Introduction

Molecular Medicine Reports

1791-2997 1791-3004

D.A. Spandidos

10.3892/mmr.2019.9973

mmr-19-04-3132

Articles

miR-17-5p regulates the proliferation and apoptosis of human trabecular meshwork cells by targeting phosphatase and tensin homolog

Wang

Xiaoyuan

1 Li

Zhijian

1 Bai

Jie

1 Song

Wuqi

2 Zhang

Fengmin

1Department of Ophthalmology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086, P.R. China 2Department of Microbiology, Harbin Medical University, Heilongjiang Provincial Key Laboratory of Infection and Immunity, Key Laboratory of Pathogenic Biology, Harbin, Heilongjiang 150086, P.R. China

Correspondence to: Dr Fengmin Zhang, Department of Microbiology, Harbin Medical University, Heilongjiang Provincial Key Laboratory of Infection and Immunity, Key Laboratory of Pathogen Biology, 157 Baojian Road, Harbin, Heilongjiang 150086, P.R. China, E-mail: dr_fengmingzhang@outlook.com

042019

20022019

19 4 3132 3138 12072017 17012019

2019

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Glaucoma is one of the leading causes of blindness. Previous studies have indicated that the oxidative stress-induced apoptosis of trabecular meshwork cells (TMCs) may serve a key role in the pathogenesis of glaucoma, and that micro RNA(miR)-17-5p may be involved in this process. However, the specific mechanisms require further investigation. The aim of the present study was to investigate the effects of miR-17-5p on the proliferation and apoptosis of human TMCs (HTMCs) in response to oxidative stress. It was observed that exposure to H₂O₂ induced a significant decrease in the proliferation and a marked increase in the apoptosis of HTMCs. H₂O₂ exposure also suppressed the expression of miR-17-5p and promoted the expression of phosphatase and tensin homolog (PTEN). Furthermore, transient overexpression of miR-17-5p induced a significant increase in the proliferation and a significant decrease in the apoptosis of HTMCs by affecting the expression of PTEN, and the apoptosis-related proteins B-cell lymphoma-associated X protein (Bax), B-cell lymphoma-extra large (Bcl-xL) and B-cell lymphoma-2 (Bcl-2). However, knockdown of miR-17-5p demonstrated the opposite results. The results of a dual luciferase reporter assay demonstrated that PTEN may be a direct target of miR-17-5p. In conclusion, miR-17-5p was downregulated in HTMCs under oxidative conditions, and miR-17-5p may regulate the apoptosis of HTMCs by targeting PTEN. These results provide a novel theoretical basis and potential therapeutic target for the treatment of glaucoma.

glaucoma micro RNA-17-5p phosphatase and tensin homolog human trabecular meshwork cells apoptosis

Introduction

Glaucoma is a common ophthalmic disease, and it is the second leading cause of blindness worldwide (1). It is estimated that by 2020, the number of patients with glaucoma will reach approximately 80 million (2). Glaucoma can be divided into open-angle glaucoma (OAG) and angle-closure glaucoma (ACG), and in Western countries, approximately 75% of glaucoma patients are diagnosed with OAG (3), while in Asian countries, ACG is more prevalent than OAG (4). In 2010, 86.5% of ACG patients were resident in Asia, and the largest number of patients affected by ACG was in China (47.5% of the total) (5). In recent years, increasing evidence has indicated that the oxidative stress-induced senescence of trabecular meshwork cells (TMCs) may play a key role in the occurrence and development of glaucoma (6,7); however, the specific mechanisms still require further investigation.

MicroRNAs (miRNAs) are short non-coding RNAs (length, ~22 nucleotides) that can bind to the 3′-UTR of target mRNAs and consequently suppress the expression of target genes. miRNAs can regulate a variety of biological activities, including embryonic development, angiogenesis, cell differentiation, proliferation and apoptosis (8–10). With the development of next-generation sequencing and bioinformatics methods, differentially expressed miRNAs that are potentially involved in the pathogenesis of diseases, including cancers (11,12) and glaucoma (13–15), have been successfully identified. The targets of these miRNAs, as well as related signaling pathways, have also been predicted. miR-17-5p is located on human chromosome 13q31, and it belongs to the miR-17-92 cluster (16). miR-17-5p is up-regulated in many tumor types and participates in the pathogenesis of different cancers as an oncogene (17–19). Besides cancer-related studies, miR-17-5p has been reported to promote oxidative stress-induced apoptosis of cardiomyocytes in ischemia/reperfusion-induced cardiac injury animal models (20). It has also been observed that knockdown of miR-17-5p can restore the expression of the very-low-density lipoprotein receptor and alleviate the symptoms of atherosclerosis in ApoE-/- mouse models (21). It has been observed that miR-17-5p is down-regulated in the trabecular meshwork under oxidative stress (22); however, the specific mechanisms have not yet been investigated.

In the present study, we focused on the roles of miR-17-5p in glaucoma and its related mechanisms. Human TMCs (HTMCs) were treated with H₂O₂ to induce oxidative stress, and the effects of miR-17-5p on the behaviors of HTMCs under oxidative stress were explored. We hypothesized that miR-17-5p would regulate the proliferation and apoptosis of HTMCs by targeting phosphatase and tensin homolog (PTEN). The present study may provide a theoretical basis and novel therapeutic target for the treatment of glaucoma.

Materials and methods <sec> <title>HTMC culture

HTMCs were purchased from Sciencell Research Laboratories (San Diego, CA, USA). According to the product information, HTMCs were isolated from the juxtacanalicular and corneoscleral regions of human eyes, and characterized by immunofluorescence methods using α-smooth muscle actin- and fibronectin-specific antibodies. Cells were cultured in a humidified incubator at 37°C with 5% CO₂ using Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplied with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin solution (Gibco; Thermo Fisher Scientific, Inc.). Oxidative stress was induced by treating HTMCs with DMEM that contained 300 µM H₂O₂ (Beyotime Institute of Biotechnology, Shanghai, China) for 3–7 h. Then, the cells were harvested for future analysis.

Cell transfection

The miR-17-5p inhibitor and miR-17-5p mimic oligonucleotides were synthesized by GenePharma Co., Ltd., (Shanghai, China). HTMCs were cultured until confluence, trypsinized and seeded onto new six-well plates at 100,000 cells/well. Cells were then transfected with either miR-17-5p inhibitor or miR-17-5p mimic using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After transfection, cells were cultured for 48 h and harvested for future analysis.

Cell proliferation analysis

An MTT assay was performed to determine the viability of HTMCs following different treatments. An MTT proliferation assay kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used, according to the manufacturer's protocol.

Cellular apoptosis analysis

For cellular apoptosis analysis, HTMCs subjected to different treatments were stained using an Annexin V-FITC apoptosis detection kit (Sigma-Aldrich; Merck KGaA). The apoptosis rate of the cells in different groups was detected and analyzed using a BD FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA), according to the manufacturer's protocol.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from HTMCs using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and reverse transcribed into cDNA using the PrimeScript™ RT Master Mix (Perfect Real Time) kit (Takara Biotechnology Co., Ltd., Dalian, China), then qPCR was performed using the SYBR ExScript RT-PCR kit (Takara Biotechnology Co., Ltd.) on an ABI 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The thermocycling profiles are shown in Table I. The relative expression of PTEN in each sample was normalized to the level of GAPDH using the 2^−ΔΔCq method. The expression of miR-17-5p was examined using the Hairpin-it™ miRNAs qPCR Quantitation Kit (GenePharma Co., Ltd.) according to the manufacturer's protocol, and U6 (RNU6B; GenePharma Co., Ltd.) was used for normalization. Primers were synthesized by GenScript (Nanjing, China), and the sequences of the primers are shown in Table II.

Western blot analysis

Cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology), and the concentration of total protein was measured using a BCA Kit (Beyotime Institute of Biotechnology). Next, SDS-PAGE was performed to separate the proteins, and the proteins were then transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk and incubated with primary antibodies (anti-Bax, anti-PTEN, anti-Bcl-2, anti-Bcl-xL and anti-GAPDH, all purchased from Abcam, Cambridge, MA, USA) overnight at 4°C. The following day, the membranes were incubated with HRP-conjugated secondary antibodies (purchased from Abcam, Cambridge, MA, USA), then washed and incubated with the enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology). Finally, the signals were detected using the ChemiDoc™XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Immunocytochemistry

HTMCs were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, then incubated with the anti-PTEN primary antibodies for 30 min. Cells were incubated with Alexa Fluor 488-conjugated secondary antibodies, then visualized using an Olympus fluorescent microscope (Olympus Corporation, Tokyo, Japan).

Computational methods

The target genes of miR-17-5p were predicted using online bioinformatics tools, TargetScan (http://www.targetscan.org) and miRanda (http://www.microrna.org).

Dual luciferase reporter assay

Fragments of either the wild-type PTEN 3′-UTR (PTEN-3′UTR) or mutant PTEN 3′-UTR (PTEN-MUT) region that contains the miR-17-5p binding site were synthesized and cloned into the pGL6-TA-reporter plasmid (Beyotime Institute of Biotechnology). The plasmids were transfected into 293 cells using Lipofectamine^® 3000 for 48 h, and the activity of the luciferase in each group was examined using the dual-luciferase reporter system (Beyotime Institute of Biotechnology), according to the manufacturer's protocol.

Statistics

All statistical analyses were conducted using SPSS v.22.0 software (IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation. Two independent sample t-tests were performed for comparisons between two groups. One-way analysis of variance followed by Dunnett's post-hoc test was performed for comparisons among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>Effect of oxidative stress on the proliferation and apoptosis of HTMCs in vitro

First, we investigated the effect of H₂O₂ treatment on the proliferation and apoptosis of HTMCs using MTT and flow cytometry methods. It was observed that the cell viability was significantly decreased in cells treated with H₂O₂ compared with the control group at 3, 5 and 7 h (P<0.01; Fig. 1A). Moreover, H₂O₂ treatment also induced a continuous increase in the apoptosis rate of HTMCs from 0 to 7 h (P<0.05; Fig. 1B and C).

miR-17-5p was down-regulated in HTMCs under oxidative stress in vitro

In order to explore the roles of miR-17-5p in HTMCs under oxidative stress, we compared the expression of miR-17-5p in control and H₂O₂ groups at different time points using RT-qPCR methods. As shown in Fig. 2A, H₂O₂ induced a significant decrease in the expression of miR-17-5p at 5 and 7 h (P<0.05; Fig. 2A).

miR-17-5p can regulate the proliferation and apoptosis of HTMCs in vitro

HTMCs were transfected with either miR-17-5p mimics or miR-17-5p inhibitor, and the effect of miR-17-5p on the proliferation and apoptosis of HTMCs was examined using MTT and flow cytometry methods. As shown in Fig. 2B, the expression of miR-17-5p was significantly increased in miR-17-5p mimic-transfected HTMCs and markedly decreased in miR-17-5p inhibitor-transfected HTMCs, suggesting that transfection had been successfully performed. Moreover, transient overexpression of miR-17-5p induced a significant increase in the proliferation (P<0.01; Fig. 3A and B) and a significant decrease in the apoptosis of HTMCs (P<0.01; Fig. 3C). Knockdown of miR-17-5p exhibited the opposite results. In addition, transfection of miR-17-5p mimics also induced a significant increase in the expression of Bcl-2 and Bcl-xL, and a marked decrease in the expression of Bax, while knockdown of miR-17-5p exhibited the opposite results (Fig. 4).

PTEN is a direct target of miR-17-5p in HTMCs

Using online bioinformatics tools, PTEN was predicted to be a direct target of miR-17-5p. Using western blot analysis, it was observed that the expression of PTEN continuously increased from 3 to 7 h under oxidative stress (Fig. 5A), suggesting that increased PTEN may be associated with the pathogenesis of glaucoma. Moreover, transient overexpression of miR-17-5p in HTMCs induced a significant decrease in the expression of PTEN, while knockdown of miR-17-5p promoted the expression of PTEN in HTMCs (Fig. 5B and C). Finally, the results from the dual luciferase reporter assay indicated that co-transfection of miR-17-5p mimics and wild-type PTEN-3′UTR plasmids significantly decreased the activity of the luciferases. Co-transfection of miR-17-5p mimic and PTEN-MUT exhibited no effect on the activity of the luciferases (Fig. 6), suggesting that PTEN is a direct target of miR-17-5p.

Discussion

Glaucoma is a common ophthalmic disorder that can cause damage to the optic nerve and loss of vision. Increasing evidence has indicated that oxidative stress-induced senescence of HTMCs is involved in the pathogenesis of glaucoma (7). H₂O₂ is an important oxidant in the human eye. It has been demonstrated that oxidative levels significantly increase in the aqueous humor of patients with glaucoma, which means that TMCs will also be exposed to high concentrations of H₂O₂. Thus, in previous studies, a high concentration of H₂O₂ has been used to induce oxidative stress in HTMCs, and this has now become a widely applied in vitro model in glaucoma-related studies (23,24).

In recent years, the effects of miRNAs on TMCs have been discussed in many studies (25,26). Li et al (22), performed miRNA array analysis to examine alterations in the expression levels of microRNAs in oxidative stress-induced cellular senescence in HTMCs. It was observed that the expression of miR-17-5p was significantly decreased by treatment with a high concentration of H₂O₂. However, the specific mechanisms underlying this remain unclear. In the present study, we identified that compared with untreated cells, H₂O₂ treatment induced a significant decrease in the proliferation and a marked increase in the apoptosis of HTMCs at each time point examined (P<0.05). Moreover, H₂O₂ treatment also induced a significant decrease in the expression of miR-17-5p, which was consistent with the finding from Li et al. These results indicate that miR-17-5p is down-regulated in HTMCs under oxidative stress, and miR-17-5p may participate in the mechanism of H₂O₂-induced increase in apoptosis and decrease in the proliferation of HTMCs.

Next, we transfected HTMCs with either miR-17-5p mimics or miR-17-5p inhibitor and evaluated the effect of miR-17-5p on the proliferation and apoptosis of HTMCs. We observed that transient overexpression of miR-17-5p induced a significant increase in the proliferation and a significant decrease in the apoptosis of HTMCs (P<0.05), while knockdown of miR-17-5p exhibited the opposite results. Moreover, Bcl-2 and Bcl-xL are known to be anti-apoptotic proteins, and Bax is a pro-apoptotic protein (27,28). Our data indicated that transfection of miR-17-5p mimics promoted the expression of Bcl-2 and Bcl-xL, and suppressed the expression of Bax, while knockdown of miR-17-5p exhibited the opposite effects. Taken together, these results indicate that miR-17-5p can regulate the proliferation and apoptosis of HTMCs in vitro.

PTEN is known to be a tumor suppressor in the field of cancer studies (29). Knockdown of PTEN can lead to an increase in the proliferation and a decrease in the apoptosis rate of many cancer cell types, leading to the incidence and development of various diseases (30–32). However, in the case of glaucoma, PTEN may play a different role. It has been observed that increased expression of PTEN can lead to the degeneration of optic neurons, and knockdown of PTEN can significantly alleviate optic nerve damage in glaucoma. In a very recent study, Tellios et al (33) demonstrated that phosphorylation of PTEN is significantly increased in TMCs upon treatment with TGF-β (increased levels of TGF-β in the aqueous humor is one of the main causes of fibrosis in OAG), and an increase in the expression of PTEN may contribute to the fibrosis of the HTMCs in glaucoma. In the present study, we observed that the expression of PTEN was markedly increased in HTMCs following exposure to H₂O₂ (P<0.01), suggesting that increased PTEN may be involved in the pathogenesis of glaucoma. Moreover, transfection of miR-17-5p mimic or inhibitor induced a significant decrease or increase in the expression of PTEN, respectively. The results from the dual luciferase reporter assay confirmed that PTEN is a direct target of miR-17-5p. In summary, these data indicated that miR-17-5p can regulate the proliferation and apoptosis of HTMCs by targeting PTEN.

The present study has certain limitations. We only performed cellular experiments, and the expression levels of miR-17-5p in tissue samples from patients and normal controls should be evaluated to confirm our findings from the clinical perspective. However, due to ethical issues, it is difficult to obtain the trabecular meshwork of healthy volunteers; thus, in vivo studies using animal models could be a better option.

In conclusion, our data demonstrated that miR-17-5p is down-regulated and PTEN is up-regulated in HTMCs under oxidative conditions. We also identified that miR-17-5p can regulate the proliferation and apoptosis of HTMCs through targeting PTEN. Our data indicated that miR-17-5p has the potential to become a novel therapeutic target for the treatment of glaucoma.

Acknowledgements

Not applicable.

Funding

The persent study was sponsored by the funds from the project of the Department of Health, Heilongjiang Province (project no. 2011-005).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

FZ designed the study and wrote the majority of the manuscript. XW performed most of the experiments and wrote part of the manuscript. ZL and JB performed some of the experiments. WS performed some of the statistical analysis.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References 1

Zhao

Yin

Wang

The effect of MTHFR ala222val polymorphism on open-angle glaucoma: a meta-analysis

Ophthalmic Genet3627302015

10.3109/13816810.2014.969379

25317717

Tham

Wong

Quigley

Aung

Cheng

Global prevalence of glaucoma and projections of glaucoma burden through 2040: A systematic review and meta-analysis

Ophthalmology121208120902014

10.1016/j.ophtha.2014.05.013

24974815

Bailey

Loomis

Kang

Allingham

Gharahkhani

Khor

Burdon

Aschard

Chasman

Igo

Genome-wide association analysis identifies TXNRD2, ATXN2 and FOXC1 as susceptibility loci for primary open-angle glaucoma

Nat Genet481891942016

10.1038/ng.3482

26752265

4731307

Liang

Wang

Rong

Thomas

Initial treatment for primary angle-closure glaucoma in China

J Glaucoma244694732015

10.1097/IJG.0000000000000075

24786102

Nongpiur

Khor

Jia

Cornes

Chen

Qiao

Nair

Cheng

George

ABCC5, a gene that influences the anterior chamber depth, is associated with primary angle closure glaucoma

PLoS Genet10e10040892014

10.1371/journal.pgen.1004089

24603532

3945113

Shen

Han

Huang

Guo

Wang

MicroRNA-483-3p inhibits extracellular matrix production by targeting Smad4 in human trabecular meshwork cells

Invest Ophthalmol Vis Sci56841984272015

10.1167/iovs.15-18036

26747772

Zhang

Guo

Shen

Wang

Guo

Yin

Wang

HES1 promotes extracellular matrix protein expression and inhibits proliferation and migration in human trabecular meshwork cells under oxidative stress

Oncotarget821818218332017

28423527

5400626

Feng

Sang

Zhu

Liu

Shi

Chai

MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro

Sci Rep586892015

10.1038/srep08689

25732513

4346788

Cai

Shen

Dong

Sun

Liu

Chen

MiRNA-29b suppresses tumor growth through simultaneously inhibiting angiogenesis and tumorigenesis by targeting Akt3

Cancer Lett3971111192017

10.1016/j.canlet.2017.03.032

28365400

Qun

Wenda

Weihong

Jianyang

Wei

Fangzhou

Zhenyao

Pingjin

miRNA-27b modulates endothelial cell angiogenesis by directly targeting Naa15 in atherogenesis

Atherosclerosis2541841922016

10.1016/j.atherosclerosis.2016.10.007

27755984

Falzone

Candido

Salemi

Basile

Scalisi

McCubrey

Torino

Signorelli

Montella

Libra

Computational identification of microRNAs associated to both epithelial to mesenchymal transition and NGAL/MMP-9 pathways in bladder cancer

Oncotarget772758727662016

10.18632/oncotarget.11805

27602581

5341942

Bagnoli

De Cecco

Granata

Nicoletti

Marchesi

Alberti

Valeri

Libra

Barbareschi

Raspagliesi

Identification of a chrXq27.3 microRNA cluster associated with early relapse in advanced stage ovarian cancer patients

Oncotarget2126512782011

10.18632/oncotarget.401

22246208

3282083

Guo

Shen

Jiang

Wang

Relationship between the pathogenesis of glaucoma and miRNA

Ophthalmic Res571941992017

10.1159/000450957

28073110

Molasy

Walczak

Szaflik

Majsterek

MicroRNAs in glaucoma and neurodegenerative diseases

J Hum Genet621051122017

10.1038/jhg.2016.91

27412874

Paylakhi

Yazdani

April

Fan

Moazzeni

Ronaghi

Elahi

Non-housekeeping genes expressed in human trabecular meshwork cell cultures

Mol Vis182412542012

22312193

3272053

Tsuchida

Ohno

Borjigin

Fujita

Aoki

Ueda

Takanashi

Kuroda

miR-92 is a key oncogenic component of the miR-17-92 cluster in colon cancer

Cancer Sci102226422712011

10.1111/j.1349-7006.2011.02081.x

21883694

Zhu

Peng

Shi

Wang

Wei

Wang

Zhang

MiR-17-5p enhances pancreatic cancer proliferation by altering cell cycle profiles via disruption of RBL2/E2F4-repressing complexes

Cancer Lett41259682018

10.1016/j.canlet.2017.09.044

28987387

Liao

Xiang

Nie

Zhou

Zhang

STAT3 is required for MiR-17-5p-mediated sensitization to chemotherapy-induced apoptosis in breast cancer cells

Oncotarget815763157742017

10.18632/oncotarget.15000

28178652

5362521

Jin

Yang

Liu

Shi

Wang

Association of the miR-17-5p variants with susceptibility to cervical cancer in a Chinese population

Oncotarget776647766552016

10.18632/oncotarget.12299

27765929

5363537

Shi

Bei

Kong

Liu

Lei

Wang

Xuan

Chen

miR-17-3p contributes to exercise-induced cardiac growth and protects against myocardial ischemia-reperfusion injury

Theranostics76646762017

10.7150/thno.15162

28255358

5327641

Tan

Meng

Shi

Knockdown of microRNA-17-5p ameliorates atherosclerotic lesions in ApoE-/- mice and restores the expression of very low density lipoprotein receptor

Biotechnol Lett399679762017

10.1007/s10529-017-2337-y

28374070

Luna

Qiu

Epstein

Gonzalez

Alterations in microRNA expression in stress-induced cellular senescence

Mech Ageing Dev1307317412009

10.1016/j.mad.2009.09.002

19782699

2795064

Fuchshofer

Kampik

Welge-Lüssen

Effects of oxidative stress in trabecular meshwork cells are reduced by prostaglandin analogues

Invest Ophthalmol Vis Sci49487248802008

10.1167/iovs.07-0984

18971427

Chen

Liu

Gao

Zhuo

Mitochondria-targeted peptide MTP-131 alleviates mitochondrial dysfunction and oxidative damage in human trabecular meshwork cells

Invest Ophthalmol Vis Sci52702770372011

10.1167/iovs.11-7524

21697135

Wang

MicroRNA-93 is overexpressed and induces apoptosis in glaucoma trabecular meshwork cells

Mol Med Rep14574657502016

10.3892/mmr.2016.5938

27878244

Luna

Qiu

Epstein

Gonzalez

MicroRNA-24 regulates the processing of latent TGFβ1 during cyclic mechanical stress in human trabecular meshwork cells through direct targeting of FURIN

J Cell Physiol226140714142011

10.1002/jcp.22476

20945401

3152464

Reshi

Wang

Hui

Hong

Anti-apoptotic genes Bcl-2 and Bcl-xL overexpression can block iridovirus serine/threonine kinase-induced Bax/mitochondria-mediated cell death in GF-1 cells

Fish Shellfish Immunol611201292017

10.1016/j.fsi.2016.12.026

28025159

Yang

Yao

JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates the crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin 1

Int J Mol Sci1625744257582015

10.3390/ijms161025744

26516844

4632824

Liu

Pan

Han

Liu

MicroRNA-216a promotes the metastasis and epithelial-mesenchymal transition of ovarian cancer by suppressing the PTEN/AKT pathway

Onco Targets Ther10270127092017

10.2147/OTT.S114318

28579808

5449156

Ronen

Abbott

Kravtsov

Abdelkader

Banerjee

Iczkowski

PTEN loss and p27 loss differ among morphologic patterns of prostate cancer, including cribriform

Hum Pathol6585912017

10.1016/j.humpath.2017.04.024

28504208

Feng

Zou

Zhou

Deng

Sun

Modulation of CASC2/miR-21/PTEN pathway sensitizes cervical cancer to cisplatin

Arch Biochem Biophys623-62420302017

10.1016/j.abb.2017.05.001

28495512

Shen

Wang

Yang

Chen

Yang

Loss of PTEN expression in breast cancer: Association with clinicopathological characteristics and prognosis

Oncotarget832043320542017

28410191

5458267

Tellios

Belrose

Tokarewicz

Hutnik

Liu

Leask

Motolko

Iijima

Parapuram

TGF-β induces phosphorylation of phosphatase and tensin homolog: Implications for fibrosis of the trabecular meshwork tissue in glaucoma

Sci Rep78122017

10.1038/s41598-017-00845-x

28400560

5429747

Figure 1.

Effect of oxidative stress on the proliferation and apoptosis of HTMCs in vitro. (A) Proliferation rate of HTMCs in different groups by MTT assay, **P<0.01 vs. the control group; (B) Apoptosis rate of HTMCs in different groups by flow cytometry method. (C) Quantification of results in (B). **P<0.01 vs. the 3 h group, *P<0.05 vs. the 5 h group.

Figure 2.

Expressions of miR-17-5p in HTMCs by RT-qPCR methods. (A) Effect of oxidative stress on the expression of miR-17-5p in HTMCs. (B) Effect of transfection of miR-17-5p mimics or inhibitor on the expression of miR-17-5p in HTMCs. *P<0.05 and **P<0.01 vs. the control group. HTMCs, human trabecular meshwork cells.

Figure 3.

Effect of miR-17-5p on the proliferation and apoptosis of HTMCs in vitro. (A) Morphological feature of HTMCs in different groups by inverted microscope (scale bar, 100 µm); (B) Proliferation rate of HTMCs in different groups by MTT assay; (C) Apoptosis rate of HTMCs in different groups by flow cytometry method. **P<0.01 vs. the control group. Control, un-transfected cells; Inhibitor, miR-17-5p inhibitor transfected cells; Mimics, miR-17-5p mimics transfected cells. HTMCs, human trabecular meshwork cells.

Figure 4.

Effect of miR-17-5p on the expression of Bcl-2, Bcl-xL and Bax in HTMCs by western blot methods. Control, un-transfected cells; Inhibitor, miR-17-5p inhibitor transfected cells; Mimics, miR-17-5p mimics transfected cells. Bcl-2, B-cell lymphoma-2; Bcl-xL, B-cell lymphoma extra-large; BAX, Bcl-2-associated X protein; HTMCs, human trabecular meshwork cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5.

Roles of PTEN in the pathogenesis of glaucoma. (A) Expression of PTEN in HTMCs under oxidative stress by western blot method; (B) Effect of overexpression and knockdown of miR-17-5p on the mRNA expression of PTEN in HTMCs by RT-qPCR method; (C) Effect of overexpression and knockdown of miR-17-5p on the protein expression of PTEN in HTMCs using ICC method (scale bar, 100 µm). **P<0.01 vs. NC. NC, un-transfected cells; Inhibitor, miR-17-5p inhibitor transfected cells; Mimics, miR-17-5p mimics transfected cells. PTEN, Phosphatase and tensin homolog; HTMCs, human trabecular meshwork cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6.

PTEN is direct target of miR-17-5p. (A) Sequence alignment of the paired 3′-UTR of miR-17-5p and PTEN by bioinformatics tool Targetscan and miRanda; (B) Results of dual luciferase reporter assay. **P<0.01. WT, Fragments of wild-type PTEN 3′-UTR; Mutant, fragments of wild-type PTEN 3′-UTR; NC, miR-17-5p negative control; PTEN, Phosphatase and tensin homolog; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Table I.

Thermocycling profiles for reverse transcription-quantitative polymerase chain reaction.

Cycle no.	Temperature	Time
1	95°C	30 Sec
40	95°C	5 Sec
	60°C	4 Sec

Table II.

Sequences of the primers used in the present study.

Gene name	Sequences of the primer
miR-17-5p	F: 5′-TCTAGATCCCGAGGACTG-3′
	R: 5′-ATCGTGACCTGAACC-3′
U6	F: 5′-CTCGCTTTGGCAGCACA-3′
	R: 5′-AACGCTTCACGAATTTGCGT-3′
PTEN	F: 5′-CGACGGGAAGACAAGTTCAT-3′
	R: 5′-AGGTTTCCTCTGGTCCTGGT-3′
GAPDH	F: 5′-GACAGTCAGCCGCATCTTCT-3′
	R: 5′-TTAAAAGCAGCCCTGGTGAC-3′

PTEN, phosphatase and tensin homolog; F, forward; R, reverse.