KLF6SV1 siRNA inhibits proliferation of human lens epithelial cells

Reagents

KLF6sv1 was purchased from Invitrogen company (Grand Island, NY). P27 kip1 antibody and proliferation cell nuclear antigen (PCNA) was purchased from Santa cruz biotechnology Inc. (Santa Cruz, CA). Eagle’s minimal essential medium (MEM), streptomycin and penicillin were purchased from Gibco (Grand Island, NY). Fetal calf serum were purchased from Invitrogen. Hanks solution was purchased from Gibco. Poly-lysine was purchased from Sigma (St. louis, MO). Culture plates were purchased from BD Biosciences (San Jose, CA). Hybond-P polyvinylidene difluoride (PVDF) membrane was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Methylthiazolyltetrazolium (MTT) was purchased from Sigma. The MTT cell proliferation kit was purchased from the ATCC (Manassas, VA).

Cell culture of HLEC

HLEC (SRA 01/04) were purchased from the ATCC and cultured in Eagle’s minimal essential medium (MEM) supplemented with 20% fetal bovine serum and gentamicin, 100 U/ml penicillin, and 100 μg/ml streptomycin in a six-well plate at 37 °C and 5% CO₂ and 95% air. All experiments were performed with HLEC cells between passages 18 and 25 [15].

KLF6SV1 siRNA plasmid construct

Vector pSuppressorNeo is a vector used to generate biologically active siRNAs from the U6promoter. Synthetic oligonucleotide primers (5′-GAT CCC CTG GCG ATG CCT CCC CCG ACt tca aga gaG TCG GGG GAG GCA TCG CCA TTT TTG GAA A-3′ and 5′-AGC TTT TCC AAA AAT GGC GAT GCC TCC CCC GAC tct ctt gaa GTC GGG GGA GGC ATC GCC AGG G-3′) were annealed and then were introduced into pSuppressor Neovector.

HLEC transfected with plasmid containing KLF6SV1 siRNA, empty vector only, and medium only served as the experimental groups, the vehicle control group, and the blank control group, respectively. Transfection was performed in 60 mm plates using 3 µg (1 µg/µl) vector in 10µl of Metafectene Pro reagent (Biontex, Martinstried, Germany). After 48 h of transfection, cells were treated with G418 (Life Technologies, Grand Island, NY) for 2 weeks for positive clone selection. After G418 treatment, several stable transfectant cells were cloned. Each clone was screened for expression of KLF6 by western blot analysis [16].

Western blot analysis

HLEC in lysis buffer containing 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 20 mM Tris-HCl, pH 7.5, and protease inhibitors was crushed by ultrasonic shaft. Protein concentration was measured using absorbance spectroscopy. Protein was separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies against KLF6SV1, p27 ^kip1, and PCNA overnight at 4 °C, followed by incubation with secondary antibodies.

The membrane was then assayed using the enhanced chemiluminescent kit (ECL, Thermo Scientific, Rockford, IL) and scanned with ChemiDoc™Doc XRS+ system (Bio-Rad, Hercules, CA). The density of each band was obtained using Quantity One 4.6.2 basic software (Bio-Rad). Values were expressed as fold change relative to control and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control [16].

3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide (MTT) assay

Quadruple samples of rLEC were grown on 96 well plates and were infected with 2 µg (1 µg/µl) of either of the two vectors in 10 µl of Metafectene Pro reagent, or were not infected. After 2, 4, 6, 8, and 14 days, wells were incubated in a medium containing yellow tetrazolium for 20 h [16]. Cell viability ratio=(1-A value of transfection group/A value of non-transfection group)×100%.

Cell proliferation and bromodeoxyuridine (BrdU) incorporation

Cells (5.0×10³) were plated onto a 24-well multiwell plate (Falcon, Becton Dickinson, Franklin Lakes, NJ) and allowed to attach for 24 h. The culture medium was then replaced with fresh medium. Cells were trypsinized and counted using a cell counter (Coulter Z1; Coulter, FL). For BrdU incorporation, cells growing on coverslips were incubated with 10 µmol/l BrdU for 3 h. After fixing in cold methanol/acetone (1:1) for 10 min, the cells were sequentially incubated in 1.5 mol/l HCl for 10 min. Then the cells were washed with PBS and incubated with the mouse anti-BrdU primary antibody (Santa Cruz, CA) for 1 h. The cells were washed four times with PBS. Cells with different BrdU-incorporation patterns were analyzed and counted with a conventional microscope [17,18].

Statistical analysis

The data were analyzed by the two-tailed Student t-test using SPSS 10.0 (IBM Inc., Beijing, China) and a p<0.05 was considered significance.

Downregulation of KLF6SV1 in HLEC transfected with pKLF6SV1 siRNA

We explored the biologic role of KLF6-SV1 using siRNA-mediated gene silencing to specifically downregulate its expression. There is expression of KLF6SV1 protein in the HLEC transfected with vehicle (Figure 1, lane 3) or without transfection (Figure 1, lane 2). Little expression of KLF6SV1 was detected in HLEC transfected with pKLFSV1 siRNA (Figure 1, lane 3).

Upregulation of p27^kip1 in HLEC transfected with pKLF6 cDNA

Upregualtion of p27^kip1 by transfection of skp2 siRNA can inhibit the proliferation of rabbit tennon’s fibroblast cells after glaucoma surgery which indicate it is important for inhibition of cell proliferation [18]. Expression of p27^kip1 increased in HLEC transfected with pKLFSV1 siRNA (Figure 2, lane 3) compared with vehicle control (Figure 2, lane 1) and blank control (Figure 2, lane 2). Upregulation of p27 ^kip1 made it possible for us to investigate the effect of p27 ^kip1 inhibition on the proliferation of HLEC.

Downregulation of PCNA in HLEC transfected with pKLF6SV1 siRNA

PCNA is important marker for cell proliferation. Expression of PCNA decreased in HLEC transfected with pKLF6SV1 siRNA (Figure 3, lane 3) compared with vehicle control (Figure 3, lane 1) and blank control (Figure 3, lane 2) which indicated that pKLF6SV1 siRNA can decrease the expression of PCNA.

KLF6SV1 siRNA decrease cell viability of HLEC