H1299, H522, HCC95 and HCC1438 lung cancer cells were maintained in RPMI-1640 medium (Gibco-BRL, Glasgow, UK) with 10% fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin and 100 g/ml streptomycin).
Tumor and corresponding normal lung tissue specimens were obtained from 27 patients with non-small cell lung cancer (NSCLC). A total of 27 patients with NSCLC (9 squamous cell carcinomas, 17 adenocarcinomas and 1 large cell carcinoma), who underwent curative resection at the Konyang University Hospital (Daejeon, Korea), were analyzed. None of the patients had received chemotherapy or radiotherapy prior to surgery. Informed consent was obtained from each patient prior to surgery. This study was approved by the Bioethics Committee of Konyang University Hospital. All of the tumor and macroscopically normal lung tissue samples were obtained at the time of surgery, and were rapidly frozen in liquid nitrogen and stored at −80°C until analysis. Tissue samples were histologically confirmed by hematoxylin and eosin staining.
microRNA precursor transfection
Cells were plated in 6-well plates at a density of 1.5×105 cells/well. The following day, cells were transfected with 50 nM pre-miR™ miRNA precursor (has-miR-99b; Ambion, Austin, TX, USA) and pre-miR™miRNA precursor-negative control #1 (negative control #1; Ambion) with Oligofectamine (Invitrogen, Carlsbad, CA, USA) based on the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction (qRT-PCR) assays
Total RNA was isolated with TRIzol reagent (Gibco-BRL) according to the manufacturer’s instructions. The first strand of complementary DNA (cDNA) was synthesized using the oligo(dT) primer system (SuperScript III First-strand Synthesis System; Invitrogen). Aliquots of the reaction mixture were used for the quantitative PCR (qPCR) amplification with the IQ5 system (Bio-Rad Laboratories, Hercules, CA, USA) using iQ SYBR-Green Supermix (Bio-Rad). PCR was run for 40 cycles of denaturation at 95°C for 15 sec, annealing at 55°C for 15 sec, and elongation at 72°C for 15 sec. Gene expression was quantified by the comparative CT method, with normalizing CT values to the housekeeping gene, β-actin. Following amplification, melting curve analysis was performed to ensure the specificity of the products.
TaqMan microRNA expression assay
qRT-PCR analysis for miRNAs was performed in duplicate with a TaqMan MicroRNA assay kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, and RNU6B was used for normalization.
We determined whether or not miR99b modulates FGFR3 expression using a luciferase assay. The dual luciferase vector, psiCHECK2, was purchased from Promega (Madison, WI, USA). The 1463 bp fragment was synthesized by PCR using cDNA of 293T cells. The forward primer with the XhoI restriction site (5′-GGGCTCGAGGGCC ACTGGTCCCCAACAATGTG-3′) and the reverse primer with the NotI restriction site (5′-GGGCGGCCGCCCAGTAA CAGTACAGAACGAACCAAC-3′) were used to amplify the FGFR 3′UTR. The PCR products were cloned into the XhoI/NotI 3′UTR site of the psiCHECK2 plasmid (Promega). The correct sequence of all the clones was verified by DNA sequencing. 293T cells were seeded in 12-well plates in Dulbecco’s modified Eagle’s medium (DMEM) medium, supplemented with 10% heat-inactivated FBS. The cells were transfected with psiCHECK2-FGFR UTR constructs containing 3′-UTR of FGFR, in the presence or absence of miR99b (Ambion) using Effectene (Qiagen, Hilden, Germany). The cells were collected 48 h following transfection, and the cell lysates were prepared according to the Promega instruction manual. The Renilla luciferase activity was measured using a Lumat LB953 luminometer (EG&G Berthhold, Bad Wildbad, Germany), and the results were normalized using the activity of luciferase. All experiments were performed in triplicate.
Westren blot analyses
Cells were lysed in Pro-Prep protein extraction solution (iNtRON Biotechnology, Gyeonnggi-do, Korea) 48 h following transfection. An equal amount of proteins was resolved by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. The primary antibodies used for the analysis were mouse anti-FGFR3 (1:1000; BD Biosciences, San Jose, CA, USA) and mouse anti-β-actin antibodies (1:2000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
Cells were seeded in 96-well plates at a density which enabled transfection per well in triplicate. Proliferation indices were measured 24, 47 and 72 h later using the CellTiter96 Aqueous One Solution Cell Proliferation Assay (MTS assay; Promega). All experiments were performed in triplicate.
Colony formation assay
We used the Cell Transformation Detection kit (Millipore, Bedford, MA, USA) to evaluate the ability to form colonies on soft agar. Briefly, 1 ml underlayers consisting of 0.8% agar medium were prepared in 6-well plates. miR-99b and negative control miRNA-transfected cells were trypsinized, centrifuged, resuspended in 0.4% agar medium (equal volumes of 0.8% Noble agar and culture medium), and plated onto the top agar at 2,500 cells per well. The cells were kept wet by adding a small amount of RPMI-1640 with 10% FBS and incubated for 3 weeks at 37°C. Fresh culture medium was replaced every 3 days. Colonies were visualized using cell staining solution (Millipore) and counted under a microscope.
Statistical differences in the expression of miRNAs were analyzed by the Student’s t-test. Statistical analysis was performed using SPSS 12.0 computer software (SPSS Inc.; Chicago, IL, USA). A value of p<0.05 was considered to be significant.