Special AT-rich Sequence-binding Protein-1 Promotes Prostate Cancer Cell Proliferation And Invasion

Background and Objection:Prostate cancer (PCa) is one of the most common cancers, and the leading noncutaneous malignancy in Chinese men. Epidemiological studies have suggested that multiple risk factors are involved in prostate carcinogenesis. Although radical prostatectomy and radiation therapy are commonly used to improving outcomes of the PCa patients, the postoperative 5-year survival rate is low. Thus, the clinical treatment of PCa patients remains a challenge.Special AT-rich sequence-binding protein-1 (SATB-1) is a tissue specific nuclear protein that has emerged as new regulators for cancer biology by participating in both oncogenic and tumor suppressing pathways. SATB-1 regulates gene expression that controls diverse cellular processes including cell apoptosis and proliferation. Previous studies have demonstrated the potential involvement of SATB-1 in various cancer with potential roles in oncogenic pathways. However, the expression and role of SATB-1 in PCa remains largely unclear. The aim of the current study was to determine whether SATB-1 affects the biological behaviors of PCa and further elucidate if this effect works through the epithelial-mesenchymal transition (EMT) pathway.This study was divided into three major sections:Part One:Experimental Research of SATB-1 in prostate cancer tissues and cell linesObjective:Through this experiment clearly SATB-1 in prostate cancer cell lines and expression, and experimental materials provide morphological basis for subsequent research.Method:1) Human tissue samples:A total of 98 pairs of PCa tissue and matched normal prostate tissues were obtained from patients who undergone surgical resection at Southern Medical University between December 2010 and November 2014. Consent was obtained from all patients, and the experimental protocols were approved by the local ethics committee of Southern Medical University. Clinical and pathologic information were collected from a retrospective review of well-documented medical records, and characteristics of patients were detailed.2) Cell Lines and Culture Conditions:The PC3, LNCaP, and 22Rv1 cell lines (ATCC, Manassas, VA, USA) were cultured in RPMI-1640, supplemented with 10% fetal bovine serum (FBS) and antibiotics. The DU145 cell line was cultured in minimum essential Eagle’s medium supplemented with 10% FBS and 2 mM L-glutamine with antibiotics. The human nontumorigenic prostate epithelial cell line RWPE-1 was cultured in keratinocyte serum-free medium supplemented with 5 ng/ml human recombinant epidermal growth factor and 30 mg/ml bovine pituitary extract (Invitrogen, Carlsbad, CA). Cultures were maintained in a 5% CO2 humidified atmosphere at 37℃.3) Immunohistochemistry:For immunohistochemical analysis of SATB-1 expression,4μm TMA-sections were automatically pre-treated using the PT Link system and then stained in an Autostainer Plus (DAKO; Glostrup, Copenhagen, Denmark) with primary antibody:anti-SATB-1 (Abcam Inc., Cambridge, MA, USA). Expression of SATB-1 was denoted as positive when there was nuclear positivity of any intensity in at least 1 percent of cancer cells. Cases denoted as positive in any of the TMA-cores of the primary tumour or lymph node metastasis were considered positive. Stromal lymphocytes served as a positive control for SATB-1.4) Quantitative real-time PCR:Total RNA was extracted from specimens or cell lines using TRIzol reagent (Invitrogen). For qRT-PCR, RNA reverse transcribed to cDNA from 1μg of total RNA was reverse transcribed in a final volume of 20μl using random primers and a Reverse Transcription Kit (Takara,Dalian, China). According to the manufacturer’s instructions, the reverse transcription was performed at 37℃ for 15 min, then at 85℃ for 5 s. qRT-PCR analyses were performed using a standard protocol from Power SYBR Green (Takara, Dalian, China). All protocols were performed according to the manufacturer’s instructions. The Act values were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers were synthe-sized by Shanghai Sangon Biological Engineering Technology Services (Shanghai, China). The primer sequences used for the studies are: SATB-1 (forward,5’-GTGGAAGCCTTGGGAATCC-3’; reverse, 5’-CTGACAGCTCTTCTTCTAGTT-3’) and GAPDH (forward, 5’-TCGGAGTCAACGGATTTGGTCGT-3’; reverse, 5’-TGCCATGGGTGGAATCATATTGGA-3’) The qRT-PCR assays and data collection were performed using an ABI 7500 instrument. Each sample was analyzed in triplicate.5) Western blot analyses:Cells were lysed using the mammalian protein extraction reagent, RIPA (Beyotime), supplemented with a protease inhibitor cocktail (Roche) and PMSF (Roche). Protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22 mm nitrocellulose (NC) or polyvinylidene difluoride membranes (Sigma). The membranes were washed, blocked, and incubated with specific primary antihuman antibodies. Primary antibodies of SATB1 and β-actin were purchased from Abeam (Cambridge, MA, USA). The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG An ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad).Result:We first examined the SATB1 expression levels in 98 pairs of PCa tissue samples and matched normal prostate tissue samples by performing immunohistochemistry, qRT-PCR and western blotting analyses. The expression of SATB-1 was found to be significantly up-regulated in in the cancerous tissues compared with the corresponding adjacent non-tumorous tissues. Our results showed that SATB-1 protein was predominantly located in nucleus. The positive staining of SATB1 was observed in 77 of 98 (78.57%) of PCa cases, and in 18 of 98 (18.36%) of adjacent nonneoplastic specimens, with statistic significance (P<0.001).Conclusion:The results of qRT-PCR and western blotting revealed that tumor tissues showed higher levels of SATB-1 mRNA and protein than adjacent nontumor tissues. Moreover, we examined the expression of SATB-1 in a panel of PCa cell lines as well as an immortalized nontumorigenic human prostate epithelial cell line RWPE-1. Both mRNA and protein levels of SATB-1 were greatly elevated in all human prostate cancer cell lines in comparison to RWPE-1 cells, but expression levels of SATB-1 varied among PCa cell lines.Part TWO:Experimental study of prostate cancer cells to promote SATB-1Objective:Discussion SATB-1 prostate cancer cell proliferationMethod:1) Cell transfection:The SATB-1 expression vector pcDNA3.1-SATB-1 and SATB-1-specific shRNA pGenesil2-SATB-1 (the shRNA sequence was designed to target SATB-1 as follows:5’-GGAATGCTCTGAAGGACTTAC-3’) were constructed, and then transfected into 60%-confluent cells, respectively, using lipofectamin 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Following transfection, the transfected cells were allowed to recover for 24 h, and then individual clones with stable transfection screened with G418 (400 mg/ml) for two weeks were isolated. Thereafter, single colony of stable cells was picked and maintained in culture medium containing 200 mg/ml G418 to further culture. All the subsequent experiments were performed using the stable cells from single colony. Cells transfected with pcDNA3.1 or pGenesil2 control vector were used as negative controls.2) MTT assay:After transfection, cells were seeded in a 96-well plate at a density of 104 cells/well. Thereafter, the media was changed and the cells were incubated with 20μl MTT (5 mg/ml; Sigma-Aldrich) for 4 h. The mixture was centrifuged at 12,000 x g at 4℃ for 10 min. The supernatant was removed,50 μl DMSO was added to each well, and the solutions were agitated on a decolorization shaker for 10 min. The optical density (OD) of each well was measured using an enzyme immunoassay analyzer (Power Wave XS; BioTek Instruments Inc., Winooski, VT, USA) at 490 nm. The inhibitory rate of cell proliferation (inhibition ratio, IR) was calculated as follows: IR= (1-mean OD value of the experimental group/mean OD value of the control group) x 100%. Using the cell proliferation inhibitory rates with the different concentrations of baicalein at 48 h, the 50% inhibitory concentration (IC50) of baicalein was calculated.Result:In order to test the oncogenic activity of SATB-1 in PCa, LNCap cells were used to establish SATB-1 overexpressing cell lines and DU145 cells were used to establish SATB-1 silencing cell lines by viral transduction. The levels of SATB-1 in the resultant cell lines with forced SATB-1 expression (designated as LNCap-SATB-1) and silenced SATB-1 expression (designated as DU145-shSATB-1) were verified by qRT-PCR and Western blot. Therefore we investigated whether SATB-1 would affect human PCa cell proliferation. MTT assay showed that LNCap-SATB-1 cell line had a significant increase in cell proliferation compared with control, in contrast, DU145-shSATB-1 cell line had lower rates of cell proliferation.Conclusion:To study whether SATB-1 could regulate EMT, western blotting was performed to show expression of EMT-relevant markers, and the results indicated that E-cadherin expression was significantly increased in DU145- shSATB-1 cells, and expression of vimentin was greatly reduced in DU145-shSATB-1 cells, indicating that SATB-1 is involved in the EMT process in PCaPart THREE:Experimental study SATB-1 regulation of migration and invasion of prostate cancer cellsObjective:To investigate the effect SATB-1 in vitro Transwell chamber prostate cancer cell migration and invasionMethod:Transwell invasion assayCell invasion assay was carried out using Transwell chambers with inserts of 8-mm pore size (Corning Costar). Briefly, cells suspended in serum-free media were plated into the upper chamber for invasion assay after transfection, and media supplemented with 10% FBS was placed into the lower chamber. After 24 hours, the cells that had invaded through the membrane to the lower surface were fixed with methanol, stained with Crystal violet for 10 minutes, and counted.Result:Migration and invasion assay were performed to investigate the effects of SATB-1 on the migratory and invasive behaviors of PCa cells in vitro by transwell assay. The results demonstrated that the number of DU145-shSATB-1 cells that transited the artificial basement membrane or/and Matrigel Matrix was significantly reduced than control cells, while the migration and invasion capability was significantly increased in LNCap-SATB-1 cells. However, no significant difference was observed between the pcDNA3.1 empty vector-transfected group and the control group.Conclusion:These data confirmed that ectopic expression of SATB-1 in LNCap cells by pcDNA3.1-SATB-1 vector could promote their capability of migration and invasion in PCa in vitro.