Antibodies against OPN (rabbit polyclonal anti-human; 1:1,500; ab8448), phosphorylated (p)-Y118-paxillin (rabbit polyclonal anti-human; 1:1,000; 2541), GAPDH [rabbit polyclonal anti-human; XP^® Rabbit mAb (HRP Conjugate); 1:2,000; 8884), paxillin (rabbit anti-human; 1:1,000; 2542), FAK (rabbit polyclonal anti-human; 1:1,000; 3285) and pY Tyr576/577-FAK (rabbit polyclonal anti-human; 1:1,000; 3281) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). All other reagents were obtained from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany) unless otherwise described. The human primary renal cortical epithelial cells (Normal; PCS-400-011) were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in DMEM containing 200 U/ml penicillin G (Gibco; Thermo Fisher Scientific, Inc.), 200 µg/ml streptomycin and 10% FBS (HyClone; GE Healthcare Life Sciences) at 37°C in a humid atmosphere of 5% CO₂ at 37°C.

siRNAs were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The siRNA sequences for mouse OPN siRNA were as follows: siRNA sense, 5′-GAGGAGCCAUUUAUUGAAACUCG-3′ and antisense, 5′-CGAGUUUCAAUAAAUGGCUCCUC-3′; and siRNA control (oligos with no matching GeneBank sequence) sense, 5′-GCGACGAUCUGCCUAAGAU-3′ and antisense, 5′-AUCUUAGGCAGAUCGUCGC-3′. The pairs of siRNA oligonucleotides were prepared as a 20 µM stock. The OPN gene was cloned into the pcDNA™3.1 (−) Mammalian Expression Vector (V79520; Invitrogen; Thermo Fisher Scientific, Inc.) as the overexpression cassette.

For transient transfection, five groups were created, consisting of the control, overexpression group transfected with pcDNA3.1-OPN-GFP (over OPN), empty carrier group with overexpression of a blank plasmid (over blank), siRNA interference group (si-OPN) and siRNA blank (si-blank). The primary renal cortical endothelial cell line was cultured in 6-well plates to 80% confluence and then transfected using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The cells were collected 24 h subsequent to transfection for other experiments.

Cell viability was determined using the MTT method (14). The cells were plated into 96-well microtiter plates for 24 h. Subsequently, 10 µl of MTT-labeling reagent was added to each well and the plates were incubated at 37°C for 4 h. The cultures were then solubilized and spectrophotometric absorbance of the samples was detected by a microtiter plate reader. The wavelength used to measure the absorbance of the formazan product was 570 nm, with a reference wavelength of 750 nm. Cell viability was expressed as a percentage of the positive control.

Wound healing experiments were performed for quantitative investigation of the migration of cells. Cells were grouped as aforementioned, consisting of the control, over OPN, over blank, si-OPN and si-blank groups. The initial cell seeding density was ~1×10⁵ cells/cm². A square wound was made using a cell scraper. The cells were washed twice with PBS and then incubated with fresh DMEM for another 24 h at 37°C. The cells were then visualized by microscopy (IX71; Olympus, Corporation, Tokyo, Japan).

A further cell migration identification was performed using Transwell assay. The cultured cells were detached with trypsin subsequent to 8 h, and the treated cells were seeded into a Millicell (Greiner Bio-One, Frickenhausen, Germany) containing pores of 8 mm in diameter at an initial seeding density of 1×10⁵ cells/cm², suspended in DMEM containing 1% fetal bovine serum. The Millicell pores were then put into the wells of a 24-well plate containing 20% fetal bovine serum in DMEM. The cells on the Millicell pores were transfected with four types of vectors (over OPN, over blank, si-OPN and si-blank). Fresh DMEM was added 4 h later, and the cells were kept at 37°C for another 24 h. The migrated cells were stained with crystal violet (0.1% wt) and the number of migratory cells was recorded using an optical microscope at ×100 magnification. The cell number was randomly counted in 12 different fields of view for each group, and the data were averaged from three parallel experiments.

Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequent to being treated with RNase-free DNase I (Roche Applied Science, Penzberg, Germany), first-strand cDNA was synthesized with Moloney murine leukemia virus (M-MLV) reverse transcriptase and Oligo-dTs (Takara Bio, Inc., Otsu, Japan). RT-qPCR was conducted using the ABI Prism 7500 Sequence Detection System, according to the manufacturer's instructions (Applied Biosystems; Thermo Fisher Scientific, Inc.). Specific primers used for RT-qPCR were as follows: OPN forward, 5′-GAAGTTTCGCAGACCTGACAT-3′ and reverse, 5′-GTATGCACCATTCAACTCCTCG-3′; FAK forward, 5′-AGTGGACCAGGAAATTGCTTTG-3′ and reverse, 5′-GTGTTTTGGCCTTGACAGAATCPCR-3′; paxillin forward, CTGCTGGAACTGAACGCTGTA-3′ and reverse, 5′-GGGGCTGTTAGTCTCTGGGA-3′; GAPDH forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTTC-3′. For the relative comparison of each gene, we analyzed the Cq value of qPCR data with the 2^−ΔΔCq method normalized by the endogenous control GAPDH (15).

The procedure for immunoblotting has been previously described (16). Cells were treated with cell lysis buffer and then protein samples were collected. Briefly, 40 µg protein/lane was loaded into the lanes of 12% denatured polyacrylamide gel. The proteins were separated by electrophoresis and transferred to nitrocellulose membranes (0.45 µm; Schleicher & Schuell; GE Healthcare Life Sciences). The membranes were blocked with 3% bovine serum albumin in 0.01 M PBS (pH 7.4) and 0.05% Tween-20 (PBST) at room temperature for 1 h. Subsequently, the membrane was incubated with the aforementioned primary antibodies against the target proteins overnight at 4°C. Following two quick washes in PBST, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase [Goat Anti-Rabbit IgG H&L (HRP); dilution, 1:4,000 in PBST; ab97051; Abcam, Cambridge, UK) for 2 h at room temperature. Following washes as aforementioned, an enhanced chemiluminescence reagent (Pierce™ DAB Substrate kit; 34002; Thermo Fisher Scientific, Inc.) was used to produce fluorescence for detection. The density of immunoblotting was quantified using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative expression of the target protein was quantified as a ratio against GAPDH.

Experiments were performed routinely with >4 samples per group, with the values expressed as the mean ± standard error of the mean. All experiments were replicated at least three times. Statistical analysis was performed using one-way analysis of variance and Student's t-test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The OPN expression was assessed by analyzing the protein and mRNA levels. The results showed that the overexpression vector was evidently effective, while the siRNA, which was selected from several alternative siRNA sequences, could efficiently disturb the expression of OPN (Fig. 1). The expression of the over OPN group was significantly increased compared with the blank group (P=0.0011). In addition, the expression of si-OPN group was significantly decreased compared with the blank (P=0.0021).

To investigate the effects of OPN on the proliferation of human epithelial cells, the MTT assay was used to examine the cell viability. However, there was no significant difference between cell viability in the si-OPN group, over OPN group and the control groups (Fig. 2). The results showed that increasing or decreasing OPN expression did not significantly affect the cell proliferation.

Macroscopic analyses of time-matched OPN overexpression vs. control wounds showed that closure was markedly accelerated (P=0.0372 and P=0.0241, respectively) at early time points (12 and 18 h). Cells transfected with OPN-overexpressing vectors exhibited a mean closure of the wound of 56% after 24 h, while cells transfected with OPN-siRNA vectors achieved a mean closure of the wound of 12% by 24 h, compared with the 24% average closure observed in the blank control (Fig. 3A). Since OPN-overexpressing cells are capable of almost complete and efficient wound closure, OPN may play a pivotal role in the repair of deep layers of skin.

Transwell assay was also used to detect the migration of epithelial cells. The number of migrated cells were counted, and their shape is shown in Fig. 3B. The numbers of migrated cells in the over-OPN and si-OPN groups were significantly increased (P=0.0223) and decreased (P=0.0421), respectively, compared with the three blank groups, indicating that OPN plays a vital role in accelerating cell migration.

To investigate whether OPN induced stronger cell attachment to the substrate, the expression of cell adhesion molecules, including FAK and paxillin were also examined by western blotting. The overexpression of OPN improved the phosphorylation of paxillin (P=0.0001) and FAK (P=0.0001). Additionally, protein expression of pY118-paxillin (P=0.0012) and pY-FAK (P=0.0002) was inhibited by OPN downregulation. However, there is no difference in the total FAK or paxillin among the five groups (Fig. 4). This result shows that the ability of cell adhesion is decreased by OPN, while OPN is involved in modulating signaling molecules associated with adhesion during healing of the deep layers of a wound.