LncRNAs are considered to be important regulators of cellular process, such as development, differentiation, and metabolism, through affecting gene expression and cell homeostasis.(15, 16) Evidence is accumulating that shows lncRNAs are involved in biological functions by interacting with other molecules, such as DNA,(17) RNA,(18) proteins,(19) and metal ions.(20) Abnormal expression of lncRNAs is involved in the pathophysiological process of many diseases, including cancer, respiratory disease, and diabetes.(21-23) Recent studies have explored the expression levels of lncRNAs in upper airway allergic diseases.(5, 10, 11) Ma et al. showed that the expression profile of lncRNAs was altered in the CD4+ T cells from AR mice.(11) A change in expression of lncRNAs has been detected in nasal mucosa from patients with AR, but no more bioinformatics analysis was provided.(10) These studies indicate that lncRNAs are involved in the pathogenesis of AR. However, the function and mechanism of action of lncRNAs in AR remain unclear.
In the present study, we assessed genome-wide lncRNA expression patterns in the nasal mucosa from patients with and without AR by microarray analysis, and predicted their possible functions by analyzing the co-expressed mRNAs. Moreover, we also used an in vitro model mimicking the allergen exposure environment of airway epithelium to verify the predicted results. Our results indicated that 57 lncRNA and 127 mRNA transcripts were identified as being differentially expressed between the two groups, including 22 upregulated and 35 downregulated lncRNAs, and 43 upregulated and 84 downregulated mRNAs, respectively. To validate the accuracy of microarray analysis, we further randomly chose and validated 4 lncRNAs and 2 mRNAs from the differentially expressed RNAs by qRT–PCR. The consistency of our verification results with those of the microarray analysis strongly suggests the reliability of the microarray results.
The functions of the lncRNAs have not yet been fully annotated and the most common method for their functional prediction is through referring to the functional annotations of their co-expressed mRNAs.(13) As shown in Figure 3, the differentially expressed lncRNAs are co-expressed with hundreds of mRNAs, which may play a vital role in the pathogenesis and development of AR, such as MUC7, IL 33, THY1, and CXCL12. We predicted the functions of the lncRNAs by GO/KEGG enrichment analysis of these co-expressed mRNAs. The most enriched GO annotations are involved in positive regulation of the integrin biosynthetic process, cell adhesion, focal adhesion, inflammatory response, extracellular matrix, T cell receptor complex, cell junction, and intracellular calcium activated chloride channel activity. Some of these functions are known to be involved in the pathogenesis of AR, such as immune cell activation, inflammatory cell migration, and inflammatory response. KEGG pathway analysis also showed that the co-expressed mRNAs regulated some signaling pathways involved in the activity and function of immune cells, including protein processing in the endoplasmic reticulum, protein export, MAPK signaling pathway, and leukocyte transendothelial migration. Interestingly, recent studies have shed light on these biological processes, molecular functions, cellular components, and signaling pathways associated with AR.(11, 24-26)
Due to the variety of functions of lncRNAs, their molecular regulatory mechanism remains unknown.(27) Previous studies have reported that lncRNAs regulate the transcription of nearby genes in a cis-regulatory manner by recruiting remodeling factors to local chromatin.(28) In this study, we explored the cis-regulatory relationships between the differentially expressed lncRNAs and their co-expressed mRNAs (Table 3). We found that the expression of tight junction proteins and chemokines, such as CLDN1 and CXCL12, were cis-regulated by lnc-TMEM207-2 and lnc-CXCL12-4, respectively. When combined with our KEGG pathway analysis, the differentially expressed CLDN1, CXCL12, and THY1 are involved in leukocyte transendothelial migration. The comprehensive analytical result provides additional information concerning immune cell migration mediated by lncRNAs in the pathogenesis of AR. We also constructed the lncRNA–TF and lncRNA–TF–mRNA network based on the results of trans-regulatory analysis. The core network (Fig. 5) shows that TFs, including STAT2, GATA2, GATA3, and ZBTB7A, regulate lncRNA expression in AR. The expression of SAMD9 is regulated by STAT2, which plays a role in regulating cell proliferation and apoptosis. The proteins encoded by GATA2 and GATA3 play essential roles in regulating the transcription of genes involved in the development and proliferation of hematopoietic cell lineages and T cells. Diseases associated with ZBTB7A include photosensitive epilepsy and lymphoma. Thus, trans-regulatory analysis provides another way to predict the functions of lncRNAs in the pathogenesis of AR.
Besides genetic and lifestyle-related factors, AR is also affected by the composition of inhaled air. The respiratory epithelial cells may mediate parts of the innate and adaptive immunity by their antigen presentation, phagocytosis, cytokine secretion, and pattern recognition abilities.(29) The epithelial surface of the respiratory tract is the “first battlefield” of allergic inflammation, where the epithelial cells interact with the inhaled allergens and trigger inflammatory cascade reactions. A recent study found that CXCL12 and the chemokine receptor CXCR4 were critical components of the inflammatory processes involved in a murine model of allergic airway disease.(26) Upon interaction with CXCR4, CXCL12 can result in the most efficacious chemoattraction of T lymphocytes. In the present study, we examined the epithelial responses to allergen exposure using a cell culture model and demonstrated that OVA/HDM exposure induced the expression of lnc-CXCL12-4, CXCL12, and CXCR4 in BEAS-2B within a short time after exposure compared to untreated cells. This is consistent with our previous clinical observations. In nasal polyps from patients with AR, the expressions of lnc-CXCL12-4, CXCL12, and CXCR4 were increased significantly compared to those from nasal polyps without AR. Taken together, these data support the potential importance of lnc-CXCL12-4 and the CXCL12/CXCR4 axis in the immune responses and inflammation in AR.