Cell culture and hypoxic culture conditions
The NSCLC cell line NCI-H520 was purchased from the American Type Culture Collection (Manassas, VA, USA). The A549, NCI-H460, and NCI-H1975 cell lines were purchased from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The NSCLC cell lines were maintained in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS) and 5 mg/mL penicillin/streptomycin at 37 °C. Human pulmonary microvascular endothelial cells (HPMEC) were purchased from ScienCell Research Laboratories (San Diego, CA, USA) and maintained in endothelial cell medium (ScienCell) supplemented with 10% FBS and 5 mg/mL penicillin/streptomycin at 37 °C. Cells under normoxic conditions were incubated at 21% O2 and 5% CO2. Cells under hypoxic conditions were incubated at 1% O2 and 5% CO2.
Western blotting analysis
To extract proteins for western blot analysis, cells were first lysed in RIPA buffer supplemented with proteinase inhibitors (Pierce Biotechnology, Rockford, IL, USA). Equivalent concentrations of protein measured by a BCA Protein Assay Kit (Thermo Fisher Scientific) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% non-fat milk for 1 h and then incubated overnight at 4 °C with primary antibodies against Tie1 (1:1000, Abcam, Cambridge, UK), BMI-1 (1:1000, Abcam), LGR5 (1:1000; Bioss, Beijing, China), CD44 (1:1000, Cell Signaling Technology, Danvers, MA, USA), HIF-1α (1:2000, Abcam), β-actin (1:5000, Abcam), and HA-tag (1:3000, Proteintech, Wuhan, China). Following incubation for 1 h with HRP-conjugated secondary antibodies (Cell Signaling Technology), immunoreactive bands were visualized with an enhanced chemiluminescence detection system (Thermo Fisher Scientific). The relative intensities of target proteins were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Quantitative reverse transcription PCR (RT-qPCR)
Total RNA was extracted from cells with Trizol reagent (Invitrogen, Carlsbad, CA, USA). A PrimeScript RT reagent kit (Takara, Kyoto, Japan) was used to synthesize cDNA and an SYBR Green Realtime PCR Premix (Takara) was used in qPCR with the primer sequences: Tie1, Forward 5′-TTGTGCCCCTGGTCATTTTG-3′, Reverse 5′-TCCAGTTCTGAGGCCATGTT-′; GAPDH, Forward 5′-TCAAGAAGGTGGTGAAGCAGG-3′, Reverse 5′-TCAAAGGTGGAGGAGTGGGT-3′. The relative abundance of mRNA was normalized to GAPDH and the 2−ΔΔCT method was used to analyze expression levels.
Cell viability assays
Cells were incubated for 48 h with a gradient of cisplatin concentrations (0−32 μM; Sigma-Aldrich, St. Louis, MO, USA). Cell viability was assessed using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, cells (~3×103) were grown under normoxic or hypoxic conditions in 96-well plates with or without cisplatin for up to 48 h. CCK-8 solution (10 μl) was added to each well and after 2 h incubation at 37 °C, formazan dye was measured at 450 nm absorbance on a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) and compared to a standard curve.
Sphere formation assay
To assess the formation of spheres, cells (1×106 per well) preincubated under normoxic or hypoxic conditions were seeded in six-well plates in complete medium. Sphere formation was induced by the addition of 2% B27, 20 ng/mL basic fibroblast growth factor, and 20 ng/mL epidermal growth factor in ultralow attachment six-well plates. After 7 days of incubation, spheres with a diameter greater than 50 μM were counted at ×40 magnification.
Cell migration assay
Cell migration was assessed with the aid of 24-well plate 8-mm pores Transwell inserts (Millipore). Cells (1× 104) were plated into the top chamber and allowed to migrate into the lower chamber for 24 h. Migrated cells were stained with 0.2% crystal violet and counted at ×40 magnification.
Immunofluorescence (IF) staining
Cells were fixed with 4% paraformaldehyde, washed with PBS, and then blocked with10% goat serum. They were then incubated with HIF-1α (1:200, Abcam) antibodies overnight at 4 °C. After incubation, cells were washed twice with PBS and stained with Cy3 (red)-conjugated secondary antibody for a further 2 h at 37 °C. Surplus antibody was removed by washing before obtaining images with an Olympus microscope (Olympus, Tokyo, Japan) at ×40 magnification. Images were captured with a DP50 camera and DP50 software (Olympus).
Plasmid constructs, lentivirus production, and transfection
The stable knockdown of Tie1 was obtained by transfecting A549 and NCI-H1975 cells with lentiviral constructs containing short hairpin RNA (shRNA) sequences. The target sequences 5′-GAGAACCTAGCCTCCAAGATT-3′ of Tie1 shRNA were cloned into the vector GV248 (Genechem, Shanghai, China). A non-silencing scrambled shRNA was used as a negative control (5′-GGCAAGACATACGCTCTCATA-3′). Small interfering RNA (siRNA) against human HIF-1α (target sequences: 5′-GACGATCATGCAGCTACTACA-3′) and negative control (5′-GGCACTATCCAACGGTAATCA-3′) were synthesized by GenePharma (Shanghai, China) and transfected into A549 and NCI-H1975 cells. To construct a HIF-1α overexpression vector, the full-length human HIF-1α was cloned into a pcDNA3.1 vector. The dominant-negative HIF-1α construct lacking DNA binding and activation domains was generated by PCR and cloned into a pcDNA3-HA vector and transfected into A549 and NCI-H1975 cells. All constructs were confirmed by sequencing and all cells were transfected with vectors or siRNA using Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer’s instructions.
Promoter luciferase reporter assays
The HRE sites in the promoter of Tie1 were assessed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The consensus sequence of the HRE sites in the 1500-bp fragment of the human Tie1 promoter (from −1500 to −1 bp relative to the translation start site) were mutated by substituting CG with AT. The DNA fragment was then cloned into vector pGL3 to generate the construct pGL3-Tie1-Luc. The luciferase reporter vector containing the Tie1 promoter was then transfected into A549 cells together with the pRL-TK Renilla luciferase plasmids. Cells were transfected using Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer’s instructions. Luciferase activity was measured after 48 h incubation under hypoxic conditions.
Chromatin immunoprecipitation (ChIP) assay
Binding between the promoter region of Tie1 and HIF-1α was assessed used a ChIP assay kit (Millipore) following the manufacturer’s instructions. Briefly, A549 cells were incubated under hypoxic conditions for 24 h and then sonicated to obtain fragmented DNA. Chromatin was immunoprecipitated with anti-HIF-1α (Novus Biologicals, Littleton, CO, USA) or rabbit IgG (Sigma-Aldrich). The region in the Tie1 promoter containing the HIF-1α-binding site (5′-CTCGTG-3′, from −1032 to −1038 bp relative to the translation start site) was detected and amplified by PCR using the following primers: forward 5′-CATCCCAACCATTCCATTCCG-3′ and reverse 5′-TTCCCAGAACGGAACAAGACC-3′.
Mouse xenograft model
All animal experiments were performed in accordance with the guidelines of the Laboratory Animal Ethical Committee at Shanghai Jiao Tong University (Approval number B-2018-010). BALB/c male nude mice (4–6 weeks old) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and acclimatized for 1 week in specific pathogen-free conditions with sterile food and water. A549 cells stably transfected by scramble or Tie1-shRNA lentiviruses were subcutaneously injected into the right flank of the nude mice. Mice were randomly divided into control (saline) or cisplatin groups (n = 6, 2 mg/kg body weight delivered by intraperitoneal injection three times a week over 3 weeks). Tumor growth was measured every 5 days. The tumor volume was determined by the formula: Volume (mm3) = Length × (Width)2. At the end of the experiment mice were euthanized by cervical decapitation and tumors were excised and weighed. Hematoxylin and eosin (H&E) staining and immunohistochemistry analysis (IHC) of paraffin sections from xenograft samples was performed using antibodies against Ki67 (1:500, Cell Signaling Technology) and LGR5 (1:150, Abcam). For the limiting dilution tumorigenicity assay, each nude mouse was injected with different concentrations of cells (1 ×106, 1 ×105, 1 ×104, or 1×103 cells in 100 μl DMEM, n = 5) subcutaneously in the flank. The volume of the tumors was observed and recorded after 5 weeks.
All data were analyzed using the statistical software package GraphPad Prism 7.0 (GraphPad, San Diego, CA, USA). Statistical significance between two groups was determined using the Student’s t test, and comparisons among more than two groups were performed using analysis of variance (ANOVA). All in vitro experiments were repeated at least three times independently. Data are presented as the mean ± standard deviation (SD) unless otherwise stated. P < 0.05 was considered significant.