Cell culture and cell treatment
Human breast cancer cell lines (MCF-7 and MDA-MB-231) were purchased from the Cell Bank of China Academy of Sciences Type Culture Collection. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) plus 10% fetal bovine serum (Gibco, Vienna, Austria), 100 µg/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were cultured at 37 ℃ in a humidified 5% CO2 incubator. All transfection assays were carried out with Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific) referring to protocol. Opti-MEM (Invitrogen) was used as the transfection medium. RNA interference was carried out by transfecting 50 nM control and si-BRWD3 respectively, from GenePharma Company (China). Silencer si-BRWD3 sequence was followed as: GCAAACGUCAGCAUACUUATT. Scrambled siRNA (UUCUCCGAACGUGUCACGUTT) was used as control. All these assays were carried out at least in triplicate.
The cells were plated on slides. The next day, cells were infected to silence BRWD3. After 48 h of transfection, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized with 0.05% Triton-X for 3 min, and then blocked with 2% bovine serum albumin for 1 h. The antibodies including FITC-labeled Phalloidin (#40735ES75, YIASEN Biotech Co.Ltd, China), β-tublin (ab6046, USA) were used to show microfilament and microtubules in the cells. After sections were washed with phosphate-buffered saline (PBS), 3×5 min, the cells were then counter-stained with 4′,6-diamidino-2- phenylindole (DAPI) for nuclei labeling. Images were collected by an Olympus fluorescence microscope at ×40 magnification.
Detection of protein expression with western blot
The cells were washed with cold PBS and then were harvested with RIPA buffer containing proteinase inhibitors. The protein was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA). After block for 1 h with 5% nonfat mike powder in PBS, the proteins were incubated o/n with anti-GFP antibody (#AG281, Beyotime Biotechnology, China) or anti-GAPDH (Signa-Aldrich, USA). The membrane was washed with PBS with Tween detergent (PBST), 3×10 min. After incubation with horseradish peroxidase-coupled secondary antibodies for 1 h at room temperature, the membrane was washed with PBST again, 3×10 min. Then, the proteins were detected and exhibited with ECL reagents (Pierece, USA).
To measure the motility ability of cells, scratch wound assay and transwell assays were carried out. For scratch wound assay, breast cancer cells were cultured into a 6-well plate. After 24 hours of RNAi, the wound was scraped with a sterile pipette tip. The 6-well plate was washed twice to clean the cells fallen off. The cells were cultured with media as above-mentioned, except with 5% FBS. The images were obtained at 0 h, 24 h, and 48 h, and analyzed with Image J. The cell motility ability was measured and evaluated with the scratch wound healing area.
To do transwell assays, according to a previous report with modification , the mixture of serum-free media and matrigel (Corning, NY, USA) was put into the insert chamber (BD Falcon, Franklin Lakes, NJ, USA) overnight. The 5×104 of control cells (siRNA-Control) and treated (si-BRWD3) of MCF-7 or MDA-MB-231 were digested and re-suspended in serum-free DMEM and seeded into the insert. The bottom chamber was filled with 600 µl of DMEM plus 10% FBS. After migration for 48 h, non-migration cells on the upper side of the filter were scraped off with a cotton swab, and cells on the bottom were fixed with 4% PFA for 10 min, and then stained with 0.2% crystal violet in 20% methanol for 15 min at room temperature, and washed twice with PBS, 2×5 min. The migration ability of cells was evaluated by counting the number of invasive cells on the bottom of the inserts with a light microscope. The assay was performed in triplicate and was repeated at least three times.
Cell proliferation and invasion detected with the xCELLigence™ viability assay
The x CELLigence® RTCA DP system (Roche Applied Science, Penzberg, Germany) was used to automatically and continuously monitor live cell proliferation, growth, viability, migration, and spreading with characters of non-invasive, close to physiological status, a higher degree of accuracy, and lab free [29, 30]. The cells transfected with siRNA-Control or si-BRWD3 were seeded on an E-plate-16 (ACEA Biosciences, San Diego, CA) at the optimal cell density (4×104 cells) with 100 µl DMEM media without FBS for proliferation assay. Cells’ growth was recorded in real-time every 30 min. The cell growth status was exhibited with normalized cell index values. The assays were repeated three times.
As previously reported , the mixture of serum-free media and matrigel (Corning, NY, USA) was put into the upper chamber of CIM (cellular invasion/migration)-Plate 16. After 4 h, the cells transfected with siRNA-Control or si-BRWD3, 4×104 cells of MCF-7 or MDA-MB-231 were digested and re-suspended in serum-free DMEM and seeded into the insert chamber with FBS- free DMEM. The bottom chamber contained 165 µl of DMEM plus 10% FBS. The x CELLigence® RTCA DP system was used to monitor cell migration in real-time every 30 min. The difference between siRNA-Control and si-BRWD3 was shown with a normalized cell index. All data (including from the growth monitor assay) were expressed as mean±SD. Statistical analysis was performed with two-way ANOVA. Statistically significant was set as p<0.05.
Chromatin immunoprecipitation combining with Sequencing (ChIP-seq) assay
Construction of plasmids and Cell transfection
No antibody was available for ChIP or even for immunoprecipitation for BRWD3, thus ChIP assay was performed with anti-GFP antibody (#AG281, Beyotime Biotechnology), which had been used in ChIP or immunoprecipitation assays [31, 32]. The EGFP from pEGFP-N1 (Clontech) was amplified with PCR, and then the amplicon was inserted into the pcDNA3.1 (+) vector (Invitrogen, Thermo Fisher Scientific) to construct pcDNA3.1-EGFP. Because of the large molecular weight of BRWD3 (203.5KDa), it’s very difficult to introduce the BRWD3 into cell lines. So，the sequences (3691-5815 bp) encoding two BRD repeats of BRWD3 was amplified with PCR and inserted into pcDNA3.1-EGFP to construct pcDNA3.1-2BRD (BRWD3)-EGFP. The primers and reaction conditions were all listed in supplementary table 1. Both the plasmid sequences were confirmed with the Sanger sequencing. Both pcDNA3.1-EGFP and pcDNA3.1-2BRD (BRWD3)-EGFP were introduced into MDA-MB-231 cells when the cell was grown to 50-60% confluence respectively. Transfection was confirmed by the assays of cell immunofluorescence and western blot. The plasmid of pcDNA3.1-2BRD (BRWD3)-EGFP was used as bait to immunoprecipitate specific DNA fragments binding to BRWD3 through 2BRD, and the pcDNA3.1-EGFP was used as the matching control.
ChIP assay The ChIP was carried out referenced to a previous report . Briefly, when the confluence of the cell transfected with target plasmids reached 80-90%, the cells were incubated with 1% formaldehyde in 1×PBS (final concentration of 0.75%) at room temperature for 10 min to chemically cross-link DNA to 2BRD(BRWD3). Glycine (final concentration of 125 mM) was added to quench the cross-link reaction. Washed twice with 1×ice-cold PBS, and harvested in cold lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl pH 8.0, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 50 μM iodoacetamide) containing 1 mM fresh phenylmethylsulfonyl fluoride (PMSF). The cell lysate was plated on ice and sonicated to 200-1,000 bp. The supernatant was obtained from the mixture with centrifuge for 10 min at 10,000 rpm at 4℃ and was diluted to 2 A260 units/ml in 1×RIPA buffer containing proteinase inhibitors. 200 μL mixture was put aside as input. The lysates were pre-cleared for 2 h at 4℃ by the protein A/G-Sepharose-salmon sperm competitor DNA. Then the supernatant was incubated with anti-GFP (#AG281, Beyotime Biotechnology, China) and 50 μL protein G sepharose (GE Healthcare, Japan) overnight at 4°C. The resin was centrifuged, 3000 rpm for 1 min，and washed three times with Low Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), High Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid sodium salt, 1mM EDTA, 10 mM Tris, pH 8.1). The beads were washed at 4℃ for 5 min at every step above-mentioned. The complexes were eluted twice from beads with 250 μl elution buffer (1% SDS, 0.1 M NaHCO3). 20 μl NaCl (5 M) was added into the supernatant (~500 μl) to reverse cross-link overnight at 65℃.
Both immunoprecipitated DNA and input DNA were purified with the QIAquick® PCR purification kit (#28104, QIAGEN, Germany). The DNA concentration was measured with QUBIT dsDNA HS ASSAY KIT (Q32851, ThermoFisher, USA), and total DNA is required to be more than 10 ng.
HiSeq 2500 Illumina sequencing All samples were performed a conventional procedure, including end repair, adaptor ligation, size selection, amplification with PCR, enriched with AMPure XP beads, to construct library expressing 2BRD (BRWD3)-EGFP and control. The quality of libraries was evaluated with a 2100 Bioanalyzer High Sensitivity DNA chip, and the quantity was evaluated with the KAPA kit (# KK4602, illumina). Finally, the samples were subjected to HiSeq 2500 Illumina sequencing in CapitalBio Technology (China).
The quality of the raw data was checked with the FastQC software. Clean data was matched to the human genome (Ensemble GRCh38.81) with the bowtie1 . Peak calling of unique mapped reads was conducted with MACS14 . Annotation of ChIP peak, comparison, and visualization were conducted with ChIPseeker . All the sites binding to pcDNA3.1-2BRD (BRWD3)-EGFP were selected through eliminating the sites binding to pcDNA3.1-EGFP. All sequencing data including will be deposited in NCBI’s Gene Expression Omnibus when the article is exhibited.
Annotation and enrichment of gene functions All Ensemble gene IDs of sites were converted into gene symbols, then performed gene function annotation and enrichment by utilizing the Gene Ontology. The unique genes were submitted and analyzed in the database (Released 20200407). Fisher’s Exact test and the correction of Bonferroni for multiple testing were set and only items with Bonferroni-corrected for P<0.05 were displayed and listed.
Quantitative Real-time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
After the selection of the core genes with ChIP-seq, the transcription level was measured with qRT-PCR, when BRWD3 was knocked down in MDA-MB-231 and MCF-7, to determine the interaction among BRWD3 and its influencing genes. Total RNA was isolated with RNAsimple Total RNAkit (Tiangen, China), according to the manufacturer's protocol. Total RNA was reverse transcribed using PrimeScriptTM RT Master Mix（Perfect Real Time）kit (TaKaRa, Japan), following the instruction. QRT-PCR was carried out with SuperReal Color Premix (Tiangen, China) on LightCycler® 480 (Roche, German). GAPDH was selected as the reference gene for the normalized of all other gene expression results. All primers and reaction conditions were listed in the supplementary table 1. The relative expression levels were calculated by 2-ΔΔCt (∆Ct = CtmRNA- CtGAPDH RNA). The experiments were carried out at least in triplicate. The average of independent analyses for each gene was calculated. The data were shown as mean±standard deviation (SD) of independent samples. SPSS 20.0 program was used to test significant differences through one-way analysis of variance (one-way ANOVA) and multiple comparisons. The value of a statistically significant difference was set as p<0.05.