Archives

  • 2018-07
  • 2020-07
  • 2020-08
  • br Fig A miR expression in prostate cancer tissues and

    2020-08-12


    Fig. 1. (A) miR-93 expression in prostate cancer tissues and human benign prostatic hyperplasia tissues detected by qRT-PCR; (B) miR-93 expression in prostate cancer tissues and human benign prostatic hyperplasia tissues detected by in situ hybridization; (C) miR-93 expression in prostate cancer Amphotericin (LNCaP, PC-3, C4-2B and DU145) detected by qRT-PCR; (D) Kaplan– Meier curves for overall survival of 45 prostate cancer patients with high miR-93 expression and 15 prostate cancer patients with low miR-93 expression. These experiments were repeated three times and the data are represented as the mean ± SD. P b 0.05, P b 0.01, P b 0.001 vs. the miR-NC group.
    Fig. 2. (A) miR-93 expression in prostate cancer PC-3 cells transfected with miR-93 mimic or miR-negative control (miR-NC); (B) miR-93 expression in prostate cancer DU145 cells transfected with miR-93 inhibitor or miR-NC; (C) cell viability of PC-3 cells transfected with miR-93 mimic or miR-NC determined by MTT assay; (D) cell viability of DU145 cells transfected with miR-93 inhibitor or miR-NC determined by MTT assay; (E) cell viability of PC-3 cells transfected with miR-93 mimic or miR-NC determined by colony formation assay; (F) cell viability of DU145 cells transfected with miR-93 inhibitor or miR-NC determined by colony formation assay. These experiments were repeated three times and the data are represented as the mean ± SD. P b 0.01, P b 0.001 vs. the miR-NC group.
    3.3. miR-93 PC cancer cell migration and invasion in vitro
    To further verify whether miR-93 inhibited the cell migration and in-vasion of PC cell lines, we also determined the effect of miR-93 on cell migration and invasion using Transwell and scratch wound-healing assay. The results of Transwell assay indicated that the ability of migra-tion and invasion were increased in the miR-93-mimics-transfected PC-
    3 cells comparing to miR-NC-transfected cells (Fig. 3A). Consistently,
    miR-93 inhibitors significantly promoted the migration and invasion of the DU145 cells (Fig. 3B). This trend was further confirmed in scratch wound-healing assay in PC-3 cells transfected with miR-93 mimic (Fig. 3C) or miR-93 inhibitor (Fig. 3D). Together, miR-93 promotes the migration and invasion of PC cells.
    3.4. miR-93 mimic injection promotes tumorigenesis in vivo
    In addition to the in vitro studies, we finally examined whether miR-93 mimic injection contributed to the growth of PC-3 xenograft tumors in vivo (Fig. 4A, B and C). It was found that miR-93 mimic treatment 
    promoted the growth of PC-3 xenograft tumors when compared with control tumors of mice injected with miR-NC from Day 1 to 28. These data suggest that miR-93 upregulated the tumor growth in vivo.
    3.5. Isolation and characterization of green tea polysaccharide
    Crude polysaccharide CGTP was obtained from Green tea with the yield of approximately 13.5% (w/w) based on the dried-defatted Green tea residue. Subsequently, CGTP was subjected to a DEAE-Sepharose CL-6B Fast Flow chromatography and eluted with distilled water, followed by 0.3 and 1.0 M NaCl at a flow rate of 3.0 ml/min to yield three fractions, namely GTPa, GTPb and GTPc, respectively. Water-soluble polysaccharide fraction GTPa was further separated through a Sephacryl S-300 HR column chromatography and eluted with 0.1 M NaCl solution at a flow rate of 1.0 ml/min, resulting in a pu-rified polysaccharide fraction GTP (with the yield of about 1.8% based on the dried-defatted Green tea residue), which was used for further chemical characterization and biological activity study.
    GTP appeared as a light-yellow powder and contains 95.78% of total carbohydrate. Negative responses to Bradford Protein Assay and no
    Fig. 3. (A) The migratory and invasive abilities of PC-3 cells transfected with miR-93 mimic or miR-NC determined by Transwell assay. (B) The migratory and invasive abilities of DU145 cells transfected with miR-93 inhibitor or miR-NC determined by Transwell assay. (C) The migratory and invasive abilities of PC-3 cells transfected with miR-93 mimic or miR-NC determined by wound-healing assay. (D) The migratory and invasive abilities of DU145 cells transfected with miR-93 inhibitor or miR-NC determined by wound-healing assay. These experiments were repeated three times and the data are represented as the mean ± SD. P b 0.01 vs. the miR-NC group.
    absorption at 280 nm indicated that the absence of protein. The polysac-charide samples were not contaminated with uronic acid as evidenced by the carbazole–sulfuric acid method.
    A single, narrow and symmetrically sharp peak on HPGPC indicated that GTP was a homogeneous polysaccharide (Fig. 5). According to the regression equation of the calibration curve made by different dextran standards (y = 11.160 − 0.4279x, R2 = 0.9984), the average molecular weight of GTP was estimated to be about 7.0 × 104 Da with retention time of 14.752 min. By comparison with the retention time of the stan-dards under the same conditions, the results of GC indicated that GTP was typical homopolysaccharide, which was only composed of glucose (Glc).