Myopia is a very common eye condition that is rapidly increasing in occurrence. It
is the most common eye disease in teenagers [1], and uncorrected ametropia is the
main cause of moderate or severe visual impairment in people aged 50 years and above
[2]. It is estimated that the number of patients with myopia will reach 4.758 billion
by 2050, accounting for 49.8% of the global population [3]. The severity of a patient’s
myopia is also associated with increased risk of visual impairment due to macular
degeneration, cataracts, retinal detachment, and glaucoma caused by high myopia [4].
A decreased visual function in the early stage of myopia [5–7] has also been noted,
but the molecular mechanism remains unclear. Due to the existence of a large number
of genes that affect biological function, retinal pigmental epithelium (RPE) single-cell
captured ribonucleic acid (RNA) sequencing technology was adopted to explore the molecular
mechanism of RPE cell apoptosis in myopia and provide a theoretical basis for clinical
guidance aimed at protecting early visual function in myopic patients.
The original data of the gene expression profiles in the samples were uploaded to
the GCBI analysis platform (https://www.gcbi.com.cn/gclib/html/index). The samples
were divided into 2 groups of 3 samples each: the negative control group and the myopic
model group. The samples of the 2 groups were compared and labelled as the model group
and the control group (i.e. the myopic model group compared with the negative control
group). The signal values of the original data expression spectrum were read respectively,
and the differences in gene expression between the myopic model group and the negative
control group were analysed. The criteria for the screening of the differential gene
were as follows: p < 0.05, Q < 0.05, fold change (FC) > 2. The up-regulated and down-regulated
genes in the expression profile in the model group vs. the control group were obtained.
The edgeR package was used to perform difference analysis. The false discovery rate
(FDR) < 0.05 and | log2 (multiple change) > 1 were set as the cut-off values for the
screening of the differentially expressed genes (DEG). The gene co-expression network
was constructed using the EBcoexpress package. The GO enrichment analysis of the DEG
in the co-expression network was carried out using the Database for Annotation, Visualisation,
and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/)
was adopted to form a path map reflecting the DEG by the KEGG pathway enrichment analysis
of DEG.
At the beginning of the experiment, there was no correlation between the ocular axial
length of the two groups (p > 0.05). Six weeks after the beginning of the experiment,
the ocular axial length of the mice in the model group was significantly longer than
that of the mice in the control group (p < 0.01). The changes in the ocular axial
length at each stage of the experiment are shown in Table I.
Table I
The changes in the ocular axial length at each stage of the experiment (
x
± s)
Group
N
At the beginning of the experiment
At the end of the experiment
t value
P-value
Control group
6
2.74 ±0.18
3.06 ±0.13
–3.556
0.005
Myopic model group
6
2.72 ±0.11
3.38 ±0.11
–10.547
< 0.001
t value
0.254
0.805
P-value
–4.687
0.001
The cluster map showed that 3900 genes were identified in the model group vs. the
control group. DEG analysis identified 617 genes, including 96 up-regulated genes
and 521 down-regulated genes. Different gene expression patterns were observed between
the two groups, indicating the reliability of DEGs.
In the differential genes of the model group vs. the control group, the genes with
a statistical significance of p < 0.05 were screened out, which indicates that there
are gene expression changes in RPE cells during myopia.
Through the functional enrichment analysis of differential genes, a total of 1801
GO items were detected in the model group vs. the control group. Among these items,
1039 GO items had a statistical significance of p < 0.05, including 774 in the biological
process, 152 in the cellular components, and 111 in molecular function.
In the model group vs. the control group, GO correlated with the negative regulation
of the neurotransmitter transportation, Tau protein binding, long-term neuronal synaptic
plasticity regulation, and GABA transport, and other biological processes related
to neuronal apoptosis and neurotransmitter regulation were found. These genes were
correlated with the neurodegenerative diseases related to β-amyloid metabolism and
with glycolysis related to fructose 6-phosphate. This suggests that the RPE cells
were in a state of anaerobic oxidation, protein dysfunction, and neuromodulation disorder
during myopia.
The results of the signal pathway enrichment analysis showed that 219 pathways were
detected in the model group vs. the control group, of which 87 were statistically
significant (p < 0.05). The 30 highest expressions of the KEGG pathway in the RPE
cells of mice in the myopic model group showed that neurotransmitter-related pathways
(such as the glutamatergic synapses and GABAergic synapses), oxidative-phosphorylation-related
pathways (such as the fructose/mannose metabolism), and protein-related pathways (such
as the oxytocin pathway) were activated (Figure 1).
Figure 1
The first 30 pathways of the enrichment of KEGG in the model group vs. the control
group. Each point in the figure represented a KEGG path, and the path name was shown
in the left coordinate axis. The horizontal ordinate was the Enrichment Factor, which
represented the ratio of the proportion of differentially expressed proteins annotated
to the pathway to that of the species. The higher the enrichment factor, the more
significant the enrichment of the differential proteins in the pathway
Through the single-cell RNA sequencing of the RPE cells, it was found that the cells
were exposed to hypoxia during the development of myopia and that hypoxia might lead
to functional limitations in the RPE cells [8]. The GO items with statistical significance,
such as adenosine triphosphate (ATP) production, oxidative phosphorylation, and glycolysis
through fructose 6-phosphate, were found in the RPE cells of the mice in the myopic
model group. Glycolysis (specifically the process of anaerobic oxidation) may have
occurred in the RPE cells of these mice [9]. Thus, it can be inferred that RPE cells
are exposed to hypoxia during myopia; as RPE cells are oxygen-consuming cells, this
hypoxia may activate glycolysis and oxidative phosphorylation in the cells [10–12].
In addition, protein phosphorylation regulation, protein localisation, endoplasmic
reticulum localisation, and negative regulation of the glycoprotein process were found
in the GO affecting the function of protein. It can be inferred that the protein function
of RPE cells might be affected during myopia, leading to protein phosphorylation and
endoplasmic reticulum stress [13].
In the analysis of the KEGG pathway, it was found that a large number of pathways
associated with amino acids, dopamine, and other neurotransmitters in the RPE cells
of the mice in the myopic model, as well as the oxidation-related pathways (such as
the fructose/mannose metabolism) and galactose metabolism pathway, were activated,
suggesting the occurrence of glycolysis [14]. This confirmed that RPE cells are in
hypoxia during the development of myopia, causing anaerobic metabolism. Some related
pathways concerning the dysfunction of neurons and neurotransmitters, such as the
dopaminergic synapses, glutamate, and cholinergic synaptic signalling pathways, were
also found (the dysfunction of neurotransmitters may affect the development of RPE)
[15].
Further analysis of INSIG1, PABPC1, PSMD11, SCD1, and other genes is helpful in further
understanding the effect of myopia on the injury of the RPE cells in mice. In the
current study, the expressions of INSIG1, SCD1, and PABPC1 were up-regulated in the
eyes of the mice in the myopic model group. The RNA sequencing revealed that PABPC1,
the substrate of caspase, was up-regulated in the mice in the myopic model group (RNA
degradation is one of the markers of apoptosis). Our previous study found that c-Jun,
an autophagy inhibitor protein, is the upstream trigger signal of apoptosis and autophagy.
Bcl-2 is the homologous domain protein of the apoptosis inhibitor Beclin-1, which
can induce autophagy. However, the results of the present study show that these two
factors were decreased in the RPE cells of the mice in the myopic model group by Western
Blot (WB) and PCR. Bcl-2 and caspase are bidirectional regulators of autophagic apoptosis.
Caspase is the main driving force of apoptosis, and activated caspase can degrade
autophagy proteins (namely Beclin-1, ATG5, and ATG7) to prevent autophagy. Caspase
can also change an autophagy protein into a pro-apoptotic protein, thus triggering
the apoptotic death of cells.
The present experiment found that the expression of INSIG1 was up-regulated in the
eyes of the mice in the myopic model group and that SREBP1 was significantly activated
after the rapid degradation of the endoplasmic reticulum protein INSIG1, leading to
neuronal apoptosis induced by glutamate excitotoxicity. It was hypothesised that the
up-regulation of INSIG1 might be a firing of the mechanism of neuronal self-protection.
The expression of TDO2 was also up-regulated in the RPE cells of the mice in the myopic
model group. TDO2 is a tryptophan 2,3-dioxygenase, which is the metabolite of tryptophan.
A strong expression of tryptophan hydroxylase was observed in the bipolar cells, amacrine
ganglion cells, ganglion cells, and photoreceptors. In the nervous system, TDO2 is
a rate-limiting enzyme in the canine urethane pathway that can induce the expression
of the neuronal survival factor. The results of Fiedorowicz et al. showed that the
kynurenine pathway is out of balance in an injured retina: during the process of retinal/optic
nerve injury, the concentration of kynurenic acid transiently increases. The up-regulation
of TDO2 therefore indicates the activation of the kynurenine pathway, while the increase
of the concentration of kynurenic acid suggests retinal injury.
In conclusion, RPE cell dysfunction was found to occur during the development of myopia,
which might be correlated with gene expression and pathway regulation related to glycolysis
and oxidation, neurodegenerative disease, and the regulatory function of protein.