Seventeen pairs of clinical cSCC samples were collected from cSCC patients operated from September 2012 to April 2015 in Department of Dermatology, The First Affiliated Hospital of China Medical University. Each sample pair contains a cSCC tissue, an adjacent tissue 0.5 cm from cSCC tissue and an adjacent tissue 1 cm from cSCC tissue. The patients had not been treated with radiotherapy or chemotherapy. Informed consent was obtained from the patients, and our collection and treatment procedures were in line with standards of the ethics committee of The First Affiliated Hospital of China Medical University.

The total RNA was extracted by total RNA rapid extraction kit (BioTeke, Beijing, China) from tissues or cells, and detailed procedure referred to the manufacturer's protocol. The obtained RNA samples were reverse transcribed into cDNA by Super M-MLV reverse transcriptase (BioTeke), with the oligo(dT) and random primers. All instruments in this section were treated by RNase Erasol (Tiandz, Beijing, China), and all reagents were RNase-free.

The cDNA was used to perform real-time PCR with 2X power Taq PCR MasterMix (BioTeke) and SYBR Green (Solarbio, Beijing, China) to test Maspin mRNA level, with β-actin as the internal control. The PCR procedure was set as follows: 95°C for 10 min, 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec, and finally 4°C for 5 min. The data were analyzed by Exicycler™ 96 (Bioneer, Daejeon, Korea), calculated by 2^−∆∆Ct method (cells) or 2^−∆Ct method (tissues) (22). Sequences of real-time PCR primers (Sangon, Shanghai, China) are shown in Table I.

The total protein was extracted from cells or tissues with RIPA lysis buffer (Beyotime, Haimen, Jiangsu, China). After measuring the concentration, the protein sample was denatured by boiling, separated by SDS-PAGE (density of gel dependent on the protein size), and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). After blocking with 5% skim milk (YILI, Hohhot, Inner Mongolia, China) for 1 h, the PVDF membrane was incubated with the following antibodies at 4°C overnight: polyclonal rabbit anti-Maspin (Abcam, Cambrige, UK) (1:1,000), polyclonal rabbit anti-cleaved caspase-3 (Abcam) (1:1,000), monoclonal rabbit anti-cleaved poly(ADP-ribose) polymerase (PARP) (Abcam) (1:1,000), polyclonal rabbit anti-Bcl-2 (Boster, Wuhan, Hubei, China) (1:400), and polyclonal rabbit anti-Bax (Boster) (1:400). After rinsing with TBST, the PVDF membrane was incubated with goat anti-rabbit IgG-HRP (Beyotime) (1:5,000) at room temperature for 45 min, and exposed with ECL reagent (7 sea, Shanghai, China). After antibodies were removed by stripping buffer (Beyotime), the PVDF membrane was incubated with monoclonal mouse anti-β-actin (Santa Cruz, CA, USA) (1:1,000) and goat anti-mouse IgG-HRP (Beyotime) (1:5,000) to test the internal control, β-actin.

To construct Maspin overexpression plasmid, Maspin coding sequence (CDS) was obtained by PCR from human cDNA, and amplified by TA cloning using UltraPower pUM-T rapid cloning kit (BioTeke). After sequencing, the Maspin CDS fragment was inserted into pcDNA3.1 vector (XhoI+HindIII), and the overexpression plasmid pcDNA3.1-Maspin was gained. The sequences of PCR primers are shown in Table II.

Three cSCC cell lines A431, SCL-1 and SCC12, were preserved in our laboratory. A431 cells and SCL-1 cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone) (SCC12 cells with 20% FBS), 100 U/ml penicillin, 100 µg/ml streptomycin (Beyotime) at 37°C incubator (Lishen, Tianjin, China) with 5% CO₂. The overexpression plasmid pcDNA3.1-Maspin and the control pcDNA3.1 were transfected into A431 cells by Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) with serum-free medium, 24 h later, G418 (Invitrogen) was added into medium with 100 µg/ml to screen the integrated cells, and the medium was refreshed every 2–3 days until the monoclonal cell lines were obtained.

Cell counting kit-8 (CCK-8) assay was performed to test the cell viability. A431 cells were seeded into 96-well plates with 3×10³ per pore beforehand. After cells adhered, CCK-8 reagent (Beyotime) was added into the 96-well plate with 10 µl per pore to incubate for 1 h, and the optical density of the solution was tested at 450 nm with a microplate reader (BioTek, VT, USA). This OD₄₅₀ value was the datum at 0 h, and the data in 24 h, 48 h, 72 h and 96 h were detected later, respectively.

Colony formation assay was performed to detect the colony formation ability of A431 cells. A431 cells were seeded into a 35-mm petri dish at 400/well, and cultured in 5% CO₂ at 37°C, medium was refreshed every 3 days. Approximately 14 days later, most of the clones had formed. The cells were fixed with 4% paraformaldehyde (Sinopharm, Beijing, China) for 20 min, stained with Wrings-Giemsa stain (Jiancheng, Nanjing, Jiangsu, China) for 5–8 min, and the clone number was counted with an inverted phase contrast microscope (Motic, Xiamen, Fujian, China). The clones with ≥50 cells were considered positive.

To measure the invasion potential of A431 cells, the Transwell assay in the presence of Matrigel (BD, Franklin Lakers, NJ, USA) was performed in Transwell chambers with 8-µm aperture polycarbonate membrane (Corning, NY, USA). Beforehand 40 µl of 2.5 mg/ml Matrigel was added into Transwell upper chamber to coat the polycarbonate membrane, and preheated at 37°C for 2 h to solidify. Then 200 µm serum-free cell suspension (2×10⁴ cells) were added into Transwell upper chamber, and 800 µm DMEM with 20% FBS was added in the lower chamber. After culturing for 24 h, the Transwell chamber was fixed in 4% paraformaldehyde (Sinopharm) for 20 min, and stained by 0.5% crystal violet for 5 min. Then the cells in upper chamber were wiped off, and the cells on reverse side of polycarbonate membrane were counted with an inverted phase contrast microscope (Motic) at ×200 magnification.

In order to observe the nuclear morphology change, Hoechst staining was performed. A431 cells were seeded onto glass slides in 12-well plates with 1×10⁵/pore. When the confluence reached 80%, the cells were treated with Hoechst Staining kit (Beyotime), observed with a fluorescence microscope (Olympus, Tokyo, Japan) at ×400 magnification.

Flow cytometry was performed to test apoptosis and cell cycle of A431 cells. A431 cells cultured in 6-well plates were collected when the confluence reached 90%. After rinsing with PBS (Double-helix, Shanghai, China) twice, the cells were treated with Annexin V-FITC/PI cell apoptosis test kit (Wanleibio, Shenyang, Liaoning, China) and detected using FACSCalibur (BD) according to the manufacturer's protocol.

For the cell cycle analysis, the collected cells were treated by Cell Cycle Analysis kit (Beyotime), and detected using FACSCalibur (BD) according to the specification. Proliferation index (PI) = (percentage of cells in S phase + G2/M phase) / percentage of cells in G1 phase (23).

The data in this study are presented as mean ± standard deviation (SD) with three individual experiments, and analyzed by one-way ANOVA test or paired Student's t-test. It was considered statistically significant at P<0.05, P<0.01, or P<0.001; NS, not significant).

First we detected the expression level of Maspin in 17 pairs of cSCC and adjacent tissues by real-time PCR and western blotting. As shown in Fig. 1, A, B and C groups represented the cSCC tissues, the adjacent tissues 0.5 cm from cSCC and the adjacent tissues 1 cm from cSCC, respectively. The results showed that the mRNA level of Maspin in cSCC tissues decreased by 55% (Fig. 1A and Table II), and the protein level decreased by 49% compared to the adjacent tissue 1 cm from cSCC tissues (Fig. 1B and C), respectively.

Because of the low expression level of Maspin in cSCC tissues, among three cSCC cell lines SCL-1, SCC12 and A431, we selected A431 cells, in which the Maspin expression level was the lowest (Fig. 2A), to measure the effect of Maspin on cSCC cells. To stably express Maspin, the overexpression plasmid pcDNA3.1-Maspin was constructed, and transfected into A431 cells. By screening with G418, monoclonal cells of pcDNA3.1-Maspin were obtained, and the pcDNA3.1 vector monoclonal cells were gained as the control. Real-time PCR and western blotting were performed to verify the expression efficiency of pcDNA3.1-Maspin. The results showed that the mRNA level increased by 3.78-fold (Fig. 2B), and the protein level increased by 3.86-fold (Fig. 2C) in contrast to the pcDNA3.1 group. Hence Maspin overexpression cell line was used for the subsequent experiments.

CCK-8 assay, colony formation assay and Transwell assay supplemented with Matrigel in vitro were performed to detect the cell viability, colony formation ability and invasion potential of A431 cells. The results showed that A431 cell viability decreased by 22%, 19%, 20% and 22% at 24 h, 48 h, 72 h and 96 h, respectively (Fig. 3A), the colony formation rate declined by 45% (Fig. 3B), and the invasion potential descended by 39% as a result of elevation of Maspin (Fig. 3C). Collectively, the results demonstrated that Maspin inhibited growth, proliferation and invasion in A431 cells.

We confirmed that Maspin expression was low in cSCC tissues, and inhibited growth, proliferation and invasion in cSCC cells, hence we were interested in how Maspin affects the phenotype of cSCC cells. In order to explore the underlying mechanism, we tested whether Maspin affected cell cycle transition and apoptosis in cSCC cells. Flow cyto-metry results showed that Maspin overexpression delayed cell cycle G1/S/G2 transition (Fig. 4A), and proliferation index (PI) decreased by 60% in A431 cells (Fig. 4B).

To detect the effect of Maspin on apoptosis of A431 cells, Hoechst staining, flow cytometry and western blotting were performed. In the Hoechst staining results, Maspin overexpressed A431 cells showed more intense fluorescence than parental and pcDNA3.1 groups (Fig. 5A), which is characteristic of apoptotic cells. Flow cytometry results showed that Maspin increased cell apoptosis rate of A431 cells 7-fold (Fig. 5B and C). In addition, we detected several apoptosis related genes. As shown in Fig. 5, cleaved caspase-3 increased 2-fold, cleaved PARP was upregulated 2.5-fold, Bcl-2 descended by 48%, and Bax was elevated 1.6-fold (Fig. 5D and E). These data suggested that Maspin enhanced apoptosis in A431 cells.

Collectively, Maspin was downregulated in cSCC tissues, and overexpression of Maspin inhibited growth, proliferation and invasion by delaying cell cycle transition and enhancing apoptosis in cSCC cells.