AMMECR1 Inhibits Apoptosis and Promotes Cell-cycle Progression and Proliferation of the A549 Human Lung Cancer Cell Line

Materials and Methods

Cell culture. Human lung cancer cell line A549, NCI-H1299, NCI-H1975 and NCI-H460 as well as human bronchial epithelial cell line HBE were obtained from the Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, PR China). The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 μg/ml streptomycin, and 100 U/ml penicillin, and incubated at 37°C with 5% CO₂.

Cell transfection. Short hairpin RNA (shRNA) targeting AMMECR1 (H. sapiens; Accession#: NM_015365) was designed and the following sequence was generated: 5’-CCG GGA GGA TAC AAA GCT CCG ATT ACT CGA GTA ATC GGA GCT TTG TAT CCT CTT TTT G-3’. A negative control shRNA sequence was also generated as follows: 5’-AAT TCA AAA AGA GGA TAC AAA GCT CCG ATT ACT CGA GTA ATC GGA GCT TTG TAT CCT C-3’. Double-stranded DNA oligonucleotides were synthesized based on the shRNA sequences and inserted into lentiviral vectors (GV115; Jikai Gene Chemical Technology Co., Ltd., Shanghai, PR China) with AgeI/EcoRI sites. A549 cells were transfected with the shAMMECR1 lentivirus or with negative control lentivirus (shCtrl) according to the manufacturer's instructions. The transfected cells were then observed under fluorescence microscopy (Olympus, Tokyo, Japan) at 72 h transfection. Transfection efficiency was confirmed based on down-regulation of AMMECR1 expression determined by real-time polymerase chain reaction (PCR) and blotting analysis.

Quantitative real-time polymerase chain reaction (qPCR). Parental A549, NCI-H1299, NCI-H1975, NCI-H460 and HBE cells were harvested when the cultured cells reached 80% confluence. The transfected A549 cells were harvested when the cells were cultured for 72 h to 80% confluence. Total RNA was extracted from parental or transfected A549 cells using TRIzol reagent (Shanghai Pufei Biotechnology, Co., Ltd., Shanghai, PR China). RNA concentration and purity were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Approximately 2 μg total RNA was reverse-transcribed using a M-MLV reverse transcriptase kit (Promega, Madison, WI, USA) according to the manufacturer's instructions, followed by PCR amplification with specific AMMECR1 primers: 5’-AACTGACCAGGTATCGTAGTG-3’ (forward) and 5’-GGGATGCCC AATGCCATTTTG-3’ (reverse). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control with the following primers: 5’-TGACTTCAACAG CGACACCCA-3’ (forward) and 5’-CACCCTGTTGCTGTAGCCA AA-3’ (reverse). The AMMECR1 and GAPDH PCR products were 98 and 121 bp, respectively. The 2^−ΔΔCt method was used to determine the relative mRNA expression level. All reactions were repeated three times for each sample.

Western blot analysis. The transfected A549 cells were harvested after culture for 72 h to 80% confluence. The cells then were washed with phosphate-buffered saline, and lysed using ice-cold 2× lysis buffer (100 mM Tris-HCl, 0.2% β-mercaptoethanol, 0.1% glycerol, and 0.1% sodium dodecylsulfate). Total proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membranes were incubated with primary antibody to AMMECR1 or GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing twice with Tris-buffered saline, the membranes were incubated with appropriate secondary antibody conjugated with horseradish peroxidase (Ausbian, Shanghai, PR China). Electrochemiluminescence was performed according to the manufacturer's instructions using enhanced chemiluminescence (Thermo, Shanghai, PR China).

Cell proliferation assay. shAMMECR1-transfected and shCtrl-transfected cells were seeded in 96-well plates at a density of 2,000 cells/μl per well and cultured at 37°C in 5% CO₂. Green fluorescent protein (GFP) was the marker of lentivirus-transfected cells (8). GFP-expressing cells in each well were counted at different time points with a Cellomics ArrayScan VT1 Reader (Cellomics, Inc., Pittsburgh, PA, USA). The cell proliferation curve was plotted.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. shAMMECR1 and shCtrl cells were seeded in 96-well plates with 5×10³/200 μI and cultured for 5 days. To measure cell viability, 10 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 μg/μl) (ATCC, Manassas, VA USA) was added to each well and incubated for 4 h at 37°C. dimethyl sulfoxide (ShiYi Pharmaceutical Group, Shanghai, PR China) (100 μl) was then added. After 10 min shaking, cell proliferation was measured at OD₄₉₀ using a microplate reader (Tecan, Durham, NC, USA).

Apoptosis assay. To determine apoptosis levels, the annexin V-allophycocyanin (APC) single-cell staining method was utilized (9). shAMMECR1 and shCtrl cells were collected and washed twice with 4°C pre-cooled D-Hanks solution (Jikai Gene Chemical Technology Co., Ltd., Shanghai, PR China) (pH 7.2-7.4). Annexin V-APC reagent (Abcam, Shanghai, China) (10 μl) was added and cells were incubated in the dark at room temperature for 10 min. Flow cytometric analysis was performed on a fluorescence-activated cell sorting (FACS) Calibur platform (Guava easyCyte HT; Millipore).

Cell-cycle assay. Cell-cycle phase distribution was measured by staining DNA with propidium iodide. shAMMECR1-transfected and shCtrl-transfected cells were seeded in 6-well plates and cultured at 37°C and 5% CO₂. When the cells reached 80% confluency, they were harvested and fixed in 70% ethanol (precooled 4°C) for at least 1 h. Then the cells were centrifuged at 300 × g for 5 min, and washed with precooled D-Hanks (4°C; pH 7.2-7.4). Based on the cell count, 0.6-1 ml cell-staining solution (40× propidium iodide, 2 mg/ml; Sigma-Aldrich, Saint Louis, MO, USA), 100× RNase A solution (10 mg/ml; Fermentas, Belgium) and 1× D-Hanks were added at a ratio of 25:10:1000. The cell suspension was analyzed using a FACS Calibur platform (Guava easyCyte HT) at a flow rate of 300-800 cells/s.

Colony-formation assay. shAMMECR1-transfected and shCtrl-transfected cells were seeded in 6-well plates at a density of 400-1,000 cells per well. The cells were then cultured and observed over 14 d. Cell colonies were imaged by GFP expression with a fluorescence microscope (Olympus IX71; Olympus, Japan). At the end of the culture period, the cells were washed with phosphate-buffered saline, and stained with Giemsa (Shanghai Dingguo Biotech Co., Ltd., Shanghai, PR China). After washing with double-distilled water, the cells were allowed to dry and were then imaged to obtain colony counts.

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Figure 1.

Expression of Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis chromosomal region gene 1 (AMMECR1) was elevated in lung cancer cell lines compared to a normal cell line. AMMECR1 expression level was examined in lung cancer cell lines A549, NCI-H1299, NCI-H1975, NCI-H460 and human bronchial epithelial cell line HBE using quantitative polymerase chain reaction. AMMECR1 was significantly elevated in all four human lung cancer cell lines compared to HBE cells. **Significantly different at p<0.01.

Statistical analysis. All data are presented as mean values±standard deviation and were analyzed using SPSS 19 (SPSS, Chicago, IL, USA). Comparisons between groups were performed using Student's t-test. p-Values of less than 0.05 are considered to indicate a statistically-significant difference.