Hsa_circ_0068307 mediates bladder cancer stem cell-like properties via miR-147/c-Myc axis regulation

Abstract

Background: Circular RNAs (circRNAs) play an essential role in the regulation of gene expression. However, the underlying mechanisms remain unknown. This study aimed to evaluate the role of hsa_circ_0068307 in bladder cancer (BCa).

Methods: Rt-qPCR was used to detect hsa_circ_0068307 expression in BCa cell lines. The CCK8, colony formation, and Transwell assays were used to evaluate the effect of hsa_circ_0068307 on BCa cell migration and proliferation. Bioinformatics and luciferase reporter experiments were used to study the regulatory mechanism. Nude mouse xenografts were generated to examine the effect of hsa_circ_0068307 on tumor growth.

Results: The results showed that hsa_circ_0068307 was upregulated in BCa cell lines. Downregulation of hsa_circ_0068307 suppressed cell migration and proliferation in T24 and UMUC3 cells. Hsa_circ_0068307 silencing suppressed cancer stem cell differentiation by upregulating miR-147 expression. Upregulation of miR-147 suppressed c-Myc expression, which is involved in cancer stem cell differentiation. Luciferase reporter assays confirmed that hsa_circ_0068307 upregulated c-Myc expression by targeting miR-147. In vivo studies showed that hsa_circ_0068307 knockdown suppressed T24 tumor growth.

Conclusions: These data indicate that downregulation of hsa_circ_0068307 reversed the stem cell-like properties of human bladder cancer through the regulation of the miR-147/c-Myc axis.

Keywords: Bladder cancer; Cancer stem cell; Hsa_circ_0068307; c-Myc; miR-147.

Fig. 1

Hsa_circ_0068307 is expressed at high levels in BCa and exerts oncogenic effects in the BCa cell lines T24 and UMUC3. a Rt-qPCR detection showing the expression of hsa_circ_0068307 in tumor tissues and adjacent normal tissues. Data are presented as the mean ± SD. ***P < 0.001. b Rt-qPCR detection showing the expression of hsa_circ_0068307 in SV-HUC-1 and BCa cell lines. Data are presented as the mean ± SD. ***P < 0.001 vs. SV-HUC-1. c The expression of hsa_circ_0068307 was detected in T24 and UMUC-3 cells transfected with siRNA hsa_circ_0068307 (si-circRNA) or negative control (NC). Data are presented as the mean ± SD. ***P < 0.001 vs. NC. d, e CCK8 detection showing that hsa_circ_0068307 knockdown suppressed cell proliferation in T24 (d) and UMUC-3 (e) cells. Data are presented as the mean ± SD. ***P < 0.001 vs. NC. f, g Colony formation assays showing the proliferation of T24 and UMUC3 cells after knockdown of hsa_circ_0068307. Data are presented as the mean ± SD. ***P < 0.001 vs. NC. h, i Transwell assays showing the migration of BCa cells after knockdown of hsa_circ_0068307. Data are presented as the mean ± SD. ***P < 0.001 vs. NC

Fig. 2

Hsa_circ_0068307 acts as a sponge for miR-147, and c-Myc is a direct target of miR-147. a The complementary sites within hsa_circ_0068307 and miR-147 were predicted by bioinformatics analysis. The mutated (Mut) version of hsa_circ_0068307 is also shown. b Dual luciferase reporter assays demonstrated that miR-147 is a direct target of hsa_circ_0068307. Data are presented as the mean ± SD. ***P < 0.001 vs. control. c, d qRt-PCR detection showing the expression of hsa_circ_0068307 and miR-147 in T24 and UMUC3 cells transfected with si-circRNA or miR-147 inhibitor. Data are presented as the mean ± SD. ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA. e The predicted binding sites of miR-147 with the 3′-UTR of c-Myc. The mutated version of the 3′-UTR-c-Myc is also shown. f Relative luciferase activity was determined 48 h after transfection with miR-147 mimic/normal control or with the 3′-UTR-c-Myc wild-type/Mut in HEK293T cells. Data are presented as the mean ± SD. ***P < 0.001 vs. control. g, h qRt-PCR detection showing the expression of miR-147 (g) and c-Myc (h) in T24 and UMUC3 cells transfected with miR-147 mimic. Data are presented as the mean ± SD. ***P < 0.001 vs. NC

Fig. 3

Knockdown of hsa_circ_0068307 inhibits cancer stem cell-mediated BCa cell proliferation and migration by regulating the miR-147/c-Myc axis in in vitro. a, b RT-qPCR assay showing hsa_circ_0068307 expression in T24 (a) and UMUC3 (b) cells after transfection with siRNA against hsa_circ_0000291, combined with or without miR-147 inhibitor or the c-Myc overexpression vector. Data are presented as the mean ± SD. ***P < 0.001 vs. NC. c, d RT-qPCR assay showing miR-147 expression in T24 (c) and UMUC3 (d) cells. Data are presented as the mean ± SD. ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA. e, f RT-qPCR detection showing c-Myc expression in T24 (e) and UMUC3 (f) cells. Relative protein levels were analyzed and data are presented as the mean ± SD. ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA. g, h CCK8 assays were performed to assess cell proliferation in T24 (g) and UMUC3 (h) cells. Data are presented as the mean ± SD. *P < 0.05, ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA. i, j Clone formation assay showing the proliferation of T24 and UMUC3 cells. Data are presented as the mean ± SD. **P < 0.01, ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA. k, l Cell migration was determined in T24 and UMUC3 cells using Transwell^® assays. Data are presented as the mean ± SD. **P < 0.01, ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA. m Western blow analysis showing the expression of cancer stem cell-related proteins OCT-4, Sox-2, and NANOG in T24 and UMUC3 cells. Relative protein levels were analyzed and data are presented as the mean ± SD. ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA

Fig. 4

c-Myc overexpression reverses the inhibitory effect of miR-147 on cancer stem cell-mediated BCa cell proliferation and migration in vitro. a, b qRT-PCR assay showing miR-147 (a) and c-Myc (b) expression in T24 and UMUC3 cells. Data are presented as the mean ± SD. ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA. c, d CCK8 assays were performed to assess cell proliferation in T24 (c) and UMUC3 (d) cells. Data are presented as the mean ± SD. *P < 0.05, ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA. e, f Clone formation assay showing the cell proliferation of T24 and UMUC3 cells. Data are presented as the mean ± SD. ***P < 0.001 vs. NC. ^##P < 0.01, ^###P < 0.001 vs. si-circRNA. g, h Cell migration was determined in T24 and UMUC3 cells using Transwell^® assays. Data are presented as the mean ± SD. ***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circRNA. i Western blot analysis showing the expression of cancer stem cell-related proteins OCT-4, Sox-2, and NANOG in T24 and UMUC3 cells

Fig. 5

Downregulation hsa_circ_0068307 suppresses tumor growth in nude mouse xenografts. a Representative photographs of T24 tumor formation in xenografts of nude mice. n = 6 per group. b Summary of tumor volumes in mice measured every week. Data are presented as the mean ± SD. **P < 0.01, ***P < 0.001 vs. NC. c Tumor weight was measured after 30 days of injection. Data are presented as the mean ± SD. ***P < 0.001 vs. NC. d qRT-PCR assay showing the expression of miR-147. Data are presented as the mean ± SD. ***P < 0.001 vs. control. e Western blot analysis of the expression of c-Myc and cancer stem cell-related proteins OCT-4, Sox-2, and NANOG in tumor tissues. Data are presented as the mean ± SD. ***P < 0.001 vs. NC