction and, in turn, SIRT1 maintains the nuclear accumulation of YAP2.

Many oncoproteins or tumor suppressors are found to be regulated by SIRT1, which raises the possibility that SIRT1 acts as an oncogene. The findings that overexpression of SIRT1 in APCmin/+ mice reduces colon cancer formation and SIRT1 deficiency causes reduced ability to repair double-strand DNA breaks and impaired genome stability suggest that SIRT1 serves as a tumor suppressor.32 The role of SIRT1 as an oncogene or a tumor repressor may depend on its substrates or the cellular location of specific cancers. The combined results indicate that SIRT1 upregulates the activity of YAP2 in HepG2 cells and that SIRT1 was overexpressed. Nuclear staining in human HCC tumor tissue leads us to speculate that YAP2 mediates the function of SIRT1 in promoting HCC tumorigenesis.

Abbreviations

HCC:

hepatocellular carcinoma

NAD:

nicotinamide adenine dinucleotide

YAP2:

Yes-associated protein 2.

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Acknowledgements

We thank Dr Depei Liu for the gift of all the SIRT1 plasmids; Dr Lei Zhang for the 3*SD binding site artificial-luciferase reporter plasmid; Dr Zhixiong Xiao for the p73 antibody; Ursula Adams for manuscript editing; and Xudong Zhao and Su Liu of IBP core facility center for technical support. This work was supported by the National Science Foundation of China (81172553, 81201564, 81125010 and 81030025), and the Ministry of Science and Technology of China (973-2009CB918704 and 973-2012CB910701).

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Author notes

  1. B Mao and F Hu: These authors contributted equally to this work.

Affiliations

  1. Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
    • B Mao
    • , J Cheng
    • , P Wang
    •  & Z Yuan
  2. Department of Orthopedics, Tianjin Hospital, Tianjin, China
    • F Hu
  3. Department of Orthopedics, Clinical Division of Surgery, Chinese PLA General Hospital, Beijing, China
    • M Xu
    • , F Yuan
    • , Y Wang
    •  & W Bi
  4. Institute of Cancer Stem Cell, Dalian Medical University Cancer Center, Dalian, China
    • S Meng
  5. The Chinese Hippo Consortium, China
    • S Meng
    •  & Z Yuan
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  9. Z Yuan
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  10. W Bi
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Correspondence to Z Yuan or W Bi.

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Supplementary Information accompanies this paper on the Oncogene website

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Mao, B., Hu, F., Cheng, J. et al. SIRT1 regulates YAP2-mediated cell proliferation and chemoresistance in hepatocellular carcinoma. Oncogene 33, 1468–1474 (2014). https://doi.org/10.1038/onc.2013.88

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Keywords

  • SIRT1
  • YAP
  • acetylation
  • transcription
  • hepatocellular carcinoma

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