This research involved collecting human tissues from Khatam-ol-Anbia and Rasule-Akram hospitals, with no experimenting on human subjects or animals. In vitro experiments on commercial cell lines and pathological samples were approved as a Ph.D. thesis proposal, by Ferdowsi University of Mashhad (code number: IR.UM.REC.1399.104).
Clinical tissue samples.
In this study, a total number of 52 pathological tissue samples with a diagnosis of primary breast cancer were collected. None of the patients had been treated with preoperative radiotherapy, chemotherapy, or other relevant modalities. Breast cancer tissue samples along with their matched adjacent apparently normal tissues were collected, immediately preserved in liquid nitrogen, and then stored at -80ºC until analysis. All patients signed informed consents and agreed to the use of their surgical specimens for research.
We used UCSC genome browser to scan the genomic area around C1orf132 (located at chr1: 207,978,592-208,052,441 (hg19)) for potential promoter activity. Histone modifications by ChIP-seq from ENCODE (H3K27ac and H3K4me3) and DNA-seq were used to detect the active area near transcription start site (TSS). Using ENCODE ChIP-seq data for different transcription factors (TFs), a list of TFs which bind to C1orf132 potential promoters was extracted. Fantom5 data were used to determine which genomic regions the reads originated from.
To evaluate the correlation of C1orf132 transcripts originating from the putative p2/distal promoter with the tumor state of breast tissues, we performed data mining of The Cancer Genome Atlas (TCGA) breast cancer subtypes to study their association with the expression level of C1orf132.
Cloning the putative promoters of C1orf132.
The potential promoter regions (p1 and p2) for C1orf132, according to the bioinformatics analysis, were amplified from genomic DNA using Accu Taq polymerase kit (Invitrogen, USA) and specific primers (supplementary Table 1). The amplified products (1203 bp for p1 and 2143 bp for p2), were cloned into the promoterless green fluorescent protein (pEGFP-1) reporter vector, using flanking sequences on primers for AgeI/SalI restriction sites (Supplementary Table 1). The accuracy of the cloned constructs was confirmed by DNA sequencing (Europhins, Italy).
Cell culture and transfection.
MCF7 and MCF12A were obtained from IIGM cell bank. MCF7 cell line was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. MCF12A cells were cultivated in a special medium containing DMEM/F12 (Thermo Fisher Scientific, USA), 5% heat inactivated horse serum (Gibco, USA), 20 ng/ml recombinant human EGF (AF100-15 Peprotech, USA), 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin (Vibrio cholerae C8052, Sigma, USA), 10 µg/ml human recombinant insulin (Zinc solution 12585-014, Gibco, USA) and 1% penicillin/streptomycin, and incubated at 37ºC with 5% humified CO2. To examine the promoter activity of the cloned regions, the recombinant vectors were used to transfect MCF7 cells. All experiments were done with complete medium in at least triplicates.
We examined the nuclear vs. cytoplasmic subcellular localization of C1orf132 in MCF12A cells by fractionation with cell fractionation buffer (Ambion, USA), according to the manufacturer’s instructions. RNA was extracted to assess the relative proportion of C1orf132 in the nuclear and cytoplasmic fractions. The transcription levels of beta 2-microglobulin (B2M) as a cytoplasmic marker, U1 as a nuclear marker and C1orf132 were then assessed using quantitative reverse transcription- polymerase chain reaction (qRT‐PCR).
Promoter activity reporter assay.
MCF7 cells were seeded in 48-well plates (SPL Life Sciences, South Korea). After complete adhesion, cells were transfected with 1 µg of vector (pEGFP-1-p1 or pEGFP-1-p2) using Lipofectamine LTX & PLUS reagent (Invitrogen, USA), according to the manufacturer’s instructions. pEGFP-1 is a promoterless vector, containing eGFP reporter. 48 h after transfection and under a fluorescent microscope, the presence of the GFP signal was monitored in MCF7 cells transfected with pEGFP-C1, as the positive control, and pEGFP-1-p1 or pEGFP-1-p2 vectors.
Promoter deletion using CRISPR/Cas9 system.
In order to suppress C1orf132 expression, we decided to delete its p2 promoter using CRISPR/Cas9 system. Three pairs of different guide RNAs (gRNAs) were designed (Supplementary Table 1) to target the putative sequences of the p2 transcription start site, using http://crispr.mit.edu. The three gRNAs where then cloned into TOPO-TA gRNA vector. Briefly, guide RNA plasmid backbone (TOPO-TA gRNA) was linearized using the BbsI enzyme (New England Biolabs, USA) and digested at 37˚C for 1 h. Annealing took place using sense and anti-sense oligonucleotides in buffer 2 (NEB), before placing the reaction in a thermocycler with the ramp of 0.1˚C/sec from 95˚C to 25˚C. To ligate the gRNA within the linearized vector, T4 DNA ligase (Thermo Fisher Scientific, USA) was used and the reaction was incubated at room temperature for 60 min. Then, E. coli cells were transformed with gRNA vectors using heat-shock protocol followed by plating the cells on LB/ampicillin (100 μg/ml) plates overnight. Colony-PCR was performed to verify the sequence of the gRNA plasmids, using the reverse oligo of guides and forward primer of TOPO vector.
Different pairs of gRNA-containing vectors were co-transfected along with wild type-Cas9 vector (PX458) into MCF12A cells by lipofectamine LTX & PLUS reagent (Invitrogen, USA). Single cells expressing GFP were selected by a cell sorter machine (BD FACSCelesta), 24 h after transfection. Edited colonies were investigated for deletion, by DNA extraction using Puregene Core kit A (Qiagen, USA), according to the manufacturer’s instructions, followed by performing PCR using flanking primers (Edited-Test) and DNA sequencing (Europhins, Italy).
Wound healing assay.
Edited and unedited MCF12A cells were seeded into 24-well plates (with 6 repeats) and grew to 90% confluency. The monolayers were scratched using a 200 μl pipette tip and then the floating cells were removed by several washes with phosphate buffered saline (PBS). Subsequently, the cells were incubated at 37ºC for 24 h, before being photographed. The migration area of wound healing was border-lined and analyzed, using the ImageJ software.
Cell cycle analysis by flow cytometry.
Triplicates of three different densities (10000, 20000, and 30000) of edited vs. unedited MCF12A cells were seeded into 12-well plates. The Vybrant™ DyeCycle™ Violet Stain kit (Invitrogen, USA), which is capable of entering living cells and staining DNA, was used to examine the cell cycle 24 h later. Briefly, cells were washed in cold PBS and then harvested, and resuspended in the complete medium. Next, flow cytometry tubes each containing 1 ml of cell suspension in complete medium at a concentration of 1×106 cells/ml were prepared. 1 μl of Vybrant™ DyeCycle™ Violet Stain was added to each tube (final concentration of 5 μM) and mixed well. After 1 h incubation at 37˚C with protection from light, the samples were analyzed without washing or fixing on a flow cytometer (BD FACSCelesta) using laser beam.
RNA extraction and qRT-PCR.
A small amount of frozen patient tissue samples was lysed for RNA extraction using RNSol (ROJE, Iran)/or QIAZOL (Qiagen, USA). Total RNA was extracted according to the manufacturer’s instructions. 1 µg of RNA was first treated with DNase I (Thermo Fisher Scientific, USA) in order to eliminate any traces of DNA contamination, and then reverse transcribed using PrimeScript first strand cDNA synthesis kit (TaKaRa, Japan). The expression of target genes was evaluated using BioFACT™ 2X real-time PCR master mix (BioFact, South Korea) through ABI StepOne real-time PCR system. The sequence of primers used for quantifying each target gene can be found in Supplementary Table 1. Relative expression of target genes to B2M was calculated according to 2-ΔΔCt method. For the cells, the expression of target genes was evaluated using SensiFAST SYBR No-ROX one-step kit (Bioline. USA Cat. No: BIO-72001), according to the manufacturer’s instructions. 30 ng of RNA was used in one step real-time PCR using Rotor-Gene instrument (Qiagen, USA).
TaqMan miR assay for miR-29c-3p detection.
Hsa-miR-29c-3p (ID 000587), and U6 snRNA (ID 001973) TaqMan miR assays were ordered from Applied Biosystems (Foster City, CA). Total RNA extraction followed by qRT-PCR assay were performed to determine the expression level of miR-29c-3p in edited versus unedited MCF12A cells, according to the manufacturer’s protocol. Briefly, total RNA extraction was performed by Qiazol (Qiagen, USA), followed by reverse transcription using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, USA), before doing qPCR using Taqman® Universal PCR Master Mix II (Thermo Fisher Scientific, USA). The samples were then incubated for 10 min at 95ºC for initial denaturation, and then subjected to 40 PCR cycles, each consisting of 95ºC for 15 sec and 60ºC for 60 sec. U6 was used as the internal control of miR-29c-3p. The 2−ΔΔCt method was used to analyze microRNA levels.
All statistical analyses were performed using GraphPad Prism 6 software. Student t-test was employed to investigate the significance of observed differences of gene expression alterations. All tests were done in 3 biological repeats and values are reported as mean± standard deviation (SD). P values less than 0.05 were considered as statistically significant.