An Interaction-Based Method for Refining Results From Gene Set Enrichment Analysis

Abstract

Purpose: To demonstrate an interaction-based method for the refinement of Gene Set Enrichment Analysis (GSEA) results.Method: Intravitreal injection of miR-124-3p antagomir was used to knockdown the expression of miR-124-3p in mouse retina at postnatal day 3 (P3). Whole retinal RNA was extracted for mRNA transcriptome sequencing at P9. After preprocessing the dataset, GSEA was performed, and the leading-edge subsets were obtained. The Apriori algorithm was used to identify the frequent genes or gene sets from the union of the leading-edge subsets. A new statistic d was introduced to evaluate the frequent genes or gene sets. Reverse transcription quantitative PCR (RT-qPCR) was performed to validate the expression trend of candidate genes after the knockdown of miR-124-3p.Results: A total of 115,140 assembled transcript sequences were obtained from the clean data. With GSEA, the NOD-like receptor signaling pathway, C-type-like lectin receptor signaling pathway, phagosome, necroptosis, JAK-STAT signaling pathway, Toll-like receptor signaling pathway, leukocyte transendothelial migration, chemokine signaling pathway, NF-kappa B signaling pathway and RIG-I-like signaling pathway were identified as the top 10 enriched pathways, and their leading-edge subsets were obtained. After being refined by the Apriori algorithm and sorted by the value of the modulus of d, Prkcd, Irf9, Stat3, Cxcl12, Stat1, Stat2, Isg15, Eif2ak2, Il6st, Pdgfra, Socs4 and Csf2ra had the significant number of interactions and the greatest value of d to downstream genes among all frequent transactions. Results of RT-qPCR validation for the expression of candidate genes after the knockdown of miR-124-3p showed a similar trend to the RNA-Seq results.Conclusion: This study indicated that using the Apriori algorithm and defining the statistic d was a novel way to refine the GSEA results. We hope to convey the intricacies from the computational results to the low-throughput experiments, and to plan experimental investigations specifically.

MicroRNA-124-3p Attenuated Retinal Neovascularization in Oxygen-Induced Retinopathy Mice by Inhibiting the Dysfunction of Retinal Neuroglial Cells through STAT3 Pathway

“…Our previous study determined 12 downstream candidate genes of miR-124-3p by RNA-sequencing and bioinformatics analysis in developing mouse retinas [18]. Consequently, we performed RT-PCR to validate the mRNA expression of these genes (Prkcd, Irf9, Stat3, Cxcl12, Stat1, Stat2, Isg15, Eif2ak2, Il6st, Pdgfra, Socs4, and Csf2ra) in the OIR+miR-NC group and OIR+miR-124-3p group.…”

“…RNA extraction of retinas at P17 and cDNA synthesis were performed using TRIzol reagent (Invitrogen, San Diego, CA, USA) and a cDNA synthesis kit (Takara, Kustatsu, Japan), respectively. The method of qRT-PCR was described in our previous study [18]. Information regarding the PCR primers used is summarized in Table 1.…”

“…Recently, several effective miRNA antagonists and mimics, and their delivery media have been developed, providing a promising prospect for miRNAs in the treatment of ocular neovascular diseases [16]. Our previous studies have shown that miR-124-3p expression in mouse retinas increases after birth, and its related pathways predicted by bioinformatics analysis are associated with retinal vascular development [17,18]. MiR-124 is mainly expressed in the inner nuclear layer (INL) and outer limiting membrane (OLM) of the retina, with some low-level expression in the ganglion cell layer (GCL) in healthy human retinal tissue [19].…”