Identification of genes involved in glaucoma pathogenesis using combined network analysis and empirical studies

Abstract

Glaucoma is a leading cause of blindness. We aimed in this study to identify genes that may make subtle and cumulative contributions to glaucoma pathogenesis. To this end, we identified molecular interactions and pathways that include transcription factors (TFs) FOXC1, PITX2, PAX6 and NFKB1 and various microRNAs including miR-204 known to have relevance to trabecular meshwork (TM) functions and/or glaucoma. TM tissue is involved in glaucoma pathogenesis. In-house microarray transcriptome results and data sources were used to identify target genes of the regulatory molecules. Bioinformatics analyses were done to filter TM and glaucoma relevant genes. These were submitted to network-creating softwares to define interactions, pathways and a network that would include the genes. The network was stringently scrutinized and minimized, then expanded by addition of microarray data and data on TF and microRNA-binding sites. Selected features of the network were confirmed by empirical studies such as dual luciferase assays, real-time PCR and western blot experiments and apoptosis assays. MYOC, WDR36, LTPBP2, RHOA, CYP1B1, OPA1, SPARC, MEIS2, PLEKHG5, RGS5, BBS5, ALDH1A1, NOMO2, CXCL6, FMNL2, ADAMTS5, CLOCK and DKK1 were among the genes included in the final network. Pathways identified included those that affect ECM properties, IOP, ciliary body functions, retinal ganglion cell viability, apoptosis, focal adhesion and oxidative stress response. The identification of many genes potentially involved in glaucoma pathology is consistent with its being a complex disease. The inclusion of several known glaucoma-related genes validates the approach used.

Insights into the regulatory molecules involved in glaucoma pathogenesis

“…FOXC1 involvement in the NFKB signaling pathway was supported by observation of FOXC1 effects on NFKB1 expression at RNA and protein levels (Moazzeni et al, 2019). FOXC1 through regulation of NFKB1 can affects apoptosis, ECM properties, immune and inflammatory responses (Lan, Petznick, Heryati, Rifada, & Tong, 2012; G. Li et al, 2007; Moazzeni et al, 2019; Palenski, Gurel, Sorenson, Hankenson, & Sheibani, 2013; L. Zhou, Li, & Yue, 1999). FOXC1 may also be linked to PAX6 and its associated proteins through mediation by FOXC1 regulated genes, MEIS2 and CLOCK (Paylakhi et al, 2011; Paylakhi et al, 2013).…”

“…FOXC1 indirectly interacts with other genes or their encoded products, most importantly, PAX6 , MYOC , CXCL6 , and CLOCK . These interactions signal the existence of complicated regulatory pathways and networks that are relevant to glaucoma pathogenesis (Banaei‐Esfahani et al, 2015; Moazzeni et al, 2019; Paylakhi et al, 2012).…”

“…The FOXC1‐MYOC connection is also important because MYOC affects RHO signaling in TM cells (X. Shen, Koga, Park, SundarRaj, & Yue, 2008). FOXC1 effects on this signaling pathway can also be mediated by its interactions with PITX2 and PLEKHG5 ; PLEKHG5 expression is regulated by FOXC1 (Moazzeni et al, 2019;Paylakhi et al, 2011; Paylakhi et al, 2013). PLEKHG5 participates in negative feedback regulation of the RHO pathway (Fleischer, 1989; Gauthier & Liu, 2017; Goh & Manser, 2010, 2012; Iyer, Maddala, Pattabiraman, & Rao, 2012; Lee, Moon, Lee, & Joo, 2010; Riento et al, 2005; J. Yang et al, 2016).…”

“…NFKB1 directly or indirectly interacts with CYP1B1, OPTN, MYOC, and TGFβ signaling, all of which are obviously related to glaucoma pathogenesis (Akizuki et al, 2013; Gingery et al, 2008; Palenski et al, 2013). Through OPTN and MYOC interactions, NFKB1 also interacts with SPARC that is involved in apoptosis and ECM properties (Chang et al, 2010; Moazzeni et al, 2019; Wei et al, 2012). NFKB1 may also affect SOD2, and thus possibly contribute to a proposed role for mitochondrial dysfunction in glaucoma (Du et al, 2013; Sacks et al, 2018).…”

“…This cytokine may affect immune functions relevant to glaucoma pathogenesis. ADAMTS5 is direct target of NFKB1 (Moazzeni et al, 2019). This gene encodes a matrix metalloproteinase that can degrade ECM proteins and thus potentially affect aqueous fluid outflow in the anterior chamber (Demircan et al, 2013; Keller, Bradley, & Acott, 2009).…”