Hepatitis B virus X protein promotes hepatocellular carcinoma transformation through interleukin-6 activation of microRNA-21 expression.

European Journal of Cancer (2014) xxx, xxx– xxx

Available at www.sciencedirect.com

ScienceDirect journal homepage: www.ejcancer.com

Original Research

Hepatitis B virus X protein promotes hepatocellular carcinoma transformation through interleukin-6 activation of microRNA-21 expression Chi Han Li a, Feiyue Xu a, Sheungching Chow a, Lu Feng a, Deling Yin b, Tzi Bun Ng a, Yangchao Chen a,c,⇑ a

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Department of Internal Medicine, College of Medicine, East Tennessee State University, Johnson City, TN 37604, USA c Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518057, China b

Received 6 March 2014; received in revised form 4 July 2014; accepted 7 July 2014

KEYWORDS Hepatocellular carcinoma Hepatitis B virus Hepatitis B X protein MicroRNA-21 Interleukin 6

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and chronic hepatitis B virus (HBV) infection is the major risk factor of HCC. The virus encodes HBV X (HBx) protein that plays a critical role in the development of HCC. Studies have revealed numerous HBx-altered genes and signalling pathways that heavily contribute to tumourigenesis of non-tumour hepatocytes. However, the role of HBx in regulating other critical gene regulators such as microRNAs is poorly understood, which impedes the exploration of a complete HBx-associated carcinogenic network. Besides, critical microRNAs that drive the transformation of non-tumour hepatocytes are yet to be identified. Here, we overexpressed C-terminal truncated HBx protein in a non-tumour hepatocyte cell line MIHA, and measured a panel of cancer-associated miRNAs. We observed that oncogenic miR-21 was upregulated upon ectopic expression of this viral protein variant. HBx-miR-21 pathway was prevalent in HCC cells as inhibition of HBx in Hep3B and PLC/PRF/5 cells significantly suppressed miR21 expression. Subsequently, we showed that the upregulation of miR-21 was mediated by HBx-induced interleukin-6 pathway followed by activation of STAT3 transcriptional factor. The high dependency of miR-21 expression to HBx protein suggested a unique viral oncogenic pathway that could aberrantly affect a network of gene expression. Importantly, miR-21 was essential in the HBx-induced transformation of non-tumour hepatocytes. Inhibition of miR-21

⇑ Corresponding author at: School of Biomedical Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Shatin, Hong Kong. Tel.: +852 39431100; fax: +852 26035123. E-mail address: [email protected] (Y. Chen).

http://dx.doi.org/10.1016/j.ejca.2014.07.008 0959-8049/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Li C.H. et al., Hepatitis B virus X protein promotes hepatocellular carcinoma transformation through interleukin-6 activation of microRNA-21 expression, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.07.008

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C.H. Li et al. / European Journal of Cancer xxx (2014) xxx–xxx

effectively attenuated anchorage-independent colony formation and subcutaneous tumour growth of MIHA cells. Our study suggested that overexpression of miR-21 was critical to promote early carcinogenesis of hepatocytes upon HBV infection. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Chronic hepatitis B virus (HBV) infection is a major risk factor of hepatocellular carcinoma (HCC) [1]. Recent studies showed that HCCs with HBV infection exhibit a higher degree of aggressiveness than noninfected tumours. HBV X protein (HBx) is encoded from the HBV genome [2,3] that is involved in the pathogenic mechanism of HBV-associated HCC [4]. It has multiple molecular functions in human hepatocytes via interacting with various transcription factors and modulating numerous cellular signalling pathways of the host [4–12]. Though studies were rigorously conducted to reveal the role of HBx protein in HCC biology, the gene expression network affected by the viral protein is not fully understood. Recently, studies showed that HBx protein induced differential expression of microRNAs (miRNAs). MiRNAs have sizes ranged from 20 to 23 nucleotides, and they participate in many biological processes including embryonic development, cell differentiation, proliferation and apoptosis [13–15]. Aberrant expression of miRNAs has been implicated in numerous cancer types including HCC. However, HBV-associated miRNAs that drive the transformation of normal hepatocytes and HCC carcinogenesis are poorly studied. Besides, the mechanism for HBx to alter the expression of miRNAs is still largely unexplored. Here, we demonstrated that induction of miR-21 was dependent on HBx-activation of interleukin-6 (IL-6)-STAT3 pathway. Expression of miR-21 was essential in transforming non-tumour hepatocytes to gain the ability to form anchorage independent colonies and in vivo tumour, which implied a critical role during early HCC development. 2. Materials and methods 2.1. Cell culture and drug treatment Human hepatoma cell lines (Hep3B and PLC/PRF/ 5), immortalised non-tumourigenic hepatocyte cells (MIHA [14] and L02) and HEK293T were maintained as previously described [16]. Cells were treated with STAT3 inhibitor, cucurbitacin (Tocris, Bristol, United Kingdom (UK)), at a dose of 0.5 lM [17] for 72 h. Cells were treated with recombinant human IL-6 (Invitrogen) at indicated concentrations.

2.2. Cancer-associated miRNA profiling in HBxexpressing stable cell line MIHA cells were transfected with pcDNA3.1/myc containing COOH-terminal truncated HBx cDNA (HBx-D35). After transfection, the cells were incubated with G418 (Invitrogen) for 2 weeks. Differential expression of cancer-associated miRNA in HBx protein expressing MIHA cells was measured by Cancer MicroRNA qPCR Array with QuantiMire (System Biosciences). Signals were normalised by U6 level. 2.3. Lentivirus packaging and transduction Lentiviruses were packaged according to our protocol [18]. The list of lentiviral vectors used, transduction and cell sorting methods were described in supplementary materials and methods. 2.4. Subcutaneous xenograft tumour models Subcutaneous injection of MIHA cells was conducted as previously described [19]. Details of the assay were described in supplementary materials and methods. 3. Results 3.1. miR-21 was upregulated by ectopic HBx expression in MIHA cells Stable MIHA cells expressing HBx-D35 were established to emulate HBV-induced transformation. Overexpression of HBx-D35 was confirmed by Western blotting (Fig. 1A). Subsequently, we profiled cancer-associated miRNA expression in MIHA cells by Cancer MicroRNA qPCR Array with QuantiMire. Differential expression of miRNAs was observed upon the HBx-D35 overexpression. MiRNAs with fold change larger than 1.5 were considered as biologically significant (Fig. 1B and C). In the subsequent validation, we attempted to identify miRNAs critical in the transformation of nontumour hepatocytes. Among upregulated miRNAs, those with a cancer promoting role such as miR-373 and miR-21 were selected (Fig. 1B). For the downregulated miRNAs, miR-137 and miR-126 were selected for validation as they were shown to have tumour suppressive functions (Fig. 1C). Although other miRNAs from the profiling might have greater alteration in level, their



D

A

B miRNA Fold increase miR-122a 4.69 miR-373 4.34 Let-7 family 4.27 miR-218 2.73 miR-154 2.71 miR-202 2.63 miR-136 2.60 miR-101-1 2.33 miR-181d 2.31 miR-221 2.18 miR-19a+b 2.02 miR-16 1.82 miR-145 1.78 miR-21 1.71 miR-29a+b+c 1.66 miR-7 1.65 miR-149 1.58 miR-22 1.57

3

C miRNA Fold decrease miR-223 6.06 miR-203 4.58 miR-155 4.04 miR-150 3.94 miR-126 3.55 miR-137 2.95 miR-296 2.74 miR-26a 2.20 miR-133a 2.12 miR-95 1.90 miR-196a 1.74 miR-153 1.70 miR-92 1.56 miR-25 1.54 miR-199a+b 1.52

E

F

Fig. 1. Profiling of cancer-associated microRNAs (miRNAs) in MIHA cells with ectopic hepatitis B virus X (HBx) expression. (A) Stable MIHA cells expressing HBx protein were established, and overexpression of HBx protein was detected by Western blotting. Differential expression of cancer-associated miRNAs was measured in MIHA cells upon overexpression of HBx protein, and (B) upregulated miRNAs including miR-21 and miR-373, and (C) downregulated miRNAs including miR-126 and miR-137 were identified. (D, E) Lentiviruses carrying full length HBx (HBx-FL) or C-terminal deleted HBx (HBx-D35) transgene were infected into MIHA cells. Overexpression of the HBx proteins was detected by (D) qRT-PCR and (E) Western blotting analysis. (F) Cell proliferation assay showed that ectopic expression of HBx-FL or HBx-D35 significantly increased the cell proliferation rate of MIHA cells compared to parental and lentivirus control cells. (*,#p < 0.05).

reported cancer-associated roles contradicted to the hypothesis of hepatocarcinogenesis process. We believed that they have an indifferent role during HBx-induced hepatocytes’ transformation. To validate the association between HBx and the miRNAs, we generated lentivirus carrying full length HBx (HBx-FL) or HBx-D35 transgene to transduce MIHA cells. After transduction, EGFP-positive cells were sorted out by flow cytometry to obtain pure lentivirus-infected populations, and the expressions of HBx-FL and HBx-D35 were confirmed by qRT-PCR (quantitative reverse transcription polymerase chain reaction) (Fig. 1D) and Western blotting (Fig. 1E). Two pairs of qRT-PCR primers that amplified either within or outside the HBx protein C-terminal region were employed to verify the expression of HBx-FL and HBx-D35. HBx-FL was detected by both primer pairs whereas HBx-D35 was only detected by the primers flanking outside the C-terminal region (Fig. 1D). We further showed that both HBx-FL and HBx-D35 significantly promoted MIHA cell proliferation (Fig. 1F). We hypothesised that HBx proteins could induce differential miRNA expression which contributed to the altered phenotypes of non-tumour hepatocytes.

Subsequently, we validated the miRNA profiling in both HBx-FL- and HBx-D35-expressing MIHA cells by qRT-PCR. MiR-21 expression was significantly increased upon ectopic HBx expression (Fig. 2A), whereas miR-126, miR-137 and miR-373 had no significant changes (Fig. 2B). In another immortalised hepatocyte L02, ectopic expression of HBx protein also induced the upregulation of miR-21 expression, which showed that HBx specifically induced miR-21 in hepatocytes (Fig 2A). To prove the presence of HBx-miR-21 pathway in HCC cells, we inhibited HBx by siRNAs in Hep3B and PLC/PRF/5 cells which were positive in HBx expression. MiR-21 expressions were significantly inhibited upon knockdown of HBx in both cell lines (Fig. 2C). The levels of miR-126, miR-137 and miR373 were then measured in Hep3B, but knockdown of HBx failed to alter their expressions (Fig. 2D). Besides, overexpression of these miRNA candidates (Sfig. 1A and B) failed to affect the proliferation rate of the MIHA cells (Sfig. 1C). Furthermore, inhibition of miR-21 activity by miRZip-21 in HBx-FL expressing MIHA cells upregulated a putative miR-21 target programmed cell death 4 (PDCD4) (Fig. 2E) [20]. Since miR-21 was the only miRNA specifically induced by


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Fig. 2. Ectopic expression of hepatitis B virus X (HBx) induced miR-21 expression in HCC. (A) Lentiviral overexpression of full length HBx (HBxFL) and HBx-D35 upregulated the level of miR-21 in MIHA and L02 cells, (B) but failed to alter the expression of miR-126, miR-137 and miR-373 in MIHA cells. (C) Knockdown of HBx inhibited the expression of miR-21 in Hep3B and PLC/PRF/5 cells, (D) while the expressions of miR-126, miR-137 and miR-373 were not significantly changed. (E) Inhibition of miR-21 activity in HBx-expressed MIHA cells through second infection of lentivirus expressing miRZip-21 significantly increased the transcript level of PDCD4. (*p < 0.05).

HBx in MIHA cells and has a well-documented cancer promoting role, we decided to investigate its role in the transformation of non-tumour hepatocytes. 3.2. Upregulation of IL-6 by HBx protein induced miR-21 expression In HCC, the underlying principle for miR-21 overexpression is not fully understood. Activation of IL-6 signalling is highly associated with HBx expression [21], which can induce miR-21 overexpression in cancer cells [22]. Here, we studied the association between HBx-IL6 pathway and miR-21 induction during early hepatocytes’ transformation. Overexpression of HBx-FL and HBx-D35 significantly increased IL-6 mRNA level in MIHA cells (Fig. 3A). HBx-D35 induced a remarkably higher IL-6 expression in the cells compared to HBxFL, suggested that such variant activated the IL-6 pathway more robustly. We further treated MIHA cells with recombinant human IL-6, and miR-21 level was increased both time-dependently (Fig. 3B) and dosedependently (Fig. 3C). The treatment also upregulated miR-21 expression in L02 (Fig. 3D), which validated the presence of IL-6-miR-21 pathway in hepatocytes.

IL-6 treatment also increased both pri-miR-21 (Fig. 3E) and pre-miR-21 levels (Fig. 3F) time-dependently in MIHA cells. The concurrent upregulation of primary, precursor and mature miR-21 suggested that IL-6 activated miR-21 gene transcription in non-tumour hepatocytes. Furthermore, knockdown of IL-6 by siRNAs significantly reduced the expression of miR-21 in Hep3B cells (Fig. 3G), which suggested that IL-6 signalling was pivotal in HBx-mediated induction of miR-21 expression in HCC cells. 3.3. Role of STAT3 in HBx-induced miR-21 expression We further delineated the molecular pathway of IL-6-mediated miR-21 upregulation. IL-6 and STAT3 pathways are prevalent in human HCC [23–26]. However, the role of HBx proteins in the regulation of IL-6-STAT3 pathway was not clear. We showed that phosphorylated STAT3 (pSTAT3) was markedly elevated after overexpression of HBx-FL and HBx-D35 (Fig. 4A). Alike the upregulation of IL-6 mRNA expression, HBx-D35 induced a higher level of phosphorylated STAT3 compared to HBx-FL (Fig. 4A). HBx was highly associated with the activation of IL-6-STAT3



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Fig. 3. Hepatitis B virus X (HBx)-mediated IL-6 regulation induced miR-21 expression. (A) The level of IL-6 mRNA was measured by qRT-PCR. Both full length HBx (HBx-FL) and HBx-D35 protein induced the expression of IL-6 mRNA in MIHA cells, while HBx-D35 induced a significantly higher IL-6 mRNA level compared to HBx-FL. Recombinant human IL-6 upregulated the expression of mature miR-21 in a (B) time-dependent (10 ng/ml, 48 and 72 h) and (C) dose-dependent (10 ng/ml and 50 ng/ml) manner in parental MIHA cells. (D) IL-6 treatment also upregulated miR21 level in L02 cells dose-dependently (100 ng/ml and 1000 ng/ml). (E) Precursor miR-21 and (F) primary miR-21 were upregulated with IL-6 treatment time-dependently. (G) Knockdown of IL-6 by siRNAs significantly inhibited the level of endogenous miR-21 in Hep3B cells. (*p < 0.05).

pathway in HCC cells, as we further showed that phosphorylation of STAT3 was effectively inhibited when IL-6 was depleted by siRNAs in Hep3B cells (Fig. 4B). In turn, lentiviral vector containing shSTAT3 was transduced in MIHA cells with or without HBx-FL and HBx-D35 expression that drastically prohibited total STAT3 levels (Fig. 4C). We then measured miR21 level in the STAT3-depleted cells, and upregulation of miR-21 was significantly attenuated in both HBxFL and HBx-D35 overexpressing cells (Fig. 4D). In contrast, depletion of STAT3 in parental and control lentivirus infected cells did not alter miR-21 expression (Fig. 4D). It implied that the regulation of miR-21 by STAT3 in non-malignant hepatocytes was dependent on HBx, which proved that aberrant miR-21 expression was driven by HBx-IL-6-STAT3 signalling.

HBx-D35 (Fig. 5A). IL-6 treatment increased STAT3 phosphorylation in MIHA cells (Fig. 5B), and significantly enriched STAT3 proteins at the promoter of miR-21 gene (Fig. 5C). To further demonstrate the importance of STAT3 activity in HBx-miR-21 pathway, a selective JAK/STAT3 signalling inhibitor, cucurbitacin I, was applied to deactivate STAT3 in HBx-FL and HBx-D35 overexpressed MIHA cells. Inhibition of STAT3 attenuated the induction of miR-21 by HBx (Fig. 5D). In addition, while exposure of recombinant IL-6 significantly increased miR-21 level in parental MIHA cells, it failed to upregulate miR-21 when STAT3 was constitutively inhibited by shRNAs (Fig. 5E). Taken together, STAT3 was the key transcriptional factor activated during HBx-induced transformation which led to the active transcription of miR-21.

3.4. Phosphorylation of STAT3 enhanced miR-21 promoter occupancy

3.5. Role of miR-21 in HBx-induced hepatocarcinogenesis

By chromatin immunoprecipitation, there was a significant increase of STAT3 occupancy in the miR21 promoter upon ectopic expression of HBx-FL or

By soft agar assay, we showed that overexpression of both full length HBx and HBx-D35 was able to induce anchorage-independent colony formation of MIHA cells, whereas neither parental MIHA cells nor the


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B

A

pSTAT3/tSTAT3 ratio

0.03

0.05

0.27

0.40

pSTAT3/tSTAT3 ratio

0.62

0.57

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Fig. 4. Role of STAT3 in hepatitis B virus X (HBx)-induced miR-21 expression. (A) Phosphorylation of STAT3 was promoted in MIHA cells expressing full length HBx (HBx-FL) and HBx-D35. (B) Knockdown of IL-6 by siRNAs inhibited the phosphorylation of STAT3 in Hep3B cells. (C) shRNAs targeting STAT3 were transduced in whole panel of MIHA cell lines, and the protein expression of STAT3 was knocked down. (D) While ectopic expression of HBx induced the expression of mature miR-21, simultaneous inhibition of STAT3 by shRNAs prohibited the expression of miR-21. (*,^p < 0.05).

lentiviral control MIHA-LIG cells could form colony (Fig. 6A, left panel). It showed that while full length HBx and HBx-D35 showed no difference in the induction of cell proliferation (Fig. 1F), MIHA cells expressing HBx-D35 could generate colonies around twice more than those expressing HBx-FL (Fig. 6A, right panel). HBx-D35 possessed greater ability to transform non-tumour hepatocytes compared to its fulllength counterpart. In order to characterise the role of miR-21 in HBxinduced cell proliferation, MIHA cells were co-infected with lentivirus carrying miRZip-21 transgene. Soft agar assay was conducted after infection of the panel of MIHA cells with lenti-CTRL or lenti-miRZip-21. Here, the colony forming capability of HBx-FL and HBx-D35 expressing MIHA cells was inhibited by miRZip-21 (Fig. 6B and C). Moreover, MIHA cells expressing HBx and HBx-D35 proteins were injected subcutaneously into nude mice (n = 5). Cells expressing HBx-FL developed tumours in three out of five mice while those expressing HBx-D35 developed tumours in four out of five mice (Fig. 6D). More importantly, no tumours were

found in mice injected with cells coexpressing HBx-FL and miRZip-21. Inhibition of miR-21 also effectively abrogated the effect of HBx-D35 to induce tumour formation, in which only two out of five mice developed tumours with obviously reduced tumour volume (Fig. 6D). The weights of the tumours developed by MIHA-HBx-FL and MIHA-HBx-D35 were obviously heavier than those developed by MIHA-D35-miRZip21 (Fig. 6E). 4. Discussion We proved that overexpression of miR-21 could happen as soon as the hepatocytes are being infected by HBV. Both HBx subtypes were able to induce an important oncomiR miR-21 upon ectopic expression in non-tumour hepatocytes. MiR-21 controls hepatocytes’ proliferation during liver regeneration as a result of chronic alcohol feeding and liver injury [27,28]. MiR-21 is universally overexpressed in majority of cancer types and phenomenally involves in approximately all tumourigenic processes. MiR-21 is able to induce cell



A

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C

D

B E

Fig. 5. Hepatitis B virus X (HBx) promoted STAT3-mediated transcriptional activation of miR-21. (A) ChIP assay showed that about twofold of miR-21 promoter was occupied by STAT3 in the presence of full length HBx (HBx-FL) or HBx-D35 in MIHA cells. (B) STAT3 was activated by IL-6 (50 ng/ml) in parental MIHA cells whereas total STAT3 expression was not influenced. (C) The IL-6 treatment was able to induce an 8-fold increase of phosphorylated STAT3 occupancy at miR-21 promoter. (D) Upregulation of miR-21 by HBx-FL and HBx-D35 was attenuated when STAT3 was inhibited by STAT3 inhibitor cucurbitacin in MIHA cells. (E) The promotional effect on miR-21 level in MIHA cells by recombinant IL-6 treatment was lost when STAT3 was silenced by shRNAs. (*p < 0.05).

transformation, enhance cancer cell growth and cell cycle, prevent apoptosis, promote metastasis and mediate drug-resistance [29–31]. It is reported that HBx protein induced upregulation of miR-21 which led to the repression of PDCD4 in HCC [32]. However, the role of miR-21 in transformation of normal hepatocytes and the underlying HBx-mediated miR-21 induction pathway were not explored. Here, HBx promoted the phosphorylation of STAT3 via the activation of IL-6 pathway, and transcriptionally upregulated miR-21 expression that led to transformation of non-tumour hepatocytes (Fig. 6F). Among various upstream pathways that regulate miR-21, IL-6-STAT3 signalling pathway is closely related to cellular transformation and oncogenesis in HCC. IL-6 is a cytokine that functions as a growth factor in various tumours. High levels of IL-6 are found in patients with HBV infection and HBV-associated HCC [33]. We showed that HBx-induced miR-21 expression was highly dependent on the activation of IL-6-STAT3 pathway, which demonstrated the specificity of miR-21 induction by HBx. Apart from overexpressing HBx in

non-tumour hepatocytes, silencing IL-6 in HBx protein expressing HCC cell line Hep3B could result in a reduction of miR-21 level, which consolidated the interactions between HBx, IL-6-STAT3 and miR-21. As miR-21 potentially regulates numerous targets which many of them facilitate cancer progression, the high dependency of miR-21 to HBx infection suggested a unique viral oncogenic pathway that aberrantly affects a network of gene expression. Our results from soft agar assay and xenograft assay suggested that HBx-induced miR-21 was pivotal in the transformation of non-tumour hepatocytes. More importantly, as inhibiting miR-21 with miRZIP-21 inhibited tumour xenograft growth, there is a high therapeutic value to target miR-21 in HBV-associated HCC. Previous studies revealed a number of miRNAs associated with HBx proteins, but they have not identified the HBx-induced miRNAs that participated in nontumour hepatocytes’ transformation. For example, expression of miR-143 was transcriptionally activated by NF-jB and was upregulated in HBx-expressing HepG2 cells [34]. MiR-29a was also upregulated in


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A

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C

D

F HBx-FL

HBx-Δ35

HBx-Δ35-miRZip21

E

Fig. 6. Role of miR-21 in hepatitis B virus X (HBx)-induced hepatocarcinogenesis. (A, left panel) Soft agar assay demonstrated that MIHA cells expressing full length HBx (HBx-FL) or HBx-D35 were capable of forming anchorage independent colonies. (A, right panel) MIHA cells expressing HBx-D35 exhibited higher tendency to form anchorage independent colonies compared with HBx-FL. (B) Co-expression of miRZip-21 with HBx proteins effectively abrogated the colony forming ability of HBx-expressed MIHA cells. (C) Inhibition of miR-21 significantly reduced the number of colony formed in both HBx-FL and HBx-D35 expressing MIHA cells. (D) Tumour xenografts were extracted from nude mice injected with MIHA cells with or without the expression of HBx or miRZip-21. There were three and four out of five nude mice, respectively injected with HBxFL and HBx-D35 expressing MIHA cells. Coexpression of miRZip-21 was able to inhibit the subcutaneous tumour formation ability of HBx expressing cells. (E) Inhibition of miR-21 by miRZIP-21 significantly reduced the tumour weight compared to HBx-FL and HBx-D35. (F) Schematic diagram illustrating the HBx-triggered miR-21 hepatocarcinogenesis pathway. (*p < 0.05).



HBx-expressing HepG2 cells and promoted cell migration [35]. Moreover, HBx protein can mediate the downregulation of several miRNAs. HBx repressed miR-132 expression by the induction of promoter DNA methylation [36], and suppressed miR-15a/miR16–1 by induction of c-myc and direct HBx transcript miRNA targeting sequence [37]. The above studies employed HCC cells as the model to study the association between HBx and miRNAs, which could not provide direct evidence on the early miRNAs induced by HBx during non-tumour cells’ transformation. A more recent study illustrated that ectopic expression of HBx in non-tumour hepatocyte cell line L02 suppressed p53-mediated activation of miR-148a. MiR-148a suppressed HCC cell proliferation, migration and invasion in vitro and inhibited HCC cell xenograft tumour growth and metastasis in nude mice [38]. However, it lacks the biological insight of miR-148a functional role in non-tumour cells. In our study, we demonstrated that inhibition of miR-21 effectively prohibited MIHA cells from the formation of both anchorage-independent colony and in vivo subcutaneous tumour, which were suggestive to the abrogation of non-tumour hepatocytes’ transformation. Somatic mutations of genes critical in HCC such as BRAF and PIK3CA have been depicted [39], and it is curious how they are associated with HBV infection. The induction of miR-21 by HBx may synergistically promote HCC development in HCC patients with certain gene mutation. MiR-21 can inhibit its target PTEN, and loss of PTEN is shown to cooperate with BRAF mutation to induce metastatic melanoma [40]. HBxmediated upregulation of miR-21 and BRAF mutation could also be an indication of highly aggressive HCC. The interaction between miR-21 and somatic gene mutation is worthy of further investigation. Taken together, miR-21 was a critical downstream effector of HBx that drove the tumourigenesis of nontumour hepatocytes. Ectopic HBx expression triggered the IL-6 pathway, promoted the phosphorylation and activation of transcriptional factor STAT3 and induced the expression of miR-21. Our in vitro and in vivo studies supported that miR-21 exhibited phenotypical change of the non-tumour cells during HBx-mediated hepatocarcinogenesis. Inhibiting miR-21 or attenuating its activation pathways (i.e. IL6-STAT3) could be promising when developing new treatment on HBV-associated HCC. Given the important association between HBx and miR-21 in early HCC development, targeting miR-21 may also be a novel preventive measure against the development of HCC in HBV carriers. Conflict of interest statement None declared.

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Acknowledgement This study was supported by the Health and Medical Research Fund, Food and Health Bureau, Hong Kong SAR Government (#11100452). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.ejca.2014.07.008. References [1] El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2557–76. [2] Kremsdorf D, Soussan P, Paterlini-Brechot P, Brechot C. Hepatitis B virus-related hepatocellular carcinoma: paradigms for viral-related human carcinogenesis. Oncogene 2006;25:3823–33. [3] Murakami S. Hepatitis B virus x protein: a multifunctional viral regulator. J Gastroenterol 2001;36:651–60. [4] Marra M, Sordelli IM, Lombardi A, Lamberti M, Tarantino L, Giudice A, et al. Molecular targets and oxidative stress biomarkers in hepatocellular carcinoma: an overview. J Transl Med 2011;9:171. [5] Caselmann WH et al.. Transactivators of HBV, signal transduction and tumorigenesis. In: Caselmann WH, Koshy R, editors. Hepatitis B virus: molecular mechanisms in disease and novel strategies for therapy, vol. 1. London: Imperial College Press; 1998. p. 161–81. [6] Feitelson MA, Lee J. Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis. Cancer Lett 2007;252:157–70. [7] Su F, Schneider RJ. Hepatitis B virus HBx protein activates transcription factor NF-jB by acting on multiple cytoplasmic inhibitors of rel-related proteins. J Virol 1996;70:4558–66. [8] Pan J, Lian Z, Allett S, Feitelson MA. The hepatitis B x antigen effector, URG7, blocks tumour necrosis factor a-mediated apoptosis by activation of phosphoinositol 3-kinase and bcatenin. J Gen Virol 2007;88:3275–85. [9] Yang B, Bouchard MJ. The hepatitis B virus X protein elevates cytosolic calcium signals by modulating mitochondrial calcium uptake. J Virol 2012;86:313–27. [10] Lee DK, Park SH, Yi Y, Choi SG, Lee C, Parks WT, et al. The hepatitis B virus encoded oncoprotein pX amplifies TGF-b family signaling through direct interaction with Smad4: potential mechanism of hepatitis B virus-induced liver fibrosis. Genes Dev 2001;15:455–66. [11] Yamashita T, Budhu A, Forgues M, Wang XW. Activation of hepatic stem cell marker EpCAM by Wnt-b-catenin signaling in hepatocellular carcinoma. Cancer Res 2007;67:10831–9. [12] Tu H, Bonura C, Giannini C, Mouly H, Soussan P, Kew M, et al. Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues. Cancer Res 2001;61:7803–10. [13] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97. [14] Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, Johnstone S, et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 2008;134:521–33. [15] Shivdasani RA. MicroRNAs: regulators of gene expression and cell differentiation. Blood 2006;108:3646–53.


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[16] Chen Y, Lin MC, Wang H, Chan CY, Jiang L, Ngai SM, et al. Proteomic analysis of EZH2 downstream target proteins in hepatocellular carcinoma. Proteomics 2007;7:3097–104. [17] Blaskovich MA, Sun J, Cantor A, Turkson J, Jove R, Sebti SM. Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/ signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice. Cancer Res 2003;63:1270–9. [18] Chen Y, Lin MC, Yao H, Wang H, Zhang AQ, Yu J, et al. Lentivirus-mediated RNA interference targeting enhancer of zeste homolog 2 inhibits hepatocellular carcinoma growth through down-regulation of stathmin. Hepatology 2007;46:200–8. [19] Li CH, To KF, Tong JH, Xiao Z, Xia T, Lai PB, et al. Enhancer of zeste homolog 2 silences miR-218 in human pancreatic ductal adenocarcinoma cells by inducing formation of heterochromatin. Gastroenterology 2013;144:1086–97. [20] Zhu Q, Wang Z, Hu Y, Li J, Li X, Zhou L, et al. MiR-21 promotes migration and invasion by the miR-21-PDCD4-AP-1 feedback loop in human hepatocellular carcinoma. Oncol Rep 2012;27:1660–8. [21] Xiang WQ, Feng WF, Ke W, Sun Z, Chen Z, Liu W. Hepatitis B virus X protein stimulates IL-6 expression in hepatocytes via a MyD88-dependent pathway. J Hepatol 2011;54:26–33. [22] Lo¨ffler D, Brocke-Heidrich K, Pfeifer G, Stocsits C, Hackermu¨ller J, Kretzschmar AK, et al. Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer. Blood 2007;110:1330–3. [23] Liu Y, Li PK, Li C, Lin J. Inhibition of STAT3 signaling blocks the anti-apoptotic activity of IL-6 in human liver cancer cells. J Biol Chem 2010;285:27429–39. [24] Choudhari SR, Khan MA, Harris G, Picker D, Jacob GS, Block T, et al. Deactivation of Akt and STAT3 signaling promotes apoptosis, inhibits proliferation, and enhances the sensitivity of hepatocellular carcinoma cells to an anticancer agent, Atiprimod. Mol Cancer Ther 2007;6:112–21. [25] Liu Y, Fuchs J, Li C, Lin J. IL-6, a risk factor for hepatocellular carcinoma: FLLL32 inhibits IL-6-induced STAT3 phosphorylation in human hepatocellular cancer cells. Cell Cycle 2010;9:3423–7. [26] Yang X, Liang L, Zhang XF, Jia HL, Qin Y, Zhu XC, et al. MicroRNA-26a suppresses tumor growth and metastasis of human hepatocellular carcinoma by targeting interleukin-6-Stat3 pathway. Hepatology 2013;58:158–70. [27] Song G, Sharma AD, Roll GR, Ng R, Lee AY, Blelloch RH, et al. MicroRNAs control hepatocyte proliferation during liver regeneration. Hepatology 2010;51:1735–43. [28] Ng R, Song G, Roll GR, Frandsen NM, Willenbring H. A microRNA-21 surge facilitates rapid cyclin D1 translation and

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

cell cycle progression in mouse liver regeneration. J Clin Invest 2012;122:1097–108. Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007;2007(133):647–58. McDaniel K, Herrera L, Zhou T, Francis H, Han Y, Levine P, et al. The functional role of microRNAs in alcoholic liver injury. J Cell Mol Med 2014;18:197–207. Tomimaru Y, Eguchi H, Nagano H, Wada H, Tomokuni A, Kobayashi S, et al. MicroRNA-21 induces resistance to the antitumour effect of interferon-a/5-fluorouracil in hepatocellular carcinoma cells. Br J Cancer 2000;103:1617–26. Qiu X, Dong S, Qiao F, Lu S, Song Y, Lao Y, et al. HBxmediated miR-21 upregulation represses tumor-suppressor function of PDCD4 in hepatocellular carcinoma. Oncogene 2013;32:3296–305. Coskun U, Bukan N, Sancak B, Gu¨nel N, Ozenirler S, Unal A. Serum hepatocyte growth factor and interleukin-6 levels can distinguish patients with primary or metastatic liver tumors from those with benign liver lesions. Neoplasma 2004;51:209–13. Zhang X, Liu S, Hu T, Liu S, He Y, Sun S. Up-regulated microRNA-143 transcribed by nuclear factor kappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression. Hepatology 2009;50:490–9. Wang Y, Lu Y, Toh ST, Sung WK, Tan P, Chow P, et al. Lethal7 is down-regulated by the hepatitis B virus x protein and targets signal transducer and activator of transcription 3. J Hepatol 2010;53:57–66. Wei X, Tan C, Tang C, Ren G, Xiang T, Qiu Z, et al. Epigenetic repression of miR-132 expression by the hepatitis B virus x protein in hepatitis B virus-related hepatocellular carcinoma. Cell Signal 2013;25:1037–43. Wang Y, Jiang L, Ji X, Yang B, Zhang Y, Fu XD. Hepatitis B viral RNA directly mediates down-regulation of the tumor suppressor microRNA miR-15a/miR-16-1 in hepatocytes. J Biol Chem 2013;288:18484–93. Xu X, Fan Z, Kang L, Han J, Jiang C, Zheng X, et al. Hepatitis B virus X protein represses miRNA-148a to enhance tumorigenesis. J Clin Invest 2013;123:630–45. Colombino M, Sperlongano P, Izzo F, Tatangelo F, Botti G, Lombardi A, et al. BRAF and PIK3CA genes are somatically mutated in hepatocellular carcinoma among patients from South Italy. Cell Death Dis 2012;3:e259. Dankort D, Curley DP, Cartlidge RA, Nelson B, Karnezis AN, Damsky Jr WE, et al. Braf(V600E) cooperates with Pten loss to induce metastatic melanoma. Nat Genet 2009;41:544–52.


ATAD5 Expression through E2F1 in Hepatocellular Carcinoma.

Hepatitis B virus X protein promotes hepatocellular carcinoma invasion and metastasis via upregulating thioredoxin interacting protein.

Expression of B7-H4 and hepatitis B virus X in hepatitis B virus-related hepatocellular carcinoma.

Hepatitis B virus X protein promotes CREB-mediated activation of miR-3188 and Notch signaling in hepatocellular carcinoma.

Hepatitis B virus X upregulates HuR protein level to stabilize HER2 expression in hepatocellular carcinoma cells.

Hepatitis B virus X protein mediates yes-associated protein 1 upregulation in hepatocellular carcinoma.

Hepatitis B X-gene expression in hepatocellular carcinoma.

Hepatitis B virus X protein promotes the stem-like properties of OV6+ cancer cells in hepatocellular carcinoma.

The impact of hepatitis B virus x protein and microRNAs in hepatocellular carcinoma: a comprehensive analysis.

Hepatitis B virus X protein disrupts the balance of the expression of circadian rhythm genes in hepatocellular carcinoma.

Hepatitis B virus and hepatocellular carcinoma.

Hepatitis B virus X protein promotes P3 transcript expression of the insulin-like growth factor 2 gene via inducing hypomethylation of P3 promoter in hepatocellular carcinoma.

Hepatocellular carcinoma and hepatitis B surface protein.

Hepatitis B virus whole-X and X protein play distinct roles in HBV-related hepatocellular carcinoma progression.

Hepatitis B Virus X Protein and Hepatocarcinogenesis.

Management of hepatitis B virus infection during treatment for hepatitis B virus-related hepatocellular carcinoma.

Inhibition of apoptosis by oncogenic hepatitis B virus X protein: Implications for the treatment of hepatocellular carcinoma.

Epigenetic modulation of insulin-like growth factor-II overexpression by hepatitis B virus X protein in hepatocellular carcinoma.

Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT.

Hepatitis B virus X protein-mediated non-coding RNA aberrations in the development of human hepatocellular carcinoma.

Hepatitis B virus X protein-induced upregulation of CAT-1 stimulates proliferation and inhibits apoptosis in hepatocellular carcinoma cells.

miR-106b promotes cancer progression in hepatitis B virus-associated hepatocellular carcinoma.

Downregulated long non-coding RNA DREH promotes cell proliferation in hepatitis B virus-associated hepatocellular carcinoma.

C-terminal-truncated hepatitis B virus X protein enhances the development of diethylnitrosamine-induced hepatocellular carcinogenesis.