Keloid disorder is a manifestation of excessive healing after skin injury, characterized by abnormal proliferation of fibroblasts and deposition of extracellular matrix, and is common in plastic surgery (Grabowski et al., 2020). Keloids are more frequent on some exposed parts of the skin with pain and itching, and improper care can induce infection and septicemia, causing considerable physical and psychological stress to the patient (Lee et al., 2019). However, the current diagnoses of keloids primarily rely on the clinical manifestations and associated scar scales. Patients with a tendency to develop keloids can be diagnosed only after the formation of keloids after surgery or trauma, which notably impacts the risks and efficacy of open surgery. Therefore, it is urgent to search for reliable diagnostic indicators for early screening and prevention of keloids to avoid unnecessary surgical stimulations.

Gene sequencing is able to determine the exact relationships among some gene loci and diseases, which has guiding significance for clinical diagnosis and treatment (Horgan et al., 2017). The advances in bioinformatics provide the ability to assist the secondary utilization of existing sequencing data, fully mine the information that is provided by the sequencing data, and identify the biomarkers for a variety of diseases (Min et al., 2017). As a highly recurrent fibrous proliferative tumor, in the absence of satisfactory drug and surgical treatment, the identification of early indicative biomarkers in keloid is of key significance for effective prevention, risk reduction and development of targeted therapy. At present, the identification of keloid biomarkers mostly relies on the reference of other fibrosis diseases, such as studying the role of scleroderma biomolecules in keloids. However, the efficiency of this method is relatively low, and it is prone to omit the specific markers of keloids. We thus utilized gene sequencing datasets and bioinformatics methods to filter keloid markers in the expectation of more efficient identification of key signatures of keloids.

In this study, public series of matrix files for keloids were downloaded from the Gene Expression Omnibus (GEO) database. Pivotal molecules were screened based on the PPI network and the diagnostic efficacy of the screening signature was validated by applying receiver operating characteristic (ROC) curves, and the association of the filtered signature with immune cell infiltration was evaluated to gain more insight into the molecular immune mechanisms involved in keloids.

Keloid disorder is very common in the clinical work of plastic surgery, yet due to the lack of clear pathogenesis and effective treatment measures, it is a refractory disease (Lee and Jang 2018). Pathologically, keloids have tumor-like growth characteristics, which chiefly manifest as excessive fibrosis that is provoked by continuous inflammation, vigorous cell proliferation, and excessive accumulation of the extracellular matrix (Tan et al., 2019). Clinically, keloids have individual tendencies, and they are liable to occur in exposed areas such as the sternum, scapula, and earlobe and are accompanied by pain and itching (Lee et al., 2004). The formation of postoperative keloids can have some negative and even devastating consequences for cosmetic surgery due to the lack of effective laboratory indicators to screen susceptible individuals. The current diagnosis of keloids relies heavily on clinical presentation, which is highly subjective and uncertain and does not provide prevention or early diagnosis (Berman et al., 2017). We aimed to discover diagnostic markers of keloids that could objectively assess the physiological status of patients and evaluate whether they have a tendency to develop keloids. This will facilitate early screening, diagnosis and prevention of keloid scars, and early control of keloid growth can lead to significant clinical outcomes.

This study identified TGM2 as a key molecule in keloids. Previous studies have suggested that keloids are closely associated with a sustained inflammatory response and infiltration of inflammatory factors and chemokines (Wang et al., 2020c). Accompanied by activation of inflammatory factors like interleukin six and infiltration of neutrophils, the perivascular dermis appears to have nodular proliferation of fibroblasts and excessive deposition of extracellular matrix (Potter et al., 2019; Wang et al., 2020c). The arrangement is intricately followed by the formation of a mass of rotating collagen fibers, which eventually leads to keloids (Andrews et al., 2016; Chen et al., 2019). This process is consistent with the results of the correlation analysis of TGM2 with extracellular matrix gene set and IL6 in this paper.

Considering that certain autoimmune diseases can result in tissue damage by initiating an inflammatory response, a long-term inflammatory response eventually leads to tissue fibrosis, such as skin fibrosis induced by scleroderma (Wynn and Ramalingam 2012; Sziksz et al., 2015; Sunda and Arowolo 2020). Therefore, we speculated that keloids might be related to immune infiltration and supplemented the comparison of keloids and normal tissues at the level of immune cells. The results of immune infiltration analysis indicated that the screened TGM2 was positively correlated with monocyte and natural killer cell. There were significant differences in the proportion of eight immune cells in keloid and non-keloid tissues, which was consistent with previous studies (Bagabir et al., 2012; Wang et al., 2021).

Currently, the exploration of biomarkers in the field of keloids is generally derived from speculation of similar diseases. By way of example, since keloids have to some extent tumor characteristics, it is speculated that some tumor biomarkers may also be exploited as keloid markers (Gál et al., 2017; Ud-Din and Bayat 2020). The genes that control apoptosis and growth in some tumors may regulate the occurrence of keloids. For example, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is differentially expressed in a variety of malignant tumors, such as lymphoma, bladder cancer, melanoma, and esophageal cancer, is capable of exerting a selective apoptotic effect (Fulda 2014; Alexiou et al., 2015). It has been confirmed that knocking down the expression of TRAIL aggravates the fibrosis of lung tissue, and upregulating TRAIL causes skin fibrosis to be considerably inhibited (Braithwaite et al., 2018; Jiang et al., 2019). Mutations in the oncogene PTEN (phosphatase and tensin homolog deleted on chromosome ten) are detected in hematological tumors, brain gliomas, osteosarcoma and many other tumors and can be the target for microRNA-21–5p to reduce the migration of keloid fibroblasts and govern autophagy (Worby and Dixon 2014; Chen et al., 2018; Yan et al., 2020). Further, some scholars have also postulated key genes in keloids based on liver injury, muscle injury, scleroderma, cardiac hypertrophy, and other diseases with similar fibrotic processes. For example, fussel-15 has the ability to regulate the fibroblasts of keloids and scleroderma (Arndt et al., 2011); microRNA-29 influences the fibrosis process of various organs, such as the heart, liver, lung and the growth of keloids (Deng et al., 2017; Gallant-Behm et al., 2019; Xu et al., 2020). Although some crucial genes in keloid can be revealed according to the above-mentioned ideas, their screening efficiency is relatively low and not accurate enough. Based on sequencing data and bioinformatics methods, the identification of possible biomolecules for diseases including keloid is more accurate and reliable, and more efficient.

TGM2 belongs to the TGM protein family, which consists of structural and functional enzymes and participates in the posttranscriptional modification process of proteins by inducing cross-linking, transamination and deamination between proteins (Li et al., 2018; Lee et al., 2021). TGM2 participates in many pathological reactions, including inflammation, chemotaxis of inflammatory factors, tumor progression, wound healing, tissue fibrosis, and immune diseases (Cho et al., 2020). The members of this family exhibit a variety of enzyme activities, such as protein kinase, GTPase and ATPase activities, disulfide isomerase activity, and participate in cell adhesion.

Keloids are the consequence of a series of intertwined pathological processes consisting of excessive fibroblast proliferation, extracellular matrix deposition, and dysregulated modulation of cell migration. TGM2 has been reported to regulate cell cycle protein expression levels in the tumor microenvironment to aid cell cycle progression and facilitate fibroblast proliferation (Nadalutti et al., 2011; Lee et al., 2015). The transamidase activity of TGM2 contributes to the maturation and stability of collagen and extracellular matrix by cross-linking with extracellular matrix proteins and is involved in wound healing and fibrosis in various tissues, such as scar tissue, liver and heart (Wang et al., 2020a; Wang et al., 2020b). During collagen lattice formation, TGM2 plays a dominant role in the initial formation of calcium-dependent morphology and is involved in early extracellular matrix formation (Li et al., 2018). In many tumors with high metastatic potential, TGM2 expressed above basal levels is found to be associated with loss of cell polarity and disruption of cell junctions, which makes epithelial cells motile and invasive (Pierce et al., 2013; Eckert et al., 2014). The action process of TGM2 is closely connected with the various stages of tumor growth, including the epithelial-mesenchymal transition, apoptosis, differentiation, and aggressive metastatic phenotype formation of cancer stem cells. TGM2 regulates the angiogenesis of digestive system tumors through the WntB-catenin pathway and interferes with the apoptosis of colon cancer cells (Zhang and McCarty 2017; Torres et al., 2019). TGM2 is overexpressed in esophageal adenocarcinoma and attaches to tumor staging (Leicht et al., 2014). Although the study of TGM2 in keloid pathogenesis lacks in-depth research, it is reasonable to believe that TGM2 is a very promising mechanistic molecule in keloids based on its regulation of fibroblast and extracellular matrix in tumors.

This study finally identified TGM2 as a promising biomolecule in keloids. However, the limitations of this study must be acknowledged. On the one hand, this is a retrospective study of expression profiling data. It is undeniable that age, gender, and race are important influencing factors in the development of keloids. The correlation between keloid biomarkers and these factors still needs to be elucidated by large sample and multicenter studies, which is a very promising research direction. On the other hand, the analysis of immune infiltration is based on limited genetic data. Therefore, it may deviate from the interactions of immune cells in specific diseases, disease-induced disorders or the actual situations of individuals, and further molecular experiments are necessary for verification.

TGM2 may be able to act as an auxiliary diagnostic indicator for keloids. TGM2 has been confirmed in previous studies to be associated with fibrosis and tumorigenesis; however, the role of TGM2 in keloids has not been adequately reported. Therefore, TGM2 may be developed as a new research hotspot and target for the prevention, diagnosis and treatment of keloids.