Patients and specimens
A total of 10 pairs of ruptured and un-ruptured intracranial aneurysm tissues were obtained from surgical resection during the aneurysm clipping surgery in Beijing Tiantan Hospital. The collection of human specimens was approved by the Medical Ethics Committee of Beijing Tiantan Hospital, Capital Medical University. Written informed consent was obtained from each patient according to the policies of the committee. All specimens were stored in liquid nitrogen, and 5 pairs of samples were used to conduct circRNA microarray analysis, other samples were used to perform qPCR.
Total RNA isolation and quality control
We extracted RNA with the use of Trizol Reagent (Invitrogrn, NY, USA) from 5 paired ruptured and un-ruptured intracranial aneurysms according to the manufacturer’s instructions. The quality and concentration of RNA was tested by the NanoDrop ND- 1000 (Thermo Fisher Scientific, Wilmington, DE, USA) (Additional file 1).
RNA labeling and hybridization
Sample labeling and array hybridization were performed according to the manufacturer’s protocol (Arraystar Inc.). Briefly, total RNAs were digested with Rnase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs (Additional file 2) were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the circRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned using the Agilent Scanner G2505C.
CircRNA microarray analysis
Agilent Feature Extraction software (version 126.96.36.199) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software limma package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering. Differentially expressed circRNAs between two samples were identified through Fold Change filtering. Hierarchical Clustering was performed to show the distinguishable circRNAs expression pattern among samples.
Real-time quantitative PCR (qPCR)
Real-time PCR was used to verify differentially expressed circRNAs obtained from microarray analysis. RNase R ( Lucigen, 20U, 37°C，3h ) was used to purify the circRNAs again. The relevant cDNAs were composed (M-MLV, promega ) and stored in -20°C. QuantStudio5 Real-time PCR System (Applied Biosystems) was used to perform qPCR. The sequence of circRNA results was acquired from the database “circBase” (http://circrna.org). Primers were produced by RiboBio (Guangzhou, China) (Additional file3). Because of the influence of concentration quantitative error and reverse transcription efficiency error, the cDNA content of every sample was different. In order to correct these errors, we regarded housekeeping gene β-actin as internal reference, as a result, we accepted the ratio of genes to be tested and internal reference, in other words, the relative content of gene to be tested.
Construction of circRNA-miRNA-mRNA network
CSCD (http://gb.whu.edu.cn/CSCD/) was used to recognize miRNAs binding on our target circRNA. Three algorithms including Targetscan18, miRDB19, and miRTarBase20 were used to analyze parental genes of miRNAs binding on the target circRNA. The circRNA-miRNA-mRNA network was visualized by Cytoscape(version3.7.1).
Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis
We assumed that our target circRNA may have molecular interactions with these genes, or our target circRNA may regulate biological functions through these genes. GO analysis on genes correlated with these miRNAs was performed by DAVID (https://david.ncifcrf.gov/). The P value after adjustment represents the significance of GO terms. We also perform KEGG pathway analysis of parental genes of circRNA-binding miRNAs, in order to reveal the biological or pathological processes which circRNAs participate in. The P value after adjustment represents the significance of pathway correlations as well.
Human brain vascular smooth muscle cells (HBVSMCs) were obtained from Bnbio (BNCC102172, Beijing, China). The cells were cultured in Smooth Muscle Cell Medium (SMCM) containing 2% fetal bovine serum (FBS), 5 ml of smooth muscle cell growth supplement (SMCGS, Cat. No. 1152), and 5 ml of penicillin/streptomycin solution (P/S, Cat. No. 0503) at 37°C in an incubator of 5% CO2.
Transduction of cells
The hsa_circ_0005505 specific shRNA, their relevant lentiviruses (LV-circRNA-RNAi (74402-1), LV-circRNA-RNAi (74403-2), LV-circRNA-RNAi (74404-1)) and negative control lentivirus were obtained from Shanghai Genechem Co, LTD (Shanghai, China). HBVSMCs were transduced with individual types of lentivirus at a multiplicity of infection (MOI) of 50 and the ideal value of infection efficiency was 80%.
MTT assay was used to measure the proliferation of HBVSMCs according to the manufacturer's instructions (Dingguo Biotech, Shanghai, China). HBVSMCs (2×103 per well) were plate in 96-well plates and treated with 20μl of 5mg/ml MTT solution, then the spectrophotometrically at 490 nm and 570nm was analyzed by automatic microplate reader (Tecan infinite).
Cell counting kit-8 (CCK-8) assay
CCK-8 assay was used to test the cell viability in order to verify the effect of the target gene on cell proliferation. The assay was performed according to the manufacturer's instructions (Dojindo Laboratories, Kumamoto, Japan). HBVSMCs (2×103 per well) were plate in 96-well plates and treated with 10μl of CCK-8 solution, then the spectrophotometrically at 450 nm was analyzed by automatic microplate reader (Tecan infinite).
Cell apoptosis was analyzed in two ways. The caspase3/7 assay was used to verify the effect of the target gene on apoptosis of cells by using Caspase-Glo 3/7 Assay reagent test kit (Promega). We also analyzed cell apoptosis by using the Annexin V-APC Apoptosis Detection Kit (eBiosciences #88-8007) according to the manufacturer’s instruction. HBVSMCs were stained with APC and then analyzed by fluorescence-activated cell sorting using FACScan (BD Biosciences).
Wound- healing assay
The wound -healing assay was used to measure the migration rate of cells. The transfected HBVSMCs were seeded into 96 well plates (5×104 per well). After 24h incubation, parallel wounds with similar width were made in each well by 96 Wounding Replicator (VP scientific). Wound closure level was monitored by Celigo (Nexcelom) in 0h, 8h, 24h after wounded and lastly analyze the migration rate.
The fold-changes were estimated by unpaired Student’s t-test and used to identify the differentially expressed circRNAs in the sample of intracranial aneurysms. CircRNAs were selected as differentially expressed with a P<0.05 and a fold-change >1.5, which means they were statistically significant. The significance of qPCR was assessed by Student’s t-test and P < 0.05 was considered statistically significant, it was analyzed by GraphPad Prism 8.4.0 (GraphPad Software, La Jolla, CA, USA). Other statistical methods such as chi-squared test, Wilcoxon signed-rank test and Mann Whitney U test were also performed. All statistical analyses were performed by SPSS 19.0 (SPSS, Inc., Chicago, IL).