Access and analysis of public data
GSE20685 was downloaded from the GEO dataset (http://www.ncbi.nlm.nih.gov/geo/). The whole genome expression profiles and clinicopathological information of human cancer were downloaded from the Human Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/). The expression of lncRNAs in the cell lines was obtained from the Cancer Cell Line Encyclopedia (CCLE) database (http://www.broadinstitute.org/ccle). The IC50 values of cell lines to drug therapy were obtained from Genomics of Drug Sensitivity in Cancer (GDSC) database. All transcripts were normalized by log2 transformation. Diana lncbase (http://carolina.imis.athena-innovation.gr/diana_tools) was used to predict the binding of miRNAs and lncRNA. Bioinformatics tool analysis miRdb (http://www.miRdb.org/) was used to predict the binding of miRNA and mRNA 3'UTR. The RNAhybrid website (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/) was used to predict the binding sites of miRNA and lncRNA. The bioinformatics website lncLocator (http://www.csbio.sjtu.edu.cn/cgi-bin/lncLocator.py) was used to predict the subcellular localization of lncRNA.
The correlation between genes was evaluated through pearson correlation test. The differences between BRCA tumor and normal samples was evaluated by unpaired t-test. Log-rank test was used to test the difference of survival rate among different groups of patients.
Patient specimens
BRCA specimens (n = 96) were obtained from hospitalized patients from November 2008 to June 2009. All patients were diagnosed with BRCA in the First Affiliated Hospital of China Medical University. Before surgery, the patient did not receive chemotherapy or radiotherapy. This study was approved by Ethics Committee of China Medical University (Approval number: AF-SOP- 07-1.1-01).
Cell culture
MCF-7, T47D, MDA-MB-231 and HEK-293T cell lines purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China), and MCF-7/ADR purchased from the Zhen Shanghai and Shanghai Industrial Co., Ltd (Shanghai, China). The cells were cultured in L-15 medium, DMEM medium or RPMI 1640medium (Hyclone, USA) and cultured in a humidified incubator at 37 ℃. Except the cells cultured in L-15 medium, the cells were cultured in the environment containing 5% carbon dioxide.
Microarray analysis
Microarray analysis was performed by Shanghai OE Biotech (China). The microarray was Agilent Human lncRNA 4*180K (Design ID: 076500). The raw microarray data were submitted to the GEO database (accession number: GSE155478). The LncRNAs were identified by gencode database (https://www.gencodegenes.org). The p-value and fold change value of t-test were used to screen differential lncRNAs. The screening criteria were up-regulated or down-regulated log2FC ≥ 2.0 and FDR ≤ 0.05.
Gene set enrichment analysis
Data of CBR3-AS1 expression were obtained from BRCA patients with TCGA. Taking the median of cbr3-as1 expression as the cut-off point, BRCA cases were divided into two groups. The index of gene sequencing was set as "Pearson", the curve of top set was set to 150, and all other parameters were the default values.
Cell transfection
The cells were seeded on a 6-well plate and incubated for 24 hours. The fusion degree of cells reached 70–80%. Lipofectamine 3000 (Thermo Fisher Scientific, MA, USA) transfection reagent (5µL) was dissolved in 125 µ l serum-free medium. 2 µ g DNA was diluted with 125 µl serum-free medium to prepare DNA (Hanheng, China) premix, and then 10µL p3000 reagent was added to mix well. The Lipofectamine 3000 was then mixed with DNA. After incubation for 5 minutes, the complex was added to the cells. After 4 hours, the fresh medium containing 10% serum was changed. The final transfection concentration of miRNA mimics (RiboBio, Guangzhou, China) was 50 µm and the final concentration of inhibitor was 100 µM. The final concentration of siRNA (RiboBio, China) transfection was 50 µM. p3000 was not used in the transfection process. The other steps were equivalent to DNA transfection.
In animal experiment, the plasmid was stably transfected. On the basis of the above steps, puromycin 1 mg/L was added to culture for one month, and stable transfected cell lines were selected for the experiment.
Western blotting
Cells were lysed on ice in RIPA buffer (Beyotime, China). The lysate was centrifuged at 15000 g at 4 ℃ for 15 minutes. SDS polyacrylamide gel electrophoresis was used to isolate proteins and transfer them onto PVDF membrane. The membrane was blocked in 5% skimmed milk and incubated with the primary antibody in TBST at 4℃ overnight. Then incubated with a horseradish peroxidase-labeled secondary antibody at a concentration of 1:10000 for one hour. Finally, chemiluminescence was carried out using micro chemi 4.2 system (DNR Bio-imaging system, Israel). The details of all antibodies used were showed in Table S1
Luciferase assays
HEK-293T cells were transfected with MEK4-3′UTR-WT, MEK4-3′UTR-MU, JNK1-3′UTR-WT and JNK1-3′UTR-MU plasmids. At the same time, the four groups cells were transfected with mimic NC and mimic-miR-25-3p to form eight experimental groups. Finally, each group of cells were transfected with Renilla Luciferase plasmid. After 36 hours, the cells were harvested for luciferase analysis using a dual luciferase reporting and detection system (Promega, USA). The result were normalized against Renilla luciferase activity.
qRT-PCR
Trizol (Invitrogen, USA) was used to extract total RNA from cultured cells. For mRNA and lncRNAs, ReverTra Ace qPCR RT Kit (Toyobo, Japan) was used, and 200 ng of total RNA was used to synthesize cDNA at 10 µl reaction volume. For miRNAs, Bulge-Loop™ miRNA qRT-PCR Starter Kit (Ribobio, China) was used, and 1 µg of total RNA was used to synthesize cDNA at 10 µl reaction volume. The qRT-PCR of mRNA and lncRNAs used SYBR Green Realtime PCR Master Mix (Toyobo, Japan) with 12.5 µl reaction volume. The qRT-PCR of miRNAs used Bulge-LoopTM miRNA qRT-PCR Starter Kit (ribobio, China) with 20 µl reaction volume. The primers were listed in Table S2.
Colony formation assay
MCF-7/ADR and MCF-7 cells (1 × 103/well) in logarithmic phase were resuspended and inoculated in a 60 mm cell culture dish. When adding drugs, 100 nM ADR was added to MCF-7 cells, and 30 µM ADR was added to MCF-7/ADR cells to culture for 48 h. After, culture with normal medium. Two weeks later, the cells were washed with PBS twice and fixed with methanol, and stained with 0.1% crystal violet (Beyotime, China) for 30 minutes.
CCK8 assay
MCF-7/ADR and MCF-7 cells were transiently transfected into 96 well plate with 3 × 103 cells per well. After 24 hours, the cells were treated with ADR (Sigma Chemical Co, St. Louis, MO) for 48 hours. After incubation with 10 µl of CCK-8 reagent (dojindo, Japan) for 1 h, the spectrophotometer was measured at 450 nm.
ADR accumulation assay
According to the method of our previous articles[22], the ADR cumulative measurement was performed. Briefly, the cells were exposed to 5 µM ADR for 2 h, then wash the cells with PBS. Next, using the flow cytometer to measure ADR fluorescence to determine the intracellular ADR level.
Flow cytometric analysis of apoptosis
The MCF-7/ADR and MCF-7 cells apoptosis after ADR treated was evaluated using the Annexin-V APC/7AAD Iodide Detection Kit (BD Biosciences, USA) according to the manufacturer’s instructions. Cells were then analyzed by MACSQuant (Miltenyi Biotec, Germany).
Animal assays
The GFP containing negative control plasmid and OE-CBR3-AS1 plasmid were transfected into MCF-7 cells, and then treated with puromycin to obtain stable expression cell lines. All BALB / c mice (4 weeks old) were purchased from Shanghai Laboratory Animal Center (Shanghai, China). MCF-7 / control group and MCF-7 / CBR3-AS1 cells (1 × 107 cells / 100 µ L / nude mice) were subcutaneously injected into the axillary region of nude mice. When the subcutaneous tumor grew to 200-250mm3 (16 days later), the treatment group nude mice were intraperitoneally injected with ADR (80 mg / kg), once every three days. All nude mice were treated for 6 times. The diameter of transplanted tumor was measured with digital caliper every week. The tumor volume was calculated as volume = 0.5 × width2 × length. All nude mice were humanly sacrificed at the end of the experiment, and the tumor tissues were collected for further study by qRT-PCR, IHC and Western blotting.
ISH (In situ hybridization) and IHC (Immunohistochemistry)
The assay was carried out according to the method mentioned in the previous study [23]. We collected BRCA tissues of patients from the First Affiliated Hospital of China Medical University for ISH assay. The ISH probes were ordered from BOSTER Biological Technology co.ltd (USA). We collected BRCA tissues from collected different groups of subcutaneous tissues for IHC assay. The details of all antibodies used were showed in Table S1.
Biotin pull-down
MCF-7 cell lysates were incubated with CBR3-AS1 or control probe (Sangon, China) conjugated to Streptavidin agarose resin beads (Thermo Fisher Scientific, USA) to generate probe bound dynabeads. After washing with buffer, add DNase I (20 U) and collect purified RNA. The purified RNA was analyzed by qRT-PCR. The whole experimental process and the buffer formulation were referred to Hsu et al[24]
Statistical analysis
Quantitative data were expressed as the mean ± SD of at least three independent experiments. All experimental values were evaluated using graphpad prism 8.3.0 (graphpad software). The unpaired t-test was used for statistical analysis between the two experimental groups. The relationship between CBR3-AS1 and clinicopathological factors was tested by chi square test. In all cases, P < 0.05 was considered statistically significant.