All culture media, except RPMI 1640 medium, were purchased from Gibco Invitrogen (Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA). H4 (human neuroglioma) and OVTW-59-4 (human ovarian adenocarcinoma) cells were cultured in DMEM (Dulbecco's modified Eagle's medium) medium, whereas U-87 MG (human brain glioblastoma) and HeLa (human cervical adenocarcinoma) cells were grown in minimum essential medium (MEM). CL1-5 (high-metastatic human lung adenocarcinoma) cells were cultured in RPMI-1640 medium (Hyclone) supplemented with 0.5 mM L-glutamine and 10% FBS. PC-12 (rat adrenal gland pheochromocytoma) cells were cultured in 85% RPMI-1640 supplemented with 5% FBS and 10% horse serum. All cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2 in air.
Northern hybridizations were carried out mainly as described previously . In brief, the probe for SERPINI1 was amplified from the FirstChoice PCR-Ready human brain cDNA (Ambion, Austin, TX, USA) using the forward primer 5'-CTGGAAGTCGCAGTTTAGGCCTGAA-3' and the reverse primer 5'-TGGATGGTCGACAATAACTTGAGGA-3' to produce a 559-bp fragment under the following conditions: 1 cycle of 94°C for 5 min; 32 cycles of 94°C for 20 sec, 55°C for 20 sec, and 72°C for 1 min; and 1 cycle of 72°C for 10 min. The probe for PDCD10 was amplified from the same cDNA using the forward primer 5'-TCCATGGTTTCTATGCCCCT-3' and the reverse primer 5'-TTTGGTGTTCAAGTGCCCTG-3' to produce a 445-bp fragment under the following conditions: 1 cycle of 94°C for 5 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec; and 1 cycle of 72°C for 10 min. All PCRs were performed on a T3 thermocycler (Whatman Biometra, Goettingen, Germany) using Ex Taq polymerase (Takara, Otsu, Singa, Japan) and subjected to sequencing on an ABI PRISM 3700 automated sequencer (Applied Biosystems, Foster City, CA, USA) by the core facility of the National Health Research Institutes (Zhunan, Taiwan). The sequence-verified probes, including a cDNA probe for ubiquitin supplied by the manufacturer and used as a control, were labeled with [α-32P]dCTP using the Rediprime II DNA labeling system (Amersham/GE Healthcare, Piscataway, NJ, USA) followed by hybridization against either the human MTE multiple tissue expression array or cancer profiling array (BD Bioscience Clontech, Palo Alto, CA, USA) in an ExpressHyb hybridization solution (BD Bioscience Clontech) for 4 h at 65°C. The arrays were then washed and exposed to X-ray film for detection. Each Northern blot analysis was repeated at least three times to confirm reproducibility of the results.
Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR)
Total RNAs were extracted from two human brain tumor cell lines, H4 and U-87 MG, using TRIZOL reagent (Gibco Invitrogen) followed by DNase (Ambion) treatment to eliminate potential genomic DNA contamination. For control, total RNA of normal brain tissue was obtained from Clontech. Complementary DNA (cDNA) was synthesized from 5 μg of total RNA using oligo(dT) priming (Roche Molecular Biochemicals, Indianapolis, IN, USA) and MMLV reverse transcriptase (Epicentre Technologies, Madison, WI, USA). After a 1-h incubation at 37°C, the reaction mixtures were heat inactivated at 92°C for 10 min and then applied as templates to amplify cDNAs specific for SERPINI1 (amplified with the forward primer 5'-CTTGCAATGGGAATGATGGAACTTG-3' and the reverse primer 5'-CGAAATCATGTCCACTTGTGTTCAT-3' at 1 cycle of 94°C for 5 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 90 sec; and 1 cycle of 72°C for 10 min) and for PDCD10 (amplified with the forward primer 5'-TCCATGGTTTCTATGCCCCT-3' and the reverse primer 5'-TTTGGTGTTCAAGTGCCCTG-3' at 1 cycle of 94°C for 5 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec; and 1 cycle of 72°C for 10 min). GAPDH serving as internal control was amplified using the primer set 5'-CCTGCACCACCAACTGCTTA-3' and 5'-ACCCTGTTGCTGTAGCCAAA-3' to allow comparison of RNA levels among different samples. The PCR conditions for GAPDH were the same as those for PDCD10.
5'-Rapid amplification of cDNA ends (RACE)
The 5'-RACE analysis was performed using the FirstChoice human brain RNA ligase-mediated (RLM)-RACE kit (Ambion). Two RNA adaptor primers were employed as forward primers according to the manufacturer's instruction. The reverse primers for the 5'-RACE of SERPINI1 were 5'-TAGGCTGTCATATCCCATTGAGTGG-3' for the outer primer and 5'-CAAGTTCCATCATTCCCATTGCAAG-3' for the inner primer. The reverse primers for the 5'-RACE of PDCD10 were 5'-CTCAGCTTCATTCTTCATCTCTTC-3' for the outer primer and 5'-CCTCATTCAAAAGCCAACTACAG-3' for the inner primer. Both the first and second rounds of PCR were performed with 1 cycle at 94°C for 3 min; 35 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec; and 1 cycle at 72°C for 7 min. The PCR products were cloned into a T-tailed vector pGEM-T-Easy (Promega, Madison, WI, USA) followed by sequencing to determine the 5'-ends of SERPINI1 and PDCD10 transcripts.
Construction of the reporter gene plasmids
All restriction enzymes were purchased from Takara. Genomic DNA was purified from human whole blood samples using the QIAamp Blood kit (Qiagen, Hilden, Germany). The various lengths of the intergenic segment between SERPINI1 and PDCD10 were obtained by PCR using the primers listed in Table 1 under the following condition: 1 cycle of 94°C for 5 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec; and 1 cycle of 72°C for 10 min. The amplified DNA fragments were then cloned into one of the three vectors: pGL3-Basic, pGL3-Promoter, or pGL3-Promoter with the original SV40 promoter being replaced by a CMV promoter.
Transient transfection and promoter assays
Cells were first seeded in 12-well culture plates at a density of 1.8 × 105 cells per well. Cells were then co-transfected with 3 μl of Lipofectamine 2000 (Gibco Invitrogen) and a mixture of 525 ng of DNA consisting of 500 ng of the tested pGL3 promoter constructs and 25 ng of the pRL-TK Renilla luciferase reporter plasmids (Promega) in a serum-free medium. After a 3-h incubation at 37°C, the transfection medium was replaced with fresh medium supplemented with 10% FBS and cells were incubated for another 24 h. After incubation, cells were washed twice with PBS and lysed by reporter lysis buffer (Promega). Cellular debris was removed after centrifugation at 10,000 × g for 5 min at 4°C. Cell extracts were then assayed for luciferase activity using a SIRIUS luminometer (Berthold, Bad Wildbad, Germany). In each experiment, cells were transfected with pGL3-Basic and pGL3-Promter plasmids that served as negative and positive control, respectively. To normalize transfection efficiency, the pRL-TK plasmids were co-transfected as an internal control.