EMT are one of the principal sources of sterility in women of childbearing age, incurring a substantial healthcare system burden worldwide. Recent studies have introduced a variety of novel biomarkers for diagnosing EMT, for instance TGFBI[30] and Hsa-mir-135a[31]. Based on the immune and Inflammation profiles of EMT, there are also no past reports to strategically investigate which IRGs can be used as prospective biomarkers for EMT. In addition, machine learning techniques and nomogram generation have not been employed in EMT diagnosis. In this study, we conducted a comprehensive set of integrated bioinformatics analyses and utilized machine learning methods to construct a nomogram, allowing us to assess the diagnostic potential of IRGs in individuals with EMT. The most significant discovery was the identification of five pivotal immune-related candidate genes (SCG2, FOS, DES, GREM1, and PLA2G2A), along with the creation of a diagnostic nomogram for individuals with EMT.
SCG2 is a novel biomarker characterized in our own study of patients diagnosed with EMT. It is a member of the tyrosine sulfated granule protein family expressed in endocrine, neuroendocrine, and neuronal tissues[32] and has been implicated in the formation of secretory vesicles and the packaging of peptide hormones into vesicles [33]. SCG2 has a critical function in augmenting endothelial cell proliferation, migration, and angiogenesis[34, 35]. It has been found that SCG-derived peptides, such as secretin (SN) and EM66, are helpful markers for neuroendocrine tumors[36]. Moreover, SCG2 was recognized as a stroma-associated gene and polluted poor outcomes in patients with colorectal cancer[37]. However, there are no studies on SCG2 in EMT, and we believe that SCG2 is highly expressed in EMT and has excellent efficacy for diagnosing patients with EMT. Thus, our findings provide a foundation for further exploration of SCG2 in EMT in the future.
FOS is an early transcription factor that can be implicated in the modulation of transcriptional processes through a nucleoprotein complex called AP-1[38, 39]. Under inflammatory conditions, many pro-inflammatory factors seem to be actively modulated by FOS[40]. Furthermore, the FOS gene has been implicated in estradiol-dependent cell proliferation[39], encoding a nuclear-associated protein that amplifies estrogenic messages by triggering the activation of transcription of other genes that control cell division. An estrogen-responsive element has been identified in the promoter area of the human FOS gene, a discovery that bolsters the hypothesis that estrogen occurs through direct irritation of FOS gene transcription to increase FOS mRNA levels[41]. In addition, it has been demonstrated in a preceding study that both FOS gene and protein expression are evoked in the human endometrium during the proliferative phase of the human cycle, and that FOS gene expression correlates markedly with cyclic estradiol concentrations[42]. Moreover, DES is a major filament in the intermediate that is particularly pronounced in cardiac, skeletal, and smooth muscle, and acts in cytoarchitecture, force transmission, and mitochondrial function[43]. DES is as well expressed in the uterus, but its role is not clear. It is expressed in the myometrium during the formation of the myometrium and is induced by estradiol in the endometrium[44, 45]. Likewise, our study argues that FOS and DES genes are overexpressed in EMT. Hence, we speculated that FOS or DES may guide the production of ectopic endometrium under abnormal estrogenic state. They can also be employed as diagnostic biomarkers.
GREM1, a highly conserved secreted protein that has a critical position in diverse aspects of early embryonic development and differentiation, antagonizes the activity of bone morphogenetic proteins by heterodimerizing with specific bone morphogenetic proteins and blocking the interaction of bone morphogenetic proteins with the TGF-β receptor[46, 47]. Angiogenesis holds a vital place in the insertion of ectopic endometrium and the subsequent formation of pathology. Groothuis et al. attributed the emergence of a new system of vasculature to the endothelium originating from either the human peritoneum or from the mouse host, which supplies oxygen and nutrients to the outgrowth of the ectopic endometriotic grafts[48]. In addition, multiple studies have also illustrated the participation of GREM1 in the early stages of embryonic development as well as in various stem cell sources, thus explicating the theory that it is involved in abnormal endometrial angiogenesis[49–53]. Sha et al.[54] exhibited that GREM1 was expressed at significantly higher mRNA and protein levels in the ectopic endothelium of patients with EMT than in healthy control women, which is compatible with the findings of our study. As such, GREM1 deserves to be further pursued as a prospective serum biomarker for EMT.
PLA2G2A is a secreted enzyme that hydrolyzes the sn-2 ester bond of glycerophospholipids to liberate free fatty acids and lysophospholipids[55]. The arachidonic acid and other polyunsaturated fatty acids derived therefrom are critical substrates for the eicosanoids (including prostaglandins), which are potent inflammatory mediators that incite cell growth and proliferation[56]. Equally, lysophospholipids are effective lipid mediators that are implicated in cell proliferation, survival, migration, and angiogenesis[57]. These mediators are exceptionally essential for the occurrence and development of EMT. To date, there were scant reports on PLA2G2A mRNA levels in endometriosis. In 11 patients with EMT, Eyster et al. recorded that PLA2G2A was the most up-regulated gene in ectopic endometrium compared to normal endometrium[58]. Lousse et al. similarly found that PLA2G2A mRNA levels were elevated 52-fold in 40 cases of peritoneal endometriotic lesions when compared with matched normal endometrium[59]. Our finding was in agreement with previously studies, and it could be inferred that PLA2G2A was instrumental in causing EMT.
We constructed a diagnostic model for EMT based on the aforementioned genes and plotted ROC curves to validate its performance in multiple datasets. The model offered a high diagnostic merit, so we built a nomogram model for EMT risk prediction relying on these genes. In clinical work, it is convenient to procure blood and tissue samples from hospitalized patients for disease diagnosis and determination of disease changes. Consequently, the nomogram may have some clinical value for EMT risk screening.
However, our study has several limitations. First, despite the fact that we pooled a single large dataset of EMT, the samples are still sparse, and the diagnostic value of the column line drawings awaits further validation due to the limited sample size. Second, our dataset involved the use of samples from the endometrium rather than peripheral blood samples, which still leaves a way to go in our pursuit of a non-invasive diagnostic index. Therefore, in the future, we need to validate these findings in peripheral blood specimens to realize the translational value of these biomarkers for clinical applications. Finally, although the five candidate hub genes are mainly recruited to modulate the immune pathway, their in-depth regulatory regimes for EMT still demand further basic research to fulfill.