Quantitative Proteomics Reveals the Regulatory Networks of Circular RNA BTBD7_hsa_circ_000563 in human coronary artery

Background: To investigate the association of BTBD7_hsa_circ_000563 expression in coronary artery segments with atherosclerotic stenosis, and to explore the proteome-wide identification of the BTBD7_hsa_circ_000563-regulated proteins in coronary artery Methods: The coronary artery samples were obtained from two autopsy cases. The epicardial coronary artery of every autopsy was divided into 10 segments, and coronary atherosclerosis grade and extent of the coronary artery segments were analysed by Haematoxylin and Eosin (H&E) staining. The BTBD7_hsa_circ_000563 expression of 8 segments from case 2 was quantified using RT-qPCR analysis. Results: The present study demonstrated that coronary artery segments with severe atherosclerotic stenosis showed extremely low expression of the BTBD7_hsa_circ_000563, compared with normal coronary artery segments. Furthermore, it was predicted that hsa-miR-155-5p, and hsa-miR-130a-3p are targets of the BTBD7_hsa_circ_000563. The results from the present study may laid an epigenetic foundation for studying the underlying mechanisms of the development and progression of coronary artery atherosclerosis. Conclusions: BTBD7_hsa_circ_000563 were involved in atherosclerotic changes in coronary artery segments of human being, and the verification study, mechanism study, and function study are necessary in order for CAD patients to benefit from the personalized medicine in the future.

the monitoring of CADs are concentrated on the late symptomatic phases. Therefore, in order for early detection and timely therapeutic intervention of CAD, there remains an urgent need for biomarkers exploring. Increasing evidence has implicated the circRNAs in the pathogenesis of CAD [2][3]. Among them, BTBD7_hsa_circ_000563 (also known as hsa_circ_000847) stands out as a clinically relevant tissue-associated circRNAs [4] and is involved in human various diseases [5][6].
In this study, reverse transcription (RT) followed by real-time quantitative (q) PCR (RT-qPCR) detection of BTBD7_hsa_circ_000563 was employed to discovery and validation of candidate biomarkers of CAD in coronary artery, sampled from subjects with coronary artery disease. And, in our present study, quantitative proteomics-based strategies were employed to identify BTBD7_hsa_circ_000563-regulated proteins in human coronary artery.

Study subjects
The coronary artery samples were obtained from two autopsy cases at department of human anatomy in Nanjing Medical University. Informed consent from the bereaved family was obtained for the research use only of samples and the autopsy was conducted according to the guideline of the university. The methods were performed in accordance with the approved guidelines, and all experimental protocols were approved by the ethics committee of the Nanjing Medical University and the First Affiliated Hospital of Nanjing Medical University. The age was 64 and 68 years respectively, and the female/male ratio 0:2. The postmortem delay varied between 1 and 2 days. At autopsy, epicardial coronary arteries were removed from the hearts. The epicardial coronary artery of every autopsy was divided into 10 segments: the proximal segment, the midsegment, the distal segment of the left anterior descending (LAD), left circumflex (LCX), right coronary artery (RCA) respectively, and the left main trunk (LM). And, every coronary artery segment was divided into three groups: RNA group, protein group and pathological group. The segments at the RNA group and protein group were snap-frozen in liquid nitrogen and stored at -80˚C for real-time PCR analysis. In addition, the segments at the pathological group were fixed overnight in 10% formalin and embedded in paraffin for histological analysis. Coronary atherosclerotic grading and extent of the segments were indepently analysed by pathologists committed to making a connection of current histological grading according to American Heart Association (AHA) classification guidelines [7].

RNA extraction and RT-qPCR
Total RNA was extracted from the coronary artery segments using Trizol (15596018,Invitrogen) following the manufacturer's instructions and checked for a RIN number by an Agilent Bioanalyzer 2100 (Agilent technologies, US).
Total RNA (1 µg) was used as a template to prepare cDNA before PCR as reported by Renyou Pan etc al.,in 2019.The BTBD7_hsa_circ_000563 expression was quantified using SYBR Green Realtime PCR Master Mix (TOYOBO, QPK-212,Japan) on the ABI 7900HT squence detection system (Applied Biosystems,7900HT USA) [9].

Protein extraction, LC-MS/MS Analysis
For the quantification of the protein, 500 µg of total protein was extracted using a multistep L3 lysis buffer/M2 lysis buffer protocol, as Ya-qing Zhou, Jinxia Yuan ect. reported in 2019. First, the Bradford Method was used for protein quantification. We took 100 µg protein and dissolve it to 1 µg/µL using Μ2 lysis buffer (without thiourea). It was diluted 6fold by adding 5 volumes of 100 mM TEAB before shaking by centrifugation.
Substantially,the protein samples were digested overnight at 37℃ by adding 2 µg trypsin.
Next, we weighed 10 mg of C18 column for each peptide sample. With discarding the supernatant by centrifugation,the sample was washed with 0.1% FA + 3% ACN twice for desalting and eluted with 1 mL 0.1% FA + 80% ACN. The eluted peptides were dried with a vacuum concentration meter. Third, the peptide sample was diluted to 1 µg/µl on the peptide sample, and the scanning mode was set to 5 µl and 120 min. The peptides in the sample with a mass-to-charge ratio of 350-1500 were scanned. The mass spectrometry data was collected using the Triple TOF 5600 + LC/MS system (AB SCIEX,USA). For IDA (Information Dependent Acquisition), a first-order mass spectrum was scanned with an ion accumulation time of 250 ms, and a secondary mass spectrum of 30 precursor ions was acquired with an ion accumulation time of 50 ms. The MS1 spectrum was acquired in the range of 350-1200 m / z, and the MS2 spectrum was acquired in the range of 100-1500 m / z. Set the precursor ion dynamic exclusion time to 15 s.

Data analysis
We performed protein identification and quantification by ProteinPilot software (https://sciex.com.cn/products/software/proteinpilot-software, version 4.5, SCIEX, Redwood City, California, USA). The MS/MS spectrum was searched for the uniprot-Homo_sapiens protein database. To identify peptides, the Paragon algorithm was employed against the uniprot-Homo_sapiens database. The parameters were set as follows: cysteine modified with iodoacetamide; biological modifications were selected as ID focus. For false discovery rate (FDR) calculation, an automatic decoy database search strategy was employed to estimate FDR using the PSPEP (Proteomics System Performance Evaluation Pipeline Software, integrated in the ProteinPilot Software). Only unique peptides with global FDR values from fit less than 1% were considered for further analysis. Skyline v4.2 software was applied for MS1 filtering and ion chromatogram extractions for peptides label-free quantification, the parameters setting for skyline MS1 filtering were as the same as. With the results of the Skyline quantification, the mean value of the ratio of each group was used to calculate the fold change.
Owing to analysis need, the eight samples were divided into four groups according to the ascending order of BTBD7_hsa_circ_000563 expression level, functioning as a comparative basis for further analysis of proteome data. After grouping eight samples according to BTBD7_hsa_circ_000563 expression level, the median quantitative value of the samples in each group was taken as the expression value of a protein in that group, so a new expression value of BTBD7_hsa_circ_000563 was obtained in each group. Next, the comparison between groups (finding the fold change) is also compared with the new expression value of each group. In order to test whether the difference between the groups was significant, the T test was carried out and the p-value was obtained. The screening criteria of significant difference proteins were as follows: significantly differentially expressed with a threshold fold change over 1.5 or less than 0.667 between groups; P < 0.05 in the comparison between groups; of the three comparison groups, at least one of them satisfies the above criteria with ratio trend of increasing or decreasing among all the three comparison groups following the increasing of the BTBD7_hsa_circ_000563 expression level.

Bioinformatics and Annotations
To determine the biological and functional properties of all identified proteins, the Gene Ontology Terms (http://geneontology.org/) were used to describe the properties of genes and gene products in organisms. To this end, homology searches were first performed on all identified sequences using the local NCBI blastp program for the NCBInr animal database. The e value was set to be less than 1e-5, and the best hit for each query sequence took into account the GO term match.Functional annotation of genes from the new genome and studies of genome evolution were investigated using the orthologous  Table 1. Table 1 Natural history and histological classification of atherosclerotic lesions of the coronary artery samples Grade 68  Male  3  3  3  1  1  1  1  1  3  1  Stage  3  3  3  0  0  1  1  2  3  The analysis of Haematoxylin and Eosin (H&E) staining indicated that atherosclerotic lesions were found at the segments of coronary artery in case 1 and case 2. However, coronary atherosclerosis grade and extent in the case 1 was more serious than that in case 2. All of the examined coronary segments in case 1 showed atherosclerotic changes of the intima, ranging from lesions classifiable as atherosclerotic tunica intima to secondary affection tunica intima, and stenosis from 51 to 100%. Conversely, atherosclerotic changes of normal tunica intima, fatty streak tunica intima, fibrous plaques tunica intima, and atherosclerotic tunica intima were found at coronary segments in case 2 with coronary stenosis from 0 to 75%.
Due to the RNA degradation, the purity and concentration of the total RNA at the all coronary artery segments in case 1 and at the midsegment and distal segment of the right coronary artery in case 2 failed to meet standards for RT-qPCR analysis. Therefore, the BTBD7_hsa_circ_000563 expression via RT-qPCR has been detected in coronary artery samples from case 2 except the midsegment and distal segment of the right coronary artery.
In the RT-qPCR assay, BTBD7_hsa_circ_000563 expression was assessed and the results revealed that the expression of BTBD7_hsa_circ_000563 were higher in normal coronary artery segments, by comparison with that in coronary artery segments with severe atherosclerosis, and the results were shown in Table 2 and Fig. 1. From the autopsy case 2, in coronary artery segment of proximal segment of the left circumflex with normal tunica intim and stenosis from 0 to 25%, BTBD7_hsa_circ_000563 expression was most significantly increased by 20-fold compared with that in the left main trunk with atherosclerotic tunica intima and stenosis from 51 to 75%.  The screening of significant difference proteins according to the

BTBD7_hsa_circ_000563 expression level
The identification and quantification of significant difference proteins according to the BTBD7_hsa_circ_000563 expression level are presented in Table 3. According to these analysis, 49 proteins were differentially abundant between the BTBD7_hsa_circ_000563 expression level groups. Among the 49 proteins, 10 proteins were found down-regulated, while 39 were determined to be up-regulated with the increasing of the BTBD7_hsa_circ_000563 expression level.

GO annotation of the Differentially Expressed Proteins
To further determine the biological and functional properties of all differentially expressed proteins, the GO annotation was employed to analyze the differentially expressed proteins, and .the annotation results were shown in Fig. 2.
GO annotation with respect to the 49 differentially expressed proteins in human coronary artery samples suggested that the cellular component of these proteins was predominantly derived from the mitochondrial matrix (GO:0005759), and mitochondrial inner membrane (GO:0005743)( Fig. 2A). Moreover, the molecular function of these proteins primarily included NAD or NADH binding, 3 iron, 4 sulfur cluster binding, FAD binding, NAD binding, and isocitrate dehydrogenase (NAD+) activity (Fig. 2B). In addition, the biological process of these proteins participating in predominantly included the respiratory electron transport chain (GO: 0022904), tricarboxylic acid cycle (GO:0006099), and mitochondrial electron transport, NADH to ubiquinone (GO:0006120) (Fig. 2C).

KEGG enrichment of the Differentially Expressed Proteins
KEGG pathways enrichment analysis was performed and that BTBD7_hsa_circ_000563-

Protein − protein interaction of the Differentially Expressed Proteins
The protein − protein interaction of the BTBD7_hsa_circ_000563-regulated proteins was generated by means of the STRINTG database. 25 of the 49 differentially expressed proteins are engaged in a PPI network based on STRING database Besides, the network contains 30 nodes and 86 edges, and the 30 proteins joining in the PPI networks were categorized into several functional groups. Therefore, the PPI networks indicated that the BTBD7_hsa_circ_000563-regulated proteins have potential to interact with each other and work in union to exercise their functions. Moreover, to evaluate the classification of the BTBD7_hsa_circ_000563-regulated proteins, we used the classification system to sort out the proteins. The 49 proteins hit by the classification system were divided into several classes (Fig. 4). Taking these results together, BTBD7_hsa_circ_000563-regulated proteins may make a difference to various biological processes particularly in regulating dehydrogenase and ATP synthase.

Discussion
In the study,we foud that BTBD7_hsa_circ_000563 were involved in atherosclerotic changes in human being coronary artery segments. And, the expression levels of Nhek, and Ag04450) [11], human different brain regions [6], and human endothelial progenitor cells [4] via RNA Sequencing analysis. However, the present study demonstrated that coronary artery segments with severe atherosclerotic stenosis showed extremely low expression of the BTBD7_hsa_circ_000563, as compared with normal coronary artery segments. Furthermore, it was predicted that hsa-miR-155-5p, and hsa-miR-130a-3p are targets of the BTBD7_hsa_circ_000563. The above results of the present study laid an epigenetic foundation for studying the underlying mechanisms of the development of coronary artery atherosclerosis. However, the proteome-wide analysis of BTBD7_hsa_circ_000563-regulated protein in coronary artery has not been reported.
To further explore the foundation and mechanism underlying the association between the BTBD7_hsa_circ_000563 and coronary artery atherosclerosis. The proteome-wide analysis was conducted in the present study. The proteomics analysis identified 49 proteins regulated by BTBD7_hsa_circ_000563 overexpression in human coronary artery segments.
And, bioinformatic analyses revealed that a large number of differentially expressed proteins were located in mitochondrion and involved in citrate cycle (TCA cycle) pathway, and the ATP synthase may be the hub of the regulatory networks of BTBD7_hsa_circ_000563 in coronary artery.
As complex dynamic organelles, mitochondria play numerous functions pertaining to cellular metabolism and homoeostasis, the hallmark of mitochondria is cellular energy generation by means of oxidative phosphorylation (OXPHOS) [12][13]. And, the mitochondrial DNA encodes only 13 OXPHOS proteins; the remaining about 1500 proteins from the mitochondrial proteome are transferred to the mitochondria and are encoded in the nuclear genome [14]. In addition, several converging metabolic pathways including the folate metabolism, TCA cycle, and sulphur metabolism consist in whole or partial components of the mitochondria house [15] Coronary artery disease has complex etiology, and mitochondrial dysfunction exerts influence on various celluar aberrations including energy deficit, deregulation of autophagy, metabolic abnormalities, excessive production of reactive species, endoplasmic reticulum stress, and activation of apoptosis. The impairment of the ATP synthesis and respiratory chain have been considered as a key of mitochondrial dysfunction. Therefore, recovering of the mitochondrial function including respiratory activity and ATP-producing capacity have been considered as a primary therapeutic target to improve the prognosis of coronary artery disease [16]. Recently, results from a study suggest that the serum concentration of mitochondrial ATP synthase inhibitory factor 1 is independently negatively associated with all-cause and cardiovascular mortality in and regulated proteins in coronary artery has not been studied; Finally, the therapeutical value of present finding has not been applied to clinical practice due to the absence of the function and mechanism study of BTBD7_hsa_circ_000563. Therefore, the verification study, mechanism study, and function study are necessary in order for the CAD patients to benefit from the personalized medicine in the future.

Conclusion
The most relevant finding of this study is that coronary artery segments with severe atherosclerotic stenosis showed extremely low expression of the BTBD7_hsa_circ_000563, as compared with normal coronary artery segments. Furthermore, it was predicted that hsa-miR-155-5p, and hsa-miR-130a-3p are the targets of the BTBD7_hsa_circ_000563. In addition, by means of a proteomics approach, 49 proteins regulated by enhanced BTBD7_hsa_circ_000563 expression were identified in human coronary artery segments.
Bioinformatic analyses revealed that a large number of differentially expressed proteins were located in mitochondrion and involved in citrate cycle (TCA cycle) pathway. The ATP synthase may be the hub of the regulatory networks of BTBD7_hsa_circ_000563 in coronary artery.

Declarations
Ethics approval and consent to participate: The coronary artery samples were obtained from two autopsy cases at department of human anatomy in Nanjing Medical University. Consent for publication: Informed consent from the bereaved family was obtained for the research use only of samples and written informed consent to publish this information was also obtained from study participants.
Availability of data and materials: The data would be supplied upon request. The datasets used and/or analysed during the current study available from the correspondence author.