Cells were passaged every 2-3 days to maintain exponential growth. qRT-PCR analysis of miRNA-21 and TIMP3 mRNA expression Total RNA from tissue and cells were isolated using TRIzol reagent (Invitrogen) to obtain both miRNA and mRNA. For analysis of miR-21 expression, the stem-loop
RT primer, real-time PCR primes and check details TaqMan MGB probe were designed as previously described . Briefly, miRNAs were reverse transcribed into cDNAs by SuperScript II reverse transcriptase. Real-time PCR was performed using a standard TaqMan PCR protocol according to manufacturer’s protocols (Applied Biosystems), and relative expression was calculated using the ΔCT method and normalized to the expression selleck chemicals of U6 RNA. Relative levels of TIMP3 mRNA were examined by SYBR green real-time quantitative
reverse transcription-PCR (qRT-PCR) (Applied Biosystems) and normalized to β-actin mRNA. BIBF 1120 nmr The primers for TIMP3 were: forward primer 5′-AGTTACCCAGCCCTATGA-3′, reverse primer 5′-GCAAAGGCTTAAACATCT-3′. All qRT-PCRs were performed in duplicate, and the data are presented as mean ± standard error of the mean (SEM). Oligonucleotide transfections For miR-21 knockdown, cells were transfected with 50 nM of oligonucleotide with Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. The sequences used were: 5′-UCAACAUCAGUCUGAUAAGCUA-3′ (anti-miR-21 oligonucleotide); and 5′-CAGUACUUUUGUAGUACAA-3′ (control oligonucleotide). For miR-21 overexpression, cells were transfected with a synthetic RNA duplex sequence corresponding Unoprostone to mature miR-21. The sequences were: 5′-UAGCUUAUCAGACUGAUGUUGA-3′ (miR-21 oligonucleotide); and 5′-UUCUCCGAACGUGUCACGUTT-3′ (control oligonucleotide). All oligonucleotides were synthesized by Genepharma Co.
Ltd. The sequences of the control oligonucleotides were analyzed by BLAST search to exclude potential hits in the human transcriptome. Migration assay BCAP-37, MCF-7, MDA-MB-231, and MDA-MB-435 cells were transfected with anti-miR-21, miR-21, or control oligonucleotide, cultured for 48 h, and transferred onto the top of matrigel-coated invasion chambers (24-well insert, 8 μm pore size; BD Biosciences) in a serum-free DMEM. DMEM containing 10% fetal calf serum was added to the lower chamber as a chemoattractant. After 20 h incubation, non-migrated cells were removed from the inner part of the insert with a cotton swab. Fixation and staining of migrated cells were performed using 0.1% crystal violet. Cells were quantified by fluorescence microscopy (100×). Western blot analysis Cell lysates were prepared in lysis buffer (0.15 M NaCl,50 mM Tris-Cl(pH7.5), 2 mM EDTA, 0.5%Triton-100, 5 mM DTT, 0.