Human tissue samples and cell lines
Primary lung cancer samples (90) were collected from the Chinese PLA General Hospital and 33 primary samples were collected from the tumor hospital of Henan. The median age of the cancer patients was 60 years old (range from 29 to 79). Fifteen cases of normal lung tissue were collected from the Chinese PLA General Hospital. Among 123 cancer samples, only 40 cases were available for paraffin samples with matched cancer and adjacent tissue. All samples were collected following the guidelines approved by the Institutional Review Board of the Chinese PLA General Hospital and the tumor hospital of Henan with written informed consent from patients. Nine lung cancer cell lines (H157, H1563, H727, H358, H446, H460, H23, H1299 and A549) were previously established from primary lung cancer and grown in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin/streptomycin solution (Sigma, St. Louis, MO).
5-Aza-2-deoxycytidine and SCH772984 treatment
For methylation regulation analysis, lung cancer cell lines were split to low density (30% confluence) 12 hours before treatment. Cells were treated with 5-Aza-2-deoxycytidine (DAC, Sigma, St. Louis, MO, USA) at a concentration of 2 µM in the growth medium, which was exchanged every 24 hours for a total of 96 hours and cultured at 37◦C in a 5% CO2 incubator. At the end of the treatment period, cells were prepared for extraction of total RNA. To verify the role of TMEM176A in ERK signaling, SCH772984, an ERK inhibitor, was added to TMEM176A knocking down H727 cell at 2μm for 24h (MedChemExpress, Monmouth Junction, USA) (28).
RNA isolation and semi-quantitative RT-PCR
Total RNA was extracted using Trizol Reagent (Life Technologies, Carlsbad,CA,USA). Agarose gel electrophoresis and spectrophotometric analysis were used to detect RNA quality and quantity. First strand cDNA was synthesized according to manufacturer’s instructions (Invitrogen, Carlsbad, CA). A total of 5µg RNA was used to synthesize first strand cDNA. The reaction mixture was diluted to 100µl with water, and then 2µl of diluted cDNA was used for 25µl PCR reaction. The PCR primer sequences for TMEM176A were as follows: 5′-GGG AAC AGC CGA CA G TGA T-3′ (F) and 5′-GCC AGC GTT AGC AGA GTC CT-3′(R). PCR cycle conditions were as follows: 95°C 5 min, 1 cycle; (95°C 30 s, 60°C 30 s and 72°C 30 s) 32 cycles; 72°C 5 min, 1 cycle. PCR product size is 369bp. GAPDH was amplified for 25 cycles as an internal control. The GAPDH primer sequences were as follows: 5′-GAC CAC AGT CCA TGC CAT CAC-3′ (F), and 5′-GTC CAC CAC CCT GTT GCT GTA-3′ (R). PCR cycle conditions were as follows: 95°C 5 min, 1 cycle; (95°C 30 s, 63°C 30 s and 72°C 30 s) 25cycles; 72°C 5 min, cycle. PCR product size is 448bp. The amplified PCR products were examined by 2% agarose gels.
DNA extraction, bisulfite modification, methylation specific PCR (MSP)
Genomic DNA from lung cancer cell lines and lung cancer tissue samples were prepared using the proteinase-K method. Normal lymphocyte DNA was prepared from healthy donor blood lymphocytes by proteinase-K method (29). Normal lymphocyte DNA (NL) was used as a control for unmethylation and in vitro methylated DNA (IVD) was used as a methylation control. IVD was prepared using SssI methylase (New England Biolabs, Ipswich, MA, USA) following the manufacturer's instructions. MSP primers were designed according to genomic sequences inside the CpG islands in the TMEM176A gene promoter region.
MSP primers for TMEM176A were designed -364 to -203bp upstream of the transcription start site (TSS) and synthesized to detect methylated (M) and unmethylated (U) alleles. The detected region has been previously reported to be hypermethylated and associated with low expression (30). MSP primers for TMEM176A were as follows: 5′-GTT TCG TTT AGG TTG CGC GGT TTT TC -3′ (MF), 5′-CCA AAA CCG ACG TAC AAA TAT ACG CG-3′ (MR); 5′-TGG TTT TGT TTA GGT TGT GTG GTT TTT T-3′ (UF),
5′-CAA CCA AAA CCA ACA TAC AAA TAT ACA CA -3′ (UR).
PCR cycle conditions were as follows: 95°C 5 min, 1 cycle; (95°C 30 s, 60°C 30 s and 72°C 30 s) 35cycles; 72°C 5 min, 1 cycle.
Bisulfite sequencing (BSSQ) primers encompassed a 231bp region upstream of the TMEM176A transcription start site (-388bp to -157bp) and included the region analyzed by MSP. BSSQ primers were designed as follows:
5′-GAG ACG GTA GAT GTA CGG GT-3′ (F);
5′- AAC RAA CRA CCC TAA AAA AAC CC -3′ (R). PCR cycle conditions were as follows: 95°C 5 min, 1 cycle; (95°C 30 s, 55°C 30 s and 72°C 30 s) 35cycles; 72°C 5 min, 1 cycle.
Immunohistochemistry
Immunohistochemistry (IHC) was performed in primary lung cancer samples and matched adjacent tissue samples. TMEM176A antibody was diluted to 1:50 (Cat: HPA008770, Sigma, St. Louis, MO, USA). The expression of MMP2, MMP9 and p-ERK1/2 was detected in H1299 cell xenografts. MMP2, MMP9 and p-ERK1/2 antibody was diluted to 1:100, 1:100 (Protein Tech Group, Chicago, IL, USA) and1:400 (Cell Signaling Technology, Danfoss, MA, USA). The procedure was performed as described previously (31). The staining intensity and extent of the staining area were scored using the German semi-quantitative scoring systems as previously described (31-33). Staining intensity of the membrane and/or cytoplasm was characterized as follows: no staining = 0, weak staining = 1, moderate staining = 2, strong staining = 3; the extent of staining was defined as: 0% = 0, 1–24% = 1, 25–49% = 2, 50–74% = 3, 75–100% = 4. The final immune-reactive score (0–12) was determined by multiplying the intensity score by the extent of staining score.
Construction of lentiviral TMEM176A expression vectors and selection of stable expression cells
The human full length TMEM176A cDNA (NM-018487.2) was cloned into the pLenti6 vector. Primers were as follows: 5′- CTT AGG ATC CGC CAC CAT GGG AAC AGC CGAC -3′ (F) and 5′- ACT TAG TCG ACC TAG ATT CCA CTC ACT TCC -3′(R). The HEK-293T cell line was maintained in DMEM (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum. TMEM176A expressing Lentiviral vector was transfected into HEK-293T cells (5.5 × 106 per 100 mm dish) using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad,CA,USA) at a ratio of 1:3 (DNA mass: Lipo mass). Viral supernatant was collected and filtered after 48 hours. H23 and H1299 cells were then infected with viral supernatant. H23 and H1299 cells stably expressing TMEM176A were selected with Blasticidin (Life Technologies, Carlsbad,CA, USA) at concentrations of 6.0μg/ml and 5.0μg/ml for 2 weeks, respectively.
RNA interference assay
Two sets of targeting siRNA for TMEM176A and one set of RNAi negative control duplex sequence are as follows:
SiTMEM176A1 duplex (sense: 5′-GGC UAC UCU UAU UAC AAC ATT-3′; antisense: UGU UGU AAU AAG AGU AGC CTT-3′),
SiTMEM176A2 duplex (sense: 5′-CUG UAC UGC UGG AGA AUG UTT-3′; antisense: 5′-ACA UUC UCC AGC AGU ACA GTT-3′),
SiTMEM176A negative control duplex (SiTMEM176ANC, sense: 5′-ACA UUC UCC AGC AGU ACA GTT-3′; antisense: 5′-ACG UGA CAC GUU CGG AGA ATT-3′). SiTMEM176A2 was found more effective than SiTMEM176A1, and SiTMEM176A2 was applied to further study (GenePharma Co. Shanghai, China).
Cell viability detection
H23 and H1299 cells were seeded into 96-well plates before and after re-expression of TMEM176A at 1×103 cells/well. H727 cell were plated into 96-well plates before and after knockdown of TMEM176A at a density of 5×103 cells/well. The cell viability was measured by MTT(3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide) assay at 0h, 24h, 48h, 72h, 96h (KeyGENBiotech, Nanjing, China). Absorbance was measured on a microplate reader (Thermo Multiskan MK3, MA, USA) at a wavelength of 490nm. Each experiment was repeated three times.
H23 and H1299 cells were seeded into 96-well plates at 4×103 cells/well and 3×103 cells/well irrespectively. The IC50 value was detected by the MTT assay before and after re-expression of TMEM176A. H727 cell were plated into 96-well plates at a density of 8×103 cells/well before and after knockdown of TMEM176A. Cells were treated with AZD0156 for 24 hours at 0, 0.001, 0.01.0.1, 1, 10, 100 and 1000 μm/L after seeded for 24 hours (34). Each experiment was repeated for three times.
Colony Formation Assay
TMEM176A unexpressed and re-expressed H23 and H1299 cells were seeded in 6-well plates at a density of 200 cells per well. Before and after knockdown of TMEM176A, H727 cells were seeded into 6-well plates at a density of 300 cells per well. After 2 weeks, cells were fixed with 75% ethanol for 30 min. Colonies were then stained with 0.5% crystal violet solution and counted. The experiment was performed in triplicate.
Flow cytometry
TMEM176A unexpressed and stably expressed H23 and H1299 cells were starved for 12 hours to synchronize, and cells were re-stimulated with 10% FBS for 24 hours. Cells were fixed with 70% ethanol and treated using the Cell Cycle Detection Kit (KeyGen Biotech, Nanjing, China). Cells were then detected using a FACS Caliber flow cytometer (BD Biosciences, CA, USA). The cell cycle was analyzed also for H727 cells with or without knocking down TMEM176A. Cell phase distribution was analyzed using the Modfit software (Verity Software House, ME, USA).
To increase the sensitivity of apoptosis detection, TMEM176A unexpressed and stably re-expressed H23 and H1299 cells were treated with Doxorubicin at 0.8µg/ml and 0.6µg/ml for 24 hours respectively (35). Apoptosis was also analyzed in H727 cell with or without knockdown of TMEMA176. The cells were prepared using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer’s instructions and then sorted by FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA). Each experiment was repeated three times.
Transwell assay
For migration study, cells were suspended in 200µl serum-free RPMI 1640 media, and 5 × 104 H23 and 2 × 104 H1299 cells were added to the upper chamber of an 8.0µm pore size transwell apparatus (COSTAR Transwell Corning Incorporated, Tewksbury,MA, USA). Cells were stained with crystal violet and counted in three independent high-power fields (×100) after incubation for 16 hours (H23 cells) or 16 hours (H1299). Each experiment was repeated for three times.
For invasion assay, H23 cells (1 × 105) and H1299 cells (5 × 104) were seeded to the upper chamber of a transwell apparatus coated with Matrigel (BD Biosciences, CA, USA) and incubated for 36 hours (H23) and 36 hours (H1299). Each experiment was repeated three times.
Western Blot
Cells were collected 48h after transfection and cell lysates were prepared using ice-cold Tris buffer (20 mmol/L Tris; pH 7.5) containing 137 mmol/L NaCl, 2 mmol/L EDTA, 1% Triton X, 10% glycerol, 50 mmol/L NaF, 1 mmol/L DTT, PMSF, and a protein phosphatases inhibitor (Applygen Tech. Beijing, China). For extracellular signal-regulated kinase (ERK) signaling analysis, cells were starved with serum free medium for 24 h after transfection. These cells were then stimulated with medium containing 10% serum for 60 min before collection. To analyze the sensitivity of AZD0156, cells were exposed to UV 20 mJ/cm2 for 2h before treatment with 0.5μm AZD0156 or ethanol (control), and cells were collected after 24h treatment (36). Western blot was performed as described previously (31).
Primary antibodies were as follows: TMEM176A (Sigma, St. Louis, MO), cleaved caspase-3 (Protein Tech Group, Chicago, IL, USA), MMP2 (Protein Tech Group, Chicago, IL, USA), MMP9 (Protein Tech Group, Chicago, IL, USA), cyclin B1 (Protein Tech Group, Chicago, IL, USA), CDC2 (Protein Tech Group, Chicago, IL, USA), ATM (HuaXingBoChuang, China),γ-H2AX (HuaXingBoChuang, China), p-CHK2(Zhengneng, China), ERK1/2 (Protein Tech Group, Chicago, IL, USA), p-ERK1/2 (Cell Signaling Technology, Danfoss, MA, USA), SAR1A (Protein Tech Group, Chicago, IL, USA) and β-actin (Beyotime Biotech, Nanjing, China).
Lung cancer cell xenograft mouse model
H1299 cell lines stably transfected with plenti6 vector or plenti6-TMEM176A vector (1× 107 cells diluted in phosphate-buffer saline) were injected subcutaneously into the dorsal left side of 4-week-old female Balb/c nude mice. Each group included six mice. Tumor volume was measured every 4 days. Tumor volume was calculated according to the formula: V=L×W2 /2, in which V represents volume (mm3), L represents biggest diameter (mm), and W represents smallest diameter (mm). Mice were sacrificed on the 24th day after inoculation and tumors were weighed. All procedures were approved by the Animal Ethics Committee of the Chinese PLA General Hospital.
Data Analysis
RNASeq data for TMEM176A gene expression in the dataset of lung cancer and normal tissues were downloaded from the Cancer Genome Atlas (TCGA) (http://xena.ucsc.edu/, 09/16/2019). Statistical analysis was performed using SPSS 17.0 software (SPSS, Chicago, IL). Chi-square test was used to evaluate the relationship between methylation status and clinicopathological characteristics. The 2-tailed independent samples t-test was applied to determine the statistical significance of the differences between the two experimental groups. Two-sided tests were used to determine the statistical difference, and P<0.05 was considered statistically significance.