miRNA |
Primer (5′–3′) |
---|---|
hsa-miR-26a-5p |
GSP:5′GGGTTCAAGTAATCCAGG3′ R:5′TGCGTGTCGTGGAGTC3′ |
hsa-miR-126-3p |
GSP:5′GGGGTCGTACCGTGAGTAAT3′ R:5′GTGCGTGTCGTGGAGTCG3′ |
hsa-miR-378a-3p |
GSP:5′GGGGTCTGGACTTGGAGTCA3′ R:5′GTGCGTGTCGTGGAGTCG3′ |
hsa-miR-146b-5p |
GSP:5′GGGGTGAGAACTGAATTCCA3′ R:5′GTGCGTGTCGTGGAGTCG3′ |
hsa-miR-128-3p |
GSP:5′GGGGAATCACAGTGAACCG3′ R:5′GTGCGTGTCGTGGAGTCG3′ |
hsa-miR-185-5p |
GSP:5′GGTGGAGAGAAAGGCAGT3′ R:5′TGCGTGTCGTGGAGTC3′ |
hsa-miR-199a-5p |
GSP:5′GGTGCCCAGTGTTCAGAC3′ R:5′CAGTGCGTGTCGTGGAGT3′ |
hsa-let-7a-5p |
GSP:5′GGGGGTGAGGTAGTAGGTTGT3′ R:5′GTGCGTGTCGTGGAGTCG3′ |
hsa-miR-93-5p |
GSP:5′GGCAAAGTGCTGTTCGTG3′ R:5′CAGTGCGTGTCGTGGAGT3′
|
Table 1. The sequences of primers used for qRT-PCR.
|
Group 1
(NDR) |
Group 2
(mild NPDR) |
Group 3
(healthy controls) |
F
|
P
|
---|---|---|---|---|---|
Participants (
|
33 |
33 |
20 |
– |
– |
Gender (M/W) |
13/10 |
12/11 |
11/9 |
– |
– |
Age (yrs) |
54.03 ± 13.25 |
55.12 ± 14.09 |
52.35 ± 15.77 |
0.237 |
0.789 |
Diabetes duration (yrs) |
7.62 ± 6.21 |
11.37 ± 4.26* |
– |
2.861 |
0.005 |
BMI (kg/m
|
24.72 ± 3.01 |
24.83 ± 3.27 |
23.19 ± 2.42 |
2.164 |
0.121 |
HbA1c (%) |
7.19 ± 2.15*** |
7.94 ± 2.65*** |
4.14 ± 1.36 |
19.27 |
0.0001 |
Total cholesterol (mmol/l) |
4.16 ± 1.20 |
4.22 ± 1.13 |
4.13 ± 1.12 |
0.042 |
0.958 |
Triglyceride (mmol/l) |
2.24 ± 1.57 |
2.08 ± 1.26 |
2.15 ± 1.17 |
0.113 |
0.893 |
LDL-C (mmol/l) |
2.51 ± 0.93 |
2.54 ± 0.85 |
2.48 ± 0.67 |
0.032 |
0.968 |
HDL-C (mmol/l) |
1.29 ± 0.44 |
1.22 ± 0.31 |
1.26 ± 0.24 |
0.325 |
0.723 |
AL of right eye (mm) |
23.39 ± 0.61 |
23.36 ± 0.48 |
23.35 ± 0.75 |
0.033 |
0.966 |
AL of left eye (mm) |
23.48 ± 0.60 |
23.42 ± 0.57 |
23.34 ± 0.58 |
0.359 |
0.699 |
IOP of right eye (mmHg) |
19.87 ± 3.96 |
18.44 ± 3.70 |
19.02 ± 3.51 |
1.203 |
0.305 |
IOP of left eye (mmHg) |
19.74 ± 3.68 |
18.99 ± 4.67 |
19.76 ± 4.05 |
0.335 |
0.715 |
Table 2. Comparisons of the characteristics of patients according to DR status.
Data represented the mean ± standard deviation (SD) of each group. For diabetes duration, * represents comparisons between groups 1 and 2. For HbA1c, * represents the comparisons of group 1 or 2 to group 3. ***
P < 0.001.
DR diabetic retinopathy,M /W man/woman,BMI body mass index,HbA1c glycated haemoglobin,HDL-C high-density lipoprotein cholesterol,LDL-C low-density lipoprotein cholesterol,RNFL retinal nerve fibre layer,ACR urinary albumin-to-creatinine ratio,AL axial length,IOP intraocular pressure.Statistically significant
p -values are in bold.
Dysregulation |
miRNA |
PRE-ACC |
miRNA expression (rpkm) |
Fold change |
P-valuee |
|
---|---|---|---|---|---|---|
NPDR |
NDR |
|||||
Up-regulated) |
hsa-miR-185-5p |
MI0000482 |
56,518.5 |
23,555 |
2.399 |
0.0071 |
Up-regulated |
hsa-miR-378a-3p |
MI0000786 |
6602 |
2820 |
2.341 |
0.0037 |
Down-regulated |
hsa-miR-199a-5p |
MI0000242 |
354.5 |
1151 |
0.308 |
0.0279 |
Down-regulated |
hsa-miR-128-3p |
MI0000447 |
405.5 |
1902.5 |
0.213 |
0.0068 |
Down-regulated |
hsa-let-7a-5p |
MI0000060 |
4430 |
7599.5 |
0.583 |
0.0361 |
Down-regulated |
hsa-miR-146b-5p |
MI0003129 |
1630 |
3919 |
0.416 |
0.0280 |
Down-regulated |
miR-26a-5p |
MI0000083 |
3569.5 |
18,071.5 |
0.198 |
0.0162 |
Down-regulated |
hsa-miR-126-3p |
MI0000471 |
13,511 |
57,936 |
0.233 |
0.0002 |
Table 3. Eight DR-related dysregulated miRNAs detected by miRNA sequencing.
PRE-ACC the pre-miRNA ACCESSION, NPDR non-proliferative diabetic retinopathy, NDR no diabetic retinopathy.
Eyes |
Group 1(>n = 33) |
>
Group 2(n = 33) |
Group 3 n = 20) |
F |
P |
---|---|---|---|---|---|
Right eye |
|||||
Averagep |
96.44 ± 10.36 |
97.98 ± 15.61 |
99.83 ± 12.88 |
0.417 |
0.6605 |
Superior quadrant |
117.24 ± 19.88
|
102.52 ± 21.24** |
128.77 ± 18.69 |
11.140 |
0.0001 |
Inferior quadrant |
128.54 ± 18.21 |
122.72 ± 25.64 |
131.42 ± 17.46 |
1.190 |
0.3094 |
Nasal quadrant |
75.33 ± 13.50 |
74.16 ± 17.44 |
76.38 ± 20.91 |
0.111 |
0.8954 |
Temporal quadrant |
69.56 ± 19.36 |
71.88 ± 25.73 |
73.22 ± 18.40 |
0.199 |
0.8244 |
Left eye |
|||||
Averagep |
99.57 ± 19.71 |
95.89 ± 17.46 |
103.88 ± 21.40 |
1.080 |
0.3443 |
Superior quadrant |
122.51 ± 12.48 |
109.63 ± 27.61* |
127.29 ± 28.39 |
4.336 |
0.0160 |
Inferior quadrant |
129.28 ± 25.18 |
131.73 ± 19.46 |
130.55 ± 17.20 |
0.108 |
0.8976 |
Nasal quadrant |
80.62 ± 22.41 |
75.79 ± 29.67 |
79.28 ± 22.05 |
0.313 |
0.7325 |
Temporal quadrant |
71.50 ± 12.69 |
68.66 ± 20.92 |
74.88 ± 16.33 |
0.834< |
0.4380 |
Table 4. Peripapillary RNFL thickness of the subjects.
Data represented mean ± standard deviation (SD) of each group; * represents comparisons of group 1 or 2 to group 3. # represents comparisons between group 1 and 2. * P < 0.05, ** P < 0.001,# P < 0.05. P < 0.05 was considered statistically significant.
RNFL retinal nerve fibre layer, DR diabetic retinopathy.
Statistically significant p-values are in bold.
Differential miRNAs |
Right eye |
Left eye |
||||||||
---|---|---|---|---|---|---|---|---|---|---|
Superior |
Inferior |
Temporal
|
Nasal |
Average
|
Superior |
Inferior |
Temporal
|
Nasal |
Average |
|
hsa-miR-185-5p |
0.038 |
−0.251 |
−0.233 |
−0.302 |
−0.208 |
0.011 |
0.251 |
0.302 |
0.237 |
−0.250 |
P value |
0.713 |
0.300 |
0.336 |
0.208 |
0.059 |
0.966 |
0.300 |
0.208 |
0.339 |
0.062 |
hsa-miR-378a-3p |
−0.233 |
−0.069 |
0.072 |
−0.324 |
0.190 |
−0.274 |
0.070 |
0.120 |
−0.103 |
0.009 |
P value |
0.054 |
0.571 |
0.491 |
0.162 |
0.097 |
0.121 |
0.522 |
0.447 |
0.256 |
0.85 |
hsa-miR-199a-5p |
0.049 |
0.069 |
0.051 |
−0.195 |
−0.163 |
0.480 |
−0.274 |
0.224 |
0.097 |
0.113 |
P value |
0.760 |
0.770 |
0.662 |
0.614 |
0.792 |
0.135 |
0.510 |
0.482 |
0.066 |
0.291 |
hsa-miR-128-3p |
0.178 |
−0.092 |
−0.113 |
0.019 |
0.274 |
0.075 |
−0.018 |
−0.196 |
0.019 |
0.120 |
P value |
0.008 |
0.391 |
0.291 |
0.853 |
0.121 |
0.481 |
0.868 |
0.067 |
0.866 |
0.219 |
hsa-let-7a-5p |
0.093 |
−0.075 |
−0.173 |
0.075 |
−0.138 |
0.048 |
0.289 |
0.076 |
0.105 |
0.020 |
P value |
0.383 |
0.485 |
0.110 |
0.490 |
0.195 |
0.651 |
0.865 |
0.479 |
0.323 |
0.846 |
hsa-miR-146b-5p |
0.072 |
0.104 |
0.004 |
−0.143 |
−0.131 |
0.099 |
0.010 |
0.188 |
0.015 |
0.102 |
P value |
0.502 |
0.349 |
0.966 |
0.201 |
0.243 |
0.472 |
0.901 |
0.103 |
0.904 |
0.212 |
miR-26a-5p |
0.616 |
−0.216 |
−0.088 |
0.150 |
−0.022 |
0.651 |
−0.121 |
−0.131 |
0.069 |
−0.004 |
P value |
*0.000 |
0.050 |
0.422 |
0.181 |
0.834 |
*0.000 |
0.205 |
0.243 |
0.517 |
0.967 |
hsa-miR-126-3p |
−0.010 |
−0.096 |
0.107 |
−0.048 |
−0.201 |
−0.207 |
−0.025 |
0.102 |
−0.092 |
−0.169 |
P value |
0.924 |
0.372 |
0.319 |
0.651 |
0.069 |
0.066 |
0.810 |
0.341 |
0.391 |
0.115 |
Table 5. Correlation analysis of RNFL thickness with differential miRNA expression.
Pearson correlation analysis was performed to analyse the association between RNFL thinning and differentially expressed miRNAs. * P < 0.05 was defined as statistically significant.RNFL retinal nerve fibre layer.
Statistically significant p-values are in bold.
Category |
Term |
P-value |
Gene counts |
---|---|---|---|
GO-CC |
Actin filament (GO:0005884) |
0.000533 |
6 |
Platelet dense granule membrane (GO:0031088) |
0.006565 |
2 |
|
RNA polymerase II transcription factor complex (GO:0090575) |
0.01909 |
7 |
|
Mitochondrial outer membrane (GO:0005741) |
0.019699 |
6 |
|
Actin cytoskeleton (GO:0015629) |
0.020151 |
11 |
|
Polymeric cytoskeletal fibre (GO:0099513) |
0.021144 |
9 |
|
Transcription factor TFIID complex (GO:0005669) |
0.027601 |
3 |
|
Cytoskeleton (GO:0005856) |
0.030491 |
16 |
|
Peroxisomal membrane (GO:0005778) |
0.038572 |
3 |
|
Nucleolus (GO:0005730) |
0.042895 |
19 |
|
GO-MF |
Protein serine/threonine kinase activity (GO:0004674) |
4.75E−06 |
21 |
Kinase activity (GO:0016301) |
1.76E−05 |
17 |
|
Protein kinase activity (GO:0004672) |
0.000209 |
22 |
|
Kinase binding (GO:0019900) |
0.000275 |
19 |
|
Protein kinase binding (GO:0019901) |
0.000848 |
20 |
|
Phosphotransferase activity, alcohol group as acceptor (GO:0016773) |
0.000868 |
13 |
|
Mannosyl-oligosaccharide mannosidase activity (GO:0015924) |
0.000892 |
3 |
|
Ubiquitin-like protein-specific protease activity (GO:0019783) |
0.002478 |
6 |
|
Phosphatidylinositol-4,5-bisphosphate binding (GO:0005546) |
0.005077 |
5 |
|
Signal sequence binding (GO:0005048) |
0.005941 |
4 |
Table 6. Top 10 predominant CC and MF terms in GO functional enrichment analysis.
GO gene ontology, CC cellular component, MF molecular function.
This activity is intended for ophthalmologists, endocrinologists, diabetologists, internists, and other clinicians caring for patients with diabetic retinopathy (DR).
The goal of this activity is to describe circulating microRNA (miRNA) targets and mechanisms underlying the pathogenic process of DR, according to a bioinformatic analysis of data from of 86 participants, including 33 patients with diabetes but without DR (group 1), 33 patients with diabetes with mild nonproliferative DR (group 2) and 20 healthy control participants (group 3).
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Diabetic retinopathy (DR) is a multifactorial progressive disease of the retina, which has been considered a leading cause of irreversible blindness in patients with diabetes mellitus [1]. The activation of the retinal glial cells, apoptotic cell death, neuroinflammation and gradual neurodegeneration induced by hyperglycaemic environment play important roles in the pathogenesis of DR [2]. Concomitantly with the development of diabetes, the metabolic disorder results in the abnormalities of the microvascular, breakdown of the blood–retinal barrier, impairments in neurovascular interactions, central vision loss, proliferative retinopathy, macular oedema and retinal layer thinning [[3,4]. These changes ultimately lead to progression into advanced DR. Many studies in recent years have paid attention to retinal neurodegeneration, which is the earliest pathology and may precede microvascular changes that are characteristic of DR[5-8]. It has been proven that the retinal nerve fibre layer (RNFL) thinning is a manifestation of diabetic optic neuropathy [9-11]. However, the potential molecular mechanisms underlying these pathologies remain elusive. Besides, although current therapies, such as laser photocoagulation and anti-vascular endothelial growth factor (VEGF) injections, are available for late stages of DR with proliferation and macular oedema, effective strategies still lacked to prevent or reduce vision loss in the fture [12]. Therefore, it is imperative to identify novel biomarkers and therapeutic targets to improve the therapeutic effects of early DR.
MicroRNAs (miRNAs) are small non-coding RNA molecules that orchestrate biological networks via modulation of protein expression by interfering with the translation and stability of mRNA. Some miRNAs are released into the circulation and can exist stably in plasma, urine, and other body fluids [13]. The availability of quantitative detection of circulating miRNAs enhances the use of miRNAs as biomarkers for physiological and pathological processes [14,15].Numerous clinical and experimental studies have demonstrated the important roles of miRNAs in the development of diabetes and its complications [16]. Additionally, accumulating evidence has indicated that various circulating miRNAs can serve as potential biomarkers or therapeutic targets for DR [17,18].However, the relationship between plasma miRNA and diabetic neurodegeneration remains unclear. We speculate that circulating miRNAs may play an important role in the pathogenesis of retinal neurodegeneration in patients with or without DR.
In the present study, we screened the plasma miRNAs in type 2 diabetic patients with or without DR and investigated the neurodegeneration-related miRNAs for correlation analysis. Then we explored the targets of miRNA and elucidated the underlying mechanism in mediating the pathogenic process of DR by bioinformatic analysis.