Circular RNA cTFRC acts as the sponge of MicroRNA-107 to promote bladder carcinoma progression

Abstract

Background: Circular RNA (circRNA) represents a broad and diverse endogenous RNAs that can regulate gene expression in cancer. However, the regulation and function of bladder cancer (BC) circRNAs remain largely unknown.

Methods: Here we generated circRNA microarray data from three BC tissues and paired non-cancerous matched tissues, and detected circular RNA-cTFRC up-regulated and correlated with tumor grade and poor survival rate of BC patients. We subsequently performed functional analyses in cell lines and an animal model to support clinical findings. Mechanistically, we demonstrated that cTFRC could directly bind to miR-107 and relieve suppression for target TFRC expression.

Results: We detected circular RNA-cTFRC up-regulated and correlated with tumor grade and poor survival rate of BC patients. Knock down of cTFRC inhibited invasion and proliferation of BC cell lines in vitro and tumor growth in vivo. Furthermore, the expression of cTFRC correlated with TFRC and negatively correlated with miR-107 both in BC cell lines and BC clinical samples. In addition, up-regulation of cTFRC promoted TFRC expression and contributed to an epithelial to mesenchymal transition phenotype in BC cells. Finally, we found that cTFRC acts as a competing endogenous RNA (ceRNA) for miR-107 to regulate TFRC expression.

Conclusions: cTFRC may exert regulatory functions in BC and may be a potential marker of BC diagnosis or progression.

Keywords: Bladder Cancer; Circular RNA; TFRC; cTFRC; miR-107.

Fig. 1

Up-regulated circRNAs in BC tumor tissues and its correlation with prognosis of patients. a cTFRC increased in BC tissues as compared to that in the matched nontumor tissues analyzed by circRNAs Arraystar Chip. b Schematic representation of the high expression level of cTFRC in 57 BC patients tissues compared with adjacent normal patients tissues by qPCR. c cTFRC upregulated in recurrent BC patients. d The high expression levels of cTFRC in BC patients with high grade. e Advanced T stage is associated with higher cTFRC levels. f The expression of cTFRC higher in patients with lymphatic metastasis. g Prognostic significance of cTFRC expression for BC patients was performed with cTFRC values by using the median value as the cutoff

Fig. 2

The effects of cTFRC on BC cell invasion. a Scheme illustrating the production of cTFRC. b The relative cTFRC level in BC cells. c Transwell assay different BC cells invasion activity. d Comparison of the abundance of cTFRC in the nuclear and cytoplasmic. Fractionation of EJ and T24 cells followed by qPCR. U1 RNA served as a positive control for nuclear gene expression. e RNA FISH for cTFRC. cTFRC probes were labeled with Cy3. Nuclei were stained with DAPI. Scale bar, 10 μm. f qPCR analysis of the transfection efficiency of shcTFRC vectors after transfection in EJ or T24 cells. g Determination of cell invasive potential of EJ, T24 or 5637 cells transfected with shcTFRC by Transwell assay. Data are the means ± SD of three independent experiments. *P < 0.05; **P < 0.01

Fig. 3

Silencing of cTFRC RNA inhibits BC cell proliferation. a-d Proliferation of EJ, T24, 5637 and HCV29 cells transfected with the above cTFRC shRNA-1 assessed using ³H-TdR incorporation at the indicated days. Left panel: Representative the growth curve of EJ, T24, 5637 and HCV29 cells transfected with shcTFRC-1. Right panel: Quantification of the expression of cTFRC at different time. Data in a-d are the mean ± SD of three experiments. e The growth curve of subcutaneous xenograft tumor from EJ or T24 cells in nude mice. shCtrl or shcTFRC-1 cells were subcutaneously injected into nude mice. Tumor size was measured every 5 days. Values were presented as the mean ± SD of 12 mice in each group. (Repeated-measures analysis of variance, **P < 0.01)

Fig. 4

Overexpression of cTFRC induces BC cell proliferation and invasion. a, b Proliferation of EJ and T24 cells transfected with cTFRC vector assessed using ³H-TdR incorporation at the indicated days. c Determination of cell invasive potential of EJ and T24 cells transfected with cTFRC vector by transwell assay. d, e Proliferation of EJ and T24 cells transfected with shCtrl, shcTFRC-1 or shcTFRC-1 + oe-cTFRC. f, g Transwell assays were used to evaluate the invasion in EJ and T24 cells after transfection with shCtrl, shcTFRC-1 or shcTFRC-1 + oe-cTFRC. Data are the means ± SD of three independent experiments. **P < 0.01

Fig. 5

TFRC induces bladder cancer cell proliferation, migration and invasion. a qPCR analysis of TFRC expression in different BC cell lines. b, c cTFRC level correlated with TFRC mRNA expression in BC cell lines (b) and BC clinical samples (c). d TFRC was silenced in EJ and T24 BC cell lines by shRNAs and the TFRC-silenced stable cell lines were established. e Proliferation of EJ and T24 cells transfected with the above TFRC shRNA assessed using ³H-TdR incorporation at the indicated days. f-h TFRC depletion causes a diminished attachment (f), migration (g) and invasion (h) capacity in three BC patients’ primary cells. i TFRC-silenced EJ and T24 cells were subcutaneously injected into BALB/c nude mice for observation of tumor growth. Results are shown as mean ± SD. n = 12 for each group. Data are shown as mean ± SD. **P < 0.01 by two-tailed Student’s t test. Data represent at least three independent experiments

Fig. 6

cTFRC mediates TGFβ-induced EMT. a-b qRT-PCR or western blot analysis of cTRFC, TFRC and E-cadherin levels: the protein expression of E-cadherin was reduced, while the expressions of cTFRC and TFRC were increased in EJ cells treated with TGF-β or LiCl, in comparison with those at 0 mM (control). β-actin was used as an internal control. c Phase contrast images of EJ or T24 cells 72 h after cTFRC-Vector transfection, showing an EMT-like morphology in cTFRC treated cells. d Western blot analysis of TFRC expression in overexpression cTFRC EJ or T24 cells. e TFRC and E-cadherin expression in shcTFRC and shCtrl-treated EJ or T24 groups. f TFRC and E-cadherin expression in shTFRC and shCtrl-treated EJ or T24 groups. β-actin was used as an internal control

Fig. 7

cTFRC targets miR-107 and inhibits its activity. a Binding sites of miR-107 in 3’UTR of cTFRC and TFRC. b EJ or T24 cells lysates were subject to RNA pull-down assay and tested by RT–PCR. Relative level of miR-107 was normalized to input. miR-107 can be pulled down by cTFRC probe. c Lysates prepared from EJ or T24 cells were subject to RNA pull-down assay and tested by RT–PCR. Relative expression of cTFRC was normalized to input. cTFRC can be pulled down by miR-107 probe. d miR-107 colocalized with cTFRC in BC primary tumor cells and adjacent normal cells were detected by FISH. Data are from three independent experiments. Magnification, 400X. e TFRC can be pulled down by miR-107 probe in EJ or T24 cells. Data are shown as mean ± SD. **P < 0.01 by two-tailed Student’s t test. Data represent at least three independent experiments

Fig. 8

cTFRC specifically rescue the suppressive effects of miR-107 on TFRC expression. a qPCR analysis of cTFRC, miR-107 and TFRC expression in xenograft tumor from BC cell transfected with shcTFRC-1 or shCtrl in nude mice. b, c miR-107 RNA pull-down assay and qPCR analysis of TFRC expression in EJ or T24 cells lysates transfected with cTFRC or vector. d, e Luciferase reporter assay showed the luciferase intensity of EJ and T24 cells transfected with TFRC (wild/mutant) and miR-107 (nc/miR-107) or co-transfected with cTFRC and miR-107 (nc/miR-107)

Fig. 9

The schematic diagram shows the mechanism underlying cTFRC as a ceRNA for miR-107. Activation of the EMT signaling pathway in bladder cancer patients promotes transcriptional activation of TFRC, and expression of TFRC and cTFRC is increased in bladder cancer cells. On the one hand, TFRC can promote the degradation of E-cadherin, forming positive feedback to promote EMT, and ultimately promote cell invasion and migration. On the other hand, TFRC acts as a receptor to initiate multiple signaling pathways that promote downstream cell proliferation. In terms of mechanism, as shown by the dotted oval box, cTFRC upregulating the expression of TFRC by absorbs miRNA-107 targeting TFRC, thereby promoting epithellal-mesenchymal transition, cell proliferation and cell invasion in bladder carcinoma