Tissue specimens and ethics statement
The date of EPB41L4A-AS1 expression levels of breast cancer was download from The Cancer Genome Atlas date portal(TCGA). To validate whether EPB41L4A-AS1 was a lncRNA, the Coding Potential Calculator 2 Tool (CPC2, http://cpc2.cbi.pku.edu.cn.) was employed to predict its protein-coding potential10. All breast cancer tissue specimens and paired was collected from the Department of Breast Surgery of the First Affiliated Hospital of Wenzhou Medical University. After surgical resection, the tissues were immediately transferred to Liquid nitrogen tank and stored at the − 80℃ refrigerator. All processes were authorized by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University.
Cell culture
The breast cancer cell lines (MCF-7, MDA-MB-231) were acquired from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The MDA-MB-231 cells were cultured in RPMI 1640 medium (Invitrogen, USA), while the MCF-7 cells were cultured in DMEM medium (Invitrogen, USA). All cells were cultured at 37°C and 5% CO2 and nourished by the medium containing 10% FBS (Gibco, Grand Island, NY, USA), as well as 100 UI/ml penicillin with 100 µg/ml of streptomycin, mixed and kept at 4°C. The experiments consisted of three groups. The group design consisted of a non-transfected blank control group (Blank group), an empty vector negative control group (NC group) and EPB41L4A-AS1 overexpression plasmid transfection group (Overexpression group).
Real-Time Polymerase Chain Reaction (qRT-PCR)
Extract RNA from cells and tissues according to the manufacturer's instructions using TRIzol reagent (Invitrogen, USA). Then, quantitative RNA was transcribed into cDNA, using Rever Tra Ace qPCR RT Kit (Toyobo). Real-time reactions run and analyzed by using a Real-Time PCR system (Applied Biosystems 7500), all data normalized to GAPDH levels in every sample. The primers sequences are presented as follow: EPB41L4A-AS1 forward༚5’- AAGACAGTGAGGATGTGAAT − 3’ and reverse: 5’- TATGGTGACAGCAGTGAATA − 3’;GAPDH༚forward: 5’- GTCTCCTCTGACTTCAACAGCG-3’ and reverse: 5’- ACCACCCTGTTGCTGTAGCCAA-3’.
Transfection
EPB41L4A-AS1 overexpression plasmid (pcDNA3.1- EPB41L4A-AS1) and a control vector were designed and constructed. The cloned plasmids were transfected into cells according to the manufacturer's instructions using Lipofectamine 2000 (Invitrogen) in Opti-MEM solution (Gibco). The negative control group and the blank group were transfected for the empty vector and without vector, but the same amount of Lipofectamine and Opti-MEM was used. The EPB41L4A-AS1 expression in cells after transfected by plasmids were confirmed by qPCR.
Colony forming assay
Transfected cells were seeded into 6-well plate, include 1500 cells each well, incubated at 37℃ with 5% CO2. Observing its growth state, harvesting when its cluster include more than 50 cells probably (about 7–10 days). At that time, colonies were fixed with 4% Paraformaldehyde Fix Solution and stained with 0.01% crystal violet solution.
Cell Counting Kit-8 - cell proliferation assay
Transfected cells (1500 cells/well) were seeded into 96-well plates and incubated at 37℃ with 5% CO2 for 24h, 48h, 72h and 96h, respectively. Then, 10 µl CCK-8 reagent was inserted into each well, repeated 5 times, and incubated for 2.5 h. The absorbance was then measured at 450 nm.
Cell cycle distribution assay
The transfected cells were collected and washed for three times with PBS. One milliliter of 70% ethanol was used to resuspend cells with soft stirring. The cells were stained with 300 µl of propidium iodide (PI), and then analyzed by Flow Cytometer.
Apoptosis detection analysis
Total cells were collected include free in medium and adhere in bottom. Centrifuging collected cells at 1000 rpm for 5 min and washing with 3ml PBS at 1000 rpm for 5 min, repeated three times. Finally, Using Annexin-V-FITC apoptosis detection kit according to the manufacturer’s instructions and analyzing by Flow Cytometer.
Transwell assay
The cells transfected and incubated in 6-well plate were trypsinized with Trypsin-EDTA Solution and ceased by 10%FBS DMEM. 3*104 for MCF-7 or MDA-MB-231 resuspended in serum-free medium(300µl)were transferred into the upper chamber of transwell, while the bottom chamber was filled with RPMI DMEM culture medium༈include 20%FBS༉, then put into incubator about 24h. After24h, cells adhered to the upper surface were cleaned and then, fixed with 4% Paraformaldehyde Fix Solution for 15min, obliterated the exceptional cells with PBS, and at last, stained for 15 min with 0.01% crystal violet solution. The pictures were collected by photomicroscope.
Gene ontology (GO) analysis and pathway analysis
GO analysis was performed to define the biology of EPB41L4A-AS1. GO annotations were obtained by downloading from NCBI and GO databases11. Pathway analysis was used to identify pathways significantly enriched for EPB41L4A-AS1 according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database12. Fisher's exact test was applied to identify significant GO categories and to select significant pathways.
Statistical analysis
Statistical analysis was established using SPSS 23.0 statistical software packages (SPSS, Inc., Chicago, IL, USA). Statistical significance between groups was analyzed using student’s t-test or a one-way ANOVA. P < 0.05 were considered statistically significant.