The BAV patients included in this study underwent surgical treatment and follow-up in Zhongshan Hospital Fudan University. According to severity of aortic dilatation, patients were defined as severe dilation (BAV-dilated, aortic diameter ≥ 45 mm, n = 13) and non-severe dilation (BAV-non, aortic diameter < 45 mm, n =11) groups, respectively. Patients in BAV-non group underwent aortic valve replacement (AVR) surgery, and those in BAV-dilated underwent AVR and concomitant ascending aorta replacement. Patients with impaired systolic ventricular function, significant coronary artery disease or aortic dissection were excluded in this study. Healthy donors with tricuspid aortic valve and normal aortic diameter (20mm-37mm), namely TAV-non, were employed as a control group (n = 6). Aortic specimens were obtained from patients with a tricuspid aortic valve but without aortic dilation who underwent coronary artery bypass graft were used as control group (n=3).
Transthoracic echocardiography was performed for all patients preoperatively. All echocardiographic studies were conducted according to standard techniques by experienced echocardiographers.
Plasma sample and aortic specimen collection
Written informed consent was obtained from all patients before participation. Human aortic specimens were utilized under approvals of Zhongshan Hospital, Fudan University Ethics Committee (NO. B2020-158) in accordance with the Declaration of Helsinki. The plasma samples involved in this study were collected in vacuum blood tubes with anticoagulant before operation and handled within one hour after collection. Aortic tissues were obtained from control patients with a tricuspid aortic valve but without aortic dilation who underwent coronary artery bypass graft, and patients from BAV-dilated and BAV-non groups (BAV-non and BAV-dilated) (n = 3, respectively). The sampling and clinic study were approved by the Human Research Ethics Committee of Shanghai Zhongshan hospital (B2018-285R). Written informed consent was obtained from all the patients according to the Declaration of Helsinki, and the methods were carried out in response to relevant guidelines.
EV RNA isolation and small RNA sequencing
The EVs were isolated by a precipitation method using ExoQuick Isolation Kit (SBI, CA, USA) according to the manufacturer's instruction. Total RNA was isolated from plasma EVs with Trizol reagent (Invitrogen, USA). Then, quantity and purity of the total RNA were defined by Nanodrop (Thermo, USA) and 1% agarose gel electrophoresis. Library was prepared with 1μg total RNA for each sample. Total RNA was purified by magnetic beads and among them small RNA regions corresponding to the 18-30 nt could be enrichment. Then the 18-30 nt small RNAs were ligated to adenylated 3’ adapters. After adding the unique molecular identifiers (UMI) labeled RT primer and performing reverse transcription, we synthesized the first and second strand respectively to amplify the cDNA. Finally, the quantitative and qualitative small RNA libraries were sequenced on BGISEQ-500 platform (BGI, China).
Proteomics with mass spectrometry
Human plasma was depleted using the high abundance top 14 mini column (Thermoscientific A36370). After measuring the depleted plasma protein concentration (BCA method, Pierce, Protein Quantification Assay Kits), 50 μg of proteins for each sample were reduced with 10 mM DTT (final concentration) for 30 min at 37 ℃ and alkylated with 55 mM iodoacetamide (final concentration) for an additional 30 min in dark at room temperature. Remaining detergent was removed by acetone precipitation. Briefly, acetone (-20 ℃) was added to 50 μg of proteins to a final concentration of 80% v/v and the proteins were precipitated overnight at -20℃. After centrifugation (15 min, -4℃, 16,000 g), the detergent-containing supernatant was removed, and the protein pellet was washed with 80% acetone (-20 ℃). Protein pellets were then resolved in 50 μl 6M urea (in 10mM HEPES, pH = 8.0) and digested with 0.5μg of LysC for 3h at room temperature. After adding four volumes of 50 mM ammonium bicarbonate, 0.5 μg of trypsin was added and tryptic digestion carried out overnight. The next day, digestion was stopped by adding 1% TFA. Peptides were finally desalted on C18 StageTips and kept at -80℃ until mass spectrometry analysis.
qPCR of aortic specimens
Aortic specimen miRNAs were isolated by using PureLinkTM miRNA Isolation Kit (Invitrogen, USA) and all the steps were followed according to the guideline. qPCR for miRNA was performed using TaqManTM Small RNA Assays and protocol was as mentioned in the guideline.
Screening of differential expression (DE) miRNAs and identification of its target genes prediction and novel miRNAs
Differential expression analysis was performed using DESeq2 (v1.16.139) for the reads count expression result with the parameters “adjusted p-value < 0.05, |Log2(foldchange)| ≥ 1”, and NOIseq (v3.18.140) 24 for the UMI expression result with the parameters “Probability > 0.8, |Log2(foldchange)| ≥ 1”. We used miRWalk 2.0 to predict the potential target genes of the differentially expressed miRNAs.
Data was analyzed with SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and presented as a style of mean ± standard deviation (SD). Student’s t test was used for comparison of two groups. A Chi squared test or a Fisher’s exact test was used to compare the frequencies of the categorical variables when appropriate. Two-way ANOVA was used for comparison among more than two groups. P < 0.05* was considered as statistically significant.