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  • br Flow cytometric analysis of apoptosis br Following treatm

    2020-08-12


    2.8.2. Flow cytometric analysis of apoptosis
    Following treatment with CTP (25, 50 and 100 μg/ml) for the indi-cated time, the supernatant was removed and the cells were harvested, washed with cold PBS, and stained with 5 μl of FITC-conjugated annexin V and 5 μl of PI (50 μg/ml) for 15 min in the dark using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences PharMingen, San Jose, CA, USA) according to the manufacturer's instructions. The cells were ana-lyzed immediately after staining on a FACScan flow cytometer (Becton Dickinson, San Jose, CA, U.S.A.) using CellQuest software at an excitation wavelength of 488 nm and emission wavelengths of 525 and 625 nm, respectively. For each sample, at least 20,000 cells were counted. The apoptosis rate (%) = (the number of apoptotic cells / the number of total cells observed) × 100% [30].
    Western blot analysis was used to determine the expression of dif-ferent proteins involved in GTP-induced apoptosis in PC-3 cells. After treatment with GTP (25, 50 and 100 μg/ml) for 48 h, the cells were har-vested, washed with ice-cold PBS, centrifuged, and then cell pellets were treated with lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 0.1% (w/v) Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1% Na3CO4, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM phenylmethanesulfonyl fluoride (PMSF)] on ice for isolation of total protein fractions. After centrifugation (14,000 ×g for 15 min), the super-natant fractions were collected and protein concentrations were quan-tified using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Munich, Germany) with bovine serum albumin (BSA) as the standard. Equal amounts of lysate protein (25–50 μg/well) were run on SDS-PAGE (8–12%) and then electrophoretically transferred to nitrocellulose
    membranes (Amersham Biosciences). The membrane was incubated with 5% skim nonfat milk in PBS for 1 h at room temperature, and then probed with the primary Tamoxifen against bcl-2, bax, procaspase-8, cleaved caspase-9, cleaved caspase-3, PARP and β-actin (Santa Cruz Biotechnology Inc.) overnight at 4 °C, followed by HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (Cell Sig-naling Technology, Beverly, MA, U.S.A.) for 1 h at room temperature. Blots were visualized with an enhanced chemiluminescence (ECL) Western Detection kit (Amersham Biosciences, GE Healthcare, UK) ac-cording to the recommended procedure.
    2.9. Statistical analysis
    All experiments were performed at least three times, and data were reported as the means ± SD. Statistical analyses were performed two-tailed Student t-test. A P-value of b0.05 was considered to be statistically significant.
    3. Results
    3.1. miR-93 expression is up-regulated in PC tissues and cells
    To determine the role of miR-93 in PC progression and to investigate its ability to predict clinical progression, its expression in human benign prostatic hyperplasia tissues, PC tissues and cells was analyzed. qRT-PCR analysis showed that miR-93 expression was significantly upregulated in PC tissues compared with human benign prostatic hyperplasia tissues (P b 0.0001) (Fig. 1A). In line with this result, in situ hybridization test showed that miR-93 expression was significantly higher than that in be-nign prostatic hyperplasia tissue (Fig. 1B).
    Next, before we explore the role of miR-93 in biological behavior of PC, the expression level of miR-93 in PC cells (LNCaP, PC-3, C4-2B and DU145) was also examined by qRT-PCR (Fig. 1C). Generally, in PC cells, miR-93 expression was significantly elevated with different extent in a descending order DU145 N LNCaPNC4-2B N PC-3, as compare with human benign prostatic hyperplasia tissues (P b 0.05, P b 0.01 or P b 
    0.001). DU145 cells had the highest expression of miR-93 and PC-3 cells had the lowest expression among the four PC cell lines. Hence, we chose DU145 and PC-3 cell lines to perform the loss- or gain-of-function.
    Overall survival curves were plotted according to the expression level of miR-93 in tumor specimens from 60 PC patients by the Kaplan–Meier method. Based on follow up data of 60 PC patients, pa-tients with high expression of miR-93 have shorter survival rate, worse prognosis, and are more likely to relapse and get other diseases than the patients with low expression of miR-93. At the end of the follow-up period, the overall 150-week survival rate of 45 patients with high miR-93 Tamoxifen expression and 415 patients with low miR-93 expres-sion were 40.0% and 70.3% (Fig. 1D), respectively, suggesting the level of miR-93 expression serve as a prognostic biomarker for PC.
    3.2. miR-93 promotes PC cell growth and colonies formation
    To further conform the role of miR-93 in cell proliferation, we per-formed cell proliferation and colony formation assays on PC cell line PC3 and DU145. qRT-PCR analysis confirmed the overexpression of miR-93 in PC-3 cells transfected with the miR-93 mimic (Fig. 2A) and downregulated the expression of miR-93 in DU145 cells using miR-93 inhibitor (Fig. 2B) compared with cells transfected with the negative control. As determined by MTT assay (Fig. 2C and D), overexpression of miR-93 significantly increased PC-3 cell proliferation (P b 0.05 vs. miR-NC cells at 48 h; P b 0.001 vs. miR-NC cells at 72 h), in contrast, miR-93 knockdown reduced cell proliferation rate especially after 48 and 72 h, which were significant from miR-NC cells (P b 0.01). In paral-lel, the colony formation assay rep-resented the similar results that overexpression of miR-93 promoted cell proliferation of PC-3 cells as shown by the increased colony number (Fig. 2E), while miR-93 knock-down significantly inhibited cell proliferation of DU145 cells as evi-denced by the decreased colony number (Fig. 2F). Together, our findings suggested miR-93 contributes to PC cells proliferation and col-onies formation.