Identification of functional networks associated with cell death in the retina of OXYS rats during the development of retinopathy

Figures & data

Figure 1. Apoptotic activity was analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. (A) Positive TUNEL straining (green) was observed under a fluorescence microscope. (B) Quantitative analysis. The number of apoptotic cells was calculated by averaging the number of positive TUNEL signals. *Significant interstrain differences, р < 0.05; ^#significant differences with the preceding age, р < 0.05. Bar 50 mkm.

Table 1. Differentially expressed genes (DEGs) with a fold change (FC) cutoff > 2.0 and adjusted p values < 0.05

Gene title term	Gene ontology term	Gene Symbol	FC	Padj
transmembrane protein 221	integral component of membrane	Tmem221	−87.46	6.78E–52
ENSRNOG00000045967	unknown	unknown	−75.01	6.71E–14
similar to Glutathione S-transferase A1	Glutathione transferase activity	GSTA6	−74.77	0.00104
ENSRNOG00000045691	unknown	unknown	−47.64	5.89E–05
ENSRNOG00000017412	unknown	unknown	−42.43	0.0058
ribosomal protein L28-like	structural constituent of ribosome	RGD1565183	−16.22	0.013
ENSRNOG00000030351	unknown	unknown	−14.96	2.5E–10
ENSRNOG00000033517	unknown	unknown	−13.24	0.021
ENSRNOG00000030548	Unknown	unknown	−10.41	5.59E–12
nitric oxide synthase 2, inducible	arginine binding	NOS2	−8.83	0.0067
RT1 class I, locus CE4	peptide antigen binding	RT1-CE4	−8.73	0.0099
ENSRNOG00000031667	unknown	unknown	−8.57	0.021
Osteopontin	extracellular matrix binding	Spp1	−8.57	4.38E–13
NLR family, apoptosis inhibitory protein 2	cysteine-type endopeptidase inhibitor activity involved in apoptotic process	Naip2	−7.33	0.033
ENSRNOG00000029389	unknown	unknown	−6.89	0.0011
SNRPN upstream reading frame protein-like	ubiquitin-protein transferase activity	Snurf	−6.86	1.48E–11
neuropeptide VF precursor	receptor binding	Npvf	−6.6	0.0089
ENSRNOG00000031454	unknown	unknown	−6.56	0.02
family with sequence similarity 3, member B	unknown	Fam3b	−6.31	0.035
ENSRNOG00000032578	unknown	unknown	−6.24	0.026
patatin-like phospholipase domain containing 1	lipid metabolic process	Pnpla1	−5.75	0.0028
coiled-coil domain containing 152	unknown	Ccdc152	−5.42	0.021
Family with sequence similarity 70, member B	unknown	Tmem255b	−5.28	0.01
thrombospondin 4	calcium ion binding	Thbs4	−5.11	0.034
lumican	extracellular space	Lum	−4.74	0.01
Cerebroside sulfotransferase	galactosylceramide sulfotransferase activity	Gal3st1	−4.67	0.022
phosphodiesterase 4D, cAMP-specific	3′,5′-cyclic-AMP phosphodiesterase activity	Pde4d	−4.61	0.00055
matrilin 3	proteinaceous extracellular matrix	Matn3	−4.48	0.01
carbonic anhydrase 9	carbonate dehydratase activity	Car9	−4.14	0.011
ENSRNOG00000033957	unknown	unknown	−3.93	0.003
ENSRNOG00000017661	unknown	unknown	−3.86	0.003
fibrinogen-like 2	peptidase activity	Fgl2	−3.83	4.23E–05
Tp53rk binding protein	tRNA processing	TPRKB	−3.7	0.00098
STEAP family member 4	cupric reductase activity	Steap4	−3.57	4.43E–05
ENSRNOG00000029070	unknown	unknown	−3.57	0.018
ligase IV, DNA, ATP-dependen	DNA ligase (ATP) activity	Lig4	−3.46	7.49E–19
gremlin 1, DAN family BMP antagonist	transmembrane receptor protein tyrosine kinase activator activity	GREM1	−3.44	0.0015
myosin VIIb	ATP binding	Myo7b	−3.331	0.012
family with sequence similarity 129, member A	negative regulation of protein phosphorylation	Fam129a	−3.26	0.036
wingless-type MMTV integration site family, member 16	frizzled binding	Wnt16	−3.05	0.031
interferon induced transmembrane protein 1	integral component of membrane	Ifitm1	−2.88	0.0033
retinol saturase (all trans retinol 13,14 reductase)	all-trans-retinol 13,14-reductase activity	Retsat	−2.87	1.81E–11
EGF-like module containing, mucin-like, hormone receptor-like 1	calcium ion binding	Emr1	−2.86	0.00076
cellular retinoic acid binding protein 1	retinal binding	Crabp1	−2.85	5.2E–18
ENSRNOG00000039025	unknown	unknown	−2.84	0.012
coiled-coil domain containing 146	unknown	Ccdc146	−2.8	9.9E–05
BAI1-associated protein 2-like 1	proline-rich region binding	Baiap2l1	−2.71	8.3E–06
sodium channel, voltage-gated, type VII, α	sodium channel activity	Scn7a	−2.64	0.031
zinc finger protein 182-like	metal ion binding	Zfp943	−2.62	7.96E–05
family with sequence similarity 65, member C	unknown	Fam65c	−2.59	3.2E–11
ENSRNOG00000032997	unknown	unknown	−2.56	0.046
ENSRNOG00000039790	unknown	unknown	−2.56	0.00045
YME1-like 1 (S. cerevisiae)	ATP binding	Yme1l1	−2.54	5.51E–10
solute carrier family 7 (anionic amino acid transporter light chain, xc- system), member 11	amino acid transmembrane transporter activity	Slc7a11	−2.53	0.0013
Slc24a5 solute carrier family 24, member 5	calcium, potassium:sodium antiporter activity	Slc24a5	−2.53	0.0049
Cd48 molecule	antigen binding	Cd48	−2.51	0.037
cubilin (intrinsic factor-cobalamin receptor)	calcium ion binding	Cubn	−2.49	0.0018
coiled-coil domain containing 80	heparin binding	Ccdc80	−2.48	0.032
mitochondrially encoded NADH 4L	NADH dehydrogenase (ubiquinone) activity	MT-ND4L	−2.46	7.58E–07
phospholipase A2, group IIC	extracellular region	Pla2g2c	−2.37	0.023
advillin	actin binding	Avil	−2.35	5.62E–05
CGRP receptor component	DNA-directed RNA polymerase activity	Crcp	−2.35	9.8E–14
asparaginase like 1	asparaginase activity	Asrgl1	−2.34	5.6E–06
testis expressed 15	fertilization	Tex15	−2.33	4.23E–05
zinc finger protein 420-like	metal ion binding	Zfp677	−2.27	0.0012
bone morphogenetic protein 6	BMP receptor binding	Bmp6	−2.25	0.0032
disabled 2, mitogen-responsive phosphoprotein	phosphatidylinositol 3-kinase binding	Dab2	−2.25	0.0098
calcitonin receptor-like	adrenomedullin receptor activity	Calcrl	−2.23	0.035
HAUS augmin-like complex, subunit 1	cell division	Haus1	−2.22	1.67E–07
lysyl oxidase	carbohydrate binding	Lox	−2.21	0.0033
sterile α motif domain containing 9-like	common myeloid progenitor cell proliferation	Samd9l	−2.2	0.0065
hypothetical LOC300751	unknown	RGD1311874	−2.15	0.033
zinc finger protein 873	metal ion binding	Zfp873	−2.16	0.013
myosin IC	actin-dependent ATPase activity	Myo1c	−2.11	4.88E–08
inositol (myo)-1(or 4)-monophosphatase 2	inositol monophosphate 1-phosphatase activity	Impa2	−2.10	0.0012
cadherin 19, type 2	calcium ion binding	Cdh19	−2.09	0.007
ENSRNOG00000048784	unknown	unknown	−2.08	1.42E–06
regulator of telomere elongation helicase 1	ATP-dependent DNA helicase activit	Rtel1	−2.07	2.19E–10
diacylglycerol kinase, gamma	ATP binding	Dgkg	−2.06	0.035
similar to Centromeric protein E (CENP-E protein)	unknown	Ccdc175	−2.05	0.00989
DNA primase, p49 subunit	DNA primase activity	Prim1	−2.02	1.12E–06
phosphoribosyl pyrophosphate synthetase 2	ADP binding	Prps2	−2.00	0.013

Figure 2. Statistically significant (p < 0.05) Gene Ontology terms that are related to the genes whose expression is changed in the retina of OXYS rats in comparison with Wistar rats at age 20 d.

Figure 3. This Venn diagram shows overlapping sets of genes at the 3 ages.

Figure 4. Illustration of the association networks of apoptosis in 20-day-old (A), 3-month-old (B), and 18-month-old rats (C). In each of these networks, black circles denote differentially expressed genes (DEG), whereas the GeneMANIA-predicted genes are shown in gray.

Table 2. Topological parameters of the nodes of association networks related to apoptosis in rats at ages 20 d and 3 and 18 months. The optimal parameters are in boldface

Gene name	Degree	Average Shortest Path Length	Betweenness Centrality	Closeness Centrality	Clustering Coefficient
20 days
Cdkn1b	15	1.68	0.064	0.59	0.27
Rasa1	14	1.74	0.0095	0.58	0.64
Kitlg	14	1.74	0.031	0.58	0.56
Ets1	14	1.71	0.08	0.59	0.41
Tgfbr1	13	1.71	0.024	0.59	0.63
Rock1	13	1.74	0.019	0.58	0.5
Ednrb	12	1.71	0.037	0.59	0.47
Erbb3	12	1.84	0.012	0.54	0.56
Nup153	11	1.87	0.034	0.54	0.51
Ngf	10	1.97	0.031	0.51	0.14
Tgfa	10	1.76	0.04	0.57	0.27
Pten	10	1.82	0.013	0.55	0.53
3 months
Casp1	22	1.92	0.077	0.52	0.52
Casp12	22	1.92	0.084	0.52	0.58
Casp8	21	2.02	0.094	0.49	0.49
Casp4	16	2.17	0.009	0.46	0.73
CFLAR	16	2.17	0.015	0.46	0.79
Birc3	15	1.96	0.091	0.51	0.44
Casp7	13	2.27	0.0	0.44	1.0
Nfkbia	13	1.88	0.189	0.53	0.2
Zc3h12a	13	2.04	0.142	0.49	0.32
Casp14	11	2.27	0.0	0.44	1.0
Alox15	10	2.42	0.127	0.41	0.39
Ripk3	10	2.10	0.057	0.48	0.33
Sgk1	10	2.17	0.030	0.46	0.31
18 months
Cdk1	21	1.94	0.20	0.51	0.09
Ngfr	17	2.11	0.08	0.47	0.26
Hspa1b	17	1.93	0.13	0.52	0.19
Erbb3	13	2.04	0.07	0.49	0.24
Adrb2	13	2.13	0.08	0.47	0.18
Ngf	12	2.217	0.03	0.45	0.29
Ntf3	12	2.26	0.03	0.44	0.38
Mal	12	2.11	0.05	0.47	0.24
Erbb2	12	2.02	0.08	0.50	0.25
Actn4	11	2.17	0.07	0.46	0.27
Casp8	10	2.43	0.01	0.41	0.33

Table 3. Cell death gene ontology (GO) terms for differentially expressed genes (DEGs) and additional related genes according to GeneMANIA analysis for each category (20 days, 3 months, and 18 months)

GO annotation	Genes/Total number of genes	p value
20 days
GO:0097191 extrinsic apoptotic signaling pathway	13/200	3.53e–11
GO:0051402 neuron apoptotic process	12/218	1.77e–9
GO:0097192 extrinsic apoptotic signaling pathway in absence of ligand	7/73	1.43e–6
GO:0043154 negative regulation of cysteine-type endopeptidase activity involved in apoptotic process	6/75	3.21e–5
GO:2001234 negative regulation of apoptotic signaling pathway	7/151	6.94e–5
GO:0008625 extrinsic apoptotic signaling pathway via death domain receptors	5/56	1.7e–4
GO:2001235 positive regulation of apoptotic signaling pathway	5/123	2.93e–3
GO:0008637 apoptotic mitochondrial changes	4/90	1.14e–2
GO:0090199 regulation of release of cytochrome c from mitochondria	3/37	1.45e–2
3 months
GO:0043525 positive regulation of neuron apoptotic process	6/52	1.82e–5
GO:0070266 necroptotic process	5/26	2.13e–5
GO:0097193 intrinsic apoptotic signaling pathway	8/215	1.26e–4
GO:0097191 extrinsic apoptotic signaling pathway	8/200	8.73e–5
GO:0097194 execution phase of apoptosis	4/60	7.24e–3
GO:0097300 programmed necrotic cell death	5/32	4.3e–5
GO:0008637 apoptotic mitochondrial changes	4/90	2.4e–2
GO:0008625 extrinsic apoptotic signaling pathway via death domain receptors	4/56	5.89e–3
GO:1901214 regulation of neuron death	7/217	1.19e–3
GO:0043280 positive regulation of cysteine-type endopeptidase activity involved in apoptotic process	9/179	1.02e–5
18 months
GO:0051402 neuron apoptotic process 15/218		6.2e–13
GO:0070997 neuron death	15/253	1.45e–12
GO:0097191 extrinsic apoptotic signaling pathway	10/200	3.06e–7
GO:0097194 execution phase of apoptosis	3/60	3.64e–2
GO:0008637 apoptotic mitochondrial changes	4/90	1.09e–2
GO:0043281 regulation of cysteine-type endopeptidase activity involved in apoptotic process	5/179	1.25e–2
GO:0070265 necrotic cell death	3/39	1.44e–2
GO:0072332 intrinsic apoptotic signaling pathway by p53 class mediator	3/60	3.64e–2

Figure 5. Clusters in the schemes of gene networks for 20-day-old (A), 3-month-old (B), and 18-month-old rats (C). In each of these networks, black circles denote differentially expressed genes (DEG), whereas the GeneMANIA-predicted genes are shown in gray. The hub genes are yellow

Table 4. Cluster analysis of gene networks for 20-day-old, 3-month-old and 18-month-old rats. Showing clusters with the number of nodes more than 3

Cluster	Genes	Biological processes
20 days
1	Pten, Rasa1, Hgf, Ets1, Kitlg, Ednrb	Regulation of signaling by interleukins (Il-2 and Il-7) and signal transduction EGFR, VEGF, FGFR, insulin receptor, NGF, PDGF, SCF-KIT, ERBB2, ERBB4, PIP3 activates AKT signaling, signaling by BMP, signaling by TGF-β receptor complex, signaling by GPCR; inositol phosphate metabolism and metabolism of lipids and lipoproteins and axon guidance.
2	Rock1, Erbb3, Tgfbr1, Tgfb2, Rb1cc1	apoptotic execution phase, CaM pathway, calmodulin induced events, immune system and signal transduction.
3	Fas, Btg2, Dnajb9, Cdkn1b, Tgfa, Ngf, Sycp3, Igf1	Caspase-8 activation by cleavage, FasL/CD95L signaling, regulation by c-FLIP, innate immune system.
3 months
1	Lig4, Ccl6, Tns4, Tp63, Casp8, Pdcd4, Casp12, Casp4, Casp7, cFlar,Birc3	TRAIL signaling, death receptor signaling, regulation by c-Flip, Caspase-8 activation by cleavage and dimerization of procaspase-8
2	Casp1, Cdk1, Litaf, Cryab, Sgk1, Pawr, Ripk3, Cd24, Nfkbia	the role of DCC in the regulation of apoptosis and the execution phase of apoptosis
3	Fgf2, Lgals7, Grk1, Alox15, Alox12	metabolism of lipids and lipoproteins (regulation of the metabolism of arachidonic acid)
18 months
1	Hspa1b, Nqo1, Mal, Ptprc, Anxa1, Sphk1, Ctnna1, Adrb2, Cdh1, Erbb2, Actn4, Casp7	FasL/CD95L signaling, TRAIL signaling, regulation of apoptosis by c-FLIP, in caspase 8 and procaspase 8 dimerization in the apoptotic pathways, and the regulation of the immune system
2	Hspb1, Phlda3, Il1rn, Erbb3, Ntf3, Fgfr3, Bcl2l10, Adipoq	apoptotic cleavage of cellular proteins (apoptotic execution phase), activation, myristolyation of BID and translocation to mitochondria, apoptotic factor-mediated response (cytochrome c-mediated apoptotic response and SMAC-mediated apoptotic response) in intrinsic pathway and caspase-8 activation by cleavage and dimerization of procaspase-8.
3	Alox12, Sh3glb1, Lig4, Bard1, Cdk1, Cd24, Perp, Nuak2, Tgfa	integration of energy metabolism, transmission across chemical synapses, potassium channels in neuronal system, signaling by NGF.
4	Ripk2, Myd88, Ngfr, Casp8, Chek2	regulation of innate immune system and metabolism of lipids and lipoproteins.