Graefes Arch Clin Exp Ophthalmol DOI 10.1007/s00417-014-2901-2

BASIC SCIENCE

Inhibition of cell proliferation and migration after HTRA1 knockdown in retinal pigment epithelial cells Xueting Pei & Kai Ma & Jun Xu & Ningli Wang & Ningpu Liu

Received: 20 August 2014 / Revised: 6 December 2014 / Accepted: 15 December 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Purpose The purpose of this study was to investigate the role of HtrA serine peptidase 1 (HTRA1) in the proliferation and migration of cells of the human retinal pigment epithelial cell line ARPE-19, and the possible mechanisms involved. Methods ARPE-19 cells were transduced by a recombinant lentiviral vector carrying HTRA1-shRNA to knockdown HTRA1 expression. Subsequent HTRA1 gene and HTRA1 protein levels in these cells and control cells were detected by quantitative real-time PCR and Western blot, respectively. Changes in cell proliferation and migration associated with the inhibition of HTRA1 expression were assessed, as well as changes in the mRNA levels of transforming growth factor beta 1 (TGFB1), bone morphogenetic protein 4 (BMP4), and bone morphogenetic protein 2 (BMP2). Results The recombinant lentivirus carrying HTRA1-shRNA was successfully generated, as evidenced by reduced levels of HTRA1 mRNA and HTRA1 protein in ARPE-19 cells. The knockdown of HTRA1 in ARPE-19 cells was associated with reduced cellular proliferation and migration, and increased mRNA levels of TGF-β1, BMP4, and BMP2. Conclusions Silence of the HTRA1 gene was associated with significantly higher levels of TGF-β1, BMP4, and BMP2 mRNA and reduction in the proliferation and migration of ARPE-19 cells.

Keywords Age-related macular degeneration . Retinal pigment epithelium degeneration . HTRA1 . ARPE-19

Introduction Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the elderly [1–3]. Retinal pigment epithelium (RPE) degeneration is an important feature of AMD. The RPE is primarily responsible for maintenance of the neurosensory retina, and the etiology of AMD is associated with structural and functional abnormalities of the RPE [4–6]. Multiple environmental and genetic risk factors contribute to the incidence of AMD [7–11]. Genome-wide studies have shown that the closely linked genes ARMS2 and HTRA1 at a locus on chromosome 10q26 are strongly associated with susceptibility to AMD [12, 13], with HTRA1 being the putative gene responsible for the pathogenesis of AMD [14]. HTRA1 is a secreted protease that regulates signaling of the transforming growth factor-beta (TGFβ) protein family [15]. RNA interference (RNAi) is widely used to inhibit specific gene expression and to investigate the roles of target genes in a variety of biological processes [16]. The lentiviral vector has been successfully used to transport RNAi into many kinds of cells [17], making it a very useful tool for silencing genes. In the present study, we transduced RPE cells with a lentiviral vector carrying a small hairpin RNA (shRNA) targeting HTRA1, to examine whether the in vitro inhibition of HTRA1 alters the mRNA expressions of TGFβ family members and the proliferation and migration of RPE cells.

Materials and methods X. Pei (*) : K. Ma : J. Xu : N. Wang : N. Liu Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Science Key Laboratory, No. 1 Dongjiaominxiang, Dongcheng District, Beijing, China e-mail: [email protected]

Design and construction of HTRA1 shRNA-expressing lentivirus The pFU-GW-RNAi plasmid (Genechem, Shanghai, China) was used to generate the lentivirus expressing HTRA1-

Graefes Arch Clin Exp Ophthalmol

shRNA. The following sequence from GenBank NM_002775 was chosen for the RNAi-targeting sequence: 5′-CAA CAG TTT GCG CCA TAA A-3′. The sequence of the negative control is 5′-TTC TCC GAA CGT GTC ACG T-3′. For each targeting sequence, two oligonucleotides were synthesized as follows: sense 5′-CCG GCA ACA GTT TGC GCC ATA AAC TCG AGT TTA TGG CGC AAA CTG TTG TTT TTG-3′, and antisense 5′-AAT TCA AAA ACA ACA GTT TGC GCC ATA AAC TCG AGT TTA TGG CGC AAA CTG TTG-3′. The two oligonucleotides were resolved together in water at 90 °C for 15 min and then placed at room temperature to cool and form double-stranded DNA. The double-stranded DNAs were ligated into the pFU-GW-RNAi plasmid and digested with AgeI and EcoRI restriction endonucleases (New England Biolabs, USA) to generate the HTRA1shRNA lentiviral vectors: LV-green fluorescent protein (GFP)-HTRA1-shRNA, and the normal control (NC) LVGFP-HTRA1-shRNA-NC. All constructs were verified by nucleotide sequencing. Recombinant lentiviruses were produced by cotransfecting 293 T cells with the recombinant lentiviral vector and packaging plasmids (pHelper 1.0 including gag/pol and pHelper 2.0 including VSVG) using the cationic lipid complex method (Lipofectamine 2000; Invitrogen, Carlsbad, CA, USA). The culture supernatants containing the tagged viruses were harvested 48 h after transfection, and concentrated by centrifugation at 4000×g at 4 °C for 10–15 min. Aliquots of the concentrated viruses were stored at −80 °C for subsequent use. The infectious titer was determined with 293 T cells and real-time quantitative PCR. Cell culture and lentivirus transfection Cells of the human ARPE-19 cell line were purchased from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium, Ham’s F-12 nutrient mixture (DMEM/F12) supplemented with 10 % fetal bovine serum (FBS, Gibco/Invitrogen, NY), and antibiotics (100 U/mL penicillin and 100 U/mL streptomycin, Sigma, MO) at 37 °C in 5 % CO2 and 95 % humidity. Cells were divided into three groups: non-transfected blank control (CON), transduced with a negative control lentiviral vector (NC), and transduced with an RNAi-HTRA1 lentiviral vector (RNAi-HTRA1). When the ARPE-19 cells were approximately 50 % confluent in 5 % FBS medium, the cells were transduced with specific or negative control lentiviral vectors, at a multiplicity of infection (MOI) of 5. The cells were further cultured in DMEM/F12 with 10 % FBS after transduction for 8 h and then selected using 200 mm/mL puromycin. The stable shRNA inhibitor lines were established when more than 90 % of the transduced cells were found to strongly express GFP under fluorescent microscopy. Seventy-two hours after lentiviral transduction, the cells were harvested for

determining the efficiency of HTRA1 silencing by quantitative real-time PCR. RNA isolation and real-time RT-PCR The total RNA was isolated from RPE cells using TRIzol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. RNA extract (2 μL) was reverse transcribed into cDNA in a total reaction volume of 20 μL using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada). Real-time quantitative PCR was performed using the ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA), with 25-μL reaction mixtures containing 1 μL cDNA, 9.5 μL sterilized water, 13.5 μL SYBR Green Real-time PCR Master Mix (TaKaRa, Japan), and 1 μL of primer (10 μM). The PCR reactive conditions were: Actb, 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C 30 s; HTRA1, 35 cycles of 94 °C for 30 s, 51 °C for 30 s, and 72 °C for 30 s; transforming growth factor beta 1 (TGFB1), 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; bone morphogenetic protein 2 (BMP2), 35 cycles of 94 °C for 30 s, 49 °C for 30 s, and 72 °C 30 s; and bone morphogenetic protein 4 (BMP4), 35 cycles of 94 °C for 30 s, 53 °C 30 s, and 72 °C 30 s. The amplification signals of HTRA1, TGF-β1, BMP2, and BMP4 were normalized to β-actin expression and evaluated using the equation: fold change=2–ΔΔCt. The primer sequences used in this study were: HTRA1, forward 5′-AAT GGA CAG CGA CAG ACA GA-3′, reverse 5′-CGG TGG TGA TGT GGT GTT G 3′; TGF-β1, forward 5′-GCC TTT CCT GCT TCT CA-3′, reverse 5′-CGG GTT ATG CTG GTT GT3′; BMP2 forward 5′-GGA ATG ACT GGA TTG TGG CT-3′, reverse 5′-TGA GTT CTG TCG GGA CAC AG-3′; BMP4, forward 5′-TAT GTG GAC TTC AGC GAT G-3′, reverse 5′GTG GTG TAT GTG GTG TGT GT-3′; and β-actin, with forward 5′-ATC ATG TTT GAG ACC TTC AAC A-3′ and reverse 5′-CAT CTC TTG CTC GAA GTC CA-3′. Protein extraction and Western blot ARPE-19 cells were washed three times with ice-cold phosphate-buffered saline (PBS, 4 °C, pH 7.4) for 5 min at room temperature and prepared using a protein extraction kit and a protease inhibitor kit (Pierce, Rockford, IL). The supernatant was collected and the protein content of each lysate was determined using a BCA Protein Assay Kit (Tianlai Shengwu Jishu, Tianlai, China) in accordance with the manufacturer’s instructions. Equal amounts of protein (40 μg) were resolved on a 12 % sodium dodecyl sulfate polyacrylamide gel and transferred onto a 0.22 mm polyvinylidene difluoride membrane (GE, Little Chalfont, UK). The primary antibodies used to probe the membranes were monoclonal anti-HTRA1 (Mouse anti-human, 1:200; R&D, Minneapolis, USA) and

Graefes Arch Clin Exp Ophthalmol

anti-β-actin (1:3000; Boster, Wuhan, China). The membranes were washed and incubated with peroxidase-conjugated secondary antibodies (goat anti-mouse, 1:3000; Boster, Wuhan, China). Enhanced Chemiluminescent Western Blot Detection Reagents (Pierce, Rockford, USA) were used to detect HTRA1 protein levels. The optical density of the target band was analyzed by Quantity One image analysis software (BioRad, CA). In vitro cell proliferation assay To determine the role of LV-GFP-HTRA1-shRNA transfection in the proliferation of ARPE-19 cells, a CCK-8 (Obio, Shanghai) assay was used to measure cell proliferation over six consecutive days. ARPE-19 cells were divided into three groups and plated at a density of 5×103 cells per well in 96well culture plates. After 16-h incubation, 10 μL CCK-8 was added, and the cells were incubated for an additional 4 h. The absorbance at 450 nm was measured using an ELISA plate reader (ZS-3, Xinfeng, Beijing, China). Each group was measured at 24, 48, 72, and 96 h post-seeding, and the experiment was performed at least three times. Cell migration Cell migration ability was analyzed by wound healing and Transwell chamber assays. For the wound healing assay, 1.5× 105 cells per well were cultured for 24 h in a 12-well plate with DMEM/F12 containing 10 % FBS. The cells were scratched with a 200-μL pipette tip, washed with Hank’s solution ≥3 times, and cultured in DMEM/F12 serum-free medium in a 5 % CO2 atmosphere at 37 °C. Photographs were taken at 12, 24, 36, and 48 h after wounding. Each experiment was repeated at ≥3 times. The Transwell chamber assay was performed using a 24well Transwell chamber (Corning 3428, New York, USA). After serum starvation for 12 h, 1×104 cells per well were seeded and cultured into the upper chamber with 150 μL of serum-free DMEM/F12 medium, and deposited into the bottom well containing 550 μL of DMEM/F12 plus 10 % FBS. After 24 and 48 h, migrated cells were fixed with 4 % paraformaldehyde at room temperature for 20 min. Migrated cells at the lower surface of the filter were stained with 100 μL of hematoxylin for about 20 min and counted under a

microscope from five random fields of view. Each experiment was repeated ≥3 times. Statistical analyses Data were expressed as mean±standard deviation. Statistical evaluations between the experimental group and control groups were performed with analysis of variance and Fisher’s least significant difference test using SPSS 16.0 software (SPSS Science, Chicago, IL). P-values less than 0.05 were considered statistically significant.

Results Silencing of HTRA1 in cultured ARPE-19 cells by shRNA-expressing virus After PCR amplification, positive clones with 1008 bp were identified. The detected sequence was identical to the known target HTRA1 shRNA sequence (Fig. 1). Total mRNA and protein extracts were prepared from ARPE-19 cells of each group. The HTRA1 mRNA and HTRA1 protein expression levels in these cells were determined by real-time RT-PCR and Western blot assays, respectively. Results of the real-time RT-PCR showed that mRNA levels of HTRA1 in the cells containing LV-GFP-HTRA1-shRNA were 28.6±2.38 % that of the blank controls, while Western blots showed that HTRA1 protein levels in cells containing LVGFP-HTRA1-shRNA were 35.2±5.41 % that of the blank controls. HTRA1 mRNA and HTRA1protein levels in ARPE19 cells transduced with LV-GFP-HTRA1-shRNA were significantly decreased when compared with these levels in the CON and NC control groups (P