Tumor samples and matched tumor-adjacent tissues were obtained from 116 patients undergoing curative resection of their primary HCC in the Department of Hepatobiliary Surgery at the First Affiliated Hospital of Xi'an Jiaotong University during January 2006 to December 2009. Clinical sample was used after obtaining informed consent from each patient. None of the patients had received any perioperative chemo- or radiotherapy. The demographic and clinicopathological data of all enrolled patients were obtained through review of hospital records, and are presented in Table I. The protocols of this study were approved by the Xi'an Jiaotong University Ethics Committee according to the Declaration of Helsinki.

The human immortalized normal hepatic cell line LO2 and HCC cell lines (HepG2, Hep3B, Huh7, SMMC-7721 and Bel-7402) were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C with 5% CO₂. Akt inhibitor MK-2206 (1 μM, Selleck Chemicals, Houston, TX, USA) was used to treat HCC cells following the manufacturer's instructions.

Total RNA was extracted from clinical specimens or HCC cells using TRIzol reagent (Invitrogen) following manufacturer's instruction. PCR amplification was performed using a TaqMan human miRNA assay kit (Applied Biosystems, Foster City, CA, USA) and a SYBR^® Premix Ex Taq™ II (Perfect Real-Time) kit (Takara Bio Inc., Shiga, Japan) with an ABI PRISM 7300 Sequence Detection System (Applied Biosystems). qPCR primer against mature miRNA hsa-miR-519a-3p (HmiRQP0588), Homo sapiens snRNA U6 qPCR Primer (HmiRQP9001), PTEN (HQP015535) and GAPDH (HQP006940) were purchased from Genecopoeia (Guangzhou, China).

Total protein was extracted from HCC cells and 40 μg of isolated protein was separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were probed with the following primary antibodies: Akt (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), p-Akt (1:1000, Cell Signaling Technology, Inc.), PTEN (1:1000, Cell Signaling Technology, Inc.), Cyclin D (1:1000, Cell Signaling Technology, Inc.), and p27 (1:1000, Cell Signaling Technology, Inc.) overnight. Then the membranes were incubated with the HRP-conjugated goat anti-mouse or anti-rabbit IgG antibody (ZSGB-BIO, China). Protein bands were visualized using an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK).

Immunohistochemistry was performed on paraformaldehyde-fixed paraffin sections. PTEN (1:100, Cell Signaling Technology, Inc.) antibody was used in immunohistochemistry by a streptavidin peroxidase-conjugated (SP-IHC) method. Detailed procedure of immunohistochemistry was performed as previously reported (18).

miRNA vectors, including miR-519a expression vector (HmiR0342-MR04), the control vector for miR-519a (CmiR0001-MR04, miR-control), miR-519a inhibitor (HmiR-AN0588-AM04, anti-miR-519a) and the negative control for the miR-519a inhibitor (CmiR-AN0001-AM04, anti-miR-NC), and PTEN expression plasmid (EX-I0450-M02-5) were purchased from Genecopoeia (Guangzhou, China). The PTEN siRNA duplex sequence, 5′-GUU AGC AGA AAC AAA AGG AGA UAU CAA-3′ (sense)/5′-UUG AUA UCU CCU UUU GUU UCU GCU AAC-3′ (antisense) and a nonspecific duplex oligonucleotide as a negative control were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Cells were transfected with oligonucleotides using Lipofectamine 2000 Reagent (Invitrogen Life Technologies) following the manufacturer's instructions.

Cells were seeded in triplicate in 24-well plate and allowed to settle for ~12 h. pGL3-PTEN was co-transfected into HCC cells with TK-Renilla plasmid as control signals using Lipofectamine 2000. Luciferase and control signals were measured at 48 h after transfection using the Dual Luciferase Reporter Assay kit (Promega, Madison, WI, USA), according to a protocol provided by the manufacturer. Three independent experiments were performed and the data are presented as the mean ± SD.

At 48 h after transfection, 5×10³ cells were seeded into 96-well plates and stained with 0.5 mg/ml sterile MTT (Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37°C. Then the culture medium was discarded and an extra 150 μl DMSO (Sigma-Aldrich) were added. The absorbance at 490 nm was measured at 24, 48 and 72 h after transfection. The colony formation assay was performed as previously described. Briefly, 500 cells per well were seeded on six-well plates. After 2 weeks, the colonies were stained with 1% crystal violet and the number of colonies was counted.

At 48 h after transfection, HCC cells were collected for cell cycle analysis. After being washed with PBS three times, the cells were fixed with 80% ethanol overnight at −20°C, and were subsequently treated with RNaseA (Sigma) for 30 min at 37°C, followed by incubation in 20 μg/ml of propidium iodide (Sigma) for 20 min at room temperature. After incubation, the cells were subjected to flow cytometry analysis using a FACS Calibur (BD Biosciences, Bedford, MA, USA). For proliferation, 5-bromo-deoxyuridine (BrdU) labeling and immunofluorescence was used. Cells grown on cover slips (Fisher Scientific, Pittsburgh, PA, USA) were incubated with BrdU for 1 h and stained with anti-BrdU antibody (Sigma) according to the manufacturer's instruction. Gray level images were acquired under a laser scanning microscope (Axioskop 2 plus, Carl Zeiss Co. Ltd., Jena, Germany).

Data are presented as the mean ± SD from at least three independent replicates. SPSS software, 16.0 (SPSS, Inc, Chicago, IL, USA) was used to conduct the analysis, and a two-tailed Student t-test was employed to analyze the differences between two groups. Pearson's correlation analysis was used to analyze the correlation between two indices. Survival curves were plotted by the Kaplan-Meier method and compared using the log-rank test. Differences were considered statistically significant at P<0.05.

We investigated the expression level of miR-519a in 116 pairs of HCC tissues and matched tumor-adjacent tissues using qRT-PCR. The results showed that the mean level of miR-519a expression in HCC tissues was significantly higher than that in the non-tumor tissues (P<0.05, Fig. 1A). Further results confirmed that miR-519a was upregulated in a panel of HCC cell lines (HepG2, Huh7, Hep3B, SMMC-7721 and Bel-7402) compared with that in non-transformed LO2 hepatic cell line (P<0.05, Fig. 1B). The results suggest that elevated expression of miR-519a may contribute to the development of HCC.

We determined the mean level of miR-519a as a cut-off value to investigate the correlation between miR-519a level and the clinical features and prognosis of HCC patients. As shown in Table I, the high expression of miR-519a was prominently associated with large tumor size (P=0.005), high Edmondson-Steiner grading (P=0.018), advanced tumor-node-metastasis (TNM) tumor stage (P=0.013) and venous infiltration (P=0.005). Furthermore, Kaplan-Meier analysis showed that high miR-519a expression was closely associated with shorter overall survival (P=0.0002, Fig. 2A) and disease-free survival (P=0.0005, Fig. 2B), which highlights the potential value of miR-519a as a predictive biomarker for the outcome of HCC.

To investigate the biological function of miR-519a in the development and progression of HCC, we transfected HCC cell line Hep3B with miR-519a mimic (P<0.01, Fig. 3A). Cell viability was measured using MTT assays and we observed that ectopic expression of miR-519a had more viability over time compared with NC group (P<0.05, Fig. 3B). Consistently, the upregulated expression of miR-519a markedly enhanced the colony formation as suggested by the increase in colony number (P<0.05, Fig. 3C). Furthermore, the level of DNA synthesis, examined with BrdU incorporation assay, was significantly elevated in miR-519a transduced Hep3B cells, whereas the vector control cells displayed relatively lower BrdU incorporation rates (P<0.05, Fig. 3D). Cell cycle analysis revealed that overexpression of miR-519a decreased the number of Hep3B cells in G0/G1 phase while increased the proportion of Hep3B cells in S-phase (P<0.05, Fig. 3E). Collectively, these results demonstrate that miR-519a functions to enhance proliferation, tumorigenicity and cell cycle progression of HCC cells.

Loss-of-function studies were further performed to confirm the biological function by anti-miR-519a vector. The expression of miR-519a was significantly decreased in SMMC-7721 cells (P<0.01, Fig. 4A) following transfection of miR-519a inhibitors. As shown in Fig. 4B, downregulation of miR-519a led to a significant reduction of cell viability (P<0.05). Furthermore, suppression of miR-519a significantly inhibited the colony formation ability (P<0.05, Fig. 4C). Moreover, BrdU incorporation assay confirmed that downregulation of miR-519a significantly inhibited the proliferation in SMMC-7721 cells (P<0.05, Fig. 4D). In addition, flow cytometry showed inhibition of miR-519a in SMMC-7721 cells significantly increased the percentage of cells in G1/G0 phase and decreased the percentage of cells in S phase (P<0.05, Fig. 4E). These results demonstrated that miR-519a regulates the proliferation, tumorigenicity and cell cycle of HCC cells.

To further explore the underlying mechanisms by which miR-519a exerted its functional effects on HCC, we used TargetScan to search for potential downstream targets of miR-519a. As shown in Fig. 5A, we found that 3′-UTR of PTEN contained the highly conserved putative miR-519a binding sites. qRT-PCR and western blot analysis showed that ectopic expression of miR-519a markedly decreased, whereas inhibition of miR-519a increased the mRNA (P<0.05, Fig. 5B) and protein (P<0.05, Fig. 5C) expression of PTEN. Moreover, when co-transfected with PTEN-3′ UTR luciferase reporter plasmid, as shown in Fig. 5D, miR-519a mimics led to significant reduction of luciferase activity of PTEN (P<0.01), while transfection of miR-519a inhibitors resulted in significant increase of luciferase activity of PTEN (P<0.01, Fig. 5D). In addition, the luciferase activity was unaffected by the miR-519a-mut (Fig. 5D). To further validate that PTEN was a direct downstream target of miR-519a, we examined the correlation between miR-519a level and PTEN expression in HCC tissues. We found PTEN mRNA level in the miR-519a high-expressing tumors was significantly lower than those in the miR-519a low-expressing tumors (P<0.05, Fig. 5E). Moreover, miR-519a level was inversely correlated with the level of PTEN mRNA in HCC tissues (r=−0.614, P<0.0001, Fig. 5F). Consistently, as indicated by the IHC results, the protein expression of PTEN was obviously decreased in patients with high level of miR-519a (P<0.05, Fig. 6). Collectively, these results strongly suggest that PTEN is a downstream target of miR-519a in HCC.

To evaluate whether PTEN could mediate the biological function of miR-519a in HCC, we inhibited PTEN expression with siRNAs in miR-519a-suppressive SMMC-7721 cells (P<0.05, Fig. 7A). We found that cell viability (P<0.05, Fig. 7B), colony formation (P<0.05, Fig. 7C) and proliferation (P<0.05, Fig. 7D) was significantly increased after PTEN downregulation. Furthermore, PTEN knockdown markedly reversed the prevention of cell cycle progression induced by miR-519a suppression (P<0.05, Fig. 7E). Accordingly, PTEN overexpression inhibited cell viability, colony formation, proliferation and promoted G0/G1 phase in miR-519a-overexpressing Hep3B cells (P<0.05, Fig. 7A–E, respectively). These data showed that PTEN is a functional downstream mediator of miR-519a in HCC.

Previous studies have demonstrated that PTEN was a negative regulator of PI3K/Akt signaling (19), so we further investigated whether dysregulation miR-519a altered the activity of PI3K/Akt signaling in HCC cells. As shown in Fig. 8A and B, overexpressing miR-519a significantly increased, but silencing miR-519a decreased, the Akt activity and the expression of phosphorylated Akt (Ser473) in HCC cells (P<0.05). Consistently, the levels of Cyclin D1 and p27, two downstream effectors of PI3K/Akt signaling, were also significantly altered in the miR-519a-deregulated HCC cells (P<0.05). These results indicate that miR-519a activates PI3K/Akt signaling. Furthermore, we examined whether activation of PI3K/Akt signaling contributed to miR-519a-mediated HCC cell proliferation and cell cycle progression. In Hep3B cells overexpressing miR-519a, inactivation of PI3K/Akt signaling by Akt inhibitor significantly decreased the cell viability (P<0.05, Fig. 8C), colony formation (P<0.05, Fig. 8D), proliferation (P<0.05, Fig. 8E) and promoted the percentage of G0/G1 phase (P<0.05, Fig. 8F). Moreover, the Cyclin D1 expression was significantly decreased, but p27 was increased after treating Hep3B cells overexpressing miR-519a with Akt inhibitor (P<0.05, Fig. 8G). Taken together, our results demonstrate that PI3K/Akt signaling plays an essential function during miR-519a-induced HCC cell proliferation and cell cycle progression.