Upregulation of miR-195 accelerates oxidative stress-induced retinal endothelial cell injury by targeting mitofusin 2 in diabetic rats

Accepted Manuscript Upregulation of miR-195 accelerates oxidative stress-induced retinal endothelial cell injury by targeting mitofusin 2 in diabetic rats Rui Zhang, Qian Garrett, Huimin Zhou, Xiaoxi Wu, Yueran Mao, Ximing Cui, Bing Xie, Zanchao Liu, Dongsheng Cui, Lei Jiang, Qingfu Zhang, Shunjiang Xu PII:

S0303-7207(17)30257-5

DOI:

10.1016/j.mce.2017.05.009

Reference:

MCE 9940

To appear in:

Molecular and Cellular Endocrinology

Received Date: 28 October 2016 Revised Date:

24 March 2017

Accepted Date: 5 May 2017

Please cite this article as: Zhang, R., Garrett, Q., Zhou, H., Wu, X., Mao, Y., Cui, X., Xie, B., Liu, Z., Cui, D., Jiang, L., Zhang, Q., Xu, S., Upregulation of miR-195 accelerates oxidative stress-induced retinal endothelial cell injury by targeting mitofusin 2 in diabetic rats, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.05.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Upregulation of miR-195 accelerates oxidative stress-induced retinal

2

endothelial cell injury by targeting mitofusin 2 in diabetic rats

3 a, e *

4

Rui Zhang

, Qian Garrett

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Ximing Cui a, Bing Xie

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Zhang e, Shunjiang Xu a, e **

b, c *

, Huimin Zhou

d, e **

, Xiaoxi Wu d, Yueran Mao d,

, Zanchao Liu f, Dongsheng Cui a, Lei Jiang a, Qingfu

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a, e

7 a

Central Laboratory, The First Hospital of Hebei Medical University, Shijiazhuang, P.

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R. China.

9 b

The University of New South Wales, Sydney, NSW 2052, Australia.

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c

The University of Notre Dame Australia, NSW 2008, Australia

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d

Department of Endocrinology, The First Hospital of Hebei Medical University, Shijiazhuang, P. R. China.

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f

Burn Engineering Center of Hebei Province, Shijiazhuang, P. R. China. Department of Endocrinology, The Second Hospital of Shijiazhuang City,

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Shijiazhuang, P. R. China.

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* These authors contributed equally to this work

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**

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Central Laboratory, The First Hospital of Hebei Medical University, No.89, Donggang

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Road, Shijiazhuang, 050031 China.

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Tel: +86 311 8591 7257; Fax: +86 311 8591 7290

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E-mail: [email protected]

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Correspondence to Shunjiang Xu and Huimin Zhou, PhD

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Abstract

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This study was performed to investigate the oxidative stress-induced miRNA

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changes in relation to pathogenesis of diabetic retinopathy (DR) and to establish a

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functional link between miRNAs and oxidative stress-induced retinal endothelial cell

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injury. Our results demonstrated that oxidative stress could induce alterations of

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miRNA expression profile, including up-regulation of miR-195 in the diabetic retina

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or cultured HMRECs after exposed to H2O2 or HG (P < 0.05). Oxidative stress also

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resulted in a significant reduction of MFN2 expression in diabetic retina or HMRECs

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(P < 0.05). Overexpression of miR-195 reduced MFN2 protein levels, and induced

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tube formation and increased permeability of diabetic retinal vasculature. The

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luciferase reportor assay confirmed that miR-195 binds to the 3′ -untranslated region

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(3′-UTR) of MFN2 mRNA. This study suggested that miR-195 played a critical role

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in oxidative stress-induced retinal endothelial cell injury by targeting MFN2 in

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diabetic rats.

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Keywords: diabetic retinopathy, oxidative stress, endothelial cell, miR-195, mitofusin

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2 (MFN2)

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Abbreviations

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ATCC

American type culture collection

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BRB

Blood retinal barrier

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DAVID

Database for Annotation, Visualization and Interrogated Discovery)

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DM

Diabetes mellitus

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DME

Diabetic macular edema

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DR

Diabetic retinopathy

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ECs

Endothelial cells

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GO

Gene Ontology

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HG

High glucose

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2

ACCEPTED MANUSCRIPT HMREC

Human retinal endothelial cell

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HUVEC

Human umbilical vein endothelial cell

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KEGG

Kyoto Encyclopedia of Genes and Genomes

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MFN2

mitofusin 2

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miRNA

MicroRNA

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miR-195A miR-195 antagomir

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NG

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OD

Optical density

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OSM

Osmotic control

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PKC

Protein kinase C

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siRNA

Small interfering RNA

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STZ

Streptozotocin

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3'-UTR

3′-untranslated region

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VEGF

Vascular endothelial growth factor retinal

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1. Introduction

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Normal glucose

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Diabetic retinopathy (DR) is the leading cause of visual impairment and blindness

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in diabetes mellitus (DM). It is characterized by gradual progressive alterations in the

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retinal microvasculature, including neovascularization, breakdown of blood retinal

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barrier (BRB), capillary dropout, and diabetic macular edema (DME). Multiple

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interconnecting molecular mechanisms have been proposed to explain the

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pathogenesis of diabetes induced complications, including DR. These mechanisms are

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as follows: inflammation, polyol pathway flux, accumulation of advanced glycation

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end products (AGEs), increased hexosamine pathway flux, and activation of protein

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kinase C (PKC) pathway. Although the precise mechanisms underlying pathogenesis

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of DR are not fully understood, a common feature to these pathways is increased

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status of oxidative stress in the body (Brownlee, 2005). In comparison to other tissues,

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retina is particularly susceptible to oxidative stress because of its high content of

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polyunsaturated fatty acids, high consumption of oxygen, and exposure to light

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(Brownlee, 2005; Kumari et al., 2008).

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Vascular endothelial cells (ECs), as the components of blood retinal barrier, are

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the major targets for oxidative stress. The injury of ECs induced by oxidative stress

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plays critical roles in several vascular disorders such as DR (Wu et al., 2016), and it is

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characterized

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endothelium-dependent control of vascular tone (Adachi et al., 2012; Zheng et al.,

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2010). However, the molecular mechanisms underlying oxidative stress signaling

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events which lead to impairment of endothelial barrier function remain unclear.

increased

endothelial

permeability

and

deregulation

of

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miRNAs are single-stranded non-coding small RNAs of about 20-22 nucleotides

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in length that have elicited immense interest in recent years. They can repress the

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translation or induce degradation of target mRNAs, ultimately resulting in gene

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silencing (Ambros, 2004; Bartel, 2004; Shukla et al., 2011). Several studies have

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demonstrated an relationship between miRNAs and the pathogenesis of DR (Kovacs

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et al., 2011; McArthur et al., 2011; Mortuza et al., 2014; Wu et al., 2012). Kovacs et al

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(Kovacs et al., 2011) have found that the p53- and vascular endothelial growth factor

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(VEGF)-responsive miRNAs were upregulated in the diabetic rat retinas and retinal

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endothelial cells (RECs), whereas the expression of NF-κB-responsive miRNAs

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(including miR-146, miR-155, miR-132 and miR-21) were only increased in the

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diabetic rat RECs. Other studies have observed the abnormal expression of miRNA in

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the retina of the streptozotocin (STZ)-induced diabetic rats (McArthur et al., 2011;

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Wu et al., 2012), further demonstrating a link between miRNA deregulation and the

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pathogenesis of DR.

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The aim of this study was to investigate the oxidative stress-induced miRNA

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alterations in relation to pathogenesis of DR and to establish a functional link between

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miRNAs and oxidative stress/diabetes-induced retinal endothelial injury. Using a

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miRNA expression profiling assay followed by quantitative real-time PCR validation,

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we detected a significant increase in the level of miR-195 in the retina of the

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STZ-induced diabetic rats or the cultured retinal endothelial cells after exposed to the

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H2O2-induced oxidative stress. We identified MFN2, a multifunctional mitochondrial

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membrane

protein

known

to

be

associated

with

oxidative

stress

and 4

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diabetes-associated complications (Tang et al., 2012; Zhong and Kowluru, 2011), as a

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direct target gene of miR-195. We further demonstrated that the oxidative

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stress-induced upregulation of miR-195 in retina mediated the MFN2 protein

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reduction, resulting in retinal endothelial cell injury in diabetic rats.

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2.1. Cell culture

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Human umbilical vein endothelial cells (HUVECs) and Endothelial Cell Medium

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(containing 5% FBS and Endothelial Cell Growth Supplement) were purchased from

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Sciencell Research Laboratories (Carlsbad, CA). Human retinal endothelial cells

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(HMRECs) were obtained from Wuhan PriCells Biomedical Technology Co., Ltd.

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(Wuhan, China). The 293A cell line was purchased from American type culture

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collection (ATCC). Cells were cultured and maintained in a humidified atmosphere of

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95% air and 5% CO2. Medium was changed every 48 hours when the cells were grown

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to 80 to 90% confluence.

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2.2. Oxidative stress and cell viability

HUVECs were used for development of an oxidative stress cell model to determine

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the effect of oxidative stress on cell viability after the cells were exposed to H2O2.

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HUVECs were cultured in a 96-well plate and exposed to different concentrations of

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H2O2 (Sigma-Aldrich, St. Louis, MO) in culture medium (100, 200, 400 or 800

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µmol/L) for 8, 16, or 24 h. The control group was treated with medium only. Cell

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viability was measured using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories,

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Japan) according to the manufacturer's instructions. Briefly, 10 µL of CCK-8 reagent

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was added to each well, and the plate was incubated at 37°C for 2 h. Optical density

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(OD) values were assessed at 450 nm with the GloMax®-Multi+ Detection System

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(Promega). Cell viability was expressed as a percentage of the control at the same time

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point. Samples were prepared in triplicate and the experiments were repeated three

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times. Exposure of cells to 400 µmol/L of H2O2, high glucose (25 mmol/L D-glucose,

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HG), normal glucose (5 mmol/l D-glucose, NG) or with osmotic control (25 mmol/l

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L-glucose,

OSM) for 24 h were used for investigating the oxidative stress-induced

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changes in miRNA expression profile and the protein levels of target genes related to

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DR.

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2.3. Microarray analysis of miRNA expression profile

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Total RNAs were extracted and purified from oxidative stressed HUVECs (n = 3)

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using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the

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manufacturer’s instructions. RNA (100 ng) were individually processed for miRNA

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expression profiling on a GeneChip miRNA Array (LC Science, Houston, TX) by

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LC-Biotech (Hangzhou, China).

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2.4. Bioinformatics analysis and prediction of miRNA target genes

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To identify the biological processes most relevant to the deregulated miRNAs in

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response to H2O2 exposure, an enrichment analysis was performed on predicted target

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genes. DAIAN Tools were used to generate a list of target genes. The list of the targets

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were sent to the bioinformatics database, DAVID (Database for Annotation,

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Visualization and Interrogated Discovery), to extract biological features associated

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with the miRNA target gene lists (Huang da et al., 2009). The software was used to

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perform an enrichment analysis of miRNA target genes with all known Gene Ontology

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(GO) (http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes

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(KEGG) (http://www.genome.jp/kegg/) pathways. The potential mRNA target sites

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within the conserved regions in the 3′ -untranslated region (3′-UTR) sequences were

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predicted using miRBase (www.mirbase.org/), TargetScan (www.targetscan.org/),

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miRanda (www.microrna.org/microrna/home.do) PicTar (http://pictar.mdc-berlin.de/),

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and miRecords ( http://mirecords.umn.edu/miRecords) .

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2.5. miRNA extraction and quantitative analysis

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miRNAs were extracted from the H2O2 treated HUVECs or HMRECs, or from

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frozen retinal tissues of diabetic rats using a miRcute miRNA Isolation Kit (Tiangen,

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Beijing, China). Reverse transcription was performed using the miRcute miRNA

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First-Strand cDNA Synthesis Kit and miRNA RT primer (Ribo Bio, Guangzhou, 6

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China). Real-time qPCR was performed using miRcute miRNA qPCR Detection kit

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(SYBR Green) (Tiangen, Beijing, China) with the ABI 7500 sequence detection system

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(Applied Biosystems, USA), and the data were normalized to U6 snRNA expression

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levels.

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2.6. mRNA extraction and quantitative analysis

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Total RNAs were extracted from the oxidative stressed HMRECs, or frozen retinal

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tissues of diabetic rats using TRIZOL Reagents (Invitrogen, Burlington, ON, Canada)

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following the manufacturer's instructions. Reverse transcription was performed using

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the FastQuant RT Kit (with gDNase) (Tiangen, Beijing, China). Real-time qPCR was

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performed with SuperReal PreMix Plus (SYBR Green) (Tiangen, Beijing, China)

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using the ABI 7500 sequence detection system (Applied Biosystems, USA), and the

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data were normalized to the housekeeping gene GAPDH.

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2.7. Protein extraction and western blot analysis

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Total proteins from the oxidative stressed HMRECs, or frozen retinal tissues of

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diabetic rats were extracted with protein lysis buffer (150 mM NaCl, 1% NP-40 and 50

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mM Tris-HCl, pH 8.0) supplemented with protease inhibitor cocktail (2 µg/mL

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phenylmethanesulfonyl fluoride, 2 µg/mL pepstain, 2 µg/mL aprotinin, and 2 µg/mL

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leupeptin) and separated by 10 % sodium dodecyl sulfate polyacrylamide gel

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electrophoresis (SDS-PAGE), and then transferred onto PVDF membrane. To identify

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the expression of MFN2 protein, the proteins on the PVDF membranes were detected

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by the rabbit anti-MFN2 (Cat. ab124773, abcam; diluted 1: 5000 in PBS) using a mouse

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monoclonal β-actin antibody (Cat. 60008-1-1g, proteintech; diluted 1:10000 in PBS) as

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a loading control. The membranes were then incubated for 1 h at room temperature with

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goat anti-rabbit (Cat. A23920, Abbkine), or goat anti-mouse (Cat. A23710, Abbkine).

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Membranes were scanned and analyzed using the LI-COR Odyssey® scanner and

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software (LI-COR Biosciences).

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2.8. Plasmids, siRNAs, and MFN2 expression vector

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(WT-MFN2 or MT-MFN2 vector) were purchased from NorClone Biotech (Shanghai,

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China). Scrambled controls, miR-195 mimics, miR-195 inhibitor, and miR-195

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antagomir were obtained from Ribo Bio (Guangzhou, China). MFN2 small interfering

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RNA (siRNA-MFN2) was a commodity (sc-43928, Santa Cruz). The MFN2 eukaryotic

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expression plasmid (pcDNA-MFN2) was a kind gift from Dr. Song GY (Department of

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Internal Medicine, Hebei General Hospital). HMRECs were transfected with miR-195

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mimics (100 nmol/L) or miR-195 mimics plus pcDNA-MFN2 (0.5 µg) using 0.4 µL

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lipofectamine2000 (Invitrogen, Burlington, ON, Canada).

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2.9. Luciferase reporter assay for targeting MFN2 3′-UTR

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Plasmid DNA (WT-MFN2 or MT-MFN2 vector) 150 ng and miR-195 mimics

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(100 nmol/L), miR-195 inhibitor (100 nmol/L), or scrambled controls (100 nmol/L)

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were co-transfected into 293A cells for 24 h. Luciferase activities were measured

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using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA).

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The experiments were performed in triplicate.

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2.10. Diabetic rat model

All animal experimental procedures were approved by the Animal Care and Use

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Committee of Hebei Medical University. Male Sprague-Dawley rats, weighing 300 g,

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were randomly divided into control and diabetic groups. Diabetes was induced by a

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single intraperitoneal injection of streptozotocin (STZ) (65 mg/kg, in citrate buffer, pH

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5.6). Rats treated with the same volume of citrate buffer were used as non-diabetic

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controls. Blood glucose levels were measured immediately before STZ injection, and

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daily for the first 3 days, then 4, 8, 12, 16, and 20 weeks after STZ injection. The rats

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were considered diabetic and used for the study if they had a blood glucose level of

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>20 mmol/L on three successive days after STZ injection.

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2.11. Retinal trypsin digestion

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Animals were sacrificed at 20 weeks post STZ injection (n = 6/group) and rat

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retinas were isolated and incubated at 37 °C in a solution of 3 % trypsin 8

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After incubation, the internal limiting membranes began to separate from the retinas.

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The retinas were transferred to PBS (pH 7.4) at room temperature. The retinal vessels

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were washed in distilled water under microscope to remove remaining neural tissues.

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The isolated retinal tissues were snap frozen and stored at −80°C for further gene

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expression and miRNA analysis (n = 6) or freshly prepared in 10 % formalin for

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embedding in paraffin for the morphology examinations (n = 6). The retina tissues were

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stained with haematoxylin and Periodic Acid Schiff (PAS) to evaluate microvascular

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lesions. Endothelial cell nuclei were large and ellipsoid, whereas pericytes nuclei were

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smaller and darker situated on the outer side of vessel wall. Pictures were taken with a

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fluorescence microscope (Nikon, Tokyo, Japan) and the histopathological changes of

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retinal vasculature were analyzed using a digital imaging system (Chou et al., 2013).

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Capillary cells were counted in the mid retinal area of each retina following PAS

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staining. Ten fields (1 mm2) were viewed under the microscope and digitally

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photographed for each of the normal and diabetic retinas. Endothelial cells with long

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flat nucleus located in the interior surface of blood vessel. Peripheral cells with circular

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nucleus located in the outside surface of blood vessel.

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2.12. Angiogenesis assay

An in vitro Angiogenesis Assay Kit (Millipore Filter Corporation, Bedford, MA)

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was used to evaluate tube formation of HMRECs. HMRECs were transfected with

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miR-195 mimics (100 nmol/L), scrambled controls (100 nmol/L), or the combination

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of miR-195 mimics (100 nmol/L) and pcDNA-MFN2 (0.5 µg) for 24 h, then the tube

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formation of HMRECs was analyzed according to the manufacturer’s instructions.

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Wells were photographed under a Nikon microscope (Nikon, Tokyo, Japan). Branch

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points in 5 random view-fields per well were independently counted by two

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independent observers in a blinded manner. The total number of branch points in the 5

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photographic fields of each plate was considered indicative of the complexity of the

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capillary network formed.

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2.13. Intravitreal injection

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rats were divided into 5 groups (6 animals/group), (1) non-diabetic group, normal

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control rats; (2) diabetic group, diabetic rat eyes with lipofectin injection; (3) scramble

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group, diabetic rat eyes with 1.5 µg of scrambled miRNA injection; (4) miR-195

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group, diabetic rat eyes with 1.5 µg of miR-195 antagomir injection; and (5)

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siRNA-MFN2 + miR-195 group, diabetic rat eyes with 1.5 µg of miR-195 antagomir

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and siRNA-MFN2 (100 nmol/L) injection. Animals received weekly intravitreal

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injections at 16 weeks post STZ intraperitoneal injection using the Lipofectin Reagent

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(Life Technologies, Carlsbad, CA) in the right eye for 4 weeks. All rats were

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anesthetized with intraperitoneal ketamine (60 mg/kg) and xylazine hydrochloride (4

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mg/kg) intramuscularly and treated with 0.5% tropicamide and 2.5% phenylephrine

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hydrochloride to dilate the pupils before the injection. The rats were sacrificed 1 week

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after the last injection, and the retinal tissues were collected for immunohistochemistry

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or in liquid nitrogen for miRNA quantitative analysis.

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2.14. Immunohistochemistry

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The animal retina tissue sections were immunohistochemically stained for albumin

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using primary anti-albumin antibody (1:100; Santa Cruz, USA) and secondary goat

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anti-rabbit Ig G (DBA, Milan, Italy). These methods have been previously described

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(McArthur et al., 2011).

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2.15. Statistical analysis

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Statistical analyses were performed by SPSS software version 16.0 (SPSS, Inc.,

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Chicago, IL, USA). Data are expressed as means ± SD of at least three independent

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experiments. Comparisons were made using the ANOVA analysis for three parametric

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groups. Student’s t test was used for two parametric groups. Differences were

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considered statistically significant at P < 0.05.

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3. Results

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3.1. H2O2 inhibited the growth of HUVECs 10

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Incubation of HUVECs with H2O2 at different concentrations for 8, 16 or 24 h

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resulted in a significant decrease in cell viability (P<0.05 compared with the

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respective control groups) except for treating cells with 100 µmol/L H2O2 for 8 and 16

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h (Fig. 1). The effects of H2O2-induced oxidative stress on the cell viability appeared

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to be dose- and time- dependent. After incubation of the cells with 400 µmol/L H2O2

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for 24 h, the cell viability dropped to 50% of that in control group (n = 3) (P< 0.05,

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Fig.1). Therefore, exposure of cells to 400 µmol/L H2O2 for 24 h was used in the

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subsequent experiments to induce oxidative stress in HUVCEs or HMRECs.

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Fig. 1. The viability of HUVECs in response to the H2O2-induced oxidative stress. Cells were

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exposed to different concentrations of H2O2 (100, 200, 400, and 800 µmol/L) for 8, 16, or 24 h (n

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= 3). * shows the significant difference compared with the respective control groups (P < 0.05).

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3.2. Expression and bioinformatics analysis of miRNAs induced by oxidative stress

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To investigate whether miRNAs modulate ECs response to oxidative stress and play

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roles in the pathogenesis of DR, the miRNA expression profile in HUVECs were

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identified by microarray analysis. There were 116 deregulated miRNAs in the

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HUVECs after exposure to H2O2-induced oxidative stress. Among them, 47 miRNAs

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were up-regulated and 69 miRNAs were down-regulated. Of the 116 deregulated

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miRNAs, 11 miRNAs (miR-15b, miR-17, miR-106b, miR-195, miR-497, miR-638,

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miR-1246, miR-1275, miR-4267, miR-4324, and miR-4734) changed above 1.5-fold

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(P < 0.05). The results of pathway enrichment analysis provided by KEGG showed that 11

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mellitus, apoptosis, MAPK signaling pathway and p53 signaling pathway. The results

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of GO enrichment revealed that the predicted target genes of deregulated miRNAs

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might be related to microtubule binding, glucose homeostasis, blood vessel

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remodeling, blood vessel development, cellular response to oxidative stress, and

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angiogenesis insulin secretion. Based on the results of bioinformatics analysis, the

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selected differentially expressed miRNAs were further verified by quantitative

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real-time RT-PCR in oxidative stressed HUVECs. As shown in Fig. 2A, miR-15b,

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miR-106b, and miR-497 were significantly downregulated (P < 0.05) whereas

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miR-195, miR-638, miR-1246, miR-4267, miR-4324, and miR-4734 were significantly

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upregulated (P < 0.05), and no differences were found in the levels of miR-17 or

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miR-1275 expression between the control and the H2O2-treated HUVECs (P > 0.05). In

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order to evaluate the roles of miRNAs in oxidative stress-induced retinal endothelial

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cell injury, the above differentially expressed miRNAs were also verified in the

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H2O2-treated HMRECs. As a result, a similar trend in the miRNA expression pattern

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was observed in the H2O2-treated HMRECs except for the up-regulation of miR-17

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expression and no effect was found on the miR-4324 expression (Fig. 2B).

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Fig. 2. Expression of selected miRNAs in endothelial cells in response to oxidative stress. The

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relative changes were detected by qRT-PCR in expression of the selected miRNAs (miR-15b,

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miR-17, miR-106b, miR-195, miR-497, miR-638, miR-1246, miR-1275, miR-4267, miR-4324, 12

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and miR-4734) in HUVECs (A) or HMRECs (B) after the cells were exposed to 400 µmol/L H2O2

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for 24 h (n = 3). *P < 0.05 vs control.

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3.3. Expression of selected miRNAs in diabetic rat retina

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The diabetic SD rat models were developed by injection of 65 mg/kg STZ. The

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body weight and blood glucose levels of diabetic and age-matched control rats are

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summarized in Table 1. All diabetic rats showed significant increases in blood glucose

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and significant decreases in body weight compared with age matched non-diabetic

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controls. In addition, decrease of pericytes was found in retinas of diabetic rats (Fig.

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3A). Further, we selected five differentially expressed miRNAs (miR-15b, miR-17,

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miR-106b, miR-195 and miR-497), which sequence is the same between human and

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rat species, to validate their expression patterns in the rat retinal tissues. The results of

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qRT-PCR revealed that miR-195 was significantly up-regulated in diabetic retinas

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with close to 4-fold changes, meanwhile, miR-15b, miR-106b, and miR-497 were

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decreased in diabetic retinas and no significant difference was found in the expression

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of miR-17 between diabetic and non-diabetic rat groups (Fig. 3B). So we focus on the

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roles of miR-195 and further detected the dynamic alterations in its expression during

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the development of DM induced by STZ and the results of qRT-PCR showed that the

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miR-195 levels were gradually increased in diabetic retinas post STZ injection (Fig.

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3C). As glucose could induce oxidative stress in the process of diabetes mellitus,

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therefore, we further confirmed that miR-195 levels were increased in HMRECs after

402

incubation cells with HG (25 mmol/L D-glucose) for 24 h when compared to NG.

403

However, miR-195 levels were unchanged after incubation cells with OSM (25

404

mmol/L L-glucose) (Fig. 3D).

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405 406

Table 1. General physiological parameters (body weight and glucose levels) in the

407

STZ-induced diabetic and non-diabetic rats. *P < 0.05 vs the non-diabetic controls.

408

13

ACCEPTED MANUSCRIPT non-diabetic rats

diabetic rats

(n = 10)

(n = 11)

body weight (g)

314.65 ± 17.87

297.15 ± 84.32*

blood glucose (mmol/L)

6.98 ± 0.40

27.48 ± 2.77*

body weight (g)

388.28 ± 21.40

266.65 ± 72.04*

blood glucose (mmol/L)

6.93 ± 0.45

23.18 ± 6.80*

body weight (g)

431.56 ± 20.13

221.87 ± 49.68*

blood glucose (mmol/L)

6.92 ± 0.55

24.88 ± 5.12*

4 wk post STZ injection

16 wk post STZ injection body weight (g)

462.52 ± 22.49

blood glucose (mmol/L)

6.94 ± 0.54

20 wk post STZ injection

blood glucose (mmol/L)

409 410 411

415 416 417 418 419 420 421

176.87 ± 53.25*

6.90 ± 0.48

26.11 ± 6.43*

EP

414

486.17 ± 28.44

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413

26.85 ± 5.99*

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412

196.74 ± 51.24*

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body weight (g)

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12 wk post STZ injection

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8 wk post STZ injection

422

Fig. 3. Expression of selected miRNAs in diabetic and non-diabetic rat retinas. (A) Representative

423

images of PAS staining of the retinal vascular. The endothelial cells and pericytes have been

424

indicated in the images by arrow heads and arrows, respectively. (B) Detection of the selected

425

miRNA expression by qRT-PCR in the retinas of diabetic and non-diabetic rats. *P < 0.05 vs 14

ACCEPTED MANUSCRIPT 426

non-diabetic rat group (n = 6). (C) Dynamic alterations of miR-195 in the diabetic rat retinas at 4-,

427

8-, 12, 16, and 20-week post STZ injection. *P < 0.05 vs non-diabetic rat group (n = 6). (D)

428

Expression levels of miR-195 in HMRECs after incubation cells with NG, HG or OSM. *P < 0.05

429

vs NG group (n = 3). NG, normal glucose; OSM: osmotic control.

431

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3.4. Expression of MFN2 in response to H2O2-induced oxidative stress

432

The putative targets of the selected miRNAs were predicted using TargetScan,

434

miRBase, miRanda, and PicTar software. Among those computational predicted

435

targets, MFN2 gene was selected for further validation due to its higher predictive

436

scores and its implications in insulin resistance, mitochondrial damages and diabetes

437

mellitus (Gao et al., 2012; Nie et al., 2014; Park et al., 2015; Tang et al., 2012). We

438

detected the changes in MFN2 expression at both mRNA and protein level in the

439

H2O2 or HG treated HMRECs, as well as in the STZ-induced diabetic rat retinas (Fig.

440

4). The real-time qRT-PCR and western blot results showed that the MFN2 mRNA

441

and protein levels significantly decreased in HMRECs after the H2O2 exposure (Fig.

442

4A, 4B, respectively) and HG exposure (Fig. 4C, 4D, respectively). In addition, the

443

MFN2 mRNA and protein levels also significantly decreased in diabetic retinas (Fig.

444

4E, 4F, respectively) at 20w post STZ injection. Further more, the MFN2 mRNA and

445

protein levels were found gradually decreased in diabetic retinas post STZ injection

446

(Fig. 4G, 4H, respectively).

448 449 450

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451 452 453 454 455 15

ACCEPTED MANUSCRIPT Fig. 4. Expression of MFN2 at mRNA and protein level in the H2O2- or HG- treated HMRECs and

457

in the retinas of rats with DM up to 20 weeks post STZ-injection. The levels of MFN2 mRNA

458

expression were detected in H2O2- (A) or HG-treated (C) HMRECs and rat retinas (E, G) by Real

459

time qRT-PCR. The levels of MFN2 protein were determined in H2O2-(B) or HG-treated (D)

460

HMRECs and rat retinas (F, H) by Western-blot analysis. The mRNA levels are expressed as a

461

ratio to GAPDH and normalized to control. β-actin was used as a control for equal protein loading.

462

*

463

ND: non-diabetic.

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P < 0.05 vs control (A), NG (C), or ND (E, G); (A-D: n = 3；E-H: n = 6). NG, normal glucose;

465

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464

3.5. miR-195 regulated MFN2 expression by targeting its 3′-UTR binding site

467

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To examine the direct binding of miR-195 with the 3′-untranslated region (3'-UTR) of MFN2 mRNA, the reporter plasmids (WT-MFN2 or MT-MFN2) were

469

cotransfected with miR-195 mimics, miR-195 inhibitor, or a scramble control into the

470

293A cells. As expected, a significant reduction in the reporter luciferase activity was

471

observed in the presence of miR-195 mimics compared with the scrambled controls

472

(Fig. 5A). When an inhibitor of miR-195 was added in place of miR-195 mimics, a

473

significant increase of luciferase activity was observed compared with the control or

474

scrambled miRNA (Fig. 5B). In addition, the mutated MFN2 3'-UTR reporter

475

plasmids abrogated the repressive effect of miR-195 on the activity of its target

476

3′-UTR as measured by the luciferase assay (Fig. 5C). Similarly, mutation of the

477

predicted miR-195 binding site in MFN2-3′UTR abrogated the stimulative effect of

478

miR-195 inhibitor on the activity of MFN2-3′UTR (Fig. 5D). These results confirmed

479

the direct binding site of miR-195 in the 3'-UTR of MFN2 mRNA and indicated that

480

miR-195 might directly regulate the endogenous MFN2 expression.

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481

To further validate whether miR-195 could regulate the protein level of MFN2,

482

miR-195 mimics, miR-195 inhibitor or a scramble control were transfected into the

483

HMRECs. The results of western blot analysis indicated that transfection with

484

miR-195 mimics (not the scrambled controls) led to a decrease in the MFN2 protein

485

levels (Fig. 5E), whereas transfection with miR-195 inhibitor resulted in an increase 16

ACCEPTED MANUSCRIPT 486

in the MFN2 protein levels compared with the scramble controls (Fig. 5F). These

487

results suggested that miR-195 could regulate the protein level of MFN2 by directly

488

targeting the 3'-UTR sequences of MFN2 mRNA.

489

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494 495

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496 497 498 499 500

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Fig. 5. miR-195 regulated the expression of MFN2 by targeting the 3′-UTR of MFN2 mRNA. The

503

relative luciferase activities of the MFN2 3′-UTR were measured in the 293A cells co-transfected

504

with WT-MFN2 3′-UTR luciferase vector and miR-195 mimic (100 nmol/L) (A) or miR-195

505

inhibitor (100 nmol/L) (B), or co-transfected with mutated MFN2 3′-UTR vector and miR-195

506

mimic (C) or miR-195 inhibitor (D). The MFN2 protein levels were detected by Western-blot

507

analysis in HMRECs transfected with miR-195 mimics (E), or with miR-195 inhibitor (F). Data

508

were shown as means ± SD of triplicate. *P < 0.05 vs control or scrambled miRNA (n = 3).

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3.6. miR-195 regulated the MFN2 expression in HMRECs in response to oxidative

511

stress

512 513

To investigate whether miR-195 could regulate the MFN2 expression in response

514

to oxidative stress, the HMREC cells were transfected with miR-195 mimics,

515

miR-195 inhibitor or a scramble control before the cells were subjected to the 17

ACCEPTED MANUSCRIPT H2O2-induced oxidative stress. As shown in Fig. 6, western-blot analysis results

517

showed a reduction in the MFN2 protein levels by miR-195 mimics (Fig. 6A) and a

518

further reduction in the MFN2 protein levels after the HMRECs were further

519

challenged by H2O2 (P < 0.05, Fig. 6A). In contrast, transfection with miR-195

520

inhibitor reversed the H2O2-induced decrease of MFN2 protein levels in HMRECs (P

521

< 0.05, Fig. 6B).

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522 523

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Fig. 6. The effects of miR-195 mimics or miR-195 inhibitor on MFN-2 protein level in HMRECs

533

in response to oxidative stress. HMRECs were transfected with miR-195 mimic (A) (100 nmol/L),

534

or miR-195 inhibitor (B) (100 nmol/L), or control scrambles for 24 h. The cells were then treated

535

with H2O2 (400 µmol/L) for 24 h. *P < 0.05 vs scramble (n = 3);

536

miR-195 (I) (n = 3).

538 539 540

﹟

P < 0.05 vs miR-195, or

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3.7. miR-195 regulated angiogenesis of HMRECs in vitro

To examine the effect of miR-195 on angiogenesis/tube formation by HMRECs,

541

miR-195 mimics and scrambled controls were transfected into HMRECs in vitro. The

542

transfection efficiency was confirmed by qRT-PCR with detection of over 15-fold of

543

increased expression of miR-195 or pcDNA-MFN2 plus miR-195 in HMRECs (Fig.

544

7A). As shown in Fig. 7B, transfection with miR-195 mimics could stimulate the tube

545

formation by HMRECs compared with control and scramble groups. Importantly, the 18

ACCEPTED MANUSCRIPT 546

inductive effect of miR-195 on tube formation (Fig. 7Bc) was counteracted when the

547

HMRECs were co-transfected with pcDNA-MFN2 plus miR-195 mimics (Fig. 7Bd).

548 549 550

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564 565 566

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Fig. 7. Overexpression of miR-195 induced the tube formation by HMRECs.

568

(A) Quantification of miR-195 in HMRECs transfected with miR-195 mimics, scrambled miRNA

569

or the combination of pcDNA-MFN2 and miR-195 mimics. *P < 0.05 vs control (n = 3); (B)

570

Representative images of the tube formation by normal cultured HMRECs (a) and the HMRECs

571

transfected with scrambled miRNAs (b) or miR-195 mimics (c) or pcDNA-MFN2 and miR-195

572

mimics (d). Upper: Original magnification, × 4. Lower: Quantitative analysis of the number of

573

branch points as a measure of the complexity of the capillary network. *P < 0.05 vs scrambled

574

miRNA group; # P < 0.05 vs miR-195 mimics group, n = 3.

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3.8. miR-195 regulated the vascular permeability of retinas in diabetic rats

577 19

ACCEPTED MANUSCRIPT To explore the regulatory effect of miR-195 on the permeability changes of

579

diabetic retinal vasculature in vivo, miR-195 antagomir (miR-195A), scrambled

580

miRNAs, or the combination of miR-195 antagomir and siRNA-MFN2 were

581

intravitreally injected into diabetic rat eyes. The transfection efficiency was confirmed

582

by qRT-PCR (Fig. 8A). Albumin permeation from the retinal vasculature was

583

evaluated using immunohistochemical staining. Albumin was detected within the

584

vessels but no extra vascular albumin was observed in non-diabetic control retinas

585

(Fig. 8Ba). Diffuse extravascular albumin was observed in the inner nuclear, outer

586

plexiform, and outer nuclear layers in diabetic retina (Fig. 8Bb), and the increased

587

vascular permeability of diabetic retinas was prevented by the miR-195 antagomir

588

intraocular injection (Fig. 8Bd). However, scrambled miRNAs had no significant

589

effect on vascular permeability of retinas compared to diabetic group (Fig. 8Bc).

590

Further more, combination of siRNA-MFN2 and miR-195 antagomir did not prevent

591

increased vascular permeability in diabetic retinas (Fig. 8Be).

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592

596 597 598 599 600 601 602 603

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Fig. 8. Regulatory effects of miR-195 on retina vascular permeability in non-diabetic or diabetic

608

rats. (A) Quantification of miR-195 in retinas of non-diabetic or diabetic rats following intravitreal 20

ACCEPTED MANUSCRIPT injection with scrambled miRNAs, miR-195 antagomir (miR-195A) or the combination of

610

siRNA-MFN2 and miR-195A. *P < 0.05 vs non-diabetic or scramble group (n = 6); (B)

611

Representative images of the albumin detected by immunohistochemical staining in retinas of

612

non-diabetic rats (a), diabetic rats (b), diabetic rats with intravitreal injection of scrambled miRNA

613

(c) or miR-195A (d) or combination of siRNA-MFN2 and miR-195 antagomir (e).

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614 615

4. Discussion

616

Oxidative stress is often defined as a pathologic condition in which there was an

618

imbalance between oxidant derivative production and antioxidant defense. Increased

619

oxidative stress was observed in retinal capillary endothelial cells (Kowluru and Chan,

620

2007; Kowluru et al., 2015) and it was considered as one of the major metabolic

621

abnormalities involved in the etiology of DR. Emerging studies have documented that

622

microvascular injury was one of the key alterations in DR and vascular ECs were

623

highly sensitive to oxidative stress (Zheng et al., 2010). However, the molecular

624

mechanisms underlying DR have not been well understood. Recently, miRNAs have

625

been a new research hotspot and a lot of reports suggested that it played significant

626

roles in multiple biological and pathological processes including diabetes and its

627

complications, such as DR (Dong et al., 2016; Shen et al., 2015; Wang et al., 2014).

628

As a unifying mechanism of diabetes, oxidative stress is involved in the pathological

629

process of diabetes and its complications by regulating the expression of miRNA. For

630

example, oxidative stress mediated the upregulation of miR-200c and its other family

631

members, which further induced endothelial cell apoptosis and senescence (Magenta

632

et al., 2011). In addition, it has been reported that unacylated ghrelin mediated EC

633

protection against ROS imbalance by rescuing miR-126 expression (Togliatto et al.,

634

2015).

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635

In the present study, we established a functional link between miR-195 and

636

oxidative stress/diabetes-induced retinal endothelial cell injury. We demonstrated that

637

miR-195 was a regulator for MFN2 known to be involved in oxidative stress and

638

diabetes-associated complications. Oxidative stress-induced overexpression of 21

ACCEPTED MANUSCRIPT 639

miR-195 caused down-expression of MFN2 protein, leading to tube formation and an

640

increase in retinal BRB permeability, two common pathogenic functional changes

641

associated with DR. In diabetes, overproduction of superoxides in the ECs leads to DNA damage,

643

transcription factor activation, and deregulation of multiple genes (Piconi et al., 2006;

644

Wu et al., 2016; Xie et al., 2008). As HUVECs were easy to obtain, it was usually

645

used as a vascular epithelial cell model for researching the mechanism of multiple

646

vascular diseases, such as DR (Wang et al., 2012; Yang et al., 2013). In the present

647

study, the oxidative stress cell model was developed by incubating HUVECs with

648

H2O2. The results of miRNA array analysis demonstrated that oxidative stress

649

mediated deregulation of multiple miRNAs in the cultured HUVECs. Among of the

650

miRNAs selected from the pathway enrichment analysis (miR-15b, miR-17,

651

miR-106b, miR-195, miR-497, miR-638, miR-1246, miR-1275, miR-4267, miR-4324,

652

and miR-4734), five differentially expressed miRNAs (miR-15b, miR-17, miR-106b,

653

miR-195 and miR-497), which had the same gene sequence between human and rats,

654

were chosen for further validation in diabetic rat retinas by real-time PCR. The

655

development of DR condition in rats by injection with STZ was confirmed by the

656

presence of capillary obliterations, as was expected in a well-established animal

657

model (Chen et al., 2012; Herrera et al., 2010; Mortuza et al., 2014; Ortega et al.,

658

2014). In the diabetic retinas, it was notable that the expression level of miR-195 was

659

increased significantly with 4-fold changes, whereas other miRNAs expression levels

660

had much less extent of changes, miR-15b, miR-106b, and miR-497 all had less than

661

1-fold changes while miR-17 remained unchanged. More importantly, miR-195

662

expression levels were increased gradually in diabetic retinas post STZ injection,

663

which implied its important roles in the pathogenesis of DR.

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664

Previous studies have demonstrated that miR-195 was upregulated in the hepatic

665

tissue and retina tissue of STZ-induced diabetic rats (Herrera et al., 2010; Mortuza et

666

al., 2014). However, Chen YQ et al. have reported a reduction in miR-195 expression

667

levels in the renal tissue of STZ-induced diabetic mice (Chen et al., 2012). In the

668

present study, we demonstrated that miR-195 was upregulated in the retina tissue of 22

ACCEPTED MANUSCRIPT STZ-induced diabetic rats, which was consistent with previous reports (Mortuza et al.,

670

2014). These results illustrated a tissue-specific differential expression and a

671

complexity of the regulation of miR-195 in diabetes. Retina, being a tissue rich in

672

polyunsaturated fatty acids and with high oxygen consumption, is sensitive to

673

oxidative stress. In this study, the upregulation of miR-195 in the diabetic rat retinas

674

and cultured HMRECs in response to H2O2 challenge, further suggested that increased

675

level of miR-195 expression during the course of DR in rats could be relate to the

676

oxidative stress condition.

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MFN2 is a newly discovered multifunctional protein which has been associated

678

with diabetes and oxidative stress. It is known to be implicated in the conditions of

679

oxidative stress, mitochondrial damage, insulin resistance, and diabetes mellitus

680

(Martorell-Riera et al., 2014; Park et al., 2015; Parra et al., 2014; Sebastian et al.,

681

2012). Deregulation of MFN2 has been detected in skeletal muscle of type 2 diabetic

682

patients as well as in the kidneys and left anterior myocardium in different stages of

683

diabetes in rats (Gao et al., 2012; Tang et al., 2012). Furthermore, MFN2 was one of

684

the down-regulated genes related to mitochondrial biogenesis and function in the

685

diabetic retina (Zhong and Kowluru, 2011). The bioinformatics analysis of miR-195

686

for its predicted targets revealed a high predictive score for MFN2. It was therefore

687

the interest of the present study to identify a link between miR-195 and MFN2, and

688

their roles in diabetic complications.

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Oxidative stress is a unifying mechanism for diabetic complications. In the present

690

study, we found a marked reduction in MFN2 expression, at both mRNA and protein

691

levels, in the STZ-induced diabetic rat retinas as well as the cultured human retinal

692

endothelial cells after exposed to the H2O2-induced oxidative stress. Up-regulation of

693

MFN2 could promote mitochondria fusion, further protecting cells against ROS

694

accumulation (Sugioka et al., 2004), whereas down-regulation of MFN2 reduced

695

glucose oxidation and induced the decrease of cell mitochondria membrane potential

696

in the presence of several oxidative substrates in L6E9 myotubes (Sebastian et al.,

697

2012). We further identified that MFN2 was directly targeted by miR-195 in STZ

698

induced diabetic rats. Our results demonstrated that the oxidative stress-mediated

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ACCEPTED MANUSCRIPT 699

MFN2 down-regulation was regulated by miR-195 through direct targeting at the

700

3’-UTR sequences of MFN2 mRNA. As ECs are major targets of oxidative stress, the EC injuries in DR include

702

increased retinal nonperfusion, enhanced vasopermeability, and pathological

703

proliferation of retinal vessels (Barber et al., 2011). Tube formation and increased

704

endothelial permeability as a result of BRB breakdown are prominent features of

705

pathological changes in DR (Abu El-Asrar et al., 2015; Goncalves et al., 2014; Sander

706

et al., 2001). We observed the presence of tube formation, a key step in

707

neovascularization of DR, in the retinal endothelial cells in response to the increased

708

level of miR-195 expression. Further, this phenomenon was counteracted by

709

overexpression of MFN2. In diabetic rats, the permeability of the BRB was indicated

710

by an increased albumin level within the retina vessels (Zhang et al., 2008). In our in

711

vivo experiment, the increased permeability of the BRB was attenuated by intravitreal

712

injection of the miR-195 antagomir into diabetic rats. After MFN2 was blocked by

713

siRNA, miR-195 antagomir did not attenuate the increase of permeability of BRB.

714

These results indicated that oxidative stress induced upregulation of miR-195 could

715

promote pathological changes in DR, and its function depended on inhibiting the

716

expression of MFN2.

719 720

5. Conclusions

Our data demonstrated that miR-195 was a regulator of MFN2 that are involved in

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721

oxidative stress-induced retinal endothelial cell injury. MiR-195 could be a potential

722

therapeutic target for intervention of DR in future.

723 724

Acknowledgments

725 726

This work was supported by grants from the international cooperation project of

727

Hebei Province (13397707D, 15397718D and 16397773D) and the Hebei Province

728

Health and Family Planning Commission program (ZL20140011). 24

ACCEPTED MANUSCRIPT 729 730

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ACCEPTED MANUSCRIPT Highlights miR-195 was upregulated in the diabetic rat retina and oxidative stressed HMRECs. miR-195 directly targeted the 3′- UTR of MFN2 mRNA.

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miR-195 regulated angiogenesis and vascular permeability by inhibiting the

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expression of MFN2.