Boinformatics analysis
TCEAL7 expression levels in melanoma tissues and normal tissues were analyzed by using the Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/). The correlation between the levels of TCEAL7, AKT1, AKT2 and c-Myc were analyzed by using starBase (http://starbase.sysu.edu.cn/). miRanda (http://microrna.org/microrna/home.do) and miRDB (http://www.mirdb.org/index.html) were applied to predict the miRNAs which target TCEAL7.
Patients and sample preparation
Ninety-eight fresh melanoma tissues were obtained from patients with melanoma. Thirty nevus tissues (benign proliferation of melanocytes) obtained from age and gender-matched individuals served as normal control. None of interventional treatment was performed before surgery.
Cell culture
A375, one human malignant melanoma cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China); WM-115, another human melanoma cell line and the normal melanocytes PIG1 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in DMEM (Hyclone, USA) with 10% fetal bovine serum (FBS) (Hyclone, USA) in a humidified incubator at 37°C with 5% CO2.
RNA interference and lentivirus construction
The small interfering RNAs (siRNAs) targeting the human TCEAL7 gene were designed and synthesized by the GenePharma Co., LTD (Sghanghai, China). The sequences are listed as follows:
si- TCEAL7-1: 5'- CCGAAGTCCTTATATTCCCGGGCTT-3';
si- TCEAL7-2: 5'- GAGCTGACGTGAACCGAAGTCCTTA-3';
si- TCEAL7-3: 5'- CCAGTCATTCGATGTTGCTGAGATT-3';
si-Scramble: 5'-CATCAATTGAACCGAGCCTTACGTA-3'. The three siRNAs-TCEAL7 and its negative control si-Scramble were transfected to cells by using the Lipofectamine 2000 (Invitrogen, MA, USA).
To upregulate TCEAL7 (Lenti-TCEAL7), miR-758-3p (mimics), AKT1 (Lenti-AKT1) and c-Myc (Lenti-c-Myc) in melanoma cells, the lentivirus vectors were constructed by GenePharma Co., LTD and infected into cells with the help of polybrene (Thermo Fisher Scientific, MA, USA). The infected cells were then probed with 6 μg/ml puromycin and/or 100 μg/ml G418 for 14 days to establish the stably transfected cells, which were used in the in vivo experiments.
Real-time Quantitative PCR (RT-PCR) analysis of mRNA levels
Total RNA isolation from tissues and cells was carried out with the help of Trizol reagent (Invitrogen, Carlsbad, CA, USA), which was then reverse-transcribed into cDNA using RT2 First Strand Kit (Qiagen, Germany) in the light of manufacturer’s instructions. Then, the mRNA levels were assayed by RT-PCR with TransStart Green qPCR SuperMix (TransGen, Beijing, China) in a DA7500 Real-time Nucleic Acid Amplification Fluorescence Detection System (Bio-Rad). The human GADPH was used to normalize the mRNA levels. The 2-△△Ct method was used to calculate the changes of relative mRNA (12). The experiments were carried out in three biological replicates. The primers were synthesized by Sangon (China) and are listed in Table 1.
RT-PCR analysis of miRNA levels
The level miR-758-3p was detected by RT-PCR according to previous study (13). Briefly, total miRNAs were isolated from tumor samples and cell lines based on the manufacturer's protocol of miRcute miRNA Isolation kit (Tiangen Biotech Co., Ltd.). After that, the cDNA was synthesized using the miRcute miRNA cDNA kit (Tiangen Biotech Co., Ltd.), and miR-758-3p was then detected by the miRcute miRNA luciferase quantitative kit (Tiangen Biotech Co., Ltd.). U6 was used as an internal reference. The primer sequences were listed in Table 1.
Western blotting analysis
Total proteins from cells and tissues were obtained with RIPA buffer (Thermo Fisher Scientific) containing protease inhibitor cocktail. Then, the protein concentrations were determined by using the BCA Protein Assay Kit (Thermo Fisher Scientific) based on the instructions. After that, equal amounts of protein were resolved using 10% SDS-PAGE gels and then transferred to the polyvinylidene fluoride (PVDF; Millipore, Dallas, Tx, USA) membranes. The 5% skim milk was used to block the PVDF membranes, which was then incubated with the primary antibodies overnight at 4 °C, including TCEAL7 (cat no. 11218-1-AP, Proteintech, Wuhan, China), AKT1 (cat no. ab235958, Abcam, Cambridge, MA, USA), AKT2 (cat no. ab175354, Abcam), c-Myc (cat no. ab32072, Abcam), STAT3 (cat no. ab68153, Abcam), β-catenin (cat no. ab16051, Abcam), TGF-β (cat no. ab179695, Abcam), YAP (cat no. ab52771, Abcam), FOXO1 (cat no. ab52857, Abcam) and GAPDH (cat no. 60004-1-Ig, Proteintech). The membranes were then incubated with secondary antibodies for 1 hour at room temperature. Western blotting luminol reagent (Millipore) was used to visualize the immunoreactive bands.
Cell proliferation assay
Cell viability was detected by Cell Counting Kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer’s instructions. In brief, 2ⅹ103 A375 and WM-115 cells were seeded into a 96-well plate overnight prior to transfection/infection. Next, 10 ml of CCK-8 and 90 ml cell culture medium were added into each well after 1, 2, 3, 4 or 5 days of cell transfection/infection, respectively. Following incubation for 2 hours at 37°C with 5% CO2, the absorbance of each well was measured at 450 nm.
Apoptosis assay
The melanoma A375 and WM-115 cells were placed into 6-well plates at a concentration of 5 × 105/mL and allowed to grow at 37 °C overnight, followed by cell transfections/infections. After 48 hours, the cells were collected for apoptosis detection using Annexin-V-(FITC) and propidium iodide (PI) kit (BD Biosciences, San Jose, CA, USA). The double-stained cells were subsequently analyzed by the BD flow cytometer. At least 1 × 105 cells were detected each time.
Scratch assay
Melanoma cells were placed in 6-well plates with adequate numbers to reach 100% confluence in the next day. Then, the wounds were made by using 20 μl pipette tips. The non-adherent cells were removed by washing with PBS slowly. After that, each well of the 6-well plates were added 2 mL culture medium containing 1% FBS. The wound width was measured at 0 and 24 hours post the scratch.
Transwell chamber assay
To assess cell invasion, melanoma cells were suspended in FBS-free DMEM and inoculated in the Matrigel pre-coated membrane of the top chamber (BD Bioscience), with 1×105 cells for each well. The lower chamber was added 600 μL of 10% FBS-DMEM. At 24 hours post incubation, the non-invaded cells were removed with cotton swabs, and the invaded cells in the lower surface were fixed with 10 % methanol and stained with 0.1 % crystal violet (Solarbio Co., Ltd, Beijing, China) for 8 min. Pictures were taken under a microscope with a magnification of 200×, and the invaded cells were counted. Five randomly selected fields were recorded for each well.
Dual luciferase gene reporter assay
The 3’UTR of TCEAL7 with the putative miR-758-3p-binding site were chemically synthesized and cloned into the Renilla luciferase gene (pLUC-REPORT vector; Promega, Madison, WI, USA). WM-115 and A375 cells were co-transfected with 200 ng luciferase reporter vector and 100 nM miR-758-3p mimics or the negative controls. Luciferase activity was examined 48 hours after the transfection using the Dual-Luciferase Assay kit (Promega) according to manufacturer’s instruction.
In vivo tumorigenesis assay
Nude mice experiment was approved by Animal Ethics Committee of China Japan Union Hospital of Jilin University. SPF grade male BALB/c nude mice were purchased from the Institute of Zoology, Chinese Academy of Sciences. The malignant melanoma cell lines A375 were re-suspended in 0.1 mL of PBS and subcutaneously injected into 6-week-old male nude mice (2×106 cells/mouse). The mice were euthanized 28 days after injection, and the tumors were took out and weighed.
Integrated analysis of miRNA target gene prediction database
Integrated analysis of the upstream miRNAs of TCEAL7 predication databases, including miRanda and miRDB (14-16), was conducted to identify the upstream miRNAs of TCEAL7.
Statistical analysis
All statistical analyses were performed by SPSS 19.0 software package (SPSS Inc., Chicago, IL, USA). Data from each group were expressed as mean ± standard error. Overall survival curves were calculated with the Kaplan-Meier method and were analyzed with the log-rank test. The Student’s t test and a one-way analysis of variance followed by Tukey's post hoc test were conducted to analyze differences between 2 and ≥ 2 groups, and P < 0.05 was thought as statistical significance.