Breast tissue specimens
There are two cohorts of clinical specimens included in current study. For in situ
hybridization assay, the tissue specimens were obtained from 90 female patients with breast cancer and 26 female patients with breast benign diseases while these patients underwent surgical treatment at the First Affiliated Hospital of Anhui Medical University from March 31 2001 to April 1 2002. For qRT-PCR assay, 18 breast cancer and 10 breast benign diseases specimens were collected from our department of Pathology between January to May 2010. These tissue specimens were placed in a cryovial, snap-frozen and stored in liquid nitrogen immediately after operation until use. The pathohistological diagnosis of the patients was according to breast tumor classification criteria of World Health Organization [29
]. Histology grade was based on the Scarff-Bloom-Richardson system [30
]. The median follow-up time for the breast cancer patients was 60 months and ranged from 8 to 64 months. The protocol for use of patient samples in this study was approved by our institutional review board and the informed consent form was signed by each patient or their guardians.
Construction of tissue microarray
The formalin-fixed and paraffin-embedded tissue blocks were retrieved from the archives of the Department of Pathology, the First Affiliated Hospital of Anhui Medical University, Hefei, China. The hematoxylin and eosin-stained tissue sections were reviewed by 2 pathologists to identify the representative regions for tissue microarray construction. After that, the area of interest in the donor blocks was cored thrice with a 1 mm diameter cylinder using a tissue microarrayer (Hengtai Instruments, Liaoning, China), and transferred to the recipient paraffin block. A total of three tissue microarray blocks were constructed and sectioned for in situ hybridization analysis of miRNA expression.
In situ hybridization
hybridization to detect miR-133a expression in breast carcinoma and benign disease tissues was performed as described previously [10
]. Briefly, tissue microarray sections with 3 μm in thickness were deparaffinized, rehydrated, and digested and then refixed in 4% paraformaldehyde. After that, the sections were prehybridized with 150 μl of hybridization solution and then incubated with a digoxigenin-labeled LNA probe (Exiqon, Copenhagen, Denmark) for miR-133a, U6 (positive control), and scrambled RNA (negative control) at 68°C for 20 h. In the next day, the sections were washed with 2× SSC, 1× SSC and 0.5× SSC and then incubated with a mouse antidigoxin antibody followed by streptavidin-biotin-peroxidase complex. For visualizing the positive signal, the sections were incubated in 3,3'-diaminobenzidine solution and counterstained with hematoxylin. The expression of miR-133a was reviewed and scored independently by 2 pathologists. The staining was scored as negative, if ≤ 10% of epithelial cells stained positively, whereas the staining was scored positively if > 10% of epithelial cells stained positively [31
Cell lines and culture
Human breast cancer ZR75-1, SKBR3, T47D, MCF-7 and MDA-MB-231 cell lines and the nontumourigenic HMEC cell line were obtained from the American Type Culture Collection (Rockville, MD). ZR75-1, SKBR3 and T47D cells were grown in Dulbecco modified Eagle medium (DMEM, Invitrogen, Carlsbad, CA), MCF-7 cells were cultured in RPMI-1640 (Invitrogen), MDA-MB-231 cells were cultured in Leibovitz's L-15 (Invitrogen) and HMEC cells were grown in Medium 171 with mammary epithelial growth supplement (Cascade Biologics) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and incubated at 37°C in a humidified incubator containing 5% CO2.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
To detect miR-133a expression, RNA from cell lines and fresh tissue samples was extracted using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions and then reversely transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen). After that, qPCR was performed using TaqMan MicroRNA Assay kits (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions with a Stratagene M × 3000P Real Time PCR machine (Agilent Technologies). The PCR amplification consisted of 40 cycles (95°C for 5 s, 60°C for 20 s) after an initial denaturation at 95°C for 10 s. U6 small nuclear RNA was used as an internal control. The threshold cycle (Ct) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. The fold change of miRNA expression was calculated using the 2-ΔΔCt method after normalization to U6 expression. All experiments were performed in triplicate. In addition, relative expression levels of FSCN1 mRNA were assessed by using SYBR Premix Ex Taq (Perfect Real Time) kit (TaKaRa, Dalian, China) and normalized to GAPDH mRNA. The primers for FSCN1 were 5'-CTCATCAACCGCCCCATCAT-3' (forward) and 5'-CTGCCCACCGTCCAGTATTT-3' (reverse) and GAPDH primers were 5'-TGCACCACCAACTGCTTAGC-3' (forward) and 5'-GGCATGGACTGTGGTCATGAG-3' (reverse).
Transient miRNA and siRNA transfection
MCF-7 and MDA-MB-231 cells were selected for miR-133a transfection. Briefly, the cells were grown overnight and then transfected with either miR-133a mimic (GenePharma, Shanghai, China), 2'-O methylated single-stranded miR-133a antisense oligonucleotide (ASO, GenePharma), or negative control miRNA (GenePharma) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The miR-133a mimic contained synthetic small duplex sequences of miR-133a RNA that was able to be bioprocessed into the mature miR-133a in the cells. The sequences of negative control and ASO negative control (GenePharma) were nonhomologous to any human genome sequences, and used to eliminate the potential nonsequence-specific effects. The sequences of miR-133a mimic were: 5'-UUUGGUCCCCUUCAACCAGCUG-3' (sense), 5'-GCUGGUUGAAGGGGACCAAAUU-3' (antisense); miR-133a ASO, 5'-CAGCUGGUUGAAGGGGACCAAA-3'; NC, 5'-UUCUCCGAACGUGUCACGUTT-3' (sense) and 5'-ACGUGACACGUUCGGAGAATT-3' (antisense); ASO NC, 5'-CAGUACUUUUGUGUAGUACAA-3'. In addition, FSCN1 siRNA or control scrambled siRNA (both from Qiagen, Valencia, CA) was transfected into MCF-7 cells that were transfected with or without miR-133a ASO transfection. The target sequence of FSCN1 siRNA was AGCCCTGGGCGTGTAGTGTAA. The efficiency of RNA transfection was confirmed by real-time PCR analysis.
Cell proliferation assay
Thirty-six hours after transient transfection, MCF-7 were harvested and sub-cultured in 96-well plates for up to 96 h. After that, cell proliferation was assessed using the CellTiter 96 AQueous MTS assay (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, the MTS reagent (20 μl) was added to each well and incubated at 37°C for 2 h. Then, the absorbance at 492 nm was measured by using a microtiter plate reader (Bio-Rad, CA). The experiments were in triplicate and repeated thrice. The data were summarized as mean ± SD.
Wound healing assay
MCF-7 cells were seeded into 6-well plates and transfected with either miR-133a ASO or ASO NC, and then the cells were allowed to grow until 100% confluency. Next, the cell layer was gently scratched through the central axis using a sterile plastic tip and loose cells were washed away. The wound healing was observed and photographed at three preselected time points (0, 24, and 48 h) in three random selected microscopic fields for each condition and time point. Sextuple assays were performed for each experiment and repeated once.
Cell migration and invasion assay
Tumor cell migration and invasion were carried out using a Transwell insert (8 μm, Corning, NY). MCF-7 and MDA-MB-231 cells were first transfected with miR-133a mimic or ASO or control RNA. 48 h later, the cells were starved in a medium without fetal bovine serum overnight, and then 1 × 105 cells resuspended in 0.1 ml serum-free medium were added to the upper chamber and RPMI-1640 containing 20% fetal bovine serum was added to the lower chamber as a chemoattractant. For the invasion assay, the inserts were pre-coated with extracellular Matrigel (BD Biosciences, Bedford, MA). To measure the effect of miR-133a mimic on MDA-MB-231 invasion and migration potential, the cells in the upper chamber were cultured for 28 h, while 20 h incubation for miR-133a ASO on MCF-7 cells. Invaded or migrated cells were fixed and stained with 0.1% crystal violet. Five low-magnification areas (× 100) were randomly selected and counted for the cell numbers. All experiments were performed in triplicate.
Western blot analysis
Seventy-two hours after gene transfection, the cells were washed twice with cold PBS and total cellular protein was extracted using a modified radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% Na-deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; Aprotinin, leupeptin, pepstatin: 1 μg/ml each; 1 mM Na3VO4; 1 mM NaF). The protein concentration was then determined by a protein assay kit (Bio-Rad) and equal amounts of protein lysates (50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to the nitrocellulose membrane (GE Healthcare, Arlington Heights, IL). For Western blotting, the membranes were blocked with 5% defatted milk and incubated with primary antibodies at 4°C overnight. In the next day, the membranes were washed with PBS and then incubated with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The protein bands were developed with chemiluminescence (ECL) reagents (Pierce). The antibodies were anti-FSCN1 (diluted at 1:1000; Santa Cruz Biotechnologies), anti-beta-actin (diluted at 1:1000; Santa Cruz Biotechnologies).
Luciferase reporter assay
A dual-luciferase reporter vector psiCHECK2 (Promega) was used to generate luciferase reporter constructs. The 3'-untranslated region (UTR) of human FSCN1 was amplified from human genomic DNA with primers of 5'-ATGATTCTCGAGCCTCGCTCTGGGAGTACTAGGG-3' (sense) and 5'-TATATATGCGGCCGCTGGGGCTGCAGACTGAGTTATT-3' (antisense), and inserted into the XhoI-NotI restriction sites in the 3'-UTR of the hRluc gene in psiCHECK2 vector. MCF-7 cells were grown in 24-well plates and cotransfected with 0.2 μg of psiCHECK2-FSCN1 3'UTR or psiCHECK2 control vector and 20 pmol miR-133a mimic or its negative control (GenePharma) by using Lipofectamine 2000. Each transfection was performed in triplicate and Luciferase activity was assessed 48 h after transfection using the Dual-Luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity accordingly.
Bioinformatical and statistical analyses
We performed bioinformatical analysis using the miRNA database TargetScan [32
] (release 5.1, http://www.targetscan.org
) to predict miR-133a target genes. Data were shown as means ± standard deviation (SD). All statistical analyses were performed using the SPSS 13.0 software. Differences/correlations between groups were compared using Pearson chi-square test for qualitative variables and Student t test for continuous variables. Patient relapse-free survival (RFS) and overall survival (OS) rates was analyzed using the Kaplan-Meier method and compared by log-rank analysis. P
-value < 0.05 was considered statistically significant.