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  • br Recent studies have highlighted the critical role of CDC

    2020-08-30


    Recent studies have highlighted the critical role of CDC20 in hemato-poietic stem 923564-51-6 and cancer stem cells. CDC20 regulates hematopoietic
    Fig. 3. CDC20 is required to maintain stem cell-like features of CD44+ prostate CSCs. (a, b) The mRNA expression levels of multiple stemness-related genes in CDC20-silenced or re-expressed C4–2B CD44+ (a) or DU145 CD44+ (b) cells were evaluated by qRT-PCR. Quantified mRNA levels were normalized to β-actin and presented relative to the controls (P value: Wilcoxon test). (c, d) Relative expression of CDC20 and a series of stemness related genes (CD44, MYC, SOX2, OCT4, KLF4) was examined in primary and secondary passaged prostate cancer cell spheroids derived from CD44+ C4–2B (c) or DU145 (d) with CDC20 knockdown or re-expression (Scale bar = 75 μm), the spheroid number was counted (P value: Wilcoxon test). (e, f) The limiting dilution assay of spheroid formation was performed in control and Lv-shCDC20 C4–2B CD44+ (e) or DU145 CD44+ (f) cells with different gradient cell numbers as indicated. (g, h) The indicated CD44+ C4–2B (g) or DU145 (h) cells were treated with docetaxel of different gradient concentration and cell viability was measured by CCK-8 assay (P value: Wilcoxon test). (i) A total of 1000, 5000 or 10,000 Con or shCDC20 CD44+ C4–2B cells were subcutaneously injected into 6-week-old, male, nude mice (n = 6/group). The tumour xenografts derived from Con or shCDC20 CD44+ C4–2B cells and the tumour incidence (inc.) in two generations were shown. (j) H&E and IHC staining of CDC20 and CD44 were examined in second generation tumours derived from Con or shCDC20 CD44+ C4–2B cells (Scale bar = 50 μm). All representative results were collected from three independent experiments and all data were represented as Mean ± SD. All p values were defined as: *p b .05, **p b .01 and ***p b .001.
    Fig. 4. CDC20 activates β-catenin signaling via collapsing the destructive complex and promoting β-catenin nuclear translocation and transactivation. (a) Signaling pathway analysis of differentially expressed genes in control relative to shCDC20 CD44+ C4–2B cells; FDR: false discovery rate. (b) The mRNA expression levels of a series of β-catenin/TCF targeted genes in CDC20-silenced or re-expressed C4–2B CD44+ cells were evaluated by qRT-PCR (P value: Wilcoxon test). (c) The qRT-PCR assay was performed to detect the mRNA expression of multiple stemness related genes in indicated groups (P value: Wilcoxon test). (d) The spheroid formation assay of CD44+ C4–2B of indicated groups was conducted and spheroid number was counted (P value: Wilcoxon test). (e) Western blotting assay of cytoplasmic and nuclear proteins were extracted from CD44+ C4–2B or DU145 cells with and without CDC20 knockdown. (f) Immunofluorescence staining of β-catenin was performed in CD44+ C4–2B or DU145 cells with and without CDC20 knockdown. (g) Interaction of Axin1 and CDC20 in CD44+ C4–2B cells was detected by western blot. (h) Western blot assay for Axin1 and CDC20 expression level was examined in CD44+ C4–2B cells with Con or CDC20 knockdown upon treatment of 50 μg/mL of cycloheximide (CHX) at various time points, and the percentage of intensity compared to 0 min was calculated. The results were come from three independent experiments and all data were represented as Mean ± SD. All p values were defined as: *p b .05, **p b .01 and ***p b .001. r> stem cell hematopoiesis and leukemia by promoting ubiquitination of MEIS1 and p21 [27]. It is preferentially expressed in the basal and supe-rior layers of the epidermis rather than differentiated cells, and serves as an effective key regulator for adult stem cell fate [37]. CDC20 also can 
    enhance the stability of SOX2 by directly interacting with SOX2, thereby conferring self-renewal and tumorigenicity in glioblastoma [38].
    So far, high expression of cancer stem-like cell marker CD44 is asso-ciated with chemical and radiotherapy resistance in breast [39],
    Fig. 5. The expression of CDC20 and β-catenin predicts malignant clinicopathological features and prognosis for prostate cancer patients. (a) H&E staining and IHC staining of CDC20 and β-catenin were shown in prostate cancer tissues (scale bar = 50 μm). (b) Correlation analysis of CDC20 and β-catenin expression was performed in different prostate cancer tissues (Spearman r2 = 0.5979; P value b.001). (c, d) BCR (c) and DFS (d) of prostate cancer patients in different groups were compared according to CDC20 and β-catenin expression using Kaplan-Meier curves (P value: log rank test). (e) The schematic diagram of underlying mechanisms was described in our study. All p values were defined as: *p b .05, **p b .01 and