Figure 7
The circTCF25 promotes proliferation and migration of bladder cancer cells via circTCF25-miR-103a-3p/miR-107-CDK6 pathway.
(A) The sketch of structure of circTCF25 and mock vector are shown. Both of them consist of cytomegalovirus promoter, front circular frame (flanking genomic sequence) and back circular frame (inverted upstream sequence). But only circTCF25 vector includes splice sites and TCF25 encoding sequence. (B) Real-time qPCR analysis shows the expression levels of circTCF25 after transfection in T24 and EJ cells. Over-expression of circTCF25 has been confirmed compared to control. Data are shown as mean ± SD, ***P < 0.001, n = 3. (C) Over-expression of circTCF25 down-regulates the expression of miR-103a-3p and miR-107 significantly compared with mock vector group in T24 and EJ cells by qPCR. Mean ± SD, **P < 0.01, ***P < 0.001, n = 3. (D) The expression levels of miR-103a-3p and miR-107 in twenty-pair samples. BC, bladder carcinomas. NC, adjacent normal controls. Mean ± SEM. *P < 0.05. (E) Western blot analysis shows the CDK6 protein level in EJ and T24 cells after transfection with circTCF25, mock and nothing. β-actin is internal control. *P < 0.05, n = 3. (F) Representative photographs show immunohistochemical staining of tissues in (D) with antibody of CDK6. Scale bar, 100 μm. Bladder tumor tissues showed stronger brown staining for CDK6 compared with para-cancer tissues. (G) Western blot analysis of CDK6 using tissues in (F), *P < 0.05. (H) Would healing assay evaluates cell migration capability. The width of wound is measured in three-independent wound sites of each group at 0 and 6 hour. Scale bar, 200 μm. Mean ± SD, **P < 0.01. (I) Edu proliferation assay determines cell proliferation ability. More red granules are observed in T24-circTCF25 and EJ-circTCF25 cells, reflecting more active DNA replication. The nuclei are stained by Hoechst 33342. Scale bar, 100 μm. Mean ± SD, *P < 0.05.
