HCC tissues and adjacent non-tumor tissues were obtained from patients diagnosed with HCC at the Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) from January 2007 to December 2009. The HCC patients did not receive any adjuvant therapy before surgery, such as chemotherapy or radiotherapy. The fresh tissues were stored in liquid nitrogen. All of the patients provided written informed consent. Xi'an Jiaotong University Ethics Committee approved the research on the basis of the Declaration of Helsinki.

The normal hepatic cell line (LO2) and five human HCC cell lines (Huh7, MHCC-97L, MHCC-97H, SMMC-7721 sand Hep3B) were obtained from the Chinese Academy of Sciences (Shanghai, China). Complete Dulbecco's modified Eagle's medium (DMEM) (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.) and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was applied to culture the cells. Cells were then placed in a humidified atmosphere (37°C, 5% CO₂).

Lipofectamine 2000 reagent (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.) was applied to conduct cell transfection on the basis of the product specification at a final concentration of 100 pmol according to previous studies (9,10). The mimic control (miR-control; CmiR0001-MR04), miR-577 mimics (miR-577, HmiR0074-MR04), inhibitor control (anti-miR-NC; CmiR-AN0001-AM02) and miR-577 inhibitors (anti-miR-577; HmiR-AN0678-AM02) were purchased from Genecopoeia (Guangzhou, China). HOXA1 clones and HOXA1 siRNAs were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). The cells were transfected with the siRNAs and clones above using Lipofectamine 2000 according to the manufacturer's instructions. After 48 h of transfection, cells were used for the following experiments.

TRIzol (Thermo Fisher Scientific, Inc.) was employed to extract total RNA from the tissues and cells according to the product manual. Then, TIANScript RT Kit (Tiangen Biotech, Beijing, China) was used to synthesize cDNAs. TaqMan Human MiRNA Assay Kit (Genecopoeia, Guangzhou, China) and the SYBR Premix Ex Taq™ Kit (Takara Bio Inc.,, Shiga, Japan) were used to conduct PCR amplifications for miR-577 and HOXA1 mRNA. The detection was performed in the ABI 7300 system (Applied Biosystems, Foster City, CA, USA). snRNA U6 qPCR Primer (HmiRQP9001), hsa-miR-577 primer (HmiRQP0678), GAPDH (HQP006940) and HOXA1 primer (HQP008966) were purchased from Genecopoeia (Guangzhou, China).

Proteins were isolated with RIPA buffer and then separated on 10% SDS-PAGE gels. After proteins were transferred to PVDF membranes, the membranes were blocked using 5% non-fat milk/TBST (Tris-buffered saline Tween-20). Subsequently, the primary antibodies rabbit anti-HOXA1 (1:1,000; cat. no. ab37563; Abcam, Cambridge, UK), mouse anti-E-cadherin (cat. no. 14472; Cell Signaling Technology, Inc. Danvers, MA, USA), rabbit anti-N-cadherin (cat. no. 13116; Cell Signaling Technology, Inc.), rabbit anti-vimentin (cat. no. 5741; Cell Signaling Technology, Inc.) were used to incubate the membranes at 4°C overnight. Secondary antibodies (anti-rabbit cat. no. 7074 and anti-mouse cat. no. 7076; Cell Signaling Technology, Inc.) were employed and the ECL reagent (Beyotime Institute of Biotechnology, Haimen, China) was applied for detection.

The bioinformation public database TargetScan and miRanda was used. Cells were seeded in triplicate in a 24-well plate and pGL3-HOXA1 was co-transfected into HCC cells with the TK-Renilla plasmid as control signals using Lipofectamine 2000. Moreover, vectors with the wild-type HOXA1 3′-UTR or mutant HOXA1 3′-UTR constructed by Sangon Biotech (Shanghai, China) and relevant mir-577 or anti-miR-577 vectors were co-transfected into HCC cells. After 48 h, the luciferase activity was measured by a Dual-Luciferase Reporter Assay system (E1910; Promega, Madison, WI, USA). Three independent experiments were performed and the data are presented as the mean ± SD.

Cell viability was detected by the 3-(4,5-dimethyl thiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) assay. Detailed protocol of the experiment was described in previous studies (18,19).

Migratory and invasion abilities of the cells were detected with Matrigel-uncoated and -coated Transwell inserts (8-µm; EMD Millipore, Billerica, MA, USA). The detailed experiment was performed similar to previous studies (20,21).

Male BALB/c nude mice (4–6 weeks of age) (Centre of Laboratory Animals, The Medical College of Xi'an Jiaotong University, Xi'an, China) were randomized into two groups (n=5). We subsequently injected the stably overexpressing miR-577 cells, MHCC-97H-miR-577, and MHCC-97H-miR-control cells (1×10⁶) into the tail veins for the establishment of a pulmonary metastatic model. Mice were sacrificed by cervical dislocation under anesthesia with ether 3 weeks post injection and examined microscopically (Axioskop 2 plus; Carl Zeiss Co., Ltd., Jena, Germany) by hematoxylin and eosin (H&E) staining for the development of lung metastatic foci. Animals were housed in cages maintained in the pathogen-free (SPF) conditions. All in vivo protocols were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.

SPSS 20.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) were employed in this study. All data are denoted as the mean ± SD. Statistical methods, such as one-way ANOVA, Student t-test, Kaplan-Meier method, Pearson's correlation analysis and the log-rank test were applied. A result with P<0.05 was regarded as having a statistically significant difference.

qPCR was conducted to explore miR-577 expression in 40 pairs of tumor tissues and corresponding adjacent non-tumor tissues. As exhibited in Fig. 1A, miR-577 was markedly downregulated in the HCC tissues when compared with that noted in the adjacent non-tumor tissues (P<0.05, Fig. 1A). Consistently, miR-577 expression was obviously lower in the HCC cell lines compared to the normal liver cell LO2 (P<0.05, Fig. 1B). The above results revealed that miR-577 expression was downregulated in HCC and may play a crucial role in HCC development.

Ninety-three patients were assigned into two subgroups (high/low miR-577 group), based on the median value of miR-577 expression in HCC tissues. We found that low miR-577 expression was notably related to venous invasion (P=0.007, Table I) as well as advanced tumor-node-metastasis (TNM) stage (P=0.018, Table I). Moreover, results from the Kaplan-Meier analysis revealed that patients with low miR-577 expression possessed worse overall survival (OS) (P=0.0001, Fig. 2A) and disease-free survival (DFS) (P=0.0001, Fig. 2B). Thus, the above data suggest that miR-577 could be used to predict the outcome of HCC patients.

miR-577 levels were manipulated by stable transfection with miR-577 mimics into MHCC-97H cells whose expression of miR-577 was the lowest, while miR-577 inhibitors were transfected into Hep3B cells which had the highest miR-577 expression (P<0.05, Fig. 3A). MTT assays revealed that the changes in miR-577 expression did not have any significant influence on HCC cell growth compared to the control groups (Fig. 3B). Then, data from Transwell assays confirmed that overexpression of miR-577 notably suppressed the migration and invasion abilities of the MHCC-97H cells (P<0.05, respectively, Fig. 3C), while silencing of miR-577 expression had the contrary effects on Hep3B cells (P<0.05, respectively, Fig. 3D). To confirm the in vitro functional effects of miR-577 on HCC, we performed in vivo metastatic experiments to examine whether miR-577 could inhibit the metastasis of HCC cells in vivo. We subsequently injected the stably overexpressing miR-577 cells, MHCC-97H-miR-577 and MHCC-97H-miR-control cells into the lateral veins of the nude mice. The results showed that injection of the miR-577 overexpressing cells resulted in fewer and smaller foci in the lungs of the nude mice through microscopic evaluation (5 vs. 16 nodules per lung in MHCC-97H-miR-577 and miR-control cells, respectively; P<0.01, Fig. 3E). Thus, we demonstrated that miR-577 exerts an anti-metastatic effect in HCC cells in vitro and in vivo.

In order to explore the association between miR-577 and EMT, western blot analysis was performed. The results revealed that, in MHCC-97H cells, overexpression of miR-577 induced E-cadherin and suppressed N-cadherin and vimentin (P<0.05, Fig. 4A). However, in Hep3B cells, miR-577 knockdown showed the opposite effects (P<0.05, Fig. 4B). Moreover, we examined the metastatic phenotype of these cells and found that lung sections of the mice injected with the miR-577-overexpressing cells in fact showed increased E-cadherin expression and conversely decreased vimentin expression (Fig. 4C). Thus, our results revealed that miR-577 acts as an inhibitor of the EMT process in HCC cells.

Two public databases (miRanda and TargetScan) were employed to predict the potential target of miR-577 in HCC cells. Then we focused on HOXA1 and speculated HOXA1 was a candidate target, whose 3′-UTR could bind to miR-577 (Fig. 5A). Additionally, HOXA1 has been identified as an oncogene in HCC by repressing migration and invasion of HCC cells (22). To investigate whether miR-577 could interact with the 3′-UTR of HOXA1, luciferase assays were conducted. The results indicated that miR-577 negatively regulated luciferase activity of wt 3′-UTR of HOXA1 (P<0.05, Fig. 5B). However, the results could not be found in the miR-577 mutant groups (Fig. 5B). Moreover, we performed luciferase assays and found that miR-577 overexpression significantly decreased the luciferase activity of wild-type (wt) HOXA1 3′-UTR while had no influence on that of the mutant (mt) HOXA1 3′-UTR (P<0.05, Fig. 5B). In contrary, miR-577 knockdown increased the luciferase activity of wt HOXA1 3′-UTR (P<0.05, Fig. 5B) but did not affect the luciferase activity of mt HOXA1 3′-UTR constructs. Furthermore, results from qPCR and western blot assays revealed that both HOXA1 mRNA and protein expression were negatively regulated by miR-577 in the HCC cells (P<0.05, respectively, Fig. 5C and D). Thus, we conclude that miR-577 directly targets HOXA1 in HCC cells.

Next, we attempted to determine the correlation of miR-577 and HOXA1 in HCC. The expression levels of HOXA1 mRNA and protein in HCC tissues with different miR-577 expression were detected. As expected, tissues with high miR-577 had obviously lower HOXA1 mRNA and protein expression compared to the tissues with low miR-577 (P<0.05, Fig. 6A and B). Furthermore, there existed a negative correlation between HOXA1 mRNA and miR-577 in the HCC tissues (R²=0.6866, P<0.001, Fig. 6C). In addition, we performed western blot analysis to confirm that HOXA1 was overexpressed in HCC tissues compared to that in the corresponding adjacent non-tumor tissues (P<0.05, Fig. 6D). These results confirmed that HOXA1 acts as a downstream target of miR-577 in HCC, and HOXA1 is negatively regulated by miR-577 in HCC.

To determine whether HOXA1 abrogates the effects of miR-577, we restored HOXA1 expression in miR-577-overexpressing MHCC-97H cells (P<0.05, Fig. 7A). Interestingly, regaining HOXA1 partially abrogated the inhibitory functions of the ovexpression of miR-577 in regards to migration, invasion and EMT of MHCC-97H cells (P<0.05, Fig. 7B and E). In contrast, HOXA1 inhibition by a specific siRNA significantly reversed the promotive effects of miR-577 knockdown in Hep3B cells (P<0.05, Fig. 7C, D and F). In brief, our findings demonstrated that HOXA1 reversed the anti-metastatic effects of miR-577 in HCC cells.