Abstract
Apoptosis of lymphocytes governs the response of the immune system to environmental stress and toxic insult. Signaling through the ubiquitously expressed glucocorticoid receptor, stress-induced glucocorticoid hormones induce apoptosis via mechanisms requiring altered gene expression. Several reports have detailed the changes in gene expression mediating glucocorticoid-induced apoptosis of lymphocytes. However, few studies have examined the role of non-coding miRNAs in this essential physiological process. Previously, using hybridization-based gene expression analysis and deep sequencing of small RNAs, we described the prevalent post-transcriptional repression of annotated miRNAs during glucocorticoid-induced apoptosis of lymphocytes. Here, we describe the development of a customized bioinformatics pipeline that facilitates the deep sequencing-mediated discovery of novel glucocorticoid-responsive miRNAs in apoptotic primary lymphocytes. This analysis identifies the potential presence of over 200 novel glucocorticoid-responsive miRNAs. We have validated the expression of two novel glucocorticoid-responsive miRNAs using small RNA-specific qPCR. Furthermore, through the use of Ingenuity Pathways Analysis (IPA) we determined that the putative targets of these novel validated miRNAs are predicted to regulate cell death processes. These findings identify two and predict the presence of additional novel glucocorticoid-responsive miRNAs in the rat transcriptome, suggesting a potential role for both annotated and novel miRNAs in glucocorticoid-induced apoptosis of lymphocytes.
Highlights
Apoptosis of lymphocytes is critical for the homeostatic balance of the immune system
Reads were generated by generation sequencing on the Illumina platform using total RNA extracted from dexamethasone (Dex) treated and untreated (Control) primary thymocytes
Ingenuity Pathways Analysis (IPA) of this combined gene list identified cell death and survival as a top predicted molecular and cellular function of these differentially expressed potential targets, as well as other functions critical to the induction and execution of glucocorticoid-induced apoptosis, including changes in cell morphology, cell cycle and cell signaling (Figure 3C). These computational findings suggest that these novel glucocorticoid-responsive miRNAs may contribute to glucocorticoid-induced apoptosis. Using both microarray and deep sequencing analysis, we described the prevalent repression of annotated miRNAs during glucocorticoid-induced apoptosis of primary rat thymocytes [1]
Summary
Apoptosis of lymphocytes is critical for the homeostatic balance of the immune system. Glucocorticoid hormones are potent inducers of lymphocyte apoptosis [1]. Endogenous glucocorticoids regulate immune development through the elimination of unwanted immature thymocytes during the T-cell selection process [2]. Given their aggressive proapoptotic properties, synthetic glucocorticoids are a mainstay of hematomalignant chemotherapeutic regimens. Glucocorticoids are a class of essential stress-induced steroid hormones regulating cardiovascular, metabolic, homeostatic and immunologic functions. Glucocorticoid-induced apoptosis of lymphocytes is a multifaceted process, requiring signaling through the GR and the altered expression of apoptotic effector genes [4,5,6]. Until recently, gene expression analysis of lymphocytes undergoing glucocorticoid-induced apoptosis has largely ignored the examination of non-coding RNAs, or miRNAs
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