DNA sequence upstream of the human miR-34a gene was amplified by PCR using the following primer sets: 34aF CTGCTCGAGTGCCGGTTCCTGGCTTTA, 34aR GCGAAGCTTGCTGCAATATCACCGTG. PCR product was cloned into pGL3-basic vector (Promega) between the HindIII and XhoI sites. Site directed mutagenesis was performed by overlap extension PCR as described in [40
]. And primers used were: 34aM53F TGCCTGGGTTTACCTGGGTTTATTCCGAGCCG, 34aM53R CGGCTCGGAATAAACCCAGGTAAACCCAGGCA; 34aM1F GGCGACGAGTCGCCGGAAGGGTCGCGAT, 34aM1R TTCCGGCGACTCGTCGCCCCTTCGCGGT; 34a M2F ATGGCCCGGGAGTCGGGGACCTCGGCTC, 34aM2R AGGTCCCCGACTCCCGGGCCATCGCGAC; 34a M3F TCGGTCTGGCGACAGCGCAGCTCCCCGGAT, 34a M3R AGCTGCGCTGTCGCCAGACCGACGGGAC. All the constructs were verified by sequencing.
NF-κB p65 and mutant IκB expression vector were kindly provided by Dr M. Cippitelli (University of Rome Sapienza, Rome, Italy) and Dr D. Fruci (Research Center, Ospedale Bambino Gesù, Rome, Italy). Wildtype p53 expression vector was a gift from Prof. Dong Wang (Third Military Medical University, Chongqing, China).
Cell culture and transfection
Human esophageal cancer cell line EC109 were purchased from Cell Bank of Chinese Academy of Sciences, Shanghai, China. KYSE450 were obtained from Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China. All cells were cultured in 1640 supplemented with 10% FBS, at 37°C in a humidified incubator containing 5% CO2.
For gene expression transfection, plasmids were transfected with lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Gene expression levels were detected 48 h after transfection.
RNA extraction and Real-Time qRT-PCR
Total RNA was extracted using RNAiso reagent (Takara) according to the manufacturer's instructions and quantified with a NanoDrop spectrophotometer (Thermo Scientific). MiR-34a expression was measured using a TaqMan MicroRNA RT-PCR assay (Applied Biosystems). 100 ng total RNA was converted to cDNA using specific primers, and amplification of the cDNA was done using Taqman Universal PCR Master Mix (Applied Biosystems). PCR conditions were 95°C for 3 minutes followed by 40 cycles of 95°C for 15 seconds, 60°C for 40 seconds. The expression of MiR-34a was normalized against U6 snRNA expression.
For total protein analysis, cells were harvested and lysised in T-PER Tissue Protein Extraction Reagent (Pierce Chemical Company) with freshly added PMSF. For nuclear protein analysis, cells were lysised with Nuclear and Cytoplasmic Protein Extraction Kit (beyotime). Proteins were separated by SDS-PAGE and electrotransferred to PVDF membranes, after blocked with 5% skimmed milk, membranes were incubated with a primary antibody and then incubated with a horseradish peroxidase-conjugated secondary antibody. Antibodies used were anti- NF-κB p65 (sc-372, Santa Cruz Biotechnology), anti- NF-κB p50 (06-886, upstate), anti-p53 (sc-126x, Santa Cruz Biotechnology), anti-lamin B1 (SC-56145, Santa Cruz Biotechnology), HRP-conjugated monoclonal mouse anti-glyceraldehyde-3-phosphate Dehydrogease (KC5G5, Kangchen Bio-Tech) and horseradish-peroxidase coupled goat antibodies against rabbit and mouse immunoglobulins (Beijing Zhongshan Golden Bridge Biotechnology). The immunoreactive proteins were detected using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific).
For reporter assay, p65 expression vectors or the control vectors were cotransfected with pGL3 reporter vectors along with pRL-TK vector (Renilla luciferase, Promega) using lipofectamine2000. Cell lysate was collected 48 h after transfection and luciferase activities were measured using Dual-Luciferase Reporter Assay System (promega). Activity was defined as Firefly/Renilla ratio and normalized to the negative control vector transfection.
ChIP was performed with a commercially available Chromatin Immuno-precipitation Kit (Upstate Biotechnology) according to the manufacturer's instructions. Briefly, cells (2 × 106/immunoprecipitation) were cross-linked in 1% formaldehyde for 10 min at room temperature and halted the cross-link with 0.125M glycine. Chromatin was captured with the following primary antibody: p65 (cell signal technology, 3987), p50 (upstate, 06-886), p53 (santa sc-126x), IgG (millipore, 12-371B). After overnight capture at 4°C, chromatin was collected, purified and then decrosslinked at 65°C. DNA was recovered using Spin Columns and the enrichment was detected by qPCR. Following primers were used in PCR assay: 34aΚB2, F CCCCCGTGGTTTCTGTTTG, R CCTGGGCTGGCGTTTC; 34aΚB3, F TGCGTGGTCACCGAGAAGCAG, R TTCAGGTGGAGGAGATGCCGC; 34a intron, F GCTCCATCCTCGGACCTGA, R GGCGGTCTGAGTTGGCTAG.
Electrophoretic mobility shift assays
EMSA was performed according to the manufacturer's instruction of (Pierce). Nuclear protein of EC109 and KYSE450 cells were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (beyotime). In brief, biotin labelled DNA probes containing candidate NF-κB binding sites from miR-34a promoter (34aΚB2 TCGCGATGGCCGGGGAGTCCGGGACCTCGGCT, 34aΚB3 CCGTCGGTCTGGGGACAGCCCAGCTCCCCGGA, only one stand shown) were mixed with 10 ug of nuclear extract in a 20 ul reaction volume containing 1X binding buffer, 5 mM MgCl2, 5% glycerol, 0.05% NP40 and 50 ng/ul poly(dI:dC). The reaction mixture was incubated on ice for 30 min and applied to a 6% nondenatured polyacrylamide gel containing 0.5X TBE buffer. For competition assays, a 200-fold molar excess of unlabeled probes and unlabeled mutated probes (34aΚB2M TCGCGATGGCCCGGGAGTCGGGGACCTCGGCT, 34aΚB3M CCGTCGGTCTGGCGACAGCGCAGCTCCCCGGA) were added prior to the labelled probe. For supershift assays, antibodies against p65, p50 and p53 were added in the binding reaction after DNA-protein incubation. After electrophoresis, DNA-protein complex was transferred to a nylon membrane, and cross-linked. Then the biotinylated-labelled DNA detected by chemiluminescence according to the manufacturer's directions.
Statistical analyses were performed with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Differences between experimental groups and control groups were assessed by Student's t-test. P < 0.05 was considered to be statistically significant.