Since early 2010s, RNA-sequencing (RNAseq) has been used to profile transcriptome of retinal neovascularization disease. Differential expression genes (DEGs) analysis can straightforwardly analysis a gene expression dataset. Through DEGs analysis, a set of highly up-regulated and down-regulated genes can be identified by comparing two groups of samples (e.g. hypoxia vs. normoxia). However, gene expression is highly dynamic, and the expression quantification may depend on the techniques (e.g. different platforms of microarray, or RNAseq), making the cross-dataset comparison difficult. The activities of hub genes cannot be directly measured by microarray or RNA-seq because these techniques only measure RNA expression level and do not consider gene activity changes by post-translational modifications. Fortunately, hub genes activity can be inferred by its regulons through VIPER, by taking DEGs data and an context-specific gene regulatory network (interactome) as inputs[16].
In present study, transcriptome analysis of EMP2 overexpressed and knock-downed RPE cells under hypoxia or normoxia elucidated eight sets of DEGs which has been used to construct TRN along with variant analysis. TRN analyses attempt to simplify the interactions of activity proteins and emphasize their role in EMP2 treated RPE cells. Activity proteins(APs) are often obscured in standard differential expression analyses (such as those including environmental stressors) and indeed many of the APs identified in our network analysis were not significant in the DEGs analysis, highlighting the strength of the network approach.(Fig. 5)
Under hypoxia condition, 21 DEGs were identified in EMP2-OE group when compared to VC group. However, when we used VIPER to clarify APs, several angiogenic and inflammatory factors (PDGFA, ALDH1L2, BAIAP3, BBOX1, ST3GAL5, ANGPT1, KAT6A) were found to be HGs in EMP2-OE groups. PDGFA and ANGPT1 were identified both in DEGs and APs. They shared some signaling pathway, including MAPK, Ras, Rap1 and PI3K-Akt signaling pathway. Platelet derived growth factor (PDGF)were proved to induce the proliferation and migration effects on RPE cells in PVR[17], and has been reported to participate in the pericytes regulated angiogenesis, vessel stabilization, and contribute to the formation of both the blood-brain and blood-retina barriers by regulating pericyte-endothelial cell communication[18]. PDGFRβ plays an essential role in patient vitreous-stimulated contraction of retinal pigment epithelial cells from epiretinal membranes[19]. PDGF-B was also proved to promote cell proliferation and angiogenesis by increasing the activity of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP-2) in RMECs[20].However, PDGF had not proved to have correlation with EMP2.
ALDH1L2 (Aldehyde Dehydrogenase 1 Family Member L2) is a protein coding gene which have function as mitochondrial folate enzyme[21]and the maintenance of mitochondrial integrity and energy balance of the cell[22], of which related pathways are one carbon pool by folate and metabolism of water-soluble vitamins and cofactors. ALDH1L2 had been proved to have correlation with EMP2 in breast cancer cells[23], but it was the first time to be identified in EMP2 treated hRPECs. We also found that PDGFA connected with ALDH1L2 by PLSCR4, which may mediate accelerated ATP-independent bidirectional transbilayer migration of phospholipids upon binding calcium ions that results in a loss of phospholipid asymmetry in the plasma membrane. It may play a central role in the initiation of fibrin clot formation, in the activation of mast cells and in the recognition of apoptotic and injured cells by the reticuloendothelial system. We raise a hypothesis that over-expression of EMP2 would activate PDGFA through ALDH1L2-PLSCR4 pathway by initializing fibrin clot and recognizing apoptosis.
We also identified that BAIAP3(BAI1 Associated Protein 3), which functions in endosome to Golgi retrograde transport, thus may regulate behavior and food intake by controlling calcium-stimulated exocytosis of neurotransmitters including NPY and serotonin and hormones like insulin, had correlation with MMP16[24], which has been proved to could degrade various components of the extracellular matrix, such as collagen type III and fibronectin in the pathogenesis of diabetic cataract [25].
ANGPT was also a HGs in OE-VC TRN and it had been proved to be upregulated and correlate with VEGF in NVMs [26]. It also showed some connection with PDGFA through PLXNA1, CLIP3 and KLF7. OTX2 functions as transcription factor, probably involved in the development of the brain and the sense organs. It was found to be upregulated in EMP2-knockdown mouse embryoid bodies by participant in gastrulation [27and it were identified in this study to have correlation with PDGFA through ST3GAL5.
Nevertheless, we found that the interaction among OE TRNs under hypoxia increased when compared with KD TRNs. The hypothesis is that when EMP2 over-expressed under hypoxia, PDGFA were upregulated and enhanced the interactions with other genes. The cascade interactions among these genes triggered angiogenesis through complex pathways.
This investigation was mainly based on ARACNe algorithm analysis of transcriptome information of hRPECs treated by hypoxia and exogenous EMP2. The TRNs constructed in this paper provides clues of interaction mechanism between these genes, which not only provides theoretical support for revealing the regulatory relationship among them, but also provides candidate genes, ie, up- and down-regulated pathway genes,transcriptional factors, and hub genes, which could be studied for future SNP discovery work to get associated with different phenotype/traits of interest. We also established a scientific exploration mode for exploring effective targets of anti-neovascularization, which makes the follow-up basic verification and clinical research more reliable. However, the results of bioinformatics analysis ultimately need to be verified by many experiments and combined with clinical practice to carry out gene function and mechanism verification for substantive exploration.