EMT plays a critical physiological and pathological role in developing the cardiovascular system, vascular tissue remodeling, and heart valve disease during the embryonic period. However, more research focused on the impact of the EMT in tumor development and treatment. In contrast, few studies have explored the diagnostic value of EMT-related genes or lncRNAs in CAD. Hence, exploring diagnostic biomarkers of EMT genes/lncRNAs in CAD is urgent.
Our analyses uncovered 32 EMT-related DEGs in CAD. KEGG pathway analysis of these DE-EMTs was mainly enriched in the PI3K/Akt signaling pathway. Several reports have shown that PI3K/Akt pathway is a canonical EMT signaling pathway[27, 28]. Meanwhile, we found this signaling pathway plays an essential role in the CAD. A recent study indicated that miRNA-26a-5p activated the PI3K/Akt pathway by targeting Phosphatase and Tensin Homolog (PTEN) and affected the proliferation and apoptosis of endothelial cells isolated from CAD mice. A comparative study also reported that miR-26a-5p could activate the PI3K/Akt signaling pathway through inhibition of PTEN, thereby protecting against myocardial defect/reperfusion injury. These studies have confirmed that activating the PI3K/Akt signaling pathway can prevent myocardial ischemia-reperfusion in animal models. Other studies have also suggested that regulation of the PI3K/Akt signaling pathway plays a vital role in inhibiting myocardial fibrosis, apoptosis, and the inflammatory response[31, 32].
In the present study, we performed a co-expression analysis between EMT genes and DElncRNAs through paired lncRNA and mRNA expression data in CAD patients from GEO. Eight differently expressed EMT-related lncRNAs were found to be diagnosis factors for CAD patients. After a literature review, we found no research had been conducted about the mechanisms of the eight lncRNAs except LINC02747. Previous studies have reported that LINC02747 can upregulate the expression of TFE3 by absorbing miR-608 and ultimately promote the proliferation of renal cell cancer cells (ccRCC). Gu et al. indicated that miR-608 exerts anti-inflammatory effects by targeting ELANE in monocytes. Our results showed that monocytes were more expressed in the CAD group, so whether the regulation of LINC02747-mir608- ELANE might achieve the reversal of inflammatory response in CAD patients. Other seven EMT-related lncRNAs have not been reported in relevant studies, and reports on how lncRNAs interact with EMT genes have been rarer. However, many “cis” and “trans” genes are involved in the formation and development of CAD in the cis-trans regulatory network. For example, EMB, as a “cis” gene, was enriched in the mTOR signaling pathway in our GSEA analysis. This pathway is closely associated with atherosclerosis, and the pro-inflammatory response of monocytes in CAD requires activation of mTOR. Among “trans” genes, some studies have reported that VDR gene polymorphisms lead to the development and formation of CAD by affecting changes in serum levels of 25(OH) vitamin D.[36, 37]. Previous study reported VDR in regulating inflammation through inhibiting the NF-ĸB pathway and activating autophagy. EBF4 gene promotes the elevation of Cu and leads to the progression of CAD by affecting copper related DNA methylation sites. CTCF gene is essential for cardiogenesis and inhibit cardiomyocytes apoptosis, and can be applied as a therapeutic target for the treatment of heart failure in future.[40, 41]. FLI1 gene is also reported to be closely related to immune dysfunction and platelet disorders. Although the lack of direct support in the literature, we speculated that these cis-trans genes, under the regulation of lncRNA, affect the formation and development of CAD through immune microenvironment, cell apoptosis, platelet dysfunction and other ways. So far, there has been no study on the role of EMT-related lncRNA in CAD diagnosis. These findings may provide valuable insights into the future diagnosis and treatment of CAD.
The presence of immune cells in the infarct area is vital to initiating the repair process of injured heart tissue. Temporal and spatial regulation of inflammation after infarction is crucial[43, 44]. We evaluate the type and fraction of immune cell infiltration between CAD patients and normal samples in the dataset using the CIBERSORT algorithm. Our results found CD8 T cells and NK cells share a decreased infiltration, and the infiltration of monocytes was increased in CAD patients, which was similar to the previous results[45–47]. In this GEO dataset, CD8 T cells and NK cells are favorable factors for preventing CAD, and monocytes likely promote the occurrence of CAD. Previous studies have suggested that the imbalance of immune regulation is an essential factor in promoting atherosclerosis, heart failure, and chronic kidney disease by monocytes cells. CD8 T cells play a dual role in atherosclerosis. On the one hand, CD8 T cells can secret many inflammatory cytokines to accelerate the inflammatory response and increase the instability of atherosclerotic plaques. On the other hand, cytotoxic activity against antigen-presenting cells and the presence of regulatory CD8 T cell subsets could suppress immunity and limit atherosclerosis. Ong et al. suggested that NK cells appear to protect the development of cardiac fibrosis by preventing the accumulation of specific inflammatory groups in the heart and directly restricting collagen formation in cardiac fibroblasts. Although the results of our study are similar to these researches, the mechanism of the immune system is still very complex, and some results in the immunotherapy of CAD are not ideal. We need a lot of clinical studies to demonstrate the underlying mechanism. Besides, we also found that except AC109460.4, the other seven lncRNAs related to EMT were significantly negatively correlated with CD8 T cell and NK cell and positively correlated with Treg and monocytes. The results of AC109460.4 were just the opposite. The association between these lncRNAs and the innate immune system is still unclear. More in vivo and in vitro studies are needed to explain the interaction mechanism between these lncRNAs and immune cells in CAD.
It is generally believed that lncRNAs can act in “trans” to regulate TFs mediated chromatin remodeling and transcription. These lncRNAs recruit protein factors to enhancer and regulate enhancer activity. We constructed cis and trans-regulatory networks based on these eight signatures. In the trans-regulatory network, we obtained 33 differentially expressed TF genes. The most surprising discovery was the screening of SNAI2, an EMT-TF gene (the gene coding product was the transcription factor Snai2). Our results indicated that SNAI2 was not only significantly highly expressed in CAD patients but also strongly positively correlated with LINC01775 and CTD-2089N3.3. The ROC curve showed that the SNAI2 could be a potential biomarker for diagnosing CAD. As a classic EMT-TF gene, SNAI2 has recently been shown to be involved in a broader range of biological processes, including tumor metastasis, heart development, cell differentiation, vascular remodeling, and DNA damage repair[52–54]. Previous studies have reported that the deletion of protein arginine methyltransferase 1 (PRMT1) leads to the accumulation of p53, and enhancing the degradation of SNAI2 can limit the formation of cardiac fibroblasts, coronary smooth muscle cells, and pericytes. Meanwhile, Cooley et al. reported that, by grafting mouse veins to the femoral artery in mice to simulate human coronary artery bypass grafting (CABG), the results showed that TGF-β/Smad2/3-Snai2 mediated EMT plays a crucial role in venous graft vessel remodeling. These studies have indicated that high expression of SNAI2 can promote the formation of vascular endothelium to EMT and vascular remodeling, which is one of the vital factors in the formation of CAD. At present, the role of SNAI2 in CAD has not been reported, several studies have proven that the vascular endothelial EMT process is involved in atherosclerosis, post-stent stenosis, pulmonary hypertension, and coronary artery remodeling[57–59]. Additionally, the role of EMT can be seen in a range of cell types involved in immunity, such as lymphocytes, NK cells, and myeloid cells, which contribute to inflammatory responses in diverse pathophysiological processes. Ricciardi et al. have reported a decreased viability and proliferation of NK cells and T cells after co-culture with cancer cell lines in which EMT had been induced. In our study, SNAI2 correlated with infiltration of monocytes, CD8 T cells, and NK cells activated. Previous studies have suggested that SNAI2 deletion in mice leads to impaired development of the T-lymphatic system. Subsequent studies also confirmed that Snai2 plays a vital influence in regulating CD8 T cells and targets genes with functions for T cells. Furthermore, our results indicated that the difference in these immune cell infiltrations in the SNAI2 high expression group was similar to the results of CAD patients. These immune cells have been researched to play a role in the formation, erosion, and rupture of coronary plaques[63, 64]. In summary, we inferred that SNAI2 might have significant roles in the occurrence of CAD by regulating innate and adaptive immunity through these immune cells. To confirm our conclusion, more experimental mechanistic research should be carried out in future studies.
Our study should acknowledge some limitations. First, the expression levels of critical lncRNAs in CAD were not verified in clinical samples. Secondly, these EMT-related lncRNAs were investigated in datasets with no access to individual patients’ characteristics; thus, we cannot adjust the ROC curve for traditional cardiovascular risk factors. A prospective cohort recruiting CAD patients is needed to confirm the predictive value of EMT-related lncRNAs. Thirdly, the molecular function details of SNAI2 and EMT-related lncRNAs in the progression of CAD have not been further studied. Therefore, molecular biological experiments and flow cytometry analysis are required to validate these findings, and another external validation based on a larger sample is needed.