Hepatocellular carcinoma (HCC) is a malignant tumor that has become a global health issue. The aim of the present study was to examine the role of transmembrane protein 9 (TMEM9) in cell progression, such as cell growth, cell cycle, cell metastasis of hepatoma cells, and to discuss the TMEM9 gene-encoding protein as a potential therapy target of hepatoma. RT-qPCR was performed to examine TMEM9 expression in tumor tissues and adjacent tissues of patients with liver cancer. siRNAs were used to interfere TMEM9 in HepG2 and 7721 cells. A CCK-8 assay was performed to evaluate cell growth at 24, 48 and 72 h. Cell cycle and apoptosis were analyzed using flow cytometry. Transwell assays were used to determine cell invasion, migration and adhesion. The results showed that TMEM9 was expressed abnormally in liver cancers. TMEM9 expression increased significantly in the 34 examined patients. TMEM9 knockdown inhibited proliferation in the HepG2 and 7721 cells. The flow cytometric analysis revealed that TMEM9 knockdown by RNA interference resulted in G1 arrest and induced apoptosis. Cell invasion, migration and adhesion ability were also decreased. Western blotting indicated that expression of the cell cycle-related proteins CDK1, EIF3H, RPL10L, S100A10, CCNB1 and CCNB2 was significantly decreased. In conclusion, TMEM9 plays an important role in the cell growth of hepatoma cells.
The incidence of cancer is on the increase and one of the main causes of global mortality (
The membrane protein is a unique structure of protein that plays an important role in cell contact, signal transduction and enzyme activity. It has various functions and becomes the ideal drug target. CD151, as a 4 transmembrane protein gene, is associated with the invasion and metastasis of HCC (
In the present study, we investigated the function of the
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
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-
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 (
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 (
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) (
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
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
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) (
HCC is one of the most common types of cancer worldwide. Of an estimated 700,000 cancer-associated mortalities that arising in 2008, 50% occurred in China (
In order to elucidate the possible mechanism involved, we identified the related protein expression. CDK1 is a highly conserved protein and a key player in the cell cycle regulation (
In summary, to the best of our knowledge, the present study provides evidence for the first time that TMEM9 is crucial in the cell proliferation, apoptosis and metastasis of HCC cells. Additionally, TMEM9 regulates these biological processes by regulating cell cycle-related proteins. As TMEM9 expression level is associated with the patient survival rate, inhibition of TMEM9 in tumor tissues provides a therapeutic strategy. However, additional investigations should be conducted to validate its therapeutic function in the future.
The present study was supported by the Natural Science Fund of Hubei Province (no. 2012FFA044), the Health Department Found of Hubei Province (no. JX6B18), and the Public Service Platform Construction Projects of Wuhan Technology Bureau (no. 2013060705010326).
High expression of TMEM9 is associated with the patient survival rate. (A) The mRNA expression level of TMEM9 was detected in tumor and adjacent tissues. (B) High expression of TMEM9 had a slightly higher survival rate. (C and D) The expression of TMEM9 was analyzed by RT-qPCR and western blotting in the six HCC cell lines. TMEM9, transmembrane protein 9; HCC, hepatocellular carcinoma.
Effects of TMEM9 on cell proliferation of HepG2 and 7721 cells. (A) Cell proliferation was detected at 0, 24, 48 and 72 h in HepG2, NC and sh-TMEM9 cells. (B) Cell proliferation was detected in 7721, NC and sh-TMEM9 cells. NC, negative control; TMEM9, transmembrane protein 9.
Effects of TMEM9 on cell cycle of HCC cells. (A) After TMEM9-siTNA transfection for 48 h, cell cycle distribution of HepG2 cells was identified by flow cytometry. (B) Cell cycle distribution of 7721, NC and shTMEM9 were detected. Data are based on at least 3 independent experiments and shown as mean ± SD. **P<0.01, compared with the negative control. NC, negative control; TMEM9, transmembrane protein 9.
Effects of TMEM9 on cell apoptosis of HCC cells. (A) After HepG2 cells were transfected for 48 h, the apoptotic rates of HepG2 cells were analyzed using flow cytometry. (B) The apoptotic rates of 7721, NC and shTMEM9 were detected. **P<0.01, compared with the negative control.TMEM9, transmembrane protein 9; HCC, hepatocellular carcinoma.
TMEM9 knockdown decreased invasion capacity of HCC cells
Knockdown of TMEM9 decreases cell migration in HCC cells. (A and B) The images of HepG2 and 7721 migrated cells were captured in 48 h after seeding. (C and D) Migrations of HCC cells were assayed using the 24-Transwell system. Data are one of three similar experiments. **P<0.01, when compared with the negative controls. TMEM9, transmembrane protein 9; HCC, hepatocellular carcinoma.
Knockdown of TMEM9 decreased cell adhesion in HCC cells. (A and B) The images of HepG2 and 7721 migrate cells were captured in 48 h after seeding. (C and D) The adhesion of HCC cells were identified after transfection. **P<0.01, when compared with the negative controls. TMEM9, transmembrane protein 9; HCC, hepatocellular carcinoma.
Western blotting results of TMEM9 influenced proteins. (A and B) After 48 h of TMEM9 siRNA treatment, the protein expression of CDK1, EIF3H, RPL10L, S100A10, CCNB1 and CCNB2 in HepG2 and 7721 cells were detected by western blotting, GADPH was detected as the control of sample loading. (C and D) Western blotting data are analyzed to identify the protein levels. **P<0.01 compared with the NC cells.