Cell Culture, Hypoxia exposure and Transfection
Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Walkersville, MD). HUVEC (passage 2-5) were cultured in endothelial basal medium (EBM2) supplemented with growth factors (Lonza). HeLa and HEK293 (ATCC, Manassas, VA) were cultured in DMEM media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS). HCT116 (gift from Bert Vogelstein, The Johns Hopkins University School of Medicine) and HT29 (ATCC) were cultured in McCoy′s 5A media supplemented with 10% FBS. Desferrioxamine (DFX) and other reagents were obtained from Sigma (St Louis, MO). To expose cells to hypoxia, cells were cultured in Billups-Rotenburg chamber with 94% N2, 1% O2, and 5% CO2 on 37 C for 24 hours.
Human colon cancer specimens (n
9) and paired non-cancerous normal colon specimen (n
9) were obtained from patients at The Johns Hopkins Hospital, Baltimore, MD, with documented informed consent in each case. The collection and analysis of human colon cancer specimen were approved by The Johns Hopkins University School of Medicine Institutional Review Board. Patients undergoing surgery for colorectal cancers provided written consent to donate tissue for analysis.
Precursor miRNAs and anti-sense oligonucleotides for miRNAs were from Applied Biosystems (Foster City, CA). For transfection of precursor miRNA, HCT116 cells were transfected with siPORT NeoFX (Applied Biosystems) with precursor miRNA 0–20 nM or with anti-sense miRNA 0–40 nM and harvested 48 hours later.
Total RNAs were extracted from cells using Trizol (Invitrogen). Human tissue RNAs were obtained from Applied Biosystems. Northern blotting for miR-22 was performed as described previously. Briefly, 10 µg of each RNA were loaded onto 15% TBU-gel (Invitrogen), transferred to nitrocellulose membrane, and hybridized with 32P-end-labeled probes specific for miR-22 at 42°C for 16 hours. The miR-22 probe, 5′-TAAAGCTTGCCACTGAAGAACT-3′ was synthesized by Integrated DNA Technologies; all other reagents were purchased from Applied Biosystems.
Quantitative Real-Time PCR (qRT-PCR)
To analyze miRNA expression, TaqMan MicroRNA assays were used to quantify levels of mature miRNAs following the manufacturer's instructions. Briefly cDNA was synthesized from purified small RNA (10 ng) and performed Real-Time PCR by using iCycler iQ (BioRad). Expression levels were normalized to U6. The primers for miRNA RT-PCR and the PCR mix were purchased from Applied Biosystems. To detect mRNA expression, cDNA synthesis was performed using High Capacity cDNA synthesis kit (Applied Biosystems). The primers for human VEGF and ß-actin were purchased from Applied Biosystems.
A fragment of the 3′ UTR of HIF-1α (starting after the TGA stop codon and extending for 401 bp) containing the miR-22 response element was cloned into pMIR-REPORT luciferase vector (Applied Biosystems). Mutation of the miR-22 response element (5′-GTTGACGG-3′ → 5′-GATCAGGG -3′) was made by QuikChange Site-Directed Mutagenesis kit (Stratagene). HCT116 cells were plated at 5×104 cells per well in 24 well plates. Next day, pMIR-REPORT Luciferase vectors including 3′ UTR of HIF-1α and precursor miR-22 or scrambled oligonucleotides were transfected into cells using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, luciferase assays were performed using the dual luciferase reporter assay system (Promega).
Cells were lysed in 0.4 ml of lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM EDTA, 1% NP40, 20 mM NaF, 1 mM orthovanadate and protease inhibitor cocktail). Lysates were separated by electrophoresis, blotted to membrane and reacted with specific antibodies. Antibody to mouse HIF-1α was from Cell Signaling Technology (Danvers, MA). All other primary antibodies and appropriate secondary antibodies were from Santa Cruz.
An ELISA was used to quantitate VEGF secreted from HCT116 cells into the media. HCT116 cells were incubated in 1 ml of media with 1% FBS in the absence (controls) or presence of DFX or under normoxia or hypoxia for 24 h at 37°C. The supernatants were assayed for VEGF production using the Human VEGF ELISA kit according to the manufacturer's protocol (R&D Systems).
BrdU incorporation assay
HCT116 or HeLa cells were transfected with Pre-miR-22 or Pre-miR-control and cultured for 72 hours. BrdU was added to each wells and incubated for 4 hours. After fixing cells, the cells were incubated with anti-BrdU antibody for 1 hour, then with Peroxidase Conjugated Goat anti-Mouse IgG for 30 min. TMB Peroxidase Substrate was added to each wells. Read the plate using a spectrophotometer microplate reader set at a wavelength of 450 nm.
Wound healing assay
HUVECs were seeded on 12 well plates and grown to confluent. A scratch was performed using a 1000 µl pipette tip and media change to condition media from HCT116 transfected with Pre-miR-control or Pre-miR-22. Images were captured at 16 h and the distances between the cells were measured.
Data were expressed as the mean ± SD. Statistical comparisons were made between two groups with the t test and between multiple groups by ANOVA. A value of P<0.05 was considered significant.