Cell culture and generation of stable cell lines. Human bronchial epithelial BEAS-2B cells [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured in DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS). Human umbilical vein endothelial cells (HUVEC) (purchased from ATCC) were cultured in EBM-2 (endothelial basal medium-2) complete medium. Arsenic-transformed BEAS-2B (AsT) cells (Carpenter et al. 2011 (link)) stably overexpressing miR-199a or miR-control were generated by infecting BEAS-2B cells with lentivirus carrying miR-199a-RFP or a negative control precursor (Applied Biosystems, Carlsbad, CA, USA), followed by selection with puromycin. To establish stable cell lines overexpressing COX-2 (cyclooxygenase-2), 293T cells were transfected with lentivirus carrying the COX-2 plasmid (GeneCopoeia, Rockville, MD, USA) or an empty vector to generate infectious virus. Then AsT cells were transduced with virus, followed by the puromycin selection. We also prepared PI3K (phosphatidylinositide 3-kinase)–transformed chicken embryo fibroblast cells as was previously described (Chang et al. 1997 (link); Jiang et al. 2000 (link)).
Animal experiment. Female CrTac:NCr-Foxn1nu mice (8 weeks of age) were purchased from Taconic (Hudson, NY, USA), and maintained in pathogen-free conditions. Animals were housed in sterilized cages (5 mice/cage) with hardwood chip bedding. Standardized commercial diets were provided, and sterilized water was available at all times. The average weight of animals on arrival was 20 ± 2 g (mean ± SD). A total of 2 × 106 AsT/miR-cont cells or AsT/miR-199a cells (AsT cells stably overexpressing miR-control or miR-199a, respectively) in 80 μL were injected subcutaneously into the flanks of nude (nu/nu) mice (n = 10/group). The animals used in research were treated humanely according to the Institutional Animal Care and Use Committee of Thomas Jefferson University. The mice were euthanized by decapitation 6 weeks after injection. Tumor tissues were removed and weighed. Parts of tissues were paraffin-embedded, and other parts were snap-frozen in liquid nitrogen and stored at –80°C for immunohistochemical analysis.
Reagents and antibodies. Sodium arsenic, catalase, and H2O2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Small interfering RNA (siRNA) Smartpools (pools of four individual siRNAs) against COX-2, HIF-1α (hypoxia inducible factor 1, alpha), and a scrambled control were purchased from Dharmacon (Lafayette, CO, USA). COX-2 antibody was from Cell Signaling Technology (Beverly, MA, USA). CD31 (platelet/endothelial cell adhesion molecule 1) antibody for analyzing paraffin-embedded tissues was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and for analyzing frozen tissues, from BD Pharmingen (San Jose, CA, USA). HIF-1α antibody was from BD Biosciences (Franklin Lakes, NJ, USA), and α-SMA (smooth muscle α-actin) antibody was obtained from Abcam (Cambridge, MA, USA). Primary antibodies used for Western analysis were diluted 1:1,000 in 5% bovine serum albumin as a working concentration and were incubated on a shaker overnight at 4°C.
RT-qPCR analysis. Total RNAs were extracted using Trizol (Life Technologies, Carlsbad, CA, USA). The synthesis of cDNA was performed using oligo (dT)18 primers and M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase (Promega, Madison, WI, USA). The amplification was performed by polymerase chain reaction (PCR). SYBR-green reverse transcription PCR (RT-qPCR) was performed to detect COX-2 and GADPH (glyceraldehyde-3-phosphate dehydrogenase) mRNA levels using the Power SYBR Green PCR Master Mix Kit (Applied Biosystems). Taqman RT-qPCR was performed to detect miRNA expression levels using the Taqman miRNA Reverse Transcription kit and Taqman universal PCR Master Mix (Applied Biosystems). The sequences of primer used for SYBR-green RT-qPCR were as follows:
Immunohistochemistry and immunofluorescence. Paraffin-embedded tissue sections and frozen tissue sections were prepared by routine methods (Fero Lab, Fred Hutchinson Cancer Research Center 2011 ). For immunohistochemistry, the Dako Envision two-step method of immunohistochemistry was used to stain CD31 (1:100 dilution) and α-SMA (1:150 dilution) in xenograft tumor tissues as described previously (He et al. 2012a (link)). Tissue sections were incubated with primary antibodies in a humid chamber overnight at 4°C. The microvessel density reflected by CD31-positive staining was counted in three different fields per section. For immunofluorescence staining, frozen sections were incubated with primary antibodies overnight. Goat anti-rabbit or anti-mouse IgG conjugated with fluorescein isothiocyanate or Texas Red (both from Santa Cruz Biotechnology) (1:200 dilution) were used as secondary antibodies and incubated for 2 hr at room temperature. Slides were mounted with anti-fade DAPI reagent (Invitrogen, Grand Island, NY, USA).
Chromatin immunoprecipation (ChIP)–quantitative PCR (qPCR). ChIP-qPCR was performed using the EpiTect ChIP OneDay Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. HIF-1α antibody (Abcam) was used to pull down the protein–chromatin complexes. Rabbit IgG was used as a negative control. The immunoprecipated DNA was quantified using SYBR Green qPCR (Applied Biosystems). All results were normalized to 1% input value of the same sample. COX-2 primers flanking the hypoxia-response elements (HRE) for SYBR Green qPCR were as follows:
Prostaglandin E2 (PGE2) ELISA. Cells were plated at 0.1 × 106 cells/well of a 24-well plate and allowed to recover overnight. The following day, the media were replaced with fresh media, and then the cells were cultured in normoxia or hypoxia (1% O2) for 24 hr. The conditioned media were then collected and cleared of cellular debris by centrifugation at 2,000 rpm for 2 min. PGE2 concentrations were determined using the Prostaglandin E2 EIA Kit–Monoclonal ELISA kit (no. 514010; Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions.
miRNA luciferase reporter constructs and luciferase activity assay. The 3´UTR-luciferase reporter constructs containing the 3´UTR regions of COX-2 with wild-type and mutant binding sites of miR-199a were amplified using the PCR method (GoTaq® G2 Flexi DNA Polymerase; Promega) according to the manufacturer’s instructions. The PCR products were cloned into the pMiR-luc luciferase reporter vector (Ambion, Grand Island, NY, USA). The mutant 3´UTR constructs were made by introducing four point mutations into the putative seed regions of COX-2. All the constructs containing 3´UTR inserts were sequenced and verified. The luciferase activity assay was performed as previously described (He et al. 2013a (link)).
Site-directed mutagenesis. The human full-length COX-2 reporter used was a generous gift from J. Li (Harvard University, Boston, MA, USA) (Wu et al. 2006 (link)). To generate the HRE-mutant COX-2 reporter, we performed site-directed mutagenesis on the wild-type COX-2 reporter at the potential HIF-1α binding sites with 3 base pair substitutions as previously described (Jiang et al. 1996 (link)). The mutant COX-2 reporter construct was validated by DNA sequencing.
Tube formation assay. HUVEC were cultured in EBM-2 complete medium, and switched to EBM-2 basal medium containing 0.2% FBS for 24 hr before performing the tube formation assay. The conditioned media were prepared from different cells by replacing normal culture medium with serum-reduced medium (1% FBS). After culture for 24 hr, the serum-reduced media were collected and stored at –20°C for later use. The HUVEC were trypsinized, counted, and resuspended in EBM-2 basic medium; then they were mixed with an equal volume of the conditioned medium and seeded on Matrigel-pretreated 96-well plates at 2 × 104 cells/well. After culture for 6-12 hr, tube formation was observed under a light microscope and photographed. The total lengths of the tubes in each well were measured using CellSens Standard software (Olympus; Hamburg, Germany).
The chorioallantoic membranes (CAM) assay. White Leghorn fertilized chicken eggs (Charles River, Malvern, PA, USA) were incubated at 37°C under constant humidity. Cells were transfected with miRNA precursors, or treated as specifically indicated in the figure legends. After transfection for 12 hr, the cells were trypsinized, counted, and resuspended in the serum-free medium. The cell suspensions were mixed with Matrigel at a 1:1 ratio, and implanted onto the CAM of chicken eggs on day-9 embryos. Tumor angiogenesis responses were analyzed 5 days after the implantation. The tumor/Matrigel plugs were trimmed off the CAM and photographed. The number of blood vessels as the index of angiogenesis was analyzed by counting the branches of blood vessels in three representative areas (each 1.0 mm2) by two observers in a double blind manner.
miRNA transfection. Cells were cultured in 6-well plates to reach a 60% confluency, and transfected using miR-199a or a negative-control precursor (both from Applied Biosystems) at 30 nM using Lipofectamine RNAiMAX reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. Total proteins and RNAs were prepared from the cells 60–70 hr after the transfection, followed by Western blotting or RT-PCR analysis.
Hypoxia treatment. A hypoxia incubator chamber (Stemcell, Vancouver, British Columbia, Canada) was used to generate the hypoxia environment for cell culture. Cells were cultured in hypoxia conditions (1% O2) for 24 hr at 37°C.
Reactive oxygen species (ROS) study. Cells were treated with ROS scavenger catalase (1,500 U) or with H2O2 (50 μM) for 12 hr. Then cells were harvested, and total proteins were extracted for Western blotting analysis.
Statistical analysis. All the results were obtained from at least three independent experiments. Results are presented as mean ± SE and were analyzed by Student’s t-test or one-way analyis of variance (ANOVA). All results were analyzed by SPSS for Windows, version 11.5 (IBM, Chicago, IL, USA). Differences were considered significant with p < 0.05.
Animal experiment. Female CrTac:NCr-Foxn1nu mice (8 weeks of age) were purchased from Taconic (Hudson, NY, USA), and maintained in pathogen-free conditions. Animals were housed in sterilized cages (5 mice/cage) with hardwood chip bedding. Standardized commercial diets were provided, and sterilized water was available at all times. The average weight of animals on arrival was 20 ± 2 g (mean ± SD). A total of 2 × 106 AsT/miR-cont cells or AsT/miR-199a cells (AsT cells stably overexpressing miR-control or miR-199a, respectively) in 80 μL were injected subcutaneously into the flanks of nude (nu/nu) mice (n = 10/group). The animals used in research were treated humanely according to the Institutional Animal Care and Use Committee of Thomas Jefferson University. The mice were euthanized by decapitation 6 weeks after injection. Tumor tissues were removed and weighed. Parts of tissues were paraffin-embedded, and other parts were snap-frozen in liquid nitrogen and stored at –80°C for immunohistochemical analysis.
Reagents and antibodies. Sodium arsenic, catalase, and H2O2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Small interfering RNA (siRNA) Smartpools (pools of four individual siRNAs) against COX-2, HIF-1α (hypoxia inducible factor 1, alpha), and a scrambled control were purchased from Dharmacon (Lafayette, CO, USA). COX-2 antibody was from Cell Signaling Technology (Beverly, MA, USA). CD31 (platelet/endothelial cell adhesion molecule 1) antibody for analyzing paraffin-embedded tissues was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and for analyzing frozen tissues, from BD Pharmingen (San Jose, CA, USA). HIF-1α antibody was from BD Biosciences (Franklin Lakes, NJ, USA), and α-SMA (smooth muscle α-actin) antibody was obtained from Abcam (Cambridge, MA, USA). Primary antibodies used for Western analysis were diluted 1:1,000 in 5% bovine serum albumin as a working concentration and were incubated on a shaker overnight at 4°C.
RT-qPCR analysis. Total RNAs were extracted using Trizol (Life Technologies, Carlsbad, CA, USA). The synthesis of cDNA was performed using oligo (dT)18 primers and M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase (Promega, Madison, WI, USA). The amplification was performed by polymerase chain reaction (PCR). SYBR-green reverse transcription PCR (RT-qPCR) was performed to detect COX-2 and GADPH (glyceraldehyde-3-phosphate dehydrogenase) mRNA levels using the Power SYBR Green PCR Master Mix Kit (Applied Biosystems). Taqman RT-qPCR was performed to detect miRNA expression levels using the Taqman miRNA Reverse Transcription kit and Taqman universal PCR Master Mix (Applied Biosystems). The sequences of primer used for SYBR-green RT-qPCR were as follows:
Immunohistochemistry and immunofluorescence. Paraffin-embedded tissue sections and frozen tissue sections were prepared by routine methods (Fero Lab, Fred Hutchinson Cancer Research Center 2011 ). For immunohistochemistry, the Dako Envision two-step method of immunohistochemistry was used to stain CD31 (1:100 dilution) and α-SMA (1:150 dilution) in xenograft tumor tissues as described previously (He et al. 2012a (link)). Tissue sections were incubated with primary antibodies in a humid chamber overnight at 4°C. The microvessel density reflected by CD31-positive staining was counted in three different fields per section. For immunofluorescence staining, frozen sections were incubated with primary antibodies overnight. Goat anti-rabbit or anti-mouse IgG conjugated with fluorescein isothiocyanate or Texas Red (both from Santa Cruz Biotechnology) (1:200 dilution) were used as secondary antibodies and incubated for 2 hr at room temperature. Slides were mounted with anti-fade DAPI reagent (Invitrogen, Grand Island, NY, USA).
Chromatin immunoprecipation (ChIP)–quantitative PCR (qPCR). ChIP-qPCR was performed using the EpiTect ChIP OneDay Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. HIF-1α antibody (Abcam) was used to pull down the protein–chromatin complexes. Rabbit IgG was used as a negative control. The immunoprecipated DNA was quantified using SYBR Green qPCR (Applied Biosystems). All results were normalized to 1% input value of the same sample. COX-2 primers flanking the hypoxia-response elements (HRE) for SYBR Green qPCR were as follows:
Prostaglandin E2 (PGE2) ELISA. Cells were plated at 0.1 × 106 cells/well of a 24-well plate and allowed to recover overnight. The following day, the media were replaced with fresh media, and then the cells were cultured in normoxia or hypoxia (1% O2) for 24 hr. The conditioned media were then collected and cleared of cellular debris by centrifugation at 2,000 rpm for 2 min. PGE2 concentrations were determined using the Prostaglandin E2 EIA Kit–Monoclonal ELISA kit (no. 514010; Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions.
miRNA luciferase reporter constructs and luciferase activity assay. The 3´UTR-luciferase reporter constructs containing the 3´UTR regions of COX-2 with wild-type and mutant binding sites of miR-199a were amplified using the PCR method (GoTaq® G2 Flexi DNA Polymerase; Promega) according to the manufacturer’s instructions. The PCR products were cloned into the pMiR-luc luciferase reporter vector (Ambion, Grand Island, NY, USA). The mutant 3´UTR constructs were made by introducing four point mutations into the putative seed regions of COX-2. All the constructs containing 3´UTR inserts were sequenced and verified. The luciferase activity assay was performed as previously described (He et al. 2013a (link)).
Site-directed mutagenesis. The human full-length COX-2 reporter used was a generous gift from J. Li (Harvard University, Boston, MA, USA) (Wu et al. 2006 (link)). To generate the HRE-mutant COX-2 reporter, we performed site-directed mutagenesis on the wild-type COX-2 reporter at the potential HIF-1α binding sites with 3 base pair substitutions as previously described (Jiang et al. 1996 (link)). The mutant COX-2 reporter construct was validated by DNA sequencing.
Tube formation assay. HUVEC were cultured in EBM-2 complete medium, and switched to EBM-2 basal medium containing 0.2% FBS for 24 hr before performing the tube formation assay. The conditioned media were prepared from different cells by replacing normal culture medium with serum-reduced medium (1% FBS). After culture for 24 hr, the serum-reduced media were collected and stored at –20°C for later use. The HUVEC were trypsinized, counted, and resuspended in EBM-2 basic medium; then they were mixed with an equal volume of the conditioned medium and seeded on Matrigel-pretreated 96-well plates at 2 × 104 cells/well. After culture for 6-12 hr, tube formation was observed under a light microscope and photographed. The total lengths of the tubes in each well were measured using CellSens Standard software (Olympus; Hamburg, Germany).
The chorioallantoic membranes (CAM) assay. White Leghorn fertilized chicken eggs (Charles River, Malvern, PA, USA) were incubated at 37°C under constant humidity. Cells were transfected with miRNA precursors, or treated as specifically indicated in the figure legends. After transfection for 12 hr, the cells were trypsinized, counted, and resuspended in the serum-free medium. The cell suspensions were mixed with Matrigel at a 1:1 ratio, and implanted onto the CAM of chicken eggs on day-9 embryos. Tumor angiogenesis responses were analyzed 5 days after the implantation. The tumor/Matrigel plugs were trimmed off the CAM and photographed. The number of blood vessels as the index of angiogenesis was analyzed by counting the branches of blood vessels in three representative areas (each 1.0 mm2) by two observers in a double blind manner.
miRNA transfection. Cells were cultured in 6-well plates to reach a 60% confluency, and transfected using miR-199a or a negative-control precursor (both from Applied Biosystems) at 30 nM using Lipofectamine RNAiMAX reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. Total proteins and RNAs were prepared from the cells 60–70 hr after the transfection, followed by Western blotting or RT-PCR analysis.
Hypoxia treatment. A hypoxia incubator chamber (Stemcell, Vancouver, British Columbia, Canada) was used to generate the hypoxia environment for cell culture. Cells were cultured in hypoxia conditions (1% O2) for 24 hr at 37°C.
Reactive oxygen species (ROS) study. Cells were treated with ROS scavenger catalase (1,500 U) or with H2O2 (50 μM) for 12 hr. Then cells were harvested, and total proteins were extracted for Western blotting analysis.
Statistical analysis. All the results were obtained from at least three independent experiments. Results are presented as mean ± SE and were analyzed by Student’s t-test or one-way analyis of variance (ANOVA). All results were analyzed by SPSS for Windows, version 11.5 (IBM, Chicago, IL, USA). Differences were considered significant with p < 0.05.
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