healthmedicinet

Cell culture and AZA treatment of breast cell lines

MCF7, MDA-MB-231, MDA-MB-453, MDA-MB-468, BT-20, BT-474, ZR-75-1, HCC1937, MDA-MB-361,
MDA-MB-157, and MCF12A cell lines were purchased from American Type Culture Collection
(ATCC). MCF10A cell line was kindly provided by Elif Erson Bensan, (METU, Ankara,
Turkey). All cell lines were grown as recommended by ATCC. AZA (5 ?M, Sigma-Aldrich,
A3656) dissolved in DMSO (Applichem, A1584.0100) was used to treat cells for 96 h,
and equivalent amount of DMSO was used as control treatment. Media was changed at
every 24th hour, and cells were harvested at 96
^th
hour.

RNA isolation, determination of RNA quality and hybridization to microchips

RNA was isolated from the cell lines with Nucleospin RNA kit (Macherey-Nagel (MN),
740955.5), quantified with NanoDrop ND-1000 spectrophotometer (Thermo Scientific,
USA) and the RNA integrity was determined with Agilent 2100 Bioanalyzer (Agilent Technologies,
Germany) and all RNA samples had RNA integrity number (RIN) between 9.8–10. Hybridization
of the processed RNA samples to the gene chips (HGU-133-Plus 2.0, Affymetrix, USA)
were done following the standard Affymetrix protocol, in the Microarray Hybridization
Facility, at Bilkent University (Ankara, Turkey).

Quality control and analysis of the microarray expression data

Data were scanned from the microchips with GCOS software (Affymetrix, USA). Raw data
were normalized with justRMA method using BRB-ArrayTools 3.8.0-beta, developed by
Dr. Richard Simon and the BRB-ArrayTools Development Team (NCI, USA). Quality control
and further analyses were performed using BrB-ArrayTools. RNA degradation plots and
3? to 5? signal ratios for probe sets corresponding to ACTB and GAPDH mRNAs were calculated for quality control assessments. Class comparison tool, which
uses random variance model of t test for small size samples, was used to find differentially expressed genes between
treatment groups. Functional annotations were done using “Functional Annotation Chart”
tool of DAVID Bioinformatics Resources 6.7 (NIH, USA).

CDNA synthesis and quantitative RT-PCR

cDNA was synthesized from 1 ?g total RNA, using oligo-dT primers and RevertAid First
Strand cDNA Synthesis Kit (Thermo Scientific, K1622) according to user’s protocol.
Specific primers for PCR amplification (available upon request) were designed using
PrimerBlast of NCBI 80]. qRT-PCR experiments were performed in duplicates, using DyNAmo™ HS SYBR® Green qPCR
Kit (Thermo Scientific, F-410 L). GAPDH as a reference gene was tested by calculating the significance of the log
₂
fold change of multiple probe sets represented in the array. Our results suggested
that GAPDH was a reliable and stable reference gene in response to AZA treatment in MCF7, MDA-231,
and MCF12A cell lines since no probe set exhibited fold change greater than 1.2 fold
(log
₂
fold change: ?0.28, FDR 0.1). Thus, GAPDH was used as an internal control gene in cell lines, and ACTB and SDHA geometric means were used 81] for internal control genes in tissue samples. Delta Ct method 82] was used for relative quantification of mRNAs, using the calculated efficiency values
of each primer pair. Statistical significance of differences between groups was tested
with Mann–Whitney test or Wilcoxon signed rank test (for paired data), using the log
₂
transformed expression values, using GraphPad Prism 6.0 (GraphPad Software Inc., USA).

DNA isolation, bisulfite treatment and sequencing

DNA was obtained using Nucleospin Tissue kit (Macherey-Nagel, 740952.5) as described
in the user protocol. Bilsulfite treatment was carried out with 1 ?g DNA, using Epitect
Bisulfite Kit (Qiagen, 59104). CpG islands were detected and bisulfite sequencing
primers for TAGLN gene were designed using the Methyl Primer Express® software (Applied Biosystems,
USA). The primers amplified the region between ?290 to +117 bp with respect to TSS
(GenBank: NM_003186.3) (Left primer: 5?-GGGGTTAGAGAATAGTGAAGTAGGAGTA-3?; Right primer:
5?-ACACTCACAAAACTTCCTCAAAACT-3?). Gel extraction was carried out with the QIAquick
Gel extraction kit (Qiagen, 28704). Specific PCR products were cloned into pGEM-T-easy
cloning vector (Promega, A1360) after which plasmid isolation of selected colonies
(5 for each sample) was performed with the PureLink Quick Plasmid Miniprep Kit (Invitrogen,
K210011) and sent for sequencing. Sequencing of the bisulfite-treated DNA inserts
was performed with SP6 primers using the dideoxy chain termination method (by Iontek,
Istanbul, Turkey). Bisulfite sequencing results were analyzed using QUMA software
83]. Statistical differences between breast cancer and NTB cell lines were tested with
Mann–Whitney test. Statistical differences between methylation percentages of paired
tissues were tested with Wilcoxon signed rank test, in GraphPad Prism 6.0.

Breast tumors and normal tissue samples

TAGLN expression in publicly available datasets was analyzed using Oncomineâ„¢ (Compendia
Bioscience, USA). Primary breast tumors and their matched normal tissues (Table 1) were surgically removed from patients (?=?21), at Numune Training and Research Hospital, Ankara, Turkey. The use of the tissue
material in this project was approved by the Ethics Committee and consents were obtained
in accordance with the Helsinki Declaration of 1975. Tumor samples used in the study
were composed 90 % of tumor cells, according to hematoxylin and eosin staining. RNA
isolation from tissues was performed with Trizol (Life Technologies, 15596–026). Breast
tumor cDNA array (OriGene, BCRT101) was composed of 41 tumor and 7 normal tissues.
Breast tumor tissue array (BioChain, Z7020005) was composed of 7 non-tumor and 68
tumor tissues of breast.

IHC staining and scoring

Immunohistochemical studies were performed automatically in the Bond Max equipment
(Leica Microsystems Inc.). Antigen retrieval steps were performed in Bond-Epitope
Retrieval Solution 1 (Leica Microsystems, Germany) with TAGLN antibody (1:500, Abcam
ab1416) at 100 °C. Detection was carried out with Bond Polymer Refine Red Detection
kit (Leica Microsystems, DS9390). Stained slides were dehydrated and covered with
mounting medium (Dako, CS70330) and cover-slips. Digital images of the slides were
evaluated using the H-score method 84]. Statistical differences between groups were calculated with Mann–Whitney test while
correlations were tested with Spearman correlation, in GraphPad Prism 6.0.

Survival analysis and analysis of publicly available methylation data

Survival analyses regarding the expression of TAGLN were performed using Kaplan-Meier (KM) plotter, breast cancer cohort 43]. Gene expression information of 205547_s_at probe set corresponding to TAGLN was used. Auto cutoff was used to generate the best performing KM curves. To perform
methylation analyses, all the methylation data published under super-series GSE20713
(GSE20712 and GSE22249) and a separate dataset (GSE31979), whose data distributions
were similar, were downloaded from GEO2R. Both of the studies were performed on GPL8490
Illumina HumanMethylation27 BeadChip arrays. One probe (cg24619694) corresponded to
a CpG at +445 position from TSS of TAGLN, and the other probe (cg06950730) corresponded to a CpG at ?561 position. Methylation
scores of two probes were correlated (Spearman coefficient?=?0.714, P??0.0001). Thus, average methylation scores of the two probes were used for the calculations.
The data were normalized by assigning 0 for the lowest average methylation scores
for two probes of TAGLN gene, and assigning the value 100 for the highest scores for each dataset, and the
scores in between are fit into a sigmoidal curve. Normalized methylation scores (NAMS)
were used for the further studies. Mann–Whitney test was used to compare two groups.
For correlation analyses, expression data (GSE20711) corresponding to 88 breast cancer
patients and 2 healthy individuals included in the methylation study (GSE20713) was
downloaded from GEO2R, and the Spearman’s rank correlation between expression and
methylation scores was calculated. For survival analyses using the NAMS values, a
range of values greater than or equal to the diagnostic cutoff (i.e., 60, 65, 70,
75, 80, 85, and 90) was tested, and the best performing cutoff was determined to be
NAMS 80 85]. Patients were divided into NAMS ?80 and NAMS 80 groups, and 10-year RFS were calculated
for each group. P values were calculated using Gehan-Breslow-Wilocoxon test in GraphPad Prism 6.0.

Western blotting

Protein lysates (20–50 mg) from cell lines were separated under reducing conditions
(5 % ?-mercaptoethanol), in 12 % Tris-glycine gels and SDS-Tris-glycine running buffer
and were blotted on nitrocellulose membranes. Membranes were incubated overnight at
4 °C, with primary antibodies (?-TAGLN, Abcam ab1416, 1:2,000; ?-Calnexin, Abcam,
1:20,000), followed by washing with Tris buffered saline –Tween-20 (0.25 %, TBS-T)
and secondary antibody (horse radish peroxidase-conjugated anti-rabbit IgG, Abcam
1:5,000) incubations for 1 h at room temperature. Signal detection was performed with
ECL prime system (Amersham Life Science, RPN2232).

siRNA transfections and overexpression of TAGLN in breast cancer cells

siGENOME Human TAGLN siRNA (SMARTpool, Thermo Scientific, M-003714-02-0020) and siGENOME Non-Targeting
siRNA #2 (Thermo Scientific, D-001210-02-20) control siRNA transfections were performed
with RNAi-max (Invitrogen, 13778150), using the reverse transcription protocol supplied
by the producer. TAGLN overexpression, TrueORF-Gold pCMV6-Entry-TAGLN (Origene, RC215789), and control pCMV6-Entry vectors (Origene, PS100001) were transfected
to MDA-MB-361 and MDA-MB-157 cells using Lipofectamine 2000 reagent (Invitrogen, 11668019).
Stable clones were generated with Geneticin (Gibco-Life Technologies, 10131–035) selection.

2D colony formation assays

Cells were seeded to be 2.000/well in 6-well plates and were grown for 2 weeks (MDA-MB-157,
MCF10A, and MCF12A) or for 4 weeks (MDA-MB-361). Colonies were then stained with 0.5 %
crystal violet and counted using Image J (NIH). Colony sizes of MDA-MB-361 were calculated
using Image J. Colony sizes of other cell lines could not be determined due to more
scattered nature of colonies of these mesenchymal types of cells. Statistical differences
were calculated with two sample t test in GraphPad Prism 6.0.

In vitro wound healing and Matrigel invasion assays

For in vitro wound healing assays, cells were plated to be confluent in 24-well plates. Next day,
media was changed to low serum media (0.5 % FBS) and at least 6 independent scratches/sample
were made with 200-?l pipette tips, and photos were taken after 24 h. Statistical
differences between groups were tested with the Mann–Whitney test in GraphPad Prism
6.0. For Matrigel invasion assays, Growth Factor Reduced Matrigel Matrix Basement
Membrane (BD, 356230) was diluted (1:5) and added to Transwell Permeable Support invasion
chambers (Corning, 3422) in 24 wells to form a thin layer. Cells (150,000) per chamber
were added to the upper chamber with 0.5 % serum containing media and the lower chamber
was supplied with 10 % serum containing medium. Cells on the lower side of the chamber
were stained with Giemsa and counted at the 48
^th
hour.