A total of 17 HCC tissues (age, 32–65; 9 males and 8 females) and paired adjacent tissues were obtained under the supervision of the ethics committee of the local hospital (Yantai City Hospital for Infectious Diseases, Shandong, China) between March 2015 and June 2015. All the patients did not receive any chemotherapy prior to surgery. The diagnoses of HCC were histologically confirmed by three independent pathologists, and the negative resection margin was confirmed during the surgery. Tissues were snap-frozen in liquid nitrogen. All patients provided written informed consent for the study.

The human HCC HepG2 and Huh-7 cell lines and human embryonic kidney HEK293-T cell line used in the present study were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare, Logan, UT, USA) with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂, and the medium was changed every 3 days. 3-MA was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and dissolved in the culture medium, and a 5 mM final concentration was applied to cells to inhibit autophagy. For autophagy flux assay, co-treatment with 20 µM chloroquine (C6628; Sigma-Aldrich; Merck KGaA) was applied.

The miRNA mimic for miR-7 (miR10004553-1-5) and its exact antisense inhibitor (miR200004553-1-5) were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The nucleotides were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at a final concentration of 50 nM, according to the manufacturer's protocol. miRNAs were diluted in DMEM and incubated with a proper amount (10 µl for one 6-well plate) of Lipofectamine 2000 at 37°C for 20 min. The Lipofectamine 2000-miRNA mixture was then added to each well of a 6-well plate; after 12 h of incubation in serum-free conditions, the medium was refreshed with complete medium containing 10% FBS.

To determine the cell proliferation, a Cell Counting Kit-8 (CCK-8; Wuhan Boster Biological Technology, Ltd., Wuhan, China) was used. The experiment was conducted according to the manufacturer's protocol. The absorbance value at 450 nm was determined using a plate reader as a measure of proliferative activity.

Following treatment, cells were collected and lysed with 1 ml TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Chloroform (0.2 ml) was then added to the tube and mixed vigorously with TRIzol reagent. RNA was precipitated using isopropyl alcohol from the upper phase and washed using 75% ethanol. The RNA samples were resolved in nuclease-free double-distilled water (Promega Corporation, Madison, WI, USA). miR-7 was then reverse-transcribed using a specific stem-loop primer, followed by a SYBR Green-based amplification protocol. Reverse transcription was performed with the GoScript reverse transcription kit (Promega Corporation), at 25°C for 5 min, and then 42°C for 1 h. PCR was performed using the GoTaq aPCR Master Mix (Promega Corporation), which included the DNA polymerase. The thermocycling conditions were as follows: 95°C for 20 sec, 58°C for 20 sec and 72°C for 20 sec, for 40 cycles. Small nuclear RNA U6 was used as an internal control. The primer sets for miR-7 and U6 were all obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China; cat. no., miRQ0000252-1-2). All experiments were repeated three times. Quantification was performed using the 2^−ΔΔCq method (22).

Total protein from the treated cells was obtained by homogenizing cells with radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). The protein concentration was quantified by a bicinchoninic acid kit (Thermo Fisher Scientific, Inc.). Protein (~40 µg) was loaded on 15% SDS-PAGE gels. Following electrophoresis, the protein was blotted onto a polyvinylidene fluoride membrane. The membrane was blocked with non-fat milk at 37°C for 1 h, and incubated with primary antibodies [LC3 (cat. no., 3868; dilution, 1:1,000), mTOR (cat. no., 2983; dilution, 1:1,000), p62 (cat. no., 8025; dilution, 1:1,000), p-mTOR (cat. no., 5536; dilution, 1:1,000) and β-actin (cat. no., TA310155; dilution, 1:2,000)] at 4°C for 16 h. Following 5 washes of 5 min with PBS containing 0.5% Tween-20, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit HRP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no., sc-2005; dilution, 1:5,000) and goat anti-mouse HRP (Santa Cruz Biotechnology, Inc.; cat. no., sc-2004; dilution, 1:5,000) at room temperature for 1 h, followed by visualization using an enhanced chemiluminescence detection kit (GE Healthcare Life Sciences, Little Chalfont, UK), according to the manufacturer's protocol. Antibodies against 1A/1B-light chain 3 (LC3), mTOR, phosphorylated mTOR (p-mTOR) and p62 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibody against β-actin was purchased from OriGene Technologies, Inc. (Beijing, China).

A 500 bp fragment containing the putative binding site of miR-7 was amplified from the cDNA of mTOR, the fragment of mTOR 3′UTR was then inserted into the 3′UTR of PGL-3 firefly reporter (Promega Corporation). HEK293-T cells grown in 24-well plates were transfected with promoter Renilla luciferase-thymidine kinase (internal control), PGL3-mTOR 3′UTR and miR-7 or miR-7 + miR-7 inhibitor, using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). The luciferase activity was detected after 36 h using the substrates provided in the Dual Luciferase Reporter Assay kit (Promega Corporation), according to the manufacturer's protocol. The firefly luciferase activity was normalized to Renilla luciferase activity, and the relative luciferase activity in each group was obtained by normalizing the value of (firefly luciferase activity)/(Renilla luciferase activity) to that of the negative control (NC)-transfected group.

Cells (2×104/ml) were seeded onto coverslips and cultured in 24-well plates. Immediately after transfection, cells were incubated at 37°C for 48 h. The cells on coverslips were then fixed with PBS containing 4% paraformaldehyde, and quickly penetrated with 0.3% Triton X-100 on ice for 5 min. The coverslips were blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Primary antibody against LC3 (Cell Signaling Technology, Inc.; cat. no., 3868; dilution, 1:200) was then added to the coverslips. Cells were incubated with primary antibody in a humid chamber overnight at 4°C, and the coverslips were then washed with PBS containing Triton X100 (0.5%). Alexa Fluor^® 594-conjugated anti-rabbit secondary antibody (cat. no., R37117; Thermo Fisher Scientific, Inc.) was added and incubated at room temperature for 1 h to label the primary antibody for LC3. Cells were counterstained with DAPI and observed by fluorescence microscopy in five fields of view (Nikon Corporation, Tokyo, Japan; magnification, ×400).

Data are expressed as the mean ± standard deviation. A paired Student's t-test was used to determine the differential expression of miR-7 in tumor tissues and normal tissues. One-way analysis of variance followed by Dunnett's test was used for multiple comparisons. P<0.05 (two-tailed) was considered to indicate a statistically significant difference. Statistical analysis was performed using SPSS version 19 software (IBM SPSS, Armonk, NY, USA).

Previous studies have demonstrated that miR-7 functions as a tumor suppressor and is downregulated in various types of cancer, including lung cancer and gastric cancer (19,20,23). The present study compared the expression of miR-7 in tumor samples and paired normal tissue. As presented in Fig. 1, a significant decrease in miR-7 was observed in tumor tissue compared with normal tissue, suggesting the potential antitumor role for miR-7 in HCC.

To ascertain the role of miR-7 in HCC, a CCK-8 assay was employed to examine whether miR-7 affects the proliferative activity of the HCC HepG2 cell line. The efficacy of transfection was confirmed using RT-PCR (Fig. 2A). A significant attenuation of cell proliferative activity was observed when miR-7 was overexpressed, and this effect was partially reversed by miR-7 inhibitor (Fig. 2B). These results confirmed that miR-7 serves an antitumor role in HCC.

Next, the autophagic activity was examined by western blot analysis of the autophagy markers LC3 and p62. Overexpression of miR-7 resulted in increased expression of LC3-phosphatidylethanolamine conjugate (LC3-II), and, accordingly, p62, which is a specific substrate of autophagy (24), was downregulated (Fig. 3A and B). Co-administration of chloroquine (CQ) was used to conduct the autophagy flux assay. Similar to the aforementioned results, the LC3-II level was also increased in the miR-7 group in the presence of CQ, indicating the increased autophagy flux (Fig. 3A and B). Immunofluorescent staining of LC3 was performed to visualize the autophagosome, and it was revealed that the autophagosome was aggregated in miR-7-transfected cells (Fig. 3C). These results suggested that overexpression enhances autophagic activity in HCC cells.

As mTOR is a canonical negative regulator of autophagy, it was hypothesized that miR-7-associated autophagy induction was associated with this pathway. A region in the 3′UTR of the mRNA of mTOR was identified to comprise the seed sequence matching miR-7 (Fig. 4A). A luciferase reporter assay demonstrated that miR-7 overexpression inhibited the luciferase activity of the reporter carrying mTOR 3′UTR, whereas co-transfection of miR-7 and miR-7 inhibitor significantly reversed this effect (Fig. 4B). Western blot analysis confirmed the inhibition of mTOR expression by miR-7, and consequently, p-mTOR, which represents the kinase activity of mTOR, was also inhibited by miR-7 (Fig. 4C and D). These data indicated that mTOR is a target of miR-7, and inhibition of mTOR may contribute to miR-7-activated autophagy.

To investigate the exact role of autophagy in HCC, autophagy was inhibited by 3-MA. Western blot analysis confirmed the autophagic inhibition by 3-MA in HepG2 and Huh-7 cells (Fig. 5A and B). 3-MA treatment did not result in significant alterations in cell proliferation in the NC-transfected group. By contrast, inhibition of autophagy by 3-MA significantly decreased the cell proliferative activity upon miR-7 overexpression (Fig. 5C). These data indicated that autophagy induced by miR-7 counteracts the proliferation inhibition effect of miR-7, and inhibition of autophagy may enhance the antitumor effect of miR-7.