Circular RNA hsa_circ_0068871 regulates FGFR3 expression and activates STAT3 by targeting miR-181a-5p to promote bladder cancer progression

Abstract

Background: FGFR3 plays an important role in the development of bladder cancer (BCa). Hsa_circ_0068871 is a circRNA generated from several exons of FGFR3. However, the potential functional role of hsa_circ_0068871 in BCa remains largely unknown. Here we aim to evaluate the role of hsa_circ_0068871 in BCa.

Methods: We selected miR-181a-5p as the potential target miRNA of hsa_circ_0068871. The expression levels of hsa_circ_0068871 and miR-181a-5p were examined in BCa tissues and paired adjacent normal tissues by quantitative real-time PCR. To characterize the function of hsa_circ_0068871, BCa cell lines were stably infected with lentivirus targeting hsa_circ_0068871, followed by examinations of cell proliferation, migration and apoptosis. In addition, xenografts experiment in nude mice were performed to evaluate the effect of hsa_circ_0068871 in BCa. Biotinylated RNA probe pull-down assay, fluorescence in situ hybridization and luciferase reporter assay were conducted to confirm the relationship between hsa_circ_0068871, miR-181a-5p and FGFR3.

Results: Hsa_circ_0068871 is over-expressed in BCa tissues and cell lines, whereas miR-181a-5p expression is repressed. Depletion of has_circ_0068871 or upregulation of miR-181a-5p inhibited the proliferation and migration of BCa cells in vitro and in vivo. Mechanistically, hsa_circ_0068871 upregulated FGFR3 expression and activated STAT3 by targeting miR-181a-5p to promote BCa progression.

Conclusions: Hsa_circ_0068871 regulates the miR-181a-5p/FGFR3 axis and activates STAT3 to promote BCa progression, and it may serve as a potential biomarker.

Keywords: Bladder cancer; FGFR3; STAT3; hsa_circ_0068871; miR-181a-5p.

Fig. 1

FGFR3 is overexpressed in BCa tissues, and hsa_circ_0068871 is produced at the FGFR3 gene locus containing exons 4–8. a The relative expression of FGFR3 in normal, low-grade, and high-grade tumour tissues (***p < 0.001) in GSE40355. b Relative expression of FGFR3 in tumour tissues compared with adjacent normal tissues (n = 32, *p < 0.05). c Venn diagram illustrating the overlap of circRNAs detected in the circBase, CircNet and CircInteractome. d Heat maps of expression fold-change in six circRNAs. Red indicates a higher fold-change and blue indicates a lower fold-change. e Expression of six circRNAs in cancer tissues and adjacent normal tissues (n = 10, **p < 0.01). f Hsa_circ_0068871 is produced at the FGFR3 gene locus containing exon 4–8, and the red splicing junction was verified by Sanger sequencing. g RT-PCR assay with divergent or convergent primers indicating the existence of hsa_circ_0068871 in the EJ cell line. h and i, RT-PCR analysis of hsa_circ_0068871, linear FGFR3 and β-actin in EJ and UMUC3 cells treated with RNase R

Fig. 2

Hsa_circ_0068871 is highly expressed in BCa and exerts oncogenic effects in the BCa cell lines EJ and UMUC3. a and b Hsa_circ_0068871 was highly expressed in tumour tissues compared with adjacent normal tissues (**p < 0.01). c Relative expression of hsa_circ_0068871 in SV-HUC-1 cell and BCa cell lines (*p < 0.05, **p < 0.01, ***p < 0.001). d Expression of hsa_circ_0068871 was confirmed by qPCR in BCa cell lines EJ and UMUC3 transfected with si-NC or si-circ_0068871 (**p < 0.01, ***p < 0.001). e and i Cell proliferation was determined in EJ and UMUC3 cell lines following transfection with circ_0068871, si-NC or si-circ_0068871 (**p < 0.01). f, g and h Cell migration assays were performed in EJ and UMUC3 cells using Transwell chambers (**p < 0.01). j, k and l Wound healing assays were performed in EJ and UMUC3 cells treated with si-NC or si-circ_0068871 (**p < 0.01). m, n and o Colony formation assays were performed in EJ and UMUC3 cells treated with si-NC or si-circ_0068871 (**p < 0.01). p, q and r Cell apoptosis of EJ and UMUC3 after si-NC or si-circ_0068871 as determined by flow cytometry (*p < 0.05, **p < 0.01)

Fig. 3

miR-181a-5p had low expression in BCa and acts as a tumour suppressor gene in BCa cell lines EJ and UMUC3. a and b miR-181a-5p had low expression in tumour tissues compared with adjacent normal tissues (***p < 0.001). c Relative expression of miR-181a-5p in SV-HUC-1 cell and BCa cell lines (*p < 0.05, **p < 0.01, ***p < 0.001). d and h Cell proliferation was determined in EJ and UMUC3 cell lines following transfection with miR-181a-5p-inhibitor, miR-181a-5p-NC or miR-181a-5p-mimics (**p < 0.01). e, f and g Cell migration assays were performed in EJ and UMUC3 cells using Transwell chambers (**p < 0.01). i, j and k, Wound healing assays were performed in EJ and UMUC3 cells treated with miR-181a-5p-NC or miR-181a-5p-mimics (**p < 0.01). l, m and n Colony formation assays were performed in EJ and UMUC3 cells treated with miR-181a-5p-NC or miR-181a-5p-mimics (**p < 0.01). o, p and q Cell apoptosis of EJ and UMUC3 after miR-181a-5p-NC or miR-181a-5p-mimics as determined by flow cytometry (**p < 0.01)

Fig. 4

Hsa_circ_0068871 acts as a sponge for miR-181a-5p, and FGFR3 is a direct target of miR-181a-5p. a and b Putative complementary sites within miR-181a-5p and hsa_circ_0068871 were predicted by bioinformatics analysis (RNA 22v2). c Correlations between hsa_circ_0068871 and miR-181a-5p expression were found with Pearson’s correlation analysis in BCa tissue samples (n = 32). d and e Dual luciferase reporter assays demonstrated that miR-181a-5p is a direct target of hsa_circ_0068871 (**p < 0.01). f miR-181a-5p was pulled down and enriched with hsa_circ_0068871 specific probe and then detected by qRT-PCR (**p < 0.01). g and h Biotin-coupled miR-181a-5p captures hsa_circ_0068871 in the complex compared with biotin-coupled NC in biotin-coupled miRNA capture by agarose gel electrophoresis and analysis products of g by qRT-PCR. i Detection of colocalization of hsa_circ_0068871 and miR-181a-5p in cytoplasm by RNA FISH assay (magnification, × 400). Nuclei were stained blue (DAPI), hsa_circ_0068871 was stained green, and miR-181a-5p was stained red. j Correlations between miR-181a-5p and FGFR3 expression were found with Pearson’s correlation analysis in BCa tissue samples (n = 32). k Putative complementary sites within miR-181a-5p and FGFR3 were predicted by bioinformatics analysis (TargetScan). l Dual luciferase reporter assays demonstrated that FGFR3 is a direct target of miR-181a-5p (**p < 0.01)

Fig. 5

Hsa_circ_0068871 activates STAT3 and regulates the miR-181a-5p/FGFR3 axis. a and d In EJ and UMUC3 cell lines, the expression of miR-181a-5p increased and the expression of FGFR3 decreased after knockdown of hsa_circ_0068871 by qRT-PCR. b and c The protein levels of FGFR3 and p-STAT3 to be decreased after transfection of si-circ_0068871 in EJ and UMUC3 cells by Western blot. e and f The protein levels of FGFR3 and p-STAT3 to be decreased after transfection of miR-181a-5p-mimics in EJ and UMUC3 cell lines by Western blot. g and j Low miR-181a-5p expression partially rescues the promotive effects of hsa_circ_0068871 expression on EJ and UMUC3 cells by CCK-8 assay. h and i, k and l Western blot showed that lowering the expression of miR-181a-5p can partly promote the low expression of FGFR3 and p-STAT3 caused by si-circ_0068871in EJ and UMUC3 cells

Fig. 6

Hsa_circ_0068871 can promote tumour formation in xenografted nude mice. a Representative images of nude mice injected with EJ cells (five mice per group). b Representative images of xenograft tumours in nude mice. c The growth curves of xenografts (**p < 0.01, ***p < 0.001). d Extract protein from tumours and measuring protein expression of FGFR3 using Western blot. e Immunohistochemistry (IHC) staining of FGFR3 in xenografts. Scale bar = 100 μm for 10 × and 100 μm for 20 ×. f The schematic diagram shows the mechanism through which hsa_circ_0068871 regulates FGFR3 expression and activates STAT3 targeting miR-181a-5p