Oral lichen planus is a chronic disorder of oral mucosa with a global prevalence of 1.01%(10), and reticular OLP is the type with the highest prevalence. OLP is now generally recognized as a T-cell dysfunction-induced autoimmune disease, while the exact mechanism of this disease still remains poorly understood. To help further explain the underlying molecular mechanism in OLP, we integrated the RNA-Seq and bioinformatics to explore the transcriptional profiling of this disease.
Validated by qRT-PCR, a total of 153 DEGs were detected by RNA-Seq in this current study, of which 72 were increased while 81 were decreased in OLP tissues. The top 5 upregulated and downregulated genes in OLP were NEB, TNC, NRIP1, DLG1, PTPN22 and SGMS1, TET2, SMO, PARD3, ATP5C1, respectively. Five co-expression modules were constructed by WGCNA method and hub genes of each modules were identified as followed: RYK, SLC8A1, WDR7, MAP3K5, GPBP1. According to the GO and KEGG pathway analysis, genes with aberrant expression in OLP were mainly associated with T cell receptor signaling pathway and the Wnt signaling pathway. These above-mentioned results were in line with the existing OLP pathogenesis, and provided new insights into the potential mechanism of OLP.
In OLP, a key pathogenesis is the activation of CD4+ T-helper and cytotoxic CD8+ lymphocytes along with the cytokines, resulting in keratinocytes apoptosis and degeneration of the epithelial basal layer (11). T cell in OLP tend to proliferate rapidly and differentiate into distinct Th cell types, including Th1, Th2, Treg, Th17, etc. Growing evidence showed that Th1/Th2 imbalance influenced by Treg subset as well as the CD4+ Th subset Th17 took great part in immunopathology of OLP (12). Nuclear receptor interacting protein 1 (NRIP1), also known as RIP140, was significantly increased in OLP. NRIP1 interacts with transcription factors and nuclear receptors, and previous studies focused on its molecular mechanism in regulating the immune response. Zhu Jun Yi et al suggested that the overexpression of NRIP1 in macrophages promoted M1-like polarization and expansion in inflammatory diseases (13). NRIP1 was found to modulate the transcription and secretion of pro-inflammatory cytokines by activating NF-κB (14) and this mechanism could also be found in autoimmune disease like psoriasis(15). Interestingly, the NF-κB p65 signaling pathway was also activated and played a key role in the inflammatory response in OLP(16). DLG1, which was one of the top high expressed DEGs in OLP tissues, is a member of the family of molecular scaffolding proteins and encoded a multi-domain scaffolding protein required for normal development. Altered expression of DLG1 is observed in several immunological disorders. Dlg1 functions as a positive regulator of immunity and facilitated T cell receptor (TCR) signal transduction and T cell function. T cells lacking proper Dlgh1 expression were deficient in coupling antigenic stimulation resulting in incapability of producing cytokine (17). TET2, which was the most significantly downregulated gene, is universally considered as a tumor-suppressor gene and could modulate Th1 and Th17 cell differentiation and T-cell cytokine production in vivo in autoimmune diseases (18). Earlier study showed that mutation of TET2 caused aberrant CD4+ T cell proliferation and disturbance of T cell homeostasis (19), which was closely related to the occurence of OLP. Besides, Tet2 downregulation was vital for 5-hydroxymethylcytocine (5hmC) regulation in adult T-cell leukemia/lymphoma (ATLL) progression (20) and Tet2-KO mice developed myeloid cancers with an incidence rate of about 30% (21). These results demonstrated that TET2 was strongly associated with immune responses. MAP3K5 (also named ASK1), which was the hub gene in turquoise module acquired from WGCNA, is an important member of mitogen-activated protein kinase (MAPK) family and plays crucial roles in physiological processes associated with autoimmune diseases. Recently, S.J. Mnich et al found that as an essential component for the development of rheumatoid arthritis, MAP3K5 participated in the tumor necrosis factor-alpha (TNF-α)-induced production of inflammatory mediators (22) and MAP3K5 deficiency could reduce neuroinflammation in experimental autoimmune encephalomyelitis (23). In addition, in aberrant ribosomal biogenesis, activated MAP3K5 pathways regulated the stability and activity of p53 protein (24), which acted as a transcription factor mediating the induction of cell apoptosis (25). Taken into consideration the fact that the expression of p53 has been reported upregulated in OLP tissues (26, 27), the interaction between MAP3K5 and p53 may play roles in the pathogenesis of OLP.
Wnt signaling played key roles during cell proliferation and differentiation and was implicated in multiple autoimmune diseases due to its participation in the development of T cells. In OLP, Wnt3 has been featured with positive expression in cytoplasm and lack of nuclear staining, suggesting a potential involvement of Wnt signaling pathway in OLP progression (28). RYK, the hub gene in blue module, belonged to the atypical receptor tyrosine kinase family and was a receptor of Wnt ligand Wnt5a. The interaction of Wnt ligands and RYK originated the downstream signaling cascades critical for immune regulation (29). Besides, Tenascin C (TNC), ranking as the 2nd top upregulated gene in OLP, encoded an extracellular matrix protein (ECM) and was a promoter of tumor metastasis with multiple functions. TNC expression positively linked to cancer progression and cell proliferation. TNC downregulated Dick-kopf-1 (DKK1), a Wnt inhibitor by blocking of actin stress fiber formation, thus activating Wnt signaling and induced Wnt target genes in tumor and endothelial cells (30). Nevertheless, the high expression of TNC induced by Wnt/beta-catenin activation, acting as a downstream target, leaded to a more tumorigenic cell state in Ewing sarcoma (31). The above researches demonstrated that the interaction between TNC and Wnt signaling participated in the modulation of tumor progression and invasion. Nevertheless, to the best of our knowledge, the rest of the key genes have not been reported highly involved in T cell regulation and inflammation or Wnt signaling pathway.
There were limitations in our study. Firstly, the samples size was small. Only reticular OLP tissues were collected and there was a lack of OLP tissues of erosive and erythematous types. Secondly, we did not validate the functions of the key genes as well as the potential signaling pathways in vitro or in vivo.
All in all, in this study, we strictly selected the matched samples of OLP and normal oral mucosal tissues and a range of genes were observed differentially expressed in OLP tissues. Subsequently, by application of the bioinformatic analysis method, our research also confirmed the key roles of biological activities associated with T cell regulation and inflammation and Wnt signaling pathway that have been reported in earlier studies on the molecular mechanisms of OLP and provided a theoretical basis for the study of novel drug targets.