HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN

Hepatitis B virus X protein (HBx) is involved in the initiation and progression of hepatocellular carcinoma (HCC). However, the mechanism is still needed to be elucidated. In this study, we explored the relationship between HBx and microRNA and their roles in hepato-carcinogenesis. Firstly, by global microarray-based microRNA profiling and qRT-PCR, we found miR-181a was strongly up-regulated in HepG2.2.15 cells (HBV positive) and pHBV1.3-expressing HepG2 cells, and HBx played a major role in it. Secondly, reduced PTEN protein expression in the presence of HBx was aslo mediated by miR-181a, and in the Luciferase reporter system, miR-181a inhibited the PTEN translation by binding the PTEN 3′-untranslated-region (UTR), and PTEN protein was decreased when epigenetic expression of miR-181a and rescued by knocking down miR-181a. Finally, HBx interrupted the balance between apoptosis and proliferation, which contributed to the development of hepatocellular carcinoma, was also related to the interaction of miR-181a and PTEN. Taken together, we presented here a novel cross-talk between miR-181a and PTEN which was raised by HBx, and this shined a new line in HBV-related hepato-carcinogenesis.

Hepatocellular carcinoma (HCC) is the fifth most commonly diagnosed cancer and the third leading cause of cancer-related death around the world 1 . Chronic hepatitis B virus (HBV) infection is the most prominent cause for HCC and high serum viral load of HBV is predictive of HCC development 2,3 . However, the mechanism by which HBV contributes to the development of HCC remains unclear. MicroRNAs (miRNAs) play important roles in many of the major biological processes including cell differentiation, proliferation, apoptosis, metabolism, development, and immunity in eukaryotic cells by regulating their target genes post-transcriptionally 4 . Thus, aberrant miRNA expression contributes to tumorigenesis and cancer progression 5 . MicroRNA-181a (MiR-181a) is a multifunction miRNA that participates in many biological processes such as apoptosis, cell proliferation and cellular invasion 6,7 . MiR-181a is critical in maintaining stemness of epithelial cell adhesion molecule (EpCAM) + AFP + hepatic cancer stem cells (HepCSCs) 8,9 . Moreover, in vivo expression studies show that miR-181a promotes tumor growth of SMMC-7721 cells in nude mice 10 . However, the regulatory mechanism and significance of elevated miR-181a in HBV-related HCC have not been fully understood.
Phosphatase and tensin homolog (PTEN) is one of the most frequently mutated tumor suppressors. PTEN is an upstream negative regulator of the survival phosphoinositide 3-kinase (PI3K)/AKT cascade; activation of the signal pathway of PI3K/AKT is frequently observed in multiple cancers due to loss of PTEN. The low expression of PTEN in HCC is associated with more aggressive biological behavior and poorer patient survival 11 .
In the present study, we sought to gain insight into the regulatory mechanisms of miR-181a and PTEN in HBV-related HCC. Our findings suggest that miR-181a is involved in the suppression of PTEN induced by HBx. We also show that aberrant expression of miR-181a is associated with HBV-related hepato-carcinogenesis through PTEN gene modulation, suggesting a possible novel therapeutic strategy. qRT-PCR analysis of miR-181a in HepG2 cells transfected with HBV1.3 vector and with the corresponding control (pCDNA3.0). (E) qRT-PCR assay for miR-181a in HBs-, HBc-, HBp-, or HBx-expressing cells and the corresponding controls. The data represent the mean ± SD. (F) Dual-luciferase assay for the miR-181a promoter activity of HBV or HBx-expressing cells relative to the corresponding control, versus the blank group (n = 3, *p < 0.05, ***p < 0.001). compared to HepG2 cells. MiR-181a was most prominently expressed in HepG2.2.15 cells (Fig. 1B). We further confirmed the elevated expression of miR-181a in HepG2.2.15 cells using qRT-PCR which demonstrated that the miR-181a level in HepG2.2.15 cells was dramatically higher compared to HepG2 cells (46 fold, p < 0.001, Fig. 1C). The miR-181a level in HepG2 cells transiently transfected with HBV1.3 vector was also higher than that of corresponding control (Fig. 1D).
To gain insight into the biological role of HBx in miRNA expression pattern, we analyzed the expression of miR-181a in human HepG2 cells transfected with HBs, HBc, HBp and HBx vector, respectively 12 . We found that HBx was the only protein encoded by HBV that promoted miR-181a expression (Fig. 1E). To further investigate the regulatory mechanisms of HBx on miR-181a, the activity of miR-181a promoter was detected in the HepG2 cells transfected with HBV1.3, pHBx and pcDNA3.0 vector by using luciferase reporter assay. The results showed that the promoter activity of miR-181a was significantly higher in HepG2-HBV cells or HepG2-HBx cells compared with the control group (1.94 or 3.55 fold, respectively, p < 0.001) (Fig. 1F) which suggested that both HBV and HBx up-regulate the expression of miR-181a through enhancing its promoter activity.
HBx is also pivotal in HBV suppressing PTEN protein expression. Western blot analysis revealed suppressed PTEN expression in HepG2.2.15 compared to HepG2 cells ( Fig. 2A). Moreover, HBx inhibited PTEN expression more remarkably contrasted to HBs-, HBc-, HBp-transfected HepG2 cells or negative control (Fig. 2B). With HBx being suppressed by RNAi in HepG2.2.15 cells, the expression of PTEN protein promoted in a dose dependent manner (Fig. 2C). All these strongly supported that HBx is the vital protein encoded by HBV that down-regulates PTEN expression.

MiR-181a down-regulates PTEN expression via 3′-untranslated-region (UTR) pairing.
To search for the miR-181a target genes, we identified the region complementary to its seed region in the 3′ UTR of human PTEN mRNA using the DIANA microT v3.0 algorithm, which was also confirmed by TargetScan and PicTar. Then, we cloned a sequence of the 3′ UTR of human PTEN mRNA with the predicted target site of miR-181a into downstream of the pGL3-control luciferase reporter gene vector (pGL-PTEN). Contrasted to the negative control (pRNAT), miR-181a inhibited the luciferase activity of the pGL-PTEN in a dose-dependent manner (Fig. 3A). Correspondingly, exogenous expression of miR-181a down-regulated PTEN expression while miR-181a inhibitor promoted PTEN protein level in a dose dependent manner in HepG2 cells (Fig. 3B,C).

MiR-181a inhibitor abolished the inhibitory effect of HBx on PTEN 3′UTR and PTEN protein expression.
Dual-luciferase assay revealed that exogenous expression of HBx led to by up to 44% reduction of pGL-PTEN luciferase activity compared with the control group in HepG2 cells (p < 0.001). However, miR-181a inhibitor reversed the decreased luciferase activity of pGL-PTEN induced by HBx (Fig. 4A). Furthermore, to verify the role of miR-181a in the regulation of PTEN by HBx, we analyzed the PTEN protein level in HepG2 cells co-transfected with pHBx and the specific miR-181a inhibitor (miR181a-IN). Contrasted to the negative control (miR181a-NC), miR-181a inhibitor rescued the suppressed PTEN expression resulted by HBx (Fig. 4B). Similarly, miR-181a inhibitor promoted PTEN expression in a dose-dependent manner in HepG2.2.15 cells (Fig. 4C).

HBx inhibits cell apoptosis through up-regulates miR-181a and in turn down-regulates PTEN.
We then investigated the biological significance of the elevated miR-181a in hepatocyte lines. Cell apoptosis is a key character during tumorigenesis. The miR-181a or its negative control vector (pRNAT) was transiently transfected into HepG2 cells. Expression of miR-181a was verified by qRT-PCR which revealed the transfection of miR-181a restoring its expression in HepG2 cells (data had not been shown). Analysis of apoptosis was performed after culturing for 48 h by using the Annexin V-PE apoptosis detection kit. The results showed that the apoptosis rate of miR-181a-transfected cells was lower than that of negative control and the blank group (4.23% ± 0.29% vs 7.73% ± 1.29% and 7.80% ± 0.70%, respectively, p < 0.001). Similar effects were found in pHBx-transfected HepG2 cells (3.20% ± 0.60%) compared with the corresponding control (7.83% ± 0.12%) (p < 0.001). However, miR-181a inhibitor reversed the decreased apoptosis rate of pHBx-transfected cells (increased from 3.07% ± 0.45% to 7.70% ± 0.72%, p < 0.001). Meanwhile, PTEN promoted the apoptosis rate of HepG2 cells while HBx inhibited the apoptotic effect of PTEN (p < 0.01). As for HBV expressing cells, miR-181a inhibitor also promoted the apoptosis rate of HepG2.2.15 cells (Fig. 5).

HBx promotes cell proliferation activity via up-regulating miR-181a and down-regulating PTEN. The proliferation activity of miR-181a on cells was detected by clone formation test and MTT assay.
The result showed that the clone formation rate of miR-181a-transfected cells was significantly higher than that of negative control (10.08% vs 1.61%). Contrasted to the mutation vector of PTEN (pPTEN-mut), PTEN reversed the proliferation activity of miR-181a on cells more significantly (10.45% vs 1.60%) (Fig. 6A,B,C,D). The clone formation rate of pHBx -transfected cells was higher than that of control group (16.72% vs 10.37%) (Fig. 6E,F). However, miR-181a inhibitor suppressed the clone formation rate of the former (7.52% vs 17.80%) (Fig. 6G,H). These results demonstrate that miR-181a play a vital role in promoting cell proliferation activity induced by HBx. Moreover, compared with pPTEN-mut, PTEN eliminated the effect of HBx on cells proliferation more remarkably (16.68% vs 1.55%) (Fig. 6I,J). MTT assay revealed that the proliferation activity of cells transfected by pHBx or miR-181a was significantly higher than that of corresponding control (p < 0.001). MiR-181a inhibitor abolished the proliferating effect of HBx on HepG2 cells. Besides, miR-181a inhibitor suppressed the proliferation activity of HepG2.2.15 cells in a dose-dependent manner (Fig. 7A,B).

Discussion
HBV is widely accepted to be a main cause of HCC. HBx, which is recruited on cellular chromatin and modulates chromatin dynamics at specific gene loci, plays an important role in the progression of HBV-associated HCC 13 HBx has been shown to induce various signaling pathways and cellular proteins that could link HCC with HBV infection. In recent years, miRNAs have been reported frequently to be involved in tumorigenesis 16 . MiR-181a was up regulated in EpCAM + AFP + hepatic cancer stem cells (HepCSCs) 8,9 . PTEN was down-regulated in HBV infected cells and HCC. PTEN under-expression is associated with larger tumor size, tumor microsatellite formation, and shorter overall survival in patients with HCC 11 . We revealed that miR-181a was the most elevated miRNA in HepG2.2.15 cells compared with HepG2 cells, and PTEN was down-regulated in HBV expressing cells. Then, we identified HBx played a major role in up-regulation of miR181a and suppression of PTEN in HBV expressing cells. Furthermore, we explored the role of miR-181a in the regulation of HBx on PTEN. We found that miR-181a involved in the effect of HBx on PTEN 3′ UTR activity and PTEN protein expression. All this indicate that HBx induces the down-regulation of PTEN, at least in part, via inducing miRNA-181a during HBV infection.  Dualluciferase assay of PTEN-3′ UTR activity in HepG2 cells transfected with miR-181a at different doses (pRNAT-miR-181a vector 1 μ g, 2 μ g, 4 μ g, respectively) and the corresponding controls (pRNAT vector 1 μ g, 2 μ g, 4 μ g, respectively), versus the blank group. (B) Western blotting analysis for PTEN in HepG2 cells transfected with miR-181a at different doses (pRNAT-miR-181a vector 1 μ g, 2 μ g, 4 μ g, respectively) and the corresponding controls (pRNAT vector 1 μ g, 2 μ g, 4 μ g, respectively), versus the blank group. (C) Western blotting analysis for PTEN in HepG2 cells transfected with miR-181a inhibitor (miR-181In10, miR-181In 20) and the corresponding controls (NC10, NC20) at different doses (10 μ l, 20 μ l, respectively), versus the blank group (n = 3, **p < 0.01, ***p < 0.001). To identify the possible mechanism of HBx up-regulating miR-181a, we designed the promoter of miR-181a according to the literature 10 and reconstructed the miR-181a promoter construct pGL3-miR-181a-P. The results revealed that HBx induced miR-181a up-expression by enhancing its promoter activity. The miR-181a promoter designed in this research contains cyclic adenosine monophosphate response element-binding protein element (CREB), a transcription factor binding site which could be bound by HBx 17,18 . It is reasonable to propose that HBx enhance the activity of miR-181a promoter via binding to the CREB site. However, further studies should be taken to identify its probability.
In colon cell line, up-regulation of miR-181a suppressed PTEN expression, activated phosphorylation of AKT (p-AKT), promoted cell viability, and inhibited apoptosis 19 . We identified PTEN as a direct target gene of miR-181a in hepatocyte line. This is the very first report demonstrating the regulatory mechanism of miR-181a and PTEN in hepatic cells and HBV expressing cells. We also found that HBx disturbs the cross-talk between miR-181a and PTEN. MiR-181a is a key modulatory factor for cell proliferation and differentiation at both transcriptional and posttranscriptional levels in gastric cancer, acute myeloid leukemia 20,21 . MiR-181a overexpression resulted in promotion of cell proliferation and migration but inhibition of apoptosis in colorectal cancer 22 . HBV plays an important role in promoting cell growth 23,24 . We revealed that HBx promoted hepatoma cell proliferation, inhibited cell apoptosis by up-regulating miR-181a and consequently suppressing PTEN, which revealed a novel mechanism for the growth-promoting effect of HBV on hepatoma cell. Next, we will explore the effect and mechanism of miR-181a on the activation of PI3K and Akt in hepatocellular cell lines to further support the pro-proliferation and anti-apoptotic activity of miR-181a.
MiR-181 family members were up-regulated in HepCSCs. Moreover, miR-181 family members were highly expressed in embryonic livers and in isolated hepatic stem cells 8 . However, the mechanism of miR-181 regulating EpCAM expression is not quite clear. EpCAM was a Wnt-beta-catenin signaling target gene and activation of the Wnt-beta-catenin pathway promoted EpCAM expression in cultured normal human hepatocytes and HCC cell lines 25 . Moreover, the loss of PTEN induced the activation of the beta-catenin pathway 26 . Our research confirmed the inhibiting effect of miR-181a on PTEN expression. We propose that the promotion of miR-181 driven by HBx in HCCs could influence the beta-catenin pathway and promote EpCAM expression by suppressing PTEN protein expression, which may have revealed a novel regulatory link between miR-181a and human EpCAM(+ ) liver cancer stem/progenitor cells.
Collectively, our results have revealed a new mechanism of HBV-related HCC. We delineate the role of HBx on the expression of miR-181a and its role in inducing the proliferation and antiapoptosis of hepatoma cells. HBx induces miR-181a expression by enhancing its promoter activity, and disturbs the cross-talk between miR-181a and PTEN. Moreover, miR-181a promotes cell proliferation and suppresses apoptosis by targeting PTEN. These findings indicate that miR-181a plays a vital role in the regulation of HCC cell proliferation and functions as an onco-miRNA in HBV-related HCC. Targeting miR-181a may provide an effective therapeutic approach to eradicate HBV-related HCC.
For transfection with plasmid DNA, cells were plated in 6-well plates in antibiotic free growth medium at a density of 1 × 10 6 cells/well. After 24 hours, the transfection was performed using Lipofectamine 2000 according to the manufacturer's instructions. The growth medium was changed after 6 h. Transfected cells were harvested at 48 hours and the total cellular RNA or protein was isolated for the next experiment.
MicroRNA microarray and qRT-PCR. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The microRNA gene microarray (Affymetrix microRNA 2.0 array) was completed by Capital Bio Corporation. Complementary DNA was synthesised from total RNA (both mRNA and microRNA) using the TaKaRa One step PrimeScript ® miRNA cDNA Synthesis Kit (Perfect Real Time) according to the manufacturer's instructions. To quantify the target mRNA or miRNAs, quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the ABI 7500 Real-Time PCR System with Takara