Participants and specimens
Both tumor tissue and plasma samples were collected from the General Hospital of Ningxia Medical University, Surgery department. This study were approved by the General Hospital of Ningxia Medical University Ethics Committee(Yinchuan, China). All patients involved in the study signed the informed consent for donating their samples. 23 BC tissues and 113 BC peripheral blood were collected from January 2017 to Augest 2018. The patients were including (1) women; (2) without previous cancer history; (3) without HIV/AIDS; (4) >18 years old; (5) after mastectomy. The age of patients ranging between 25-75 years old (average 49.94±9.97 years). All the patients are not received radiotherapy or chemotherapy treatment before specimen collection.
peripheral blood samples were collected from the median cubital vein with an EDTA anticoagulated vacutainer(2mL) after overnight fasting. BC tissues and para-carcinoma tissue were collected from patients who underwent surgical breast resection. The para-carcinoma tissue were located >5 cm from the tumors. Clinical and cancer pathological characteristics were collected including age, Healthy person(as control) were recruited from physical examination department in General Hospital of Ningxia Medical University. Collect the clinical, pathological, and molecular characterization data of all participants.
Human breast cancer cell line MDA-MB-231 were purchased from The Typical Culture Preservation Committee Cell Bank of Chinese Academy of Sciences. The cells were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA) at 37 °C in 5% CO2.
Tissue RNA were isolated from BC tissue and adjacent normal tissue with Trizol reagent (Invitrogen, Inc., USA). Peripheral blood RNA were isolated with Total RNA Rapid Extraction Kit (Bioteke, Inc., China). The quality of RNA were measured using Nanodrop 2000 (Thermo Scientific, USA). RNA integrity was determined by 1.3% agarose gel electrophoresis (120V, 15 min, 1×FA buffer ).
Human CircRNA Array v2 microarray(Beijing Capital Bio Biotechnology Corporation, China) has been used for circRNA microarray expression profiling. GeneSpring 13.0 software(Agilent) were used for circRNA array data analysis. Three pairs of BC tissues and para-carcinoma tissues were collected for circRNA microarray analysis. The labelled RNAs were hybridized on the microarray containing 162351 human circRNA probes. In order to improve the screening efficiency, differentially expressed of circRNAs were screened by the filter criteria FC ≥4, P-value<0.05, Original Fluorescence value ≥100.
The cDNA were synthesized by the RevertAid First strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., USA). qPCR were performed using LightCycler480II quantitative system(Roche, USA). The primers were synthesized Sangon biotech(Shanghai) Co.,Ltd, all circRNA primer was list in Table S1. The reaction in a total volume of 20 μL system including 1 µl total RNA (500 ng), 1 µl random primer, 10 µl ddH2O, 2 µl dNTP, 4µl Reaction buffer, 1µl Ribolock RNase Inhibitor, 1µl RevertAid M-MuLV RT. The cycling program is 25℃ 5min, 42℃ 60min, 70℃ 5min. GAPDH Expression were used as an internal control. Each reaction was repeated three times and the mean relative expression of genes were calculated using ΔΔCT method. The PCR production was stored at -80°C before use.
Microarray data analysis
The circRNA microarray data were extracted using Feature Extraction software(CapitalBio). Normalization, Fold change and P value were performed using GeneSpring software V13.0 (Agilent). Heat map were analysed using Cluster 3.0 software. ScatterPlot and VolcanoPlot were analysed using ggPlot2 software(R). CircRNA structure were performed by circPrimer1.2 software. Miranda v3.3a (http://miranda.org.uk/) and RNAhybrid 2.1 (https://directory.fsf.org/wiki/ RNAhybrid) were utilized to predict the target miRNAs of CIRC6969. The graphs of circRNA-miRNA interaction networks were drawn by cytoscape 3.6.0 (https://cytoscape.org/). GO analysis and genecards database(https://www.genecards.org/) were used to annotate the genesymbol of all differentially expressed circRNA, to explain their biological processes, cellular components and molecular functions. 这里需要插入文献
Data statistical analysis were performed by SPSS23.0 software (IBM,USA), GraphPad Prism version 8.0 (GraphPad Software, USA), data were presented as mean ±SD. The categorical variables were tested by the Chi-square test, and the continuous variables were tested by the t-test. ROC were performed to verify the clinical diagnostic value of circRNA. The correlation between circRNA expression and the breast cancer pathological characteristics were evaluated by Pearson`s correlation test and Curve regression analysis. Differences were considered significant if p<0.05 (* p < 0.05; **p < 0.01).