HCC tissues and the normal adjacent liver tissues were collected from 20 patients in Nanfang Hospital, Southern Medical University (Guangzhou, China). All the tissues were collected before any therapy. The Ethics Committee of Nanfang Hospital, Southern Medical University approved the research. Each patient signed the informed consent.

The human hepatocellular carcinoma cell line (HepG2) was purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 µg/ml streptomycin and 100 U/ml penicillin (Gibco, Carlsbad, CA, USA). The cells were incubated at 37°C in a moist atmosphere containing 5% CO₂.

RNA extraction from the tissues or cells was by TRIzol reagent obtained from Invitrogen (Carlsbad, CA, USA). The TaqMan MicroRNA Assays (Invitrogen) were applied to quantitate the relative expression of miR-217 and the standard SYBR Green RT-PCR kit (Takara Shuzo, Kyoto, Japan) was used to detect the mRNA expression of MTDH via qRT-PCR. Primers assigned for miR-217 and MTDH were obtained from GeneCopoeia (Rockville, MD, USA). Values were normalized to either small nucleolar RNA U6 or GAPDH.

GenePharma (Jiangsu, China) provided the miR-217 mimics and negative control (NC) RNA oligonucleo-tides. Cells were transfected with miR-217 or NC mimics (100 nM) by Lipofectamine 2000 (Invitrogen) when 70–80% cell confluence in 6-well plates. After the transfection for 36 h, cells were collected for further tests.

The expression plasmid for MTDH 3′-UTR wild-type or mutation and miR-217 were transfected into HepG2 cells. The firefly and renilla (the internal control) luciferase activities were examined by the dual-luciferase reporter assay system (Promega, Madison, WI, USA).

Proteins were extracted from HCC tissues or cells with RIPA lysis buffer and quantitated by the BCA protein assay kit (Bio-Rad, Hercules, CA, USA). Proteins were subjected to 12% SDS-PAGE gels and electrophoretically transfered onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Immunoblots were exposed to primary antibodies against MTDH or GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. Then HRP-conjugated secondary antibodies were added to analyze the immunoreactive bands with the chemiluminescence reagent (Western Lightning, Perkin Elmer Life Sciences, Boston, MA, USA).

Cells were seeded in a 96-well plate and cultured for 24, 48, 72 or 96 h at 37°C with 5% CO₂. The cell counting kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) was used to assess the cell proliferation. The numerical values obtained on an enzyme-labeled instrument (Thermo Fisher Scientific, Bonn, German) with 450 nm wavelength were used to evaluate the cell viability.

Cells were trypsinised, collected in PBS and then fixed in 70% ethanol (4°C, overnight), then washed with PBS and collected by centrifugation (1000 rpm, 5 min). The Annexin V-FITC apoptosis detection kit (Beyotime, Jiangsu, China) was used to analyze apoptotic cells via flow cytometry (BD FACSCalibur).

Sells were seeded on coverslips (Nalge Nunc International, Penfield, NY, USA) in a 24-well plate and allowed to adhere overnight. then fixed with 4% paraformaldehyde and incubated with primary anti-MTDH antibody (Invitrogen) overnight at 4°C. Cy3-conjugated secondary antibody was added and maintained at room temperature for 1 h. After washing the slides were counterstained with DAPI (Sigma-Aldrich). A confocal microscope (Olympus Corp., Tokyo, Japan) was used to obtain the fluorescence images.

Boyden chamber Transwells (8 µm, Millipore) was used to assess cell migration and invasion. The upper chamber with uncoated membrane (migration assay) or the membrane pre-coated with 100 µg Matrigel (invasion assay) was added with 200 µl cell suspension (1×10⁵ cells/ml). The lower chamber was added with 600 µl DMEM and 10% FBS as a chemoattractant. The nonfiltered cells were gently removed and fixed with 4% paraformaldehyde after incubation for 24 h at 37°C. Crystal violet (0.1%) (Sigma-Aldrich) was applied to stain the lower chamber with filtered cells. Quantitation was performed with a microscope (Olympus Corp.).

The data are presented as mean ± SD. Student's t-test was used to evaluate differences between the stimulated sample and the respective control. For multiple comparisons, statistically significant differences were assessed via one-way ANOVA. P-value <0.05 was deemed statistically significant.

As depicted in Fig. 1A, compared with the matched normal tissues, miR-217 expression level in HCC tissues significantly decreased (P<0.0001). Moreover, the expression of MTDH was examined via qRT-PCR and western blotting in HCC tissues. Compared with the normal tissues, the mRNA expression of MTDH was significantly upregulated in HCC tissues (Fig. 1B), as well as the protein expression of MTDH (Fig. 1C).

As miR-217 and MTDH expressed negatively in HCC, we investigated whether miR-217 can target MTDH. As shown in Fig. 2A, miR-217 bound to the 3′-UTR of MTDH gene. The dual luciferase reporter assay was further performed to confirm their relationship. As displayed in Fig. 2B, after cotransfection with the 3′-UTR of wild-type MTDH and miR-217, the relative luciferase activity was remarkably decreased in HepG2 cells (P<0.05). While the luciferase activity of the mutant construct showed little change, indicating that miR-217 inhibited MTDH transcription via directly targeting the 3′-UTR of MTDH.

To further validate the prediction of the suppression effect of miR-217 on MTDH gene, we explored whether miR-217 could regulate the expression of endogenous MTDH in HepG2 cells. As depicted in Fig. 3A and B, compared with the control or mimics NC, the miR-217 expression in the cells transfected with miR-217 mimics was notablely upregulated (P<0.01), indicating that the transfection efficiency was satisfactory. We next examined the MTDH mRNA expression and observed a remarkably downregulation after transfection with miR-217 mimics (P<0.01). Furthermore, we found by western blotting that the MTDH expression in the HepG2 cells with miR-217 overexpressed was markedly reduced compared with the control or mimics NC (Fig. 3C). Similar results were obtained by immunofluorescence assays (Fig. 4).

miR-217 or NC mimics were transfected into HepG2 cells to explore the role of miR-217 in HCC cells. As shown in Fig. 5A, the proliferation of miR-217-overexpressed cells was inhibited compared with the control or mimics NC. Moreover, the cell cycle showed similar results (Fig. 5B). These data suggest that miR-217 played a proliferation suppressing role in HCC. In addition, we explored the effect of miR-217 upregulation on apoptosis in HCC. Compared with the control groups, the cell apoptosis notably increased in miR-217-upregulated HepG2 cells (Fig. 5C and D), indicating that miR-217 expression promoted apoptosis of HCC cells.

As displayed in Fig. 6A, the HepG2 cells overexpressing miR-217 had lower migration and invasion compared with the control groups. Moreover, the statistical results demonstrated that miR-217 overexpression markedly suppressed migration and invasion in HCC cells (Fig. 6B and C).