All of the patient samples for immunohistochemical (IHC) analysis were obtained from the Department of Urinary Surgery, Shanghai Changzheng Hospital, Shanghai, China. This study was approved by the Clinical Research Ethics Committee of Shanghai Changzheng Hospital, and written informed consents were obtained from all the subjects.

For IHC analysis, tissue samples were paraffin-embedded, cut into 5-μm-thick sections and pasted onto glass slides. After deparaffinizing in xylene and dehydration with graded ethanol washes, the specimens were sequentially incubated with blocking solution for 10 min and 1:100 dilution of anti-SMC1A antibody (SAB4300451; Sigma-Aldrich) at 4°C overnight, and stained using UltraSensitive™ SP (mouse/rabbit) IHC kit (KIT9730; Fuzhou Maixin Biotechnology, Co., Ltd., Fuzhou, China) according to the user's manual.

The clinical significance of SMC1A expression in prostate cancer were analyzed using the online Oncomine database (www.onocomine.org) consisting of previously published and publicly available microarray data. Welsh prostate dataset (19) and Singh prostate dataset (20), which have a total of 136 samples, were used to compare the differential expression of SMC1A between normal (59 cases) and cancerous tissue (77 cases). Glinsky prostate dataset (21) (79 cancer cases) was used to analyze the correlation of SMC1A expression level with cancer biochemical recurrence. Moreover, three independent datasets including Holzbeierlein prostate dataset (22), LaTulippe prostate dataset (23), and Chandran prostate dataset (24), which have a total of 31 cancer tissues, were used to explore the relationship between SMC1A expression level and distant metastasis.

Dulbecco's modified Eagle's medium (DMEM; cat. no.12430-054), F-12 (cat. no. 21127022) and Roswell Park Memorial Institute 1640 (RPMI-1640; cat. no. 11875-093) medium and fetal bovine serum (FBS; cat. no. 10099-141) were purchased from Gibco (Grand Island, NY, USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Giemsa was from Chemicon International (Temecula, CA, USA). M-MLV reverse (cat. no. M5301) transcriptase was purchased from Promega (Madison, WI, USA). Oligo-dT₍₁₈₎ was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Terra™ qPCR Direct SYBR^® Premix (638318) was from Takara Bio (Shiga, Japan). Anti-SMC1A antibody (SAB4300451) was from Sigma-Aldrich (Munich, Germany). Mouse anti-GAPDH (sc-32233), goat anti-mouse IgG (sc-32233) and goat anti-rabbit IgG (sc-2030) were from Santa Cruz Biotechnology (Dallas, TX, USA). All the other chemicals were of analytical grade from Sangon Biotech.

Human embryonic kidney (HEK) 293T cells and human prostate cancer cell lines PC-3, DU145, LNCap and 22RV1 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China). 293T cells were cultured in DMEM containing 10% FBS. PC-3 and DU145 cells were maintained in F-12 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. 22RV1 and LNCap cells were incubated with RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. All cells were cultured in a humidified incubator at 37°C with 5% CO₂.

To silence the expression of SMC1A in PCa cell lines, the short hairpin (shRNA) sequence identified to target human SMC1A gene was 5′-TAGGAGGTTCTTCTGAGTACA-3′. The sequence of the negative control shRNA was 5′-TTCTCC GAACGTGTCACGT-3′. The oligos were annealed and ligated into pFH-L vector (Holly Lab, Shanghai, China) through NheI/PacI restriction sites to generate pFH-Lv-shSMC1A and pFH-Lv-shCon. Finally, the sequencing was performed to confirm the results of construction.

Lentiviruses were generated by triple transfection with modified pFH-shRNA plasmid and pVSVG-I and pCMVΔR8.92 helper plasmids into HEK-293T cells using Lipofectamine 2000 according to the manufacturer's instructions. The lentiviral particles were then harvested by centrifugation, filtered through a 0.45 μm filter and then stored at −80°C.

PC-3 or DU145 cells were seeded at the concentration of 5×10⁴ cell/well in 6-well plates. After 24 h of culture, lentivirus containing shRNA targeting SMC1A (shSMC1A) or the negative control (shCon) were added at a multiplicity of infection (MOI) of 50 into F-12 basic medium. After 6-h incubation, PC-3 and DU145 cells were cultured in complete medium replacing the basic medium containing the lentivirus. Then, after 5 days post-transfection, the green fluorescent protein (GFP) expression was examined using fluorescent microscopy (Olympus; cat. no.CKX41) to assess the infection efficiency.

The total RNA was extracted using TRIzol reagent according to the manufacturer's instruction and synthesized complementary DNA (cDNA) by using M-MLV reverse transcriptase. Real-time PCR reactions using Terra™ qPCR Direct SYBR^® Premix were run on Takara TP800 Thermal Cycler Dice™ real-time system. The following primers were used: SMC1A: 5′-AGCGAAAGGCA GAGATAATGG-3′ (forward) and 5′-GGTAGTCAAGAGGC AAGAAGG-3′ (reverse); β-actin: 5′-GTGGACATCCGCAA AGAC-3′ (forward) and 5′-AAAGGGTGTAACGCAACTA-3′ (reverse). Thermal cycling conditions were as follows: initial denaturation 1 min at 95°C, followed by 40 cycles of denaturation for 5 sec at 95°C, extension for 20 sec at 60°C and absorbance value was read at the extension stage. The data were analyzed with Takara Thermal Dice Real-Time system software ver3.0. SMC1A relative mRNA levels were calculated using the 2^−ΔΔCt method with normalization to β-actin.

Cells were washed twice with ice-cold PBS and lysed in 2X sodium dodecyl sulfate (SDS) sample buffer (2% mercaptoethanol, 20% glycerol, 4% SDS in 100 mM Tris-HCl buffer, pH 6.8), and incubated for 15 min on ice. The supernatants were collected by centrifugation at 12,000 × g for 15 min at 4°C, and a BCA protein assay kit was used to measure the protein content. Equal amounts of protein samples (30 μg) were loaded and separated in 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. Whereafter, the membrane was blocked with TBST buffer containing 5% non-fat milk at room temperature for 1 h, and incubated with the primary antibodies in the blocking solution at 4°C overnight. After being washed three times with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000) at room temperature for 1 h. The objective bands were detected by Pierce ECL western blotting detection kit (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as an internal control.

To evaluate the effect of SMC1A in the proliferation of prostate cancer cells, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. Four days after lentivirus infection, both PC-3 and DU145 cells were reseeded in 96-well plates at an ultimate density of 2000 cells/well and the number of active cells was measured for five consecutive days. Subsequently, MTT (10 μl, 5 mg/ml) was added to each well at a fixed time-point. After incubation at 37°C for 4 h, the acidic isopropanol (150 μl/well) was added and then incubated at 37°C. Optical density (OD) of each well was measured at 570 nm using an ELx808 Absorbance Reader (BioTek Instruments, Inc., Winooski, VT, USA).

To examine the effect of SMC1A in the colony formation of a single prostate cancer cell, the colony formation assay was executed. Four days after lentivirus infection, both PC-3 and DU145 cells were reseeded in 6-well plates at a density of 200 cells/well and cultured for 14 days in the humidified incubator at 37°C with 5% CO₂. Culture medium was replaced at 3-day intervals. Then, cells were washed in PBS, fixed in 4% paraformaldehyde for 30 min and stained with Giemsa for 15 min at room temperature. The stained colonies were washed with ddH₂O and air-dried. Finally, the ability of colony formation was observed through a light/fluorescence microscope and the colonies (>50 cells/colony) were counted.

Cell cycle distribution was analyzed by propidium iodide (PI) staining. Briefly, both PC-3 and DU145 cells were reseeded at a density of 1×10⁵ cells/well in 6-cm dishes after lentivirus infection. After the incubation period cells were harvested and fixed in 70% ethanol overnight at 4°C. The next day, cells were washed thrice and resuspended in PBS containing 100 μg/ml RNase A and 50 μg/ml PI, and then incubated in the dark at room temperature for 30 min. Cells were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). The percentage of the cells in sub-G1, G0/G1, S and G2/M phases were analyzed using ModFit software (Verity Software House, Inc., Topsham, ME, USA).

To explore the effect of SMC1A in the migration of prostate cancer cells, a 24-well Transwell chamber with 8.0 μm pore polycarbonate filter inserts (Corning; cat. no. #3422) was performed. After lentivirus infection, both PC-3 and DU145 cells were reseeded at a density of 1×10⁵ cells/well in serum-free F-12 containing 0.2% BSA in the upper chamber of each Transwell. In addition, F-12 supplemented with 10% FBS was added in the lower chamber. Then, the migration installation was incubated at 37°C with 5% CO₂ overnight and the non-migrated cells on the upper surface of the filter were lightly removed using cotton buds. The migrated cells on the lower surface were fixed in 4% paraformaldehyde for 10 min, stained in crystal violet for 2 min, and counted (five random fields per well) under a bright-field microscope. Additionally, the migrated cells were dissociated by 33% acetic acid and quantified at 570 nm using the Epoch microplate spectrophotometer (BioTek Instruments).

The impact of SMC1A silencing on the tumor development of prostate cancer in vivo was examined. DU145 (Con group), DU145-Lv-shCon (shCon group) or DU145 Lv-shSMC1A (shSMC1A group) at a density of 5×10⁶ per mouse were injected subcutaneously into 4-week-old BALB/c nude mice (n=10 per group; Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Shanghai, China). The development and growth of solid tumors were monitored by measuring tumor size using a vernier caliper every three days for a 28-days period. The tumor volume was calculated using a standard formula: tumor volume (mm³) = width (mm)² × length (mm) × 0.5. At the end of the experiment, all mice were sacrificed and individual tumor weight was measured using an electronic balance. All the animal experiments were approved by the Animal Care Committee of the Second Military Medical University.

GraphPad Prism 5.0 software was used to perform the statistical analyses. Data are presented as mean ± SD from at least three independent experiments. The Student's t-test was used to compare the differences between the groups. P<0.05 was considered to indicate a statistically significant result.

Through a lentiviral shRNA library-based screening on PC-3 cells, we identified SMC1A as a novel oncogenic candidate. To validate and further explore the function of SMC1A in prostate cancer, we first examined the expression of SMC1A in different PCa cell lines. Notably, we found that the expression of SMC1A was significantly increased in androgen-independent prostate cancer cell lines PC-3 and DU145 compared with androgen-sensitive cell lines LNCap and 22RV1, showed SMC1A expression was negatively correlated with the expression status of androgen receptor (AR) in both the protein and mRNA levels (Fig. 1A–C). Considering that SMC1A expression was much higher in androgen-independent prostate cancer cells PC-3 and DU145, they were used for further investigation.

Both PC-3 and DU145 cells were untreated or transfected with shCon or shSMC1A. The transfection efficiencies were >90% in both cells confirmed by fluorescent microscope (Fig. 2A). Western blot analysis demonstrated that shSMC1A efficiently knocked down SMC1A expression in protein levels in PC-3 and DU145 cells (Fig. 2B and C). The results of real-time PCR indicated that SMC1A was downregulated >80 and 90% in mRNA levels in PC-3 and DU145 cells, respectively (Fig. 2D and E; P<0.001, P<0.01).

Effect of SMC1A silencing on prostate cancer cell viability was assessed by MTT and colony formation assay. As shown in Fig. 3A and B, the growth rates of PC-3 and DU145 cells were significantly suppressed in shSMC1A group in comparison to the Con or shCon group (P<0.001). Furthermore, SMC1A depletion significantly inhibited colony formation ability of PC-3 and DU145 cells in size and number compared to the Con or shCon group (Fig. 3C–E; P<0.01, P<0.001). The results suggested that both PC-3 and DU145 cells showed impaired cell proliferation and colony formation abilities after SMC1A knockdown, indicating a pivotal role of SMC1A in regulation of prostate cancer cell vitality.

When culturing shSMC1A transfected PC-3 and DU145 cells, we noted that these cells showed more non-adherent cells during passages. We decided to confirm how SMC1A affected the proliferation of prostate cancer cells, and whether it functioned through the cell cycle distribution. To verify this hypothesis, PC-3 and DU145 cells were stained using PI and analyzed with FACS. As expected, the results showed that knockdown of SMC1A expression caused G0/G1 and G2/M-phase cell population increase (P<0.05, P<0.001) while S-phase cell population reduction (P<0.01) that indicated cell cycle arrest at G2/M-phase in PC-3 cells (Fig. 4A). Moreover, SMC1A silencing presented G0/G1-phase cell population increase (P<0.05) and S-phase cell population reduction (P<0.001) that showed cell cycle arrest at S-phase in DU145 cells (Fig. 4B). In addition, the rate of cells in the Sub-G1 phase, representing apoptotic cells indirectly, was remarkably increased in shSMC1A group compared with Con and shCon groups in PC-3 and DU145 cells (Fig. 4C and D; P<0.05, P<0.01). These results indicated that SMC1A might be involved in cell apoptotic and cell cycle progression.

PC-3 and DU145 cells were transfected with indicated lentivirus for 96 h, and then subjected to Transwell assay for the cell migration ability. As shown in Fig. 5, the cell numbers of PC-3 and DU145 cells in shSMC1A group which migrated to the lower chamber were less than the Con and shCon groups (P<0.01, P<0.001). These results indicated that knockdown of SMC1A significantly inhibited the migration ability of PC-3 and DU145 cells.

To further study the function of SMC1A in vivo, DU145 cells were untreated, or transfected with shCon or shSMC1A, and subcutaneously injected into the nude mice to investigate the impact of SMC1A on tumor growth. As shown in Fig. 6A, knockdown of SMC1A inhibited subcutaneous tumor growth of DU145 cells. At day 28, the mice were euthanized and the tumors were removed. At the end of the experiment, we found that the tumor volumes were significantly reduced in time-dependent manner, while the tumor weighs were markedly decreased by SMC1A silencing (Fig. 6B and C; P<0.001).

To illustrate the expression level of SMC1A and the clinical significance of SMC1A in PCa patients, we detected the specimens from patients and performed the data mining of the publicly available Oncomine datasets. In the present study, we found that the protein expression of SMC1A is significantly upregulated in PCa tissues by IHC staining and western blot assay (Fig. 7A and B). Similar results were also observed that the mRNA expression level of SMC1A was remarkably higher in PCa tissues than the normal tissues using Singh prostate (Fig. 7C; n=102, P<0.001) and Welsh rostate databases (Fig. 7D; n=34, P=0.015). Notaby, we found that the expression of SMC1A was obviously upregulated in the patients with postoperative biochemical recurrence (BCR) at 3 years by analyzing Glinsky prostate database (Fig. 7E; n=79, P=0.0408). Furthermore, we also detected that SMC1A expression was significantly and positively associated with distant metastasis in Holzbeierlein prostate database (Fig. 7F; n=48, P=0.0307). This finding was verified by other two independent databases including LaTulippe prostate (n=32) and Chandran prostate (n=31) databases. Specifically, comparing with primary site, SMC1A was visibly upregulated in three metastasis tissues (Fig. 7G, lymph node, n=5; bone, n=2, P=0.0067; soft tissues, n=2, P=0.0267; total metastasis, P=0.0087) in LaTulippe prostate database and also obviously elevated in lymph node (n=13, P<0.0001), adrenal gland (n=2, P=0.0303), liver (n=5, P=0.0047) and lung (n=1) metastasis as revealed in Chandran prostate database (Fig. 7H; total metastasis, P=0.0047).