Free
Retinal Cell Biology  |   October 2013
Inhibitory Effect of MicroRNA-34a on Retinal Pigment Epithelial Cell Proliferation and Migration
Author Affiliations & Notes
  • Qiang Hou
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Jiang Tang
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Zhenlian Wang
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Chao Wang
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Xiaogang Chen
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Ling Hou
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Xiang Da Dong
    Department of Surgery, Stamford Hospital – Affiliate of Columbia University, Stamford, Connecticut
  • LiLi Tu
    School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health of the People's Republic of China, Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Correspondance: LiLi Tu, School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, 270 Xueyuan Road, Wenzhou, Zhejiang 325027, China; tulili@mail.eye.ac.cn
Investigative Ophthalmology & Visual Science October 2013, Vol.54, 6481-6488. doi:10.1167/iovs.13-11873
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Qiang Hou, Jiang Tang, Zhenlian Wang, Chao Wang, Xiaogang Chen, Ling Hou, Xiang Da Dong, LiLi Tu; Inhibitory Effect of MicroRNA-34a on Retinal Pigment Epithelial Cell Proliferation and Migration. Invest. Ophthalmol. Vis. Sci. 2013;54(10):6481-6488. doi: 10.1167/iovs.13-11873.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: Retinal pigment epithelial (RPE) cells play important roles in ophthalmologic diseases such as proliferative vitreoretinopathy, AMD, and diabetic retinopathy. MicroRNA-34a (miR-34a) has been reported to be important in the regulation of cell proliferation, migration, differentiation, and apoptosis. In this study, we explored the effects of miR-34a on RPE cells.

Methods.: The expression level of miR-34a in subconfluent and postconfluent ARPE-19 cells was investigated with quantitative real-time PCR. MicroRNA mimic and small interfering RNA (siRNA) were transiently transfected into RPE cells. Transfected RPE cells were analyzed with WST-1 proliferation assay, and their migration was analyzed with transwell assay and in vitro scratch study. The expression or activation of target proteins was detected by Western blotting.

Results.: MicroRNA-34a was significantly downregulated in subconfluent ARPE-19 cells compared with postconfluent cells. Introduction of miR-34a inhibited the proliferation and migratory ability of RPE cells without obvious cell apoptosis. In miR-34a transfected cells, many important proliferation and/or migration related molecules such as c-Met, CDK2, CDK4, CDK6, E2F1, and phosphorylated-Cdc2 (p-Cdc2) were downregulated. Small interfering RNA designed to target c-Met also inhibited the proliferation and migration of RPE cells and downregulated CDK2, CDK6, E2F1, and p-Cdc2.

Conclusions.: MicroRNA-34a is downregulated in subconfluent RPE cells. MicroRNA-34a can inhibit the proliferation and migration of RPE cells through downregulation of its targets c-Met and other cell cycle–related molecules. Our results indicated that miR-34a is involved in the regulation of RPE cells.

Introduction
The retinal pigment epithelium is composed of a monolayer of hexagonal cells that are quiescent without proliferation and migration under normal conditions. 1 The proliferative and migratory phenotype of RPE cells occurs in pathologic conditions such as proliferative vitreoretinopathy (PVR), AMD, and diabetic retinopathy. 1 Retinal pigment epithelial cells have been considered one of the key cells implicated in PVR. 2 Uncontrolled RPE cell proliferation leads to its migration into the vitrea and retinal layers resulting in formation of epiretinal membranes that can contract and cause retinal detachment and visual impairment. 3 Among potential regulators of this process, microRNAs (miRNAs) are excellent candidates since they are involved in a variety of cellular functions such as cellular proliferation and migration. 4  
MicroRNAs are an abundant class of endogenously expressed, nonprotein-coding, short (20–25) nucleotide RNAs that regulate gene expression. 5,6 Although several reports addressed the expression profile of miRNAs in RPE cells, the exact roles of miRNA in these cells have not been well defined. 7,8 Adijanto et al. compared the miRNAs expression in human fetal RPE cells and found miR-204/211 to be significantly downregulated in dedifferentiated RPE cells. 9 Further investigation suggested that miR-204/211 promote RPE differentiation in a microphthalmia-associated transcription factor (MITF)-dependent manner. 9 In another report, Chen et al. found that miR-328 can inhibit RPE cell proliferation by downregulating PAX6. 10  
MicroRNA-34a, a gene product from chromosomal locus 1p36.22, is well known as a tumor suppressor in many tumors. 11 As a consequence, several groups have investigated the inhibitory mechanism and therapeutic potential of miR-34a in various types of cancers. 11 Direct targets of miR-34a include silent information regulator 1 (SIRT1), Bcl-2, CD44, various cyclins and CDKs, and the proto-oncoproteins MYC and MYCN. 12 MicroRNA-34a has also been implicated in many other normal biological processes such as cell proliferation, inhibition, cell cycle arrest, and senescence. 12 Specifically, we have previously reported that miR-34a is downregulated in uveal melanoma, and ectopic expression of miR-34a inhibits the proliferation and migration of uveal melanoma cells through direct downregulation of c-Met. 13  
In this study, we investigated the expression pattern of miR-34a in subconfluent ARPE-19 retinal pigment epithelial cells as compared with postconfluent cells in vitro. MicroRNA-34a was also ectopically introduced into ARPE-19 cells to determine its effects on growth, apoptosis, and migration. Subsequent experiments indicated that miR-34a downregulated c-Met levels, and direct inhibition of c-Met expression with specific siRNA inhibited the proliferation and migration of ARPE-19 cells. Overall, our data suggested that miR-34a may be involved in the regulation of RPE cell proliferation and migration. 
Materials and Methods
Cell Culture
A-retinal pigment epithelia-19 (ARPE-19) cells were purchased from American Type Culture Collection (ATCC; Manassas, VA). The cells were used at passages 10 to 20. ARPE-19 cells were cultured under two conditions: (1) Subconfluent, condition where cells were seeded and grown in 6-well plates for 3 to 5 days in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) at 37°C in 10% CO2; and (2) postconfluent, condition where cells were seeded and grown in 6-well plates for 4 weeks with the culture media being changed every 3 days under the same culture conditions as subconfluent cells. 
Immunofluorescence Staining
ARPE-19 cells (1 × 105) were seeded in 6-well plates and grown to 70% to 80% confluence or to postconfluent condition. Cells were then fixed in 4% paraformaldehyde solution for 1 hour at room temperature and permeabilized with 0.4% Triton X-100 in Tris buffered saline (TBS) for 10 minutes. Cells were blocked in TBS containing 5% BSA for 1 hour, followed by incubation with the primary antibody, Ki67, in blocking solution overnight. After washing with TBS, Cy3-conjugated secondary antibody was added to the cells for 1 hour. Cells were washed and then stained for 10 minutes with 4′,6-diamidino-2-phenylindole (DAPI) to display the nuclei. Cells were mounted in a fluorescent mounting medium, and images were captured using a fluorescence microscope (Imager Z1; Zeiss, Oberkochen, Germany). 
RNA Extraction and Real-Time PCR
ARPE-19 cells (1 × 105) were seeded in 6-well plates and grown to 80% to 90% confluence or to postconfluent condition. Total RNA was then extracted from collected cells with Trizol reagent (Invitrogen). RNA integrity was confirmed using spectrophotometry and agarose gel electrophoresis. For the detection of miR-34a expression in subconfluent and postconfluent ARPE-19 cells, 10 ng of total RNA from each culture condition were used for cDNA synthesis with a microRNA reverse transcription kit (TaqMan; Applied Biosystems, Foster City, CA), and miR-34a expression level was quantified by the microRNA Assay (TaqMan; Applied Biosystems), according to the manufacturer's instructions. Real-time PCR was performed (7500 Real-Time PCR System; Applied Biosystems). The expression level of miR-34a in postconfluent ARPE-19 cells was set as the normal control, whereas the miR-34a expression level in subconfluent cells was compared with that in postconfluent cells. U6 small nuclear RNA (snRNA) was used as internal control. 14  
Cell Proliferation Assay
ARPE-19 cells were plated at 2000 cells per well in 96-well plates (Costar, High Wycombe, UK) for each transfection. After 24 hours, transfections were performed with reagent (Lipofectamine 2000; Invitrogen). All transfections were performed in triplicates. For each well, 100 nM miR-34a mimic molecule (GenePharma, Shanghai, China) or a negative control miRNA (GenePharma) was employed. After 72 hours of culture, cell proliferation was assessed by WST-1 assay (Roche Applied Science, Mannheim, Germany). Briefly, solution reagent was added to each well and incubated at 37°C for 2 hours. Cell proliferation was assessed by measuring the absorbance at 450 nm with a reference wavelength of 600 nm using a microtiter plate reader (Molecular Devices, Sunnyvale, CA). c-Met–specific siRNA (5′-GCCAGAUUCUGCCGAACCA; GenePharma), negative control siRNA (GenePharma), or mock was used to downregulate c-Met expression in ARPE-19 cells. c-Met–specific siRNA (100 nM), negative control siRNA (100 nM), or mock was transfected into ARPE-19 cells with reagent (Lipofectamine 2000; Invitrogen). WST-1 assay was performed on indicated times after transfection, as described. 
TUNEL Assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed with the In Situ Cell Death Detection Kit (Roche Applied Science) according to the manufacturer's instructions. Briefly, ARPE-19 cells were transfected with miR-34a mimic or a negative control (NC). Four days after transfection, cells were washed three times with PBS, fixed with 4% paraformaldehyde in PBS for 1 hour at room temperature, and permeabilized with freshly prepared 0.1% Triton X-100 for 2 minutes on ice. Fifty microliters TUNEL reaction mixtures were added into each sample and incubated for 1 hour at 37°C in a humidified atmosphere in dark. Cells were further incubated with DAPI for 10 minutes and visualized through immunofluorescent microscopy. As positive control, ARPE-19 cells were treated with 1 mM H2O2 for 2 hours and then subjected to TUNEL assay. 
Transwell Migration Assays
ARPE-19 cells were grown in DMEM-F12 containing 10% FBS to approximately 60% confluence and transfected with 100 nM miR-34a mimic molecule, a negative control or mock transfection. After 48 hours, the cells were harvested by trypsinization and washed once with D-Hanks solution (Invitrogen). To measure cell migration, 8-μm pore size culture inserts (Transwell; Costar) were placed into the wells of 24-well culture plates, separating the upper and the lower chambers. In the lower chamber, 400 μL DMEM-F12 containing 10% FBS were added. Then, 5 × 104 cells were added to the upper chamber. After 20 hours of incubation at 37°C with 5% CO2, the cells that had migrated through the pores were stained with crystal violet and five vision fields were counted under the microscope (Zeiss) using a 20× objective. 
In Vitro Scratch Assay
ARPE-19 cells were grown in DMEM-F12 containing 10% FBS to approximately 60% confluence and transfected with 100 nM miR-34a molecule, a negative control, or mock transfection. After 48 hours, the cell monolayers were scratched using a 200-μL pipette tip, washed twice with Hanks medium to remove the floating cells, then cultured in 2-mL fresh serum free medium. Photographs were taken immediately after scratching and at 24 or 48 hours after culture (Imager Z1; Zeiss). The ability of migration was evaluated by comparing the cell migration rate toward the center of the gap of four vision fields in every group. 
Western Blot Analysis
ARPE-19 cells (1 × 105) were seeded in 6-well plates and grown in DMEM-F12 with 10% FBS for 24 hours prior to transfection. Seventy-two hours after transfection, the cells were washed with cold PBS and subjected to lysis in a lysis buffer (50 mM Tris-Cl, 1 mM EDTA, 20 g/L sodium dodecyl sulfate [SDS], 5 mM dithiothreitol, and 10 mM phenylmethylsulfonyl fluoride). Equal amounts of protein lysates (20 μg each) and rainbow molecular weight markers (GE Healthcare Life Sciences, Piscataway, NJ) were added, followed by separation using 8% SDS-PAGE, then electrotransferred onto polyvinylidene difluoride membranes. The membranes were blocked with a buffer containing 5% nonfat milk in PBS with 0.05% Tween-20 for 2 hours and incubated overnight with antibody at 4°C. After a second wash with PBS containing 0.05% Tween-20, the membranes were incubated with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and developed with an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). Beta-actin was used as a loading control. Antibodies for total CDK2, CDK4, CDK6, p-Cdc2 (threonine 161), E2F1, and c-Met were from Cell Signaling Technology (Beverly, MA). Caspase-3 and β-actin were from Santa Cruz Biotechnology. All primary antibodies were used at a dilution of 1:1000, whereas the secondary antibodies were used at a dilution of 1:5000. 
Statistical Analysis
All data were shown as the mean ± SEM. Differences between experimental groups and mock groups were analyzed using the Student's t-test. Statistical significance was accepted at P less than 0.05. 
Results
miR-34a Expression Was Downregulated in Subconfluent ARPE-19 Cells
Retinal pigment epithelial ARPE-19 cells were cultured for 4 weeks after they reached confluence to form a monolayer to simulate the quiescent RPE cells in situ as reported previously. 15 Compared with subconfluent cells, ARPE-19 postconfluent cells rarely displayed Ki67 staining, which is associated with cellular proliferation 16 (Fig. 1A). The expression level of miR-34a in subconfluent ARPE-19 cells was significantly decreased when compared with postconfluent ARPE-19 cells (80.8 ± 6.3%, n = 3, Fig. 1B). These data indicated that miR-34a may be involved in the regulation of RPE cell proliferation and migration. 
Figure 1. 
 
MicroRNA-34a expression was downregulated in subconfluent ARPE-19 cells. ARPE-19 cells were cultured under normal conditions (subconfluent) or 4 weeks after they reach confluence (postconfluent). (A) The proliferation status was analyzed with phase-contrast image Ki67 staining. Images represent those obtained in three independent experiments (magnification, ×100). (B) The expression of miR-34a and U6 snRNA was analyzed with quantitative real-time PCR. The data were normalized to the level of miR-34a/U6 in postconfluent state. Results are expressed as mean ± SEM. n = 3, *P < 0.01.
Figure 1. 
 
MicroRNA-34a expression was downregulated in subconfluent ARPE-19 cells. ARPE-19 cells were cultured under normal conditions (subconfluent) or 4 weeks after they reach confluence (postconfluent). (A) The proliferation status was analyzed with phase-contrast image Ki67 staining. Images represent those obtained in three independent experiments (magnification, ×100). (B) The expression of miR-34a and U6 snRNA was analyzed with quantitative real-time PCR. The data were normalized to the level of miR-34a/U6 in postconfluent state. Results are expressed as mean ± SEM. n = 3, *P < 0.01.
miR-34a Inhibited Proliferation and Migration of RPE Cells
As miR-34a expression was decreased in subconfluent ARPE-19 cells, we sought to determine whether the introduction of miR-34a had any biological effect on RPE cells. ARPE-19 cells transfected with the miR-34a mimic showed inhibition of cell growth as compared with negative control based on the WST-1 assay (Fig. 2A). A reduction in absorbance was detected by day 3 after miR-34a transfection. At day 5, the absorbance decreased 36.7 ± 6.5% in miR-34a transfected ARPE-19 cells compared with mock group (n = 3, P < 0.01, Fig. 2A). 
Figure 2. 
 
Transfection of miR-34a inhibited the proliferation and migration of ARPE-19 cells without triggering apoptosis. (A) WST-1 cell proliferation assay was performed at indicated days after transfection with either 100 nM miR-34a mimic or a negative control (NC). Data at each time point were expressed as mean ± SEM based on results obtained from triplicates. Results represent those obtained in three independent experiments. Transwell (B) and in vitro scratch assays (D) were performed to evaluate the migration potential of ARPE-19 cells. (C) The number of cells migrated in Transwell assay was quantified by counting five independent vision fields with a 20× microscope objective. Results were expressed as mean ± SEM (n = 3, *P < 0.05). Western blot assay of caspase-3 activation (E) and TUNEL assay (F) were performed 4 days after transfection with either 100 nM miR-34a mimic or NC. H2O2-treated cells were used as positive control. All images are representative of at least three independent experiments. Magnification is ×200 for transwell assay and ×100 for both in vitro scratch and TUNEL assays.
Figure 2. 
 
Transfection of miR-34a inhibited the proliferation and migration of ARPE-19 cells without triggering apoptosis. (A) WST-1 cell proliferation assay was performed at indicated days after transfection with either 100 nM miR-34a mimic or a negative control (NC). Data at each time point were expressed as mean ± SEM based on results obtained from triplicates. Results represent those obtained in three independent experiments. Transwell (B) and in vitro scratch assays (D) were performed to evaluate the migration potential of ARPE-19 cells. (C) The number of cells migrated in Transwell assay was quantified by counting five independent vision fields with a 20× microscope objective. Results were expressed as mean ± SEM (n = 3, *P < 0.05). Western blot assay of caspase-3 activation (E) and TUNEL assay (F) were performed 4 days after transfection with either 100 nM miR-34a mimic or NC. H2O2-treated cells were used as positive control. All images are representative of at least three independent experiments. Magnification is ×200 for transwell assay and ×100 for both in vitro scratch and TUNEL assays.
We then performed transwell assay and in vitro scratch assay to determine whether miR-34a was involved in the regulation of migration of RPE cells. A dramatic reduction of migration toward the lower chambers was observed in miR-34a transfected ARPE-19 cells (Mock: 82 ± 10, NC: 75 ± 14, miR-34a: 30 ± 10 cells/vision field, n = 3, Figs. 2B, 2C). Similar results were observed in the in vitro scratch assay (Fig. 2D). 
To determine whether the growth retardation of ARPE-19 cells is caused by apoptosis after miR-34a transfection, caspase-3 activation assay and TUNEL assay were carried out. Western blot results showed that miR-34a transfection had no effect on procaspase 3, whereas H2O2 treatment can cause extensive procaspase 3 activation (Fig. 2E). In TUNEL assay, ARPE-19 cells transfected with miR-34a were negative similar to both mock and negative control group. H2O2-treated cells appeared TUNEL positive with strong staining (Fig. 2F). These results indicate that miR-34a inhibits the proliferation and migration of ARPE-19 cells without triggering apoptosis. 
Introduction of miR-34a Downregulated c-Met Expression and Other Cell Cycle–Related Proteins
c-Met is a direct target of miR-34a. 13 Western blot analysis confirmed that c-Met expression was reduced by miR-34a transfection in ARPE-19 cells (Fig. 3). Concurrently, ectopic miR-34a delivery also downregulated the expression of CDK2, CDK4, CDK6, p-Cdc2, and E2F1 (Fig. 3). 
Figure 3. 
 
Introduction of miR-34a downregulated c-Met expression and other cell cycle–related proteins. ARPE-19 cells were transfected with either miR-34a or a negative control. Cell lysates were prepared and used for Western blot analysis of c-Met, CDK2, CDK4, CDK6, E2F1, and p-Cdc2. Beta-actin was used as a loading control. The band intensity was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD), and the fold change was normalized to the level of mock group. These results are representatives of at least three independent experiments.
Figure 3. 
 
Introduction of miR-34a downregulated c-Met expression and other cell cycle–related proteins. ARPE-19 cells were transfected with either miR-34a or a negative control. Cell lysates were prepared and used for Western blot analysis of c-Met, CDK2, CDK4, CDK6, E2F1, and p-Cdc2. Beta-actin was used as a loading control. The band intensity was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD), and the fold change was normalized to the level of mock group. These results are representatives of at least three independent experiments.
Downregulation of c-Met Inhibited RPE Cell Proliferation and Migration
To determine whether miR-34a inhibits RPE cell proliferation and migration through downregulation of c-Met, we altered the expression level of c-Met with specific siRNA in ARPE-19 cells (Fig. 4A). WST-1 assays showed that transfection of c-Met siRNA significantly inhibited growth of ARPE-19 cells from day 3, and the absorbance decreased 52.9 ± 5.3% (n = 3, P < 0.01, Fig. 4B) at day 5 compared with mock cells. Furthermore, migration of ARPE-19 cells transfected with miR-34a was affected after c-Met specific siRNA introduction. In both transwell (Figs. 4C, 4D) and in vitro scratch assays (Fig. 4E), c-Met downregulation effectively hampered the migration rate of ARPE-19 cells (in transwell assay, Mock: 171 ± 24, NC: 162 ± 23, c-Met: 67 ± 20 cells/vision field, n = 3, P < 0.05, Fig. 4D). 
Figure 4. 
 
Downregulation of c-Met inhibited RPE cell proliferation and migration. ARPE-19 cells were transfected with either miR-34a or a scrambled negative control. (A) The knockdown effect of c-Met was detected by Western blot. (B) WST-1 cell proliferation assay was performed to determine the effect of c-Met specific siRNA on the proliferation of ARPE-19 cells. Data at each time point were expressed as mean ± SEM of the results obtained from triplicates in one experiment. Results represent those obtained in three independent experiments. Transwell (C) and in vitro scratch assays (E) were performed to evaluate the migration potential of ARPE-19 cells after transfection with either c-Met specific siRNA or a scrambled negative control. (D) The number of cells migrated in Transwell assay was quantified by counting five independent vision fields with a 20× microscope objective. Results were expressed as mean ± SEM. (n = 3, *P < 0.05). All images are representative of at least three independent experiments. Magnification is ×200 for transwell assay and ×100 for scratch assay.
Figure 4. 
 
Downregulation of c-Met inhibited RPE cell proliferation and migration. ARPE-19 cells were transfected with either miR-34a or a scrambled negative control. (A) The knockdown effect of c-Met was detected by Western blot. (B) WST-1 cell proliferation assay was performed to determine the effect of c-Met specific siRNA on the proliferation of ARPE-19 cells. Data at each time point were expressed as mean ± SEM of the results obtained from triplicates in one experiment. Results represent those obtained in three independent experiments. Transwell (C) and in vitro scratch assays (E) were performed to evaluate the migration potential of ARPE-19 cells after transfection with either c-Met specific siRNA or a scrambled negative control. (D) The number of cells migrated in Transwell assay was quantified by counting five independent vision fields with a 20× microscope objective. Results were expressed as mean ± SEM. (n = 3, *P < 0.05). All images are representative of at least three independent experiments. Magnification is ×200 for transwell assay and ×100 for scratch assay.
Knockdown of c-Met Downregulated the Expression of Cell Cycle–Related Molecules
We further explored if direct knockdown of c-Met had similar effect on the expression of its downstream molecules. Western blot analysis showed that the expression of CDK2, CDK6, phosphorylation of Cdc2, and E2F1 was significantly decreased by c-Met siRNA transfection compared with mock ARPE-19 cells, whereas the expression of CDK4 was unaffected (Fig. 5). 
Figure 5. 
 
Direct knockdown of c-Met downregulated the expression of cell cycle–related molecules. After transfection with either c-Met specific siRNA or a negative control, ARPE-19 cell lysates were prepared and probed with CDK2, CDK4, CDK6, p-Cdc2, and E2F1 antibodies. Beta-actin was used as a loading control. The band intensity was analyzed with ImageJ software, and the fold change was normalized to the level of mock cells. These results are representative of at least three independent experiments.
Figure 5. 
 
Direct knockdown of c-Met downregulated the expression of cell cycle–related molecules. After transfection with either c-Met specific siRNA or a negative control, ARPE-19 cell lysates were prepared and probed with CDK2, CDK4, CDK6, p-Cdc2, and E2F1 antibodies. Beta-actin was used as a loading control. The band intensity was analyzed with ImageJ software, and the fold change was normalized to the level of mock cells. These results are representative of at least three independent experiments.
Discussion
The retinal pigment epithelium is a cellular monolayer sandwiched between the vessels of the choriocapillaris and light-sensitive outer segments of the photoreceptors. Embryologically, RPE development coincides with the maturation of photoreceptors. RPE cells secretes factors that promote photoreceptor differentiation, which in turn, guides RPE maturation. 3 The proper development of the retina is dependent on the melanogenesis pathway of the RPE. 17 Potential RPE cells derived from neuroectodermal tissue are dependent on the expression of transcription factors homeodomain-containing transcription factor (OTX2) and MITF. 3 With the onset of melanogenesis, neuroectodermal cells differentiate into the RPE layer. 17 Being a part of the blood–retina barrier, the RPE serves multiple functions, including absorption of light energy, transportation of ions, water, and metabolic products, and reisomerization of transretinal to 11-cis-retinal to support the photoreceptors. 3 Therefore, RPE plays essential roles for visual function. 3 However, the potential for RPE cells to transition from a quiescent to a proliferative state can result in pathologic conditions. 3  
Proliferative vitreoretinopathy is a process involving scarring that develops following cases of retinal detachments, and is the most common cause of surgical failure. 2 The physiologic basis of PVR has been described. One of the most important cells involved are RPE cells, which are present in almost 100% of epiretinal membranes. 18 Uncontrolled proliferation and migration of RPE cells eventually form pathologic membranes on both surfaces of the neural retina that can contract and cause retinal detachment and visual impairment. 2 Wu et al. showed that the clinical outcome of patients after surgery is related to the presence of viable or active RPE cells. 19 The underlying mechanisms related to the RPE cell transition from a quiescent state to a proliferative and migratory state remain unclear. 
MicroRNAs affect essential biological processes such as metabolism, cell proliferation, migration, differentiation, and development through negatively regulating the translation and stability of their target mRNAs. 20 Although RPE cells express many miRNAs, only a few miRNAs have been explored in terms of their effects. 20 Studies have looked into the expression of miR-328 in RPE cells and found that treatment with retinoic acid led to increased expression of miR-328. 10 This also leads to increased RPE cell proliferation through direct downregulation of PAX6. 10 Others showed that miR-204/211 directly regulated the differentiation of RPE cells, with increased expression of miR-204/211 leading to differentiation and vice versa. 9 MicroRNA-34a is among the most well defined miRNAs, being a direct target of p53. 21 It displayed an inhibitory effect on tumors' aggressive biological behaviors in various types of cancers. 11 In this study, we tried to address if miR‐34a participated in the regulation of RPE functions. Analysis of expression of miR-34a in subconfluent and postconfluent ARPE-19 cells revealed that miR-34a levels were significantly downregulated in subconfluent cells compared with postconfluent cells. Introduction of miR-34a mimic into subconfluent ARPE-19 cells profoundly inhibited cell proliferation and migration without triggering obvious apoptosis. These results suggest that miR-34a may be involved in the regulation of migration and proliferation of RPE cells. 
Among those direct targets of miR-34a, c-Met is known as a hepatocyte growth factor (HGF) receptor and in turn participates in cell growth, migration, and the regenerative process. 22 HGF/c-Met signal pathway has been shown to be involved in RPE proliferation and migration. 23,24 Using transgenic models, Kasaoka et al. found that expression of HGF and c-Met was induced by laser injury. 22 Constitutive activation of c-Met induced robust RPE migration into the outer retina of laser-injured eyes, whereas c-Met conditional knockout reduced RPE migration. 22 To our knowledge, there are no reports about the effects of c-Met knockdown in ARPE-19 cell in vitro. Using a c-Met knockdown approach, we demonstrated that miR-34a is involved in the regulation of ARPE-19 cell proliferation and migration through c-Met. In addition, we found direct c-Met knockdown decreased the expression of E2F1, CDK2, CDK6, and p-Cdc2, whereas the level of CDK4 was unaffected. As CDK4 has been proven to be a direct target of miR-34a, 25 this suggests that miR-34a mainly targeted c-Met to regulate the proliferation and migration of RPE cells. 
Besides c-Met, other cell cycle–related molecules, including E2F1, CDK2, CDK4, CDK6, and p-Cdc2, were downregulated in miR-34a transfected RPE cells. These molecules have been reported to be directly targeted or indirectly regulated by miR-34a. 13,26,27 Specifically, E2F1 is widely viewed as an essential positive cell cycle regulator with expression of E2F1 capable of transitioning quiescent cells from G1 to the S phase. 28 CDK2, CDK4, and CDK6 are all members of the CDK family, which are essential for the G1-S transition. 29 In TGF-β1 treated human RPE cells, the expression levels of CDK2 and CDK4 were both decreased. 30 The use of antiangiogenic antibody, bevacizumab, can also inhibit the proliferation of ARPE-19 cells through downregulation of CDK2, CDK4, CDK6, and cyclin D and E. 31  
MicroRNAs are believed to have potential therapeutic roles in many diseases. Although the miRNA expression profiles in RPE cells have been studied, 7,8 there are few reports about miRNAs in the regulation of RPE functions. Therefore, we investigated the role of miRNAs in RPE cells and their related mechanisms. Our results suggest miR-34a participated in the regulation of proliferation and migration of RPE cells, and likely has important clinical consequences in the treatment of retinal diseases. 
Acknowledgments
Supported by National Natural Science Foundation of China Grants 81100671 and 81201657, Zhejiang Provincial Natural Science Foundation of China Grant Y2110609, and Wenzhou Science & Technology Bureau Grant Y20080096. 
Disclosure: Q. Hou, None; J. Tang, None; Z. Wang, None; C. Wang, None; X. Chen, None; L. Hou, None; X.D. Dong, None; L. Tu, None 
References
He S Kumar SR Zhou P Soluble EphB4 inhibition of PDGF-induced RPE migration in vitro. Invest Ophthalmol Vis Sci . 2010; 51: 543– 552. [CrossRef] [PubMed]
Pastor JC de la Rua ER Martin F. Proliferative vitreoretinopathy: risk factors and pathobiology. Prog Retin Eye Res . 2002; 21: 127– 144. [CrossRef] [PubMed]
Strauss O. The retinal pigment epithelium in visual function. Physiol Rev . 2005; 85: 845– 881. [CrossRef] [PubMed]
Gommans WM Berezikov E. Controlling miRNA regulation in disease. Methods Mol Biol . 2012; 822: 1– 18. [PubMed]
He L Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet . 2004; 5: 522– 531. [CrossRef] [PubMed]
Lee RC Feinbaum RL Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell . 1993; 75: 843– 854. [CrossRef] [PubMed]
Li WB Zhang YS Lu ZY Development of retinal pigment epithelium from human parthenogenetic embryonic stem cells and microRNA signature. Invest Ophthalmol Vis Sci . 2012; 53: 5334– 5343. [CrossRef] [PubMed]
Kutty RK Samuel W Jaworski C MicroRNA expression in human retinal pigment epithelial (ARPE-19) cells: increased expression of microRNA-9 by N-(4-hydroxyphenyl)retinamide. Mol Vis . 2010; 16: 1475– 1486. [PubMed]
Adijanto J Castorino JJ Wang ZX Maminishkis A Grunwald GB Philp NJ. Microphthalmia-associated transcription factor (MITF) promotes differentiation of human retinal pigment epithelium (RPE) by regulating microRNAs-204/211 expression. J Biol Chem . 2012; 287: 20491– 20503. [CrossRef] [PubMed]
Chen KC Hsi E Hu CY Chou WW Liang CL Juo SH. MicroRNA-328 may influence myopia development by mediating the PAX6 gene. Invest Ophthalmol Vis Sci . 2012; 53: 2732– 2739. [CrossRef] [PubMed]
Hermeking H. The miR-34 family in cancer and apoptosis. Cell Death Differ . 2010; 17: 193– 199. [CrossRef] [PubMed]
Li L Yuan L Luo J Gao J Guo J Xie X. MiR-34a inhibits proliferation and migration of breast cancer through down-regulation of Bcl-2 and SIRT1. Clin Exp Med . 2013; 13: 109– 117. [CrossRef] [PubMed]
Yan D Zhou X Chen X MicroRNA-34a inhibits uveal melanoma cell proliferation and migration through downregulation of c-Met. Invest Ophthalmol Vis Sci . 2009; 50: 1559– 1565. [CrossRef] [PubMed]
Hu G Huang K Yu J Identification of miRNA signatures during the differentiation of hESCs into retinal pigment epithelial cells. PLoS One . 2012; 7: e37224. [CrossRef] [PubMed]
Chang JH Kang SW Ham DI. Sensitivity of CD95-induced apoptosis in different proliferative status of human retinal pigment epithelial cells. Korean J Ophthalmol . 2001; 15: 74– 80. [CrossRef] [PubMed]
Scholzen T Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol . 2000; 182: 311– 322. [CrossRef] [PubMed]
Jeffery G. The retinal pigment epithelium as a developmental regulator of the neural retina. Eye . 1998; 12: 499– 503. [CrossRef] [PubMed]
Casaroli-Marano RP Pagan R Vilaro S. Epithelial-mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells. Invest Ophthalmol Vis Sci . 1999; 40: 2062– 2072. [PubMed]
Wu WC Kao YH Hu DN. Relationship between outcome of proliferative vitreoretinopathy and results of tissue culture of excised preretinal membranes. Kaohsiung J Med Sci . 2000; 16: 614– 619. [PubMed]
Yan D Dong XD Chen X Role of microRNA-182 in posterior uveal melanoma: regulation of tumor development through MITF, BCL2 and cyclin D2. PLoS One . 2012; 7: e40967. [CrossRef] [PubMed]
Tan J Fan L Mao JJ Restoration of miR-34a in p53 deficient cells unexpectedly promotes the cell survival by increasing NFkappaB activity. J Cell Biochem . 2012; 113: 2903– 2908. [CrossRef] [PubMed]
Kasaoka M Ma J. Lashkari K. c-Met modulates RPE migratory response to laser-induced retinal injury. PLoS One . 2012; 7: e40771. [CrossRef] [PubMed]
Xu KP Yu FS. Cross talk between c-Met and epidermal growth factor receptor during retinal pigment epithelial wound healing. Invest Ophthalmol Vis Sci . 2007; 48: 2242– 2248. [CrossRef] [PubMed]
Jun EJ Kim HS Kim YH. Role of HGF/c-Met in serum-starved ARPE-19 cells. Korean J Ophthalmol . 2007; 21: 244– 250. [CrossRef] [PubMed]
Navarro F Gutman D Meire E miR-34a contributes to megakaryocytic differentiation of K562 cells independently of p53. Blood . 2009; 114: 2181– 2192. [CrossRef] [PubMed]
Zauli G Voltan R di Iasio MG miR-34a induces the downregulation of both E2F1 and B-Myb oncogenes in leukemic cells. Clin Cancer Res . 2011; 17: 2712– 2724. [CrossRef] [PubMed]
Sun F Fu H Liu Q Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Lett . 2008; 582: 1564– 1568. [CrossRef] [PubMed]
Johnson DG Schwarz JK Cress WD Nevins JR. Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature . 1993; 365: 349– 352. [CrossRef] [PubMed]
Nebreda AR. CDK activation by non-cyclin proteins. Curr Opin Cell Biol . 2006; 18: 192– 198. [CrossRef] [PubMed]
Pillaire MJ Casagrande F Malecaze F Manenti S Darbon JM. Regulation by transforming growth factor-beta 1 of G1 cyclin-dependent kinases in human retinal epithelial cells. Exp Eye Res . 1999; 68: 193– 199. [CrossRef] [PubMed]
Kuo CN Chen CY Lai CH Cell cycle regulation by bevacizumab in ARPE-19 human retinal pigment epithelial cells. Mol Med Rep . 2012; 6: 701– 704. [PubMed]
Figure 1. 
 
MicroRNA-34a expression was downregulated in subconfluent ARPE-19 cells. ARPE-19 cells were cultured under normal conditions (subconfluent) or 4 weeks after they reach confluence (postconfluent). (A) The proliferation status was analyzed with phase-contrast image Ki67 staining. Images represent those obtained in three independent experiments (magnification, ×100). (B) The expression of miR-34a and U6 snRNA was analyzed with quantitative real-time PCR. The data were normalized to the level of miR-34a/U6 in postconfluent state. Results are expressed as mean ± SEM. n = 3, *P < 0.01.
Figure 1. 
 
MicroRNA-34a expression was downregulated in subconfluent ARPE-19 cells. ARPE-19 cells were cultured under normal conditions (subconfluent) or 4 weeks after they reach confluence (postconfluent). (A) The proliferation status was analyzed with phase-contrast image Ki67 staining. Images represent those obtained in three independent experiments (magnification, ×100). (B) The expression of miR-34a and U6 snRNA was analyzed with quantitative real-time PCR. The data were normalized to the level of miR-34a/U6 in postconfluent state. Results are expressed as mean ± SEM. n = 3, *P < 0.01.
Figure 2. 
 
Transfection of miR-34a inhibited the proliferation and migration of ARPE-19 cells without triggering apoptosis. (A) WST-1 cell proliferation assay was performed at indicated days after transfection with either 100 nM miR-34a mimic or a negative control (NC). Data at each time point were expressed as mean ± SEM based on results obtained from triplicates. Results represent those obtained in three independent experiments. Transwell (B) and in vitro scratch assays (D) were performed to evaluate the migration potential of ARPE-19 cells. (C) The number of cells migrated in Transwell assay was quantified by counting five independent vision fields with a 20× microscope objective. Results were expressed as mean ± SEM (n = 3, *P < 0.05). Western blot assay of caspase-3 activation (E) and TUNEL assay (F) were performed 4 days after transfection with either 100 nM miR-34a mimic or NC. H2O2-treated cells were used as positive control. All images are representative of at least three independent experiments. Magnification is ×200 for transwell assay and ×100 for both in vitro scratch and TUNEL assays.
Figure 2. 
 
Transfection of miR-34a inhibited the proliferation and migration of ARPE-19 cells without triggering apoptosis. (A) WST-1 cell proliferation assay was performed at indicated days after transfection with either 100 nM miR-34a mimic or a negative control (NC). Data at each time point were expressed as mean ± SEM based on results obtained from triplicates. Results represent those obtained in three independent experiments. Transwell (B) and in vitro scratch assays (D) were performed to evaluate the migration potential of ARPE-19 cells. (C) The number of cells migrated in Transwell assay was quantified by counting five independent vision fields with a 20× microscope objective. Results were expressed as mean ± SEM (n = 3, *P < 0.05). Western blot assay of caspase-3 activation (E) and TUNEL assay (F) were performed 4 days after transfection with either 100 nM miR-34a mimic or NC. H2O2-treated cells were used as positive control. All images are representative of at least three independent experiments. Magnification is ×200 for transwell assay and ×100 for both in vitro scratch and TUNEL assays.
Figure 3. 
 
Introduction of miR-34a downregulated c-Met expression and other cell cycle–related proteins. ARPE-19 cells were transfected with either miR-34a or a negative control. Cell lysates were prepared and used for Western blot analysis of c-Met, CDK2, CDK4, CDK6, E2F1, and p-Cdc2. Beta-actin was used as a loading control. The band intensity was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD), and the fold change was normalized to the level of mock group. These results are representatives of at least three independent experiments.
Figure 3. 
 
Introduction of miR-34a downregulated c-Met expression and other cell cycle–related proteins. ARPE-19 cells were transfected with either miR-34a or a negative control. Cell lysates were prepared and used for Western blot analysis of c-Met, CDK2, CDK4, CDK6, E2F1, and p-Cdc2. Beta-actin was used as a loading control. The band intensity was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD), and the fold change was normalized to the level of mock group. These results are representatives of at least three independent experiments.
Figure 4. 
 
Downregulation of c-Met inhibited RPE cell proliferation and migration. ARPE-19 cells were transfected with either miR-34a or a scrambled negative control. (A) The knockdown effect of c-Met was detected by Western blot. (B) WST-1 cell proliferation assay was performed to determine the effect of c-Met specific siRNA on the proliferation of ARPE-19 cells. Data at each time point were expressed as mean ± SEM of the results obtained from triplicates in one experiment. Results represent those obtained in three independent experiments. Transwell (C) and in vitro scratch assays (E) were performed to evaluate the migration potential of ARPE-19 cells after transfection with either c-Met specific siRNA or a scrambled negative control. (D) The number of cells migrated in Transwell assay was quantified by counting five independent vision fields with a 20× microscope objective. Results were expressed as mean ± SEM. (n = 3, *P < 0.05). All images are representative of at least three independent experiments. Magnification is ×200 for transwell assay and ×100 for scratch assay.
Figure 4. 
 
Downregulation of c-Met inhibited RPE cell proliferation and migration. ARPE-19 cells were transfected with either miR-34a or a scrambled negative control. (A) The knockdown effect of c-Met was detected by Western blot. (B) WST-1 cell proliferation assay was performed to determine the effect of c-Met specific siRNA on the proliferation of ARPE-19 cells. Data at each time point were expressed as mean ± SEM of the results obtained from triplicates in one experiment. Results represent those obtained in three independent experiments. Transwell (C) and in vitro scratch assays (E) were performed to evaluate the migration potential of ARPE-19 cells after transfection with either c-Met specific siRNA or a scrambled negative control. (D) The number of cells migrated in Transwell assay was quantified by counting five independent vision fields with a 20× microscope objective. Results were expressed as mean ± SEM. (n = 3, *P < 0.05). All images are representative of at least three independent experiments. Magnification is ×200 for transwell assay and ×100 for scratch assay.
Figure 5. 
 
Direct knockdown of c-Met downregulated the expression of cell cycle–related molecules. After transfection with either c-Met specific siRNA or a negative control, ARPE-19 cell lysates were prepared and probed with CDK2, CDK4, CDK6, p-Cdc2, and E2F1 antibodies. Beta-actin was used as a loading control. The band intensity was analyzed with ImageJ software, and the fold change was normalized to the level of mock cells. These results are representative of at least three independent experiments.
Figure 5. 
 
Direct knockdown of c-Met downregulated the expression of cell cycle–related molecules. After transfection with either c-Met specific siRNA or a negative control, ARPE-19 cell lysates were prepared and probed with CDK2, CDK4, CDK6, p-Cdc2, and E2F1 antibodies. Beta-actin was used as a loading control. The band intensity was analyzed with ImageJ software, and the fold change was normalized to the level of mock cells. These results are representative of at least three independent experiments.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×