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Table 1.  

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.

Table 2.  

 

Group 1

 

(NDR)

Group 2

 

(mild NPDR)

Group 3

 

(healthy controls)

F

 

P

 

Participants ( n )

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 2)

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.

Table 3.  

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.

Table 4.  

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.

Table 5.  

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.

Table 6.  

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.

CME / ABIM MOC

Plasma miR-26a-5p Is a Biomarker for Retinal Neurodegeneration of Early Diabetic Retinopathy

  • Authors: Rui Shi, MD; Li Chen, MD; Weirong Wang, MD; Ying Deng, MD; YiZhen Liu, MD; Haiyan Zhou, MD; Rong Lin, MD, PhD
  • CME / ABIM MOC Released: 4/30/2021
  • THIS ACTIVITY HAS EXPIRED FOR CREDIT
  • Valid for credit through: 4/30/2022, 11:59 PM EST
Start Activity


Target Audience and Goal Statement

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).

Upon completion of this activity, participants will:

  • Describe plasma microRNAs (miRNAs) validated with qualitative reverse-transcriptase polymerase chain reaction in patients with type 2 diabetes with or without diabetic retinopathy (DR), and their correlation with neurodegeneration, according to retinal nerve fiber layer (RNFL) thickness measured using spectral-domain optical coherence tomography
  • Determine gene targets of miRNA and mechanisms underlying the pathogenic process of DR, according to bioinformatic analysis used to predict potential targets of miRNA associated with RNFL thickness and to investigate the functions of the potential target genes
  • Identify clinical implications of bioinformatic analysis of miRNAs in DR and their gene targets


Disclosures

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Medscape, LLC, encourages Authors to identify investigational products or off-label uses of products regulated by the US Food and Drug Administration, at first mention and where appropriate in the content.


Faculty

  • Rui Shi, MD

    Department of Pharmacology
    School of Basic Medical Sciences
    Xi'an Jiaotong University Health Science Center
    Department of Ophthalmology
    Shaanxi Provincial People’s Hospital
    Xi’an, Shaanxi, China

    Disclosures

    Disclosure: Rui Shi, MD, disclosed has no relevant financial relationships.

  • Li Chen, MD

    Department of Neurology
    Shaanxi Provincial People’s Hospital
    Xi’an, Shaanxi, China

    Disclosures

    Disclosure: Li Chen, MD, disclosed has no relevant financial relationships.

  • Weirong Wang, MD

    Department of Medical Laboratory Animal Science
    School of Basic Medical Sciences
    Xi’an Jiaotong University Health Science Center
    Xi’an, Shaanxi, China

    Disclosures

    Disclosure: Weirong Wang, MD, disclosed has no relevant financial relationships.

  • Ying Deng, MD

    Department of Pharmacology
    School of Basic Medical Sciences
    Xi'an Jiaotong University Health Science Center
    Xi’an, Shaanxi, China

    Disclosures

    Disclosure: Ying Deng, MD, disclosed has no relevant financial relationships.

  • YiZhen Liu, MD

    Department of Pharmacology
    School of Basic Medical Sciences
    Xi'an Jiaotong University Health Science Center
    Xi’an, Shaanxi, China

    Disclosures

    Disclosure: YiZhen Liu, MD, disclosed has no relevant financial relationships.

  • Haiyan Zhou, MD

    Department of Ophthalmology
    Shaanxi Provincial People’s Hospital
    Xi’an, Shaanxi, China

    Disclosures

    Disclosure: Haiyan Zhou, MD, disclosed has no relevant financial relationships.

  • Rong Lin, MD, PhD

    Department of Pharmacology
    School of Basic Medical Sciences
    Xi'an Jiaotong University Health Science Center
    Xi'an, Shaanxi, China

    Disclosures

    Disclosure: Rong Lin, MD, PhD, disclosed has no relevant financial relationships.

CME Author

  • Laurie Barclay, MD

    Freelance writer and reviewer
    Medscape, LLC

    Disclosures

    Disclosure: Laurie Barclay, MD, has disclosed no relevant financial relationships.

Editor

  • Sobha Sivaprasad, MD

    Editor, Eye

    Disclosures

    Disclosure: Sobha Sivaprasad, MD, has disclosed the following relevant financial relationships:
    Served as an advisor or consultant for: Allergan, Inc.; Apellis; Bayer AG; Boehringer Ingelheim Pharmaceuticals, Inc.; Heidelberg Pharma GmbH; Novartis Pharmaceuticals Corporation; Oculis; Optos; Oxurion; Roche
    Served as a speaker or a member of a speakers bureau for: Allergan, Inc.; Bayer AG; Novartis Pharmaceuticals Corporation; Optos
    Received grants for clinical research from: Allergan, Inc.; Bayer AG; Boehringer Ingelheim Pharmaceuticals, Inc.; Novartis Pharmaceuticals Corporation; Optos

CME Reviewer

  • Stephanie Corder, ND, RN, CHCP

    Associate Director, Accreditation and Compliance Medscape, LLC

    Disclosures

    Disclosure: Stephanie Corder, ND, RN, CHCP, has disclosed no relevant financial relationships.

CE Reviewer

  • Hazel Dennison, DNP, RN, FNP-BC, CHCP, CPHQ, CNE

    Associate Director, Accreditation and Compliance
    Medscape, LLC

    Disclosures

    Disclosure: Hazel Dennison, DNP, RN, FNP-BC, CHCP, CPHQ, CNE, has disclosed no relevant financial relationships.

Medscape, LLC staff have disclosed that they have no relevant financial relationships.


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From Eye
CME / ABIM MOC

Plasma miR-26a-5p Is a Biomarker for Retinal Neurodegeneration of Early Diabetic Retinopathy

Authors: Rui Shi, MD; Li Chen, MD; Weirong Wang, MD; Ying Deng, MD; YiZhen Liu, MD; Haiyan Zhou, MD; Rong Lin, MD, PhDFaculty and Disclosures
THIS ACTIVITY HAS EXPIRED FOR CREDIT

CME / ABIM MOC Released: 4/30/2021

Valid for credit through: 4/30/2022, 11:59 PM EST

processing....

Abstract and Introduction

Abstract

Purpose  Retinal neurodegeneration is an early pathological change in diabetic retinopathy (DR). Early-stage retinal neurodegeneration is usually asymptomatic. This study aims to identify circulating microRNAs (miRNAs) as sensitive biomarkers for early retinal neurodegeneration.
Methods We profiled the plasma miRNA expression in three mild nonproliferative diabetic retinopathy (NPDR) cases and three matched non-DR patients using RNA sequencing. The differential miRNAs were validated with qRT-PCR. The retinal nerve fibre layer (RNFL) thickness of the eyes was measured using spectral-domain Optical coherence tomography (SD-OCT). The association between differential miRNAs and RNFL thickness was analysed using the Pearson correlation analysis. Bioinformatics tools were used to predict potential targets of miRNA associated with RNFL thickness and investigate the functions of the potential target genes.
Results RNA sequencing identified 69 differential miRNAs and eight of them were reported to be associated with DR. The qRT-PCR for these eight miRNAs validated the down-regulation of circulating miR-26a-5p and miR-126-5p in a larger validating cohort. A positive correlation between plasma miR-26a-5p level and the RNFL thickness of the superior quadrant of both eyes was identified in another cohort, including 33 mild NPDR cases, 33 matched non-DR patients and 20 healthy controls. Furthermore, 367 candidate targets of miR-26a-5p were predicted. The functional studies revealed that these target genes are profoundly involved in various cellular functions and signalling pathways.
Conclusions Circulating miR-26a-5p is a potential biomarker for early-stage retinal neurodegeneration and it may be involved in the development of DR via profoundly influencing the functions of retinal cells.

Introduction

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.