The role of UBE2C on self?renewal ability of stem?like cells in prostate cancer

ZHANG Qin; LI Jiang; WU Hongping; CHEN Lei; SU Changqing.

Abstract：investigate the regulatory role of ubiquitin?conjugating enzyme 2C(UBE2C)in how prostate cancer stem cell?like cells maintain self?renewal ability. Methods The ratio of CD44+cells in DU145 and C4?2B cell lines of prostate cancer was tested by magnetic activated cell selection. The sequences of si?UBE2C1#, si?UBE2C2# and si?UBE2C3#were transfected into DU145?CD44+and C4?2B?CD44+cell lines, and negative control group was set. Real time fluorescence quantitative PCR (QPCR) was used to detect the knock?down efficacy of the 3 sequences. Western blotting was used to detect the expression of UBE2C protein after transfection. The expression of cancer stem cell?related gene ( KLF4, SOX2, Oct4, Myc, NANOG, ABCG2, CTNNB1, UBE2C and ACTB) expression was detected by QPCR, and self?renewal ability was assessed by spheroid formation and limited dilution assay. Cell proliferation assay was performed to test the sensitivity of cancer stem cells to docetaxel. Results Magnetic activated cell selection showed that the percentage of CD44+cells was 90. 3% and 98. 3% respectively in C4?2B and DU145 cell lines. QPCR showed that the expression of UBE2C decreased compared with control group after the transfection of 3 sequences(P＜0. 01), and the knock?down effi?cacy of si?UBE2C1# and si?UBE2C2# was better than si?UBE2C3#, so they were chosen for the following experiments. Western blotting showed that the expression of UBE2C protein decreased compared with control group after the transfection( P＜0. 01). Knock?down of UBE2C expression significantly inhibited the expression of stemness?related genes (Oct4, Myc, NANOG, ABCG2, CTNNB1, UBE2C and ACTB) in prostate cancer CD44+stem?like cells. The spheroid formation and frequency test showed that the first and second gener?ation of spheroid formation numbers of C4?2B?CD44+and DU145?CD44+cell lines were inhibited after the knock?down of UBE2C, and the spheroid formation frequency was also inhibited compared with control group ( P＜0. 05). In addition, knock?down of UBE2C ex?pression did increase the sensitivity of prostate cancer CD44?positive stem?like cells to docetaxel. In C4?2B?CD44+cells, the IC50 of do?cetaxel in control group, si?UBE2C1# group and si?UBE2C2# group was 42. 3, 7. 5 and 3. 6 nmol／L, and in DU145?CD44+cells, they were 56. 2, 11. 2 and 5. 0 nmol／L.Conclusion UBE2C plays an important role in the maintenance of stem cell characteristics in pros?tate cancer CD44+stem?like cells, and is expected to be a therapeutic target for inhibiting prostate cancer stem?like cells.