This study was approved by the Ethics Committee of The Second Affiliated Hospital of Harbin Medical University and written informed consent was provided by all patients enrolled in the research. A total of 32 pairs of HCC tissues and pair-matched adjacent normal tissues were collected from The Second Affiliated Hospital of Harbin Medical University. The clinicopathological features of the 32 patients [mean age, 57; (range, 45–70 years); 12 female and 20 male patients)] recruited to the study are shown in Table I. The collection dates of these tissue specimens were from July 2015 to May 2017. None of the patients had received chemotherapy or radiotherapy before surgical resection. All tissues were stored in liquid nitrogen until further RNA isolation.

Two human HCC cell lines (Huh7 and Hep3B) and an immortalized normal human liver epithelial cell line (L-O2) were obtained from the Cell Bank of the Chinese Academy of Biological Science (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to culture all cell lines mentioned above. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

miR-466 mimics and negative control miRNA mimics (miR-NC) were chemically produced by RiboBio Co., Ltd. (Guangzhou, China). Specific small interfering RNA (siRNA) targeting the MTDH gene (si-MTDH) and the negative control siRNA (si-NC) were constructed by Guangzhou GeneCopoeia Co., Ltd. (Guangzhou, China). MTDH overexpression plasmid (pcDNA3.1-MTDH) lacking the 3′-UTR and negative control plasmid (pcDNA3.1) were purchased from Genepharma Co., Ltd. (Shanghai, China). Cells were placed into 6-well plates, grown to approximately 70% confluence, and transfected with miRNA mimics, siRNA or plasmid using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Transfected cells were incubated at 37°C under 5% CO₂. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was applied to determine the transfection efficiency of the miR-466 mimics 48 h after transfection. At 72-h post-transfection, western blot analysis was performed to detect the efficiency of si-MTDH and pcDNA3.1-MTDH plasmid transfection.

TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used to extract total RNA from the tissue specimens and cultured cells. The concentration of total RNA was detected using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.). To quantify miR-466 expression, reverse transcription was performed using a TaqMan^® MicroRNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Subsequently, the synthesized complementary DNA (cDNA) was subjected to quantitative polymerase chain reaction (qPCR) using a TaqMan MicroRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling conditions for qPCR were as follows: 50°C for 2 min, 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 sec; and annealing/extension at 60°C for 60 sec.

To analyze the MTDH mRNA level, cDNA was produced from total RNA using a PrimeScript RT Reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China). Afterwards, qPCR was carried out using a SYBR Premix Ex Taq™ kit (Takara Biotechnology, Co., Ltd.). The cycling conditions for qPCR were as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. U6 small nuclear RNA and GAPDH served as internal references for miR-466 and MTDH mRNA, respectively. The primers were designed as follows: miR-466, 5′-ATGGTTCGTGGGATACACATACACGCA-3′ (forward) and 5′-GCAGGGTCCGAGGTATTC-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse); MTDH, 5′-TGCTCTCTCACAGACAA-3′ (forward) and 5′-TCGCTCTGCAGATGAGATAG-3′ (reverse); and GAPDH, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (forward) and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ (reverse). The 2^−ΔΔCq method (24) was used to calculate gene expression.

Cell proliferation was assessed using the CCK-8 assay in accordance with the supplied protocol and instructions. In detail, transfected cells were harvested and inoculated into 96-well plates at a density of 3×10³ cells/well. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. A total of 10 µl of CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added into every well at four time points (0, 24, 48 and 72 h after inoculation). After incubation at 37°C for another 2 h, the optical density (OD) values were measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 490 nm.

Cells were collected at 48 h after being transfected, and the apoptosis rate was determined using an Annexin V fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Biolegend, San Diego, CA, USA) according to the manufacturer's instruction. Briefly, transfected cells were washed twice with cold phosphate-buffered saline, suspended in 100 µl of binding buffer, and incubated with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI). The cells were then incubated in the dark at room temperature for 30 min. The rate of apoptosis was assessed by FACScan flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

The invasive and migratory properties of HCC cells were examined using 24-well Transwell plate cell culture inserts (Corning, New York, NY, USA) covered with or without Matrigel (BD Biosciences, San Jose, CA, USA), respectively. After 48 h of transfection, cells were harvested and suspended with FBS-free DMEM. A total of 5×10⁴ transfected cells were plated in the upper compartments, and the lower compartments were maintained with 500 µl of DMEM that was supplemented with 10% FBS. The transfected cells were then cultured for 24 h at 37°C with 5% CO₂. Cells remaining on the upper surface of the membranes were gently removed by swabbing the top layer. The migrated or invaded cells, which were attached to the lower surface of the membranes, were fixed with 100% methanol and stained with 0.05% crystal violet. The number of migrated or invaded cells was counted under an inverted microscope (×200 magnification; Olympus Corp., Tokyo, Japan). Five randomly selected fields were analyzed for each insert.

microRNA.org (www.microrna.org/microrna/) and TargetScan (www.targetscan.org) were adopted to search for the putative targets of miR-466. MTDH was predicted as a candidate for miR-466. To verify this, luciferase reporter plasmids, including psi-CHECK-MTDH-3′-UTR wild-type (Wt) and psi-CHECK-MTDH-3′-UTR mutant (Mut), were constructed by GenePharma Co., Ltd., and were cotransfected with miR-466 mimics or miR-NC into the cells using Lipofectamine 2000. The cotransfection was performed according to the manufacturer's instructions. After approximately 48 h of post-transfection, luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega Corps, Madison, WI, USA) following the manufacturer's protocol. The firefly luciferase activities were normalized to the Renilla luciferase activities.

Tissue specimens and cells were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate and 1 mg/ml aprotinin. A BCA Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was then utilized to determine the concentration of total protein. An equal amount of protein (30 µg) was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred into polyvinylidene difluoride membranes (Bio-Rad Laboratories). After that, the membranes were blocked in Tris-buffered saline-Tween (TBST) containing 5% non-fat milk, followed by incubation with the primary antibodies at 4°C overnight. Subsequent to washes with TBST thrice, the membranes were probed with appropriate horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; cat. no. sc-516102; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The protein blots were detected by BM Chemiluminescence Western blotting kit (Sigma-Aldrich; Merck KGaA). The primary antibodies used in this study included mouse anti-human monoclonal MTDH (1:1,000 dilution; cat. no. sc-517220; Santa Cruz Biotechnology, Inc.) and mouse anti-human monoclonal GAPDH (1:1,000 dilution; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.). GAPDH was used as the internal reference. Protein expression was quantified using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc).

All data are shown as the mean ± standard deviation and were subjected to SPSS software version 18 (SPSS, Inc., Chicago, IL, USA) for statistical analysis. Differences between groups were analyzed using Student's t-test, paired Student's t-test or one-way ANOVA, followed by the Student-Newman-Keuls (SNK) multiple comparison test. Spearman's correlation analysis was performed between miR-466 and the mRNA levels of MTDH in HCC tissues. Differences were defined as statistically significant if P<0.05.

miR-466 has been reported to be aberrantly expressed in several types of human malignancies (21–23). However, the expression pattern of miR-466 in HCC remains unknown. First, we detected miR-466 expression in 32 pairs of HCC tissues and pair-matched adjacent normal tissues. The data of RT-qPCR showed that miR-466 was lowly expressed in HCC tissues in contrast to that noted in the pair-matched adjacent normal tissues (Fig. 1A, P<0.05). In addition, RT-qPCR was performed to assess the miR-466 expression in HCC cell lines. Compared with the miR-466 expression in an immortalized normal human liver epithelial cell line (L-O2), miR-466 was downregulated in the two HCC cell lines, including Huh7 and Hep3B (Fig. 1B, P<0.05).

Given the downregulation of miR-466 in HCC, we hypothesized that miR-466 may play tumor suppressive roles in the progression of HCC. To confirm this, we transfected miR-466 mimics or miR-NC into Huh7 and Hep3B cells which possessed much lower miR-466 levels among the two HCC cell lines (data not shown). The expression of miR-466 was successfully overexpressed in Huh7 and Hep3B cells by the transfection of miR-466 mimics (Fig. 2A, P<0.05). CCK-8 assay was conducted to assess the effect of miR-466 restoration on cell proliferation in HCC. The results revealed that miR-466 upregulation significantly suppressed the proliferation of Huh7 and Hep3B cells (Fig. 2B, P<0.05). Alterations in cell proliferation are mostly related with the change in cell apoptosis; therefore, flow cytometric analysis was performed to detect the apoptosis rate of Huh7 and Hep3B cells that were transfected with the miR-466 mimics or miR-NC. It was demonstrated that the apoptosis rate of Huh7 and Hep3B cells was higher in the miR-466 overexpression group than that in the miR-NC group (Fig. 2C, P<0.05). We then examined the possible regulatory role of miR-466 in the migratory and invasive abilities of HCC cells. Transwell chamber assay indicated that ectopic miR-466 expression led to a significant suppression of Huh7 and Hep3B cell migration (Fig. 2D, P<0.05) and invasion (Fig. 2E, P<0.05). These results suggest that miR-466 may play a suppressive role in the proliferation and metastatic activity of HCC cells.

To identify the mechanisms underlying how miR-466 exerts its tumor-suppressor activity in HCC cells, we used bioinformatic analysis to search for the putative targets of miR-466. Complementary sequences were observed between miR-466 and the 3′-UTR of MTDH (Fig. 3A). MTDH was previously reported to be implicated in the oncogenesis and development of HCC (25–32) and was therefore selected for further identification. Luciferase reporter assay was applied to confirm whether MTDH is a bona fide target gene of miR-466 in HCC cells. Fig. 3B illustrates that the restoration of miR-466 expression was able to suppress the luciferase activity of the wild-type (Wt) construct of MTDH 3′-UTR (P<0.05), while modulating the miR-466 level exhibited unaltered luciferase activity of mutant (Mut) construct of MTDH 3′-UTR in Huh7 and Hep3B cells. Next, we detected the MTDH expression in HCC tissues and explored its possible relationship with miR-466. RT-qPCR analysis indicated that the mRNA level of MTDH was higher in HCC tissues than that in pair-matched adjacent normal tissues (Fig. 3C, P<0.05). Meanwhile, an inverse correlation between miR-466 and MTDH mRNA expression was validated in HCC tissues using Spearman's correlation analysis (Fig. 3D; r=−0.6714, P=0.0002). Furthermore, results of RT-qPCR and western blot analysis revealed that miR-466 re-expression decreased the mRNA (Fig. 3E, P<0.05) and protein (Fig. 3F, P<0.05) levels of MTDH in Huh7 and Hep3B cells. Collectively, our data suggest that MTDH is a direct target of miR-466 in HCC cells.

MTDH was identified as a direct target gene of miR-466 in HCC cells; hence, our subsequent analyses were focused on the functional roles of MTDH in HCC cells. To this end, siRNA against the expression of MTDH (si-MTDH) was employed to knock down endogenous MTDH expression in Huh7 and Hep3B cells. MTDH protein expression was obviously downregulated by si-MTDH transfection relative to its expression in the si-NC group (Fig. 4A, P<0.05). Analysis of cell proliferation and apoptosis using CCK-8 assay and flow cytometric analysis, respectively, showed that MTDH knockdown restricted the cell proliferation (Fig. 4B, P<0.05) and promoted the apoptosis (Fig. 4C, P<0.05) of Huh7 and Hep3B cells compared with these parameters in the si-NC group. Transwell chamber assays revealed that transfection of si-MTDH in Huh7 and Hep3B cells caused significant suppression of migration (Fig. 4D, P<0.05) and invasiveness (Fig. 4E, P<0.05) compared with these abilities in the cells transfected with si-NC. Taken together, these observations demonstrated that inhibition of MTDH exhibits similar tumor suppressive roles as miR-466 upregulation in HCC cells, suggesting that MTDH is a downstream target of miR-466 in HCC.

To further determine whether MTDH downregulation is essential for the tumor-suppressing roles of miR-466 in HCC, rescue experiments were performed in Huh7 and Hep3B cells by co-transfection with miR-466 mimics and empty pcDNA3.1 or pcDNA3.1-MTDH plasmid lacking the 3′-UTR. The downregulation of MTDH protein induced by miR-466 overexpression was restored in Huh7 and Hep3B cells after co-transfection with pcDNA3.1-MTDH (Fig. 5A, P<0.05). MTDH overexpression blunted the tumor-suppressor activity of miR-466 in regards to Huh7 and Hep3B cell proliferation (Fig. 5B, P<0.05), apoptosis (Fig. 5C, P<0.05), migration (Fig. 5D, P<0.05) and invasion (Fig. 5E, P<0.05) in vitro. In summary, these results suggest that the antitumor effects of miR-466 overexpression in HCC cells were, at least partly, mediated by inhibition of MTDH.