Between 2008 and 2013, 70 HCC patients presenting to the Zhongnan Hospital of Wuhan University (Hubei, China) were enrolled in the present study. All the patients had complete clinical and pathological follow-up data. Adjacent normal hepatocellular tissues were also collected as negative controls. These normal hepatocellular tissues were resected within at least 5 cm of the tumor margin when the patients underwent definitive surgery. Clinical fresh tissue samples were detected by qPCR. Approval for the study was provided by the independent Ethics Committee of the Zhongnan Hospital of Wuhan University. Informed and written consent was obtained from all the patients or their advisers according to the ethics committee guidelines.

Human 97H, 97L, HepG2, 7721, 7404 and HuH7 HCC cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% double antibiotics (penicillin/streptomycin) and maintained in a 37°C incubator with a 5% CO₂ humidified atmosphere. Transfections were performed using the Lipofectamine™ 2000 reagent according to the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). After 48 h of transfection, the cells were used for cell proliferation assays, cell cycle analysis, and apoptosis, Matrigel invasion, migration and adhesion assays. Silencer negative control siRNA was used as a negative control.

Cellular RNA was isolated using the TRIzol kit (Invitrogen Life Technologies). SYBR-Green RT-qPCR was performed to detect the mRNA expression. GADPH was used to normalize the RNA inputs. The primers used were: TMEM9 sense, 5′-GGGCACATTTACAACCAG-3′ and antisense, 5′-ATCAGGAAGGCCATGTAG-3′; GADPH sense, 5′-CACCCACTCCTCCACCTTTG-3′ and antisense, 5′-CCACCACCCTGTTGCTGTAG-3′.

Viability of cells 72 h after transfection was assessed using the Cell Counting Kit-8 (CCK-8) (Qihai, Shanghai, China). Briefly, cells were seeded at a density of 3×10⁴ in each 96-well plate and cultured for 0, 24, 48 and 72 h, respectively. CCK-8 reagent (100 µl/well) was added to each well and incubated for 1 h at 37°C. The optical density (OD) values were determined at 450 nm using a microplate reader. Three different experiments were performed for each experimental condition.

The cell cycle was assessed by flow cytometric analysis at different time points using a propidium iodide (PI) cell cycle detection kit (Beyotime, Shanghai, China). The cells were collected, treated and stained with PI according to the manufacturer's instructions. The cell cycle was detected using a flow cytometer (BD Biosciences, Heidelberg, Germany).

Apoptotic cells were visualized using an Annexin V-FITC/PI kit (BD Biosciences, San Jose, CA, USA). The apoptosis of HCC-transfected cells were determined by flow cytometric (FCM) analysis using a FACSCalibur.

After transfection, the cells were detached and washed twice in PBS. Then, 1×10⁵ cells/ml were seeded in the upper chamber of a Transwell insert (8-µm pore size) coated (invasion) or not coated (migration) with 80 µl Matrigel (BD Biosciences). The lower chamber was filled with 0.75 ml of DMEM. After a 48-h (invasion) or 24-h (migration) incubation period, the non-migrated cells in the upper chamber were scraped away, and adherent cells were stained with formaldehyde solution. Any cells on the underside were counted and photographed under ×200 microscope fields.

To determine the adhesion cells, 12-well plates were used. Cell suspension (1×10⁵ cells/ml) was added to the well and incubated for 1 h at 37°C. Adherent cells were fixed with 4% methanol and stained with crystal violet for 20 min. The number of adherent cells were photographed and counted from three random selected ×200 fields of microscope.

To detect the role of TMEM9 in liver cancer cells, we selected the proteins CCNB1, CCNB2, CDK1, PRL10A, S100A10 and EIF3H to detect the protein expression using western blotting. Protein lysates were prepared. Equal amounts of samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then blocked with 5% low-fat milk for 1 h or overnight at 4°C; incubated with CCNB1, CCNB2, CDK1, PRL10A, S100A10 and EIF3H with primary antibodies for 2 h, followed by secondary antibodies for 1 h at room temperature; and analyzed. GADPH protein levels were determined as a loading control.

Statistical significances were determined using the GraphPad Prism v5.0 software (GraphPad Software, La Jolla, CA, USA). Kaplan-Meier analysis was used to determine that the overall survival time between low and high expression of HCC. Data are presented as the mean ± SD of at least three independent replicates. Differences were considered significant when P<0.05 or P<0.01.

To investigate the expression of TMEM9, we used RT-qPCR to investigate in the HCC tissues of 30 patients. The results showed a higher level of TMEM9 expression (Fig. 1A). Then we investigated the correlation between TMEM9 expression and prognosis of the patients with HCC. As shown in Fig. 1B, Kaplan-Meier analysis showed that the overall survival time of lower-TMEM9-expressing patients was notably higher than that of higher-TMEM9-expressing patients. The expression levels of TMEM9 in the six HCC cells were also evaluated by qPCR and western blotting (Fig. 1C and D). The HepG2 and 7721 cell lines had a higher TMEM9 mRMA and protein expression.

To assess the potential effects of RNAi silencing TMEM9 on proliferation, CCK-8 analysis was performed 72 h after transfection. The proliferative ability of HepG2 and 7721 cells was significantly inhibited at 24, 48 and 72 h (Fig. 2).

Moreover, we investigated the effects of TMEM9 on cell cycle and apoptosis in HCC. The flow cytometric analysis revealed that the population of G0/G1 phase was significantly increased but that of S and G2/M phase was decreased in HepG2 and 7721 cells, when compared with the negative control (NC) (P<0.01) (Fig. 3).

In addition, we assessed the apoptotic function of TMEM9 in HepG2 and 7721 cells using the Annexin V-FITC/PI staining assay. As shown in Fig. 4, the results showed that knockdown of TMEM9 in HCC cells markedly induced the cell apoptotic rate compared with NC (P<0.01).

Cell metastasis plays an important role in cancer progression. We determined whether TMEM9 regulated metastasis of HCC cells. Cell invasion, migration and adhesion assays were then used to detect the metastatic capacity. As shown in Fig. 5, the cell invasion ability was reduced when compared with NC (P<0.01). The migration and adhesion cells were also decreased (P<0.01) (Figs. 6 and 7).

Signaling pathways are often activated in tumor cells. We assessed the protein expression using western blotting. The results showed that the protein expression of CDK1, EIF3H, RPL10L, S100-A10, CCNB1 and CCNB2 was decreased compared to the control group (P<0.01) (Fig. 8).