1. Introduction[A1]

The Structural Maintenance of Chromosomes 1 alpha (SMC1A) gene is located in Xp11.22-p11.21, consisting of 25 exons and 24 intron. SMC1A gene encoding a core subunit of the cohesin complex, which is essential to sister chromatid cohesion. SMC1, SMC3, SCC1 (also known as MDC1 and RAD21) and SCC3 (also known as SA2 and STAG2) subunits could interact with each other and form a ring-shaped cohesin complex [1-3]. As is known, central component of the cohesin and condensin complexes are required for conversion of interphase chromatin into mitotic-like condense chromosomes[4]. Structural Maintenance of Chromosomes (SMC) proteins are core component of the cohesin and condensin complex and essential for chromosome condensation during DNA replication and chromatid segregation of the genome in all organisms. They are also involved in checkpoint responses and epigenetic silencing of gene expression[5].

Cornelia de Lange syndrome is a dominantly developmental disorder with multisystem abnormalities including slow growth before and after birth, characteristic facial features, upper extremity defects, hirsutism, gastroesophageal dysfunction and cognitive retardaion. The incidence is as high as one in 10,000 to 30,000 newborns. Both sexes have the same phenotypic variability. To date, the three genes, NIPBL, SMC1A, and SMC3 involving in chromosome function, gene regulation and double-stranded DNA repair, could cause CdLS when mutated[6, 7]. Six in ten of the probands with CdLS have heterozygous mutations in NIPBL gene, whereas 5% have mutations in SMC1A and SMC3 genes [6, 8]. Eleven different SMC1A mutations in 14 unrelated patients have been reported. All patients had a mild to moderate CdLS phenotype [8-10].

In several decades, we focus on the relationship between SMC1A and chromosome related genetic disease. In recent years, we found that SMC1A may play a key role in tumorigenesis. Sun. M et al. determined that the effects of SMC1A knockdown on the cell cycle and apoptosis of lung adenocarcinoma cells. The results indicated that SMC1A is a novel oncogene, which modulates lung cancer cells in their proliferation and migration capabilities through arresting cell cycle at G0/G1 phase and promoting apoptosis [11]. The similar conclusion also was found in glioblastoma cells [12, 13]. However, SMC1A functions as a novel oncogene in human prostate cancer metastasis and progression has still not been reported.

2. Materials and Methods

2.1. Patient Samples

All of the patient samples were obtained from the Urinary Surgery Department of Shanghai Changzheng Hospital, Shanghai, China. This study was approved by the Clinical Research Ethics Committee of Shanghai Changzheng Hospital, and the informed consents were acquired from all of the subjects.

2.2. Reagents and antibodies

DMEM (cat no.12430-054), F12 (cat no. 21127022) and RPMI-1640(cat no. 11875-093) medium and fetal bovine serum (cat no. 10099-141) were purchased from GIO (Grand Island, NY). TRIzol Reagent was from Invitrogen (Carlsbad, CA, USA). Giemsa was from Chemicon International (Temecula, CA). M-MLV Reverse (cat no. M5301)Transcription was purchased from Promega (Madison, WI, USA). Oligo-dT₍₁₈₎ was synthesized by Sangon Biotech (Shanghai, China). Terra™ qPCR Direct SYBR® Premix (cat no. 638318) was from Takara (Otsu, Japan). Anti-SMC1A antibody (cat no. SAB4300451) was from Sigma-Aldrich (Munich, Germany). Mouse anti-GAPDH (cat no. sc-32233), Goat anti-Mouse IgG (cat no. sc-32233) and goat anti-rabbit IgG (cat no. sc-2030) were from Santa Cruz Biotechnology (Texas, USA). All the other chemicals were of analytical grade.

2.3. Cell culture

Human embryonic kidney cells 293T, 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 Sciences (Shanghai, China). 293T were cultrued in DMEM containing 10%FBS. PC-3 and DU145 cells were maintained in F-12 medium supplemented with 10% FBS, 100U/ml penicillin and 100μg/ml streptomycin. 22RV1 and LNCap cells were incubated with RPMI-1640 supplemented with 10% FBS, 100U/ml penicillin and 100μg/ml streptomycin. LNCap cells were maintained in Corning Corning^® CellBIND^® Surface cell culture flasks (Corning, cat no. #3289) for a better attachment efficiency. All cells were cultured in a humidified incubator at 37^oC under 5% CO₂ atmosphere and used for analysis during exponential phase of growth.

2.4. RNA interference

The synthesized 21-bp oligonucleotides encoding SMC1A-specific shRNA held the sequence 5’-TAGGAGGTTCTTCTGAGTACA-3’. The sequence of the negative control shRNA oligonucleotides was 5’-TTCTCCGAACGTGTCACGT-3’. The oligos were annealed and ligated into pH-L vector (Hollylab, Shanghai, China) through NheI/PacI to generate pH-Lv-shSMC1A and pH-Lv-shCon. The resulting constructs were confirmed by sequencing.

2.5. Recombinant lentivirus Transduction

PC-3 or DU145 cells were plated at 5×10⁴ cell/well in 6-well plates. After 24 h of culture, lentivirus recombinant encoding shRNA against SMC1A was added at a multiplicity of infection (MOI) of 50 into F-12 basic medium. After 6h incubation, the cells were added with complete growth medium replacing the basic medium containing the lentivirus. Then, 5 days post-transfection, gene reporter (EGFP) expression was examined using fluorescent microscopy (Olympus, cat no. CKX41).

2.6. Quantitative real-time RT-PCR analysis of SMC1A mRNA expression

Total RNA was extracted using TRIzol reagent according to the manufacturer’s instruction. 2μg total RNA was used to synthesize the first strand of cDNA using M-MLV Reverse Transcriptase. Real-time PCR reactions using Terra™ qPCR Direct SYBR® Premix were run on Takara TP800-Thermal Cycler Dice^TM Real-Time System. The following primers were used: SMC1A: 5’- AGCGAAAGGCAGAGATAATGG-3’ and 5’-GGTAGTCAAGAGGCAAGAAGG-3’; β-actin: 5’- GTGGACATCCGCAAAGAC-3’ and 5’-AAAGGGTGTAACGCAACTA-3’. Thermal cycling condition were 1 min at 95°C followed by 45 cycles of 95 °C for 5 s, 60°C for 20 s, read absorbance value at the extension stage. The data was analyzed with Takara Thermal Dice Real Time System software Ver3.0. SMC1A relative mRNA levels was calculated using the 2^-ΔΔCt method with normalization to β-actin. And the conditions

2.7. Western blot analysis of SMC1A protein expression

Cells were washed twice with ice-cold PBS and suspended in cell lysis buffer (2% Mercaptoethanol, 20% Glycerol, 4% SDS in 100mM Tris-HCl buffer, pH 6.8), and incubated for 15 min on ice. After centrifugation at 12,000 g for 15 min at 4^oC, the supernatants were collected, and the protein content were measured using BCA protein assay kit. Equal amounts of protein were subjected to SDS-PAGE. After electrophoresis, blots were transferred onto PVDF membrane using an electro-blotting apparatus (Tanon, Shanghai, China). The membrane was blocked with TBST buffer containing 5% nonfat milk at room temperature for 1 h, and incubated with the primary antibodies in the blocking solution at 4^oC overnight. After 3 washes with TBST buffer, the membrane was incubated with horseradish peroxidase (HRP) -conjugated secondary antibody (1:5000) at room temperature for 1 h. The signals of detected proteins were visualized by Pierce ECL western blotting detection kit (thermo scientific, USA). GAPDH protein level was used as an internal control to verify equal protein loading.

2.8. MTT assay

Cell proliferation was evaluated by MTT assay. Exponential growth phase cells were plated at a final concentration of 2000 cells/well in 96-well plates and cultured for five consecutive days. MTT (10µl, 10mg/ml) was then added, followed by incubation for another 4 hr at 37^oC under humidified 5% CO2 atmosphere. The MTT was removed and addition of 150μl DMSO. Optical density (OD) of each well was measured at 490 nm using an ELx808 Absorbance Reader (Bio-Tek Instruments, USA).

2.9. Colony formation assay

Cell growth and survival ability was also determined by the plate-colony-formation assay. In brief, 200 transfected cells were plated in 6-well plates. Cells were cultured for 14 days at 37^oC under humidified 5% CO2 atmosphere. Culture medium was changed at 3-day intervals. Afterward, cells were incubate in 4% paraformaldehyde for 30 min at room temperature. The colonies were stained with Giemsa for 15 min, then washed with ddH₂O and air-dried. The number of colonies (>50 cells/colony) was counted.

2.10. Flowcytometry Analysis

Cell cycle distribution was assessed by propidium iodide (PI) staining. Briefly, the transfectedcells were harvested by trypsinization, centrifuged at 250 g for 5 min, washed twice with ice-cold PBS, and fixed in 70% ethanol at 4^oC or -20^oC for at least 1 h. Cells were collected and resuspended in PBS containing 100μg/ml RNase A and 40μg/ml PI, and then incubated at 4^oC for 30 min, in dark. Cells were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA). The percentage of the cells in sub-G1, G0/G1, S, and G2/M phases were analyzed using ModFit (Verity Software House, Maine, USA) software.

2.11. Migration assay

To explore the effect of SMC1A in the migration of prostate cancer cells, 24-well transwell chamber with 8.0μm pore polycarbonate filter inserts (Corning, cat no. #3422) was performed. In the upper chamber of each transwell, cells were suspended in serum-free F-12 containing 0.2% BSA. And F-12 supplemented with 10% FBS was added in each lower chamber. Then, the inserts were incubated at a 37°C, 5% CO₂/95% air incubator for overnight and the cells that had not penetrated the filters were removed. The migrated cells attached to the bottom side were fixed in 4% paraformaldehyde for 10 min and stained in0.1% crystal violet for 30 min, rinsed in PBS and examined under a bright-field microscope.

2.12. Tumorigenesis assay

The influence of SMC1A silence on the tumor development of prostate tumor in vivo was examined. DU145, DU145-Lv-shCon or DU145 Lv-shSMC1A at 5 × 10⁶ per mouse were injected subcutaneously into 4 weeks old Balb/c nude mice (n = 10 per group, Shanghai Laboratory Animal Center, Chinese Academy of Sciences, China). The development and growth of solid tumors were monitored by measuring tumor size using a vernier caliper in a blinded fashion every five days for a 27-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 weights were measured using a electronic balance. All the animal experiments were approved by the Animal Care Committee of the Second Military Medical University.

2.13. Statistical analysis

The statistical analyses were performed with Graphpad Prism 5.0 software. The values are expressed as the mean of at least three different experiments ± S.D. The results were analyzed by Student’s t-test, and P<0.05 was considered statistically significant.

3. Results

3.1. Expression of SMC1A in prostate cancer tissue and prostate cancer cells.

To study the function of SMC1A in prostate cancer, we first analyzed its expression pattern in prostate cancer tissues. As shown in Fig 1A, SMC1A was strongly stained in prostate cancer tissue with a clear subcellular localization in the cytoplasm and nucleus of abnormal prostate epithelial cells. Then, the expression level of SMC1A was further analyzed by western blot, which showed that SMC1A was upregulated in prostate cancer tissues (Ca) compared to the adjacent normal tissues (N) (Fig 1B), implying a possible correlation between SMC1A and prostate cancer. To find a cell model for further investigation of SMC1A’s function in prostate cancer, we first analyzed the expression of SMC1A in four commonly used prostate cancer cell lines. Both WB and qPCR indicated that SMC1A expressions were elevated in PC-3, DU145 and 22RV1 cells in comparison to the androgen-sensitive LNCap cells (Fig 1C and D), which had less aggressiveness than the other three cell lines. We also found that SMC1A expression level was negatively correlated to AR expression level (Fig 1C and E), suggesting that SMC1A might be correlated with the malignancy of prostate cancer and involved in AR signaling.

3.2. Lentivirus-mediated knockdown of SMC1A in prostate cancer cells.

As PC-3 and DU145 cells expressed much higher levels of SMC1A, they were used for further investigation. Both PC-3 and DU145 cells were untreated or transducated with Lv-shCon or Lv-shSMC1A. The transduction efficiencies were above 90% in both cells confirmed by fluorescent microscope (Fig 2A). WB analysis demonstrated that Lv-shSMC1A efficiently knocked down SMC1A expressions in PC-3 and Du145 cells (Fig B and C). Q-PCR results indicated that SMC1A was down-regulated more than 80% and 90% in PC-3 and DU145cells respectively.

3.3 Down-regulation of SMC1A inhibited cell proliferation in prostate cancer cells.

After confirming the knocked down efficiency of SMC1A, PC-3 and DU145 cells were analyzed for cell growth rate with MTT assay. As shown in Fig 3A and E, cells transducted with Lv-shSMC1A displayed suppressed growth rate in comparison to the control or Lv-shCon transducted cells. The cells were then seeded onto 6-well plates for the analysis of colony formation ability. PC-3 and DU145 cells transducted with Lv-shSMC1A formed colonies with much smaller sizes compared to the control and Lv-shCon transducted cells (Fig 3B and F).The colonies formed in 6-well plates were photographed (Fig 3C and G), and counted (Fig 3D and H). The results suggested that both PC-3 and Du145 cells showed impaired colony formation abilities after SMC1A knockdown,indicating a pivotal role of SMC1A in regulation of prostate cancer cells proliferation.