CBX1 Expression Was Altered in Human TAD tissues
To determine whether CBX1 is abnormally regulated in AD, we examined CBX1 expression in 12 human AD and NA tissues. Quantitative reverse transcription polymerase chain reaction analysis showed that the RNA expression level of CBX1 in TAD samples was significantly higher than that in NA tissues (Figure 1A).
The overexpression and suppression of lncRNA Ftx in HUVECs
To investigate whether LncRNA Ftx plays a role in the progression of aortic dissection, we examined the function of lncRNAFtx in the HUVECs. We constructed a functional loss model by transfecting SH-Ftx into HUVECs and using SH-NC transfected HUVECs as a simulated control. Ftx transfected HUVECs were used as gain - function model, and Ftx-NC transfected cells were used as simulation control. The successful overexpression and knockout of lncRNAFtx was confirmed by real-time polymerase chain reaction (Fig. 1B).
Profiles of Proteome in Loss and Gain-of-Function Models of HUVECs
In order to further clarify the molecular mechanism by which lncRNA Ftx regulates the occurrence of aortic dissection, quantitative proteomics studies were conducted to analyze the differentially expressed proteins in the models of HUVECs function loss and function gain, respectively, and to further identify the key molecules and pathways involved in the carcinogenesis of lncRNA Ftx. A total of 4348 proteins were identified by high resolution liquid chromatography-tandem mass spectrometry (HPLC-MS),of which 3308 proteins were quantified. A quantifiable protein with a quantitative ratio of >1.3 was considered to be upregulated, while a quantifiable protein with a ratio of <0.769 was considered to be down-regulated. Support information 1 and 2 list all quantifiable proteins identified with increased (≥ 1.3-fold) or decreased (≤0.769) expression levels (P <0.05).
Compared with SH-NC transfected HUVECs, deletion of lncRNA Ftx induced 247 differentially expressed proteins, including 100 up-regulated proteins and 147 down-regulated proteins. Meanwhile, lncRNA Ftx overexpression resulted in 167 differentially expressed proteins, consisting of 64 up-regulated proteins and 103 down-regulated proteins, compared with Ftx-NC transfected HUVECs. Therefore, these data suggest that lncRNA Ftx does cause changes in the protein profile, which may lead to changes in the development of aortic dissection.
Functional annotation and classification of differential quantitative proteins
In order to screen for biofunctional classification of differentially expressed proteins in cells induced by lncRNAFtx knockout or overexpression, we analyzed quantifiable proteome data by GO analysis. Thus, these differentially expressed proteins are annotated and grouped into three categories: biological processes, cellular components, and molecular functions. The changes of biological function and the number of related proteins in lncRNA Ftx knockout or overexpression groups were analyzed and compared. Table 1 summarizes the first five affected biological functions and the number of differentially expressed proteins. In addition, subcellular localization analysis showed that differential quantification of proteins was widely distributed in subcellular structures. In summary,our GO analysis showed that differentially quantified proteins are associated with numerous biological processes, including cellular and metabolic processes, involve a variety of cellular components, and have a wide range of molecular functions in HUVECs.
Functional enrichment and clustering analysis of differential quantitative proteins
Enrichment and cluster analysis were then performed to identify the characteristics of proteins that were differentially expressed due to lncRNAFtx knockout or overexpression (Figure 2). First, we conducted a cluster analysis based on GO enrichment. As for the biological process, upon knockdown of lncRNA Ftx, upregulated proteins were related to alpha-amino acid biosynthetic process, whereas the downregulated proteins was related to response to dexamethasone (Figure 2a). Meanwhile, while lncRNA Ftx was overexpressed, the upregulated proteins appeared to be related to biological process including ncRNA metabolic process and ncRNA processing, and the downregulated proteins was related to actin filament bundle organization(Figure 2a). In the cellular component, the upregulated components including endoplasmic reticulum lumen was significantly enriched due to lncRNA Ftx knockdown, whereas large ribodomsl and rough endoplasmic reticulum were enriched with downregulated proteins (Figure 2b). On account of lncRNA Ftx overexpression, proteins related to small ribodomsl subunit was enriched in the upregulated proteins, while proteins related to actin cytoskeleton was enriched in the downregulated proteins (Figure 2b). On the ontology of molecular function, as lncRNA Ftx knockdown, proteins related to structural constituent of ribosome and structural molecule protein activity were enriched in the downregulated proteins (Figure 2c). While lncRNA Ftx was overexpressed, proteins related to C3HC4-type RING finger domain binding and heat shock protein binding were enriched in the upregulated proteins (Figure 2c).
Then, based on the pathways identified by KEGG analysis, we further performed a cluster-based analysis of the lncRNA Ftx response proteome. It appeared that proteins expression involved in PPAR signaling pathway and cysteine and methionine metabolism were upregulated pathways after knockdown of lncRNA, while Rap1 signaling pathway were downregulated pathways (Figure 3a). Meanwhile, overexpression of lncRNA Ftx led to upregulated pathways including Estrogen signaling pathway and legioneosis,and downregulated pathways such as hypertrophic cardiomyopathy(HCM) and dilated cardiomyopathy(DCM) (Figure 3a).
In order to determine the domain characteristics of differentially quantified proteins induced by lncRNAFtx knockout and overexpression, we further conducted domain enrichment analysis on the basis of previous domain analysis. The results implicated that the upregulated proteins affected by lncRNA Ftx knockdown contained SH3 domain,while downregulated proteins contained thioredoxin-like domain (Figure 3b). After overexpression of lncRNA Ftx, the upregulated proteins contained DnaJ C terminal domain and zinc finger (LIM-type), whereas downregulated proteins contained spectrin repeat and FYVE zinc finger (Figure 3b).
Validation of CBX1 inHUVECs as a lncRNA Ftx Regulated Protein
According to the proteomic quantitative results, in order to focus on the key proteins responsible for the action of LncRNA Ftx, we paid great attention to proteins that showed significant changes in both the knockout and overexpression of LncRNA Ftx, such as CBX1, whose fold changes were 0.735 and 1.301 respectively, when lncRNA Ftx was knocked out and overexpression. Cell experiments was conducted for CBX1 to verify the regulatory effect by Ftx. In cells, CBX1 abundance at the transcriptional level was verified using real-time polymerase reaction. Our data showed that overexpression of lncRNA Ftx significantly reduced the gene abundance of CBX1 (Figure 4). In contrast, the knockout of lncRNA Ftx significantly increased CBX1 gene expression (Figure 4). In conclusion, these data suggest that lncRNA Ftx regulates the abundance of CBX1 and may play an apoptotic role by negatively regulating the expression of CBX1.