Circular RNA circABCC4 as the ceRNA of miR-1182 facilitates prostate cancer progression by promoting FOXP4 expression

Abstract

In recent years, circular RNAs (circRNAs) have been identified to be essential regulators of various human cancers. However, knowledge of the functions of circRNAs in prostate cancer remains very limited. The correlation between circABCC4 and human cancer is largely unknown. This study aims to investigate the biological functions of circABCC4 in prostate cancer progression and illustrate the underlying mechanism. We found that circABCC4 was remarkably up-regulated in prostate cancer tissues and cell lines and promoted FOXP4 expression by sponging miR-1182 in prostate cancer cells. CircABCC4 knockdown markedly suppressed prostate cancer cell proliferation, cell-cycle progression, migration and invasion in vitro. Furthermore, silencing of the circRNA also delayed tumor growth in vivo. Taken together, our findings indicated that circABCC4 facilitates the malignant behaviour of prostate cancer by promoting FOXP4 expression through sponging of miR-1182. The circABCC4-miR-1182-FOXP4 regulatory loop may be a promising therapeutic target for prostate cancer intervention.

Keywords: FOXP4; circABCC4; migration; proliferation; prostate cancer.

Figure 1

Relative expression of circABCC4 in prostate cancer tissues. A, Heatmap of differentially expressed circRNAs in pairs of prostate cancer and adjacent normal tissues according to a microarray dataset (GSE77661). B, Relative expression of circABCC4 in 47 pairs of prostate cancer and adjacent normal tissues determined by qRT‐PCR. C, Expression patterns of circABCC4 determined by qRT‐PCR in prostate cancer cell lines. D, Kaplan–Meier survival analysis based on the expression of circABCC4 in prostate cancer tissues. *P < 0.05

Figure 2

CircABCC4 knockdown inhibits prostate cancer cell proliferation, migration and invasion. A, Relative expression of circABCC4 in PC3 and DU145 cells transfected with specific siRNAs against circABCC4. B, C, CCK8 assays were used to measure cell proliferation in PC3 and DU145 cells. D, CircABCC4 knockdown reduced colony numbers. E, CircABCC4 knockdown arrested cell‐cycle progression in PC3 and DU145 cells. F, G, Transwell assay was used to determine cell migration and invasion in PC3 and DU145 cells. *P < 0.05

Figure 3

Effects of circABCC4 knockdown on cell proliferation in vivo. A, Tumor volume was measured at the indicated time points. B, Tumor weight was calculated at the end point of the xenograft experiment. C, Relative expression of circABCC4 in formed tumor tissues. D, Protein levels of Ki67 were analysed by Western blot analysis in formed tumor tissues. *P < 0.05

Figure 4

CircABCC4 is a sponge of miR‐1182. A, Predicted binding site of miR‐1182 in circABCC4. B, Luciferase reporter assay was used to verify the direct interaction between circABCC4 and miR‐1182. C, CircABCC4 knockdown promoted miR‐1182 expression in PC3 and DU145 cells. D, Correlation between circABCC4 and miR‐1182 expression in prostate cancer tissues as determined by qRT‐PCR. E, Relative expression of miR‐1182 in 47 pairs of prostate cancer and adjacent normal tissues. *P < 0.05

Figure 5

FOXP4 is a target of miR‐1182. A, Predicted binding site of miR‐1182 in the FOXP4 3’‐UTR. B, Luciferase reporter assay was used to determine the direct interaction between miR‐1182 and FOXP4 3’‐UTR. C, Overexpression of miR‐1182 suppressed the mRNA level of FOXP4 in PC3 and DU145 cells. D, Overexpression of miR‐1182 suppressed the protein level of FOXP4 in PC3 and DU145 cells. E, Correlation between miR‐1182 and FOXP4 expression in prostate cancer tissues. F, Relative expression of FOXP4 in 47 pairs of prostate cancer and adjacent normal tissues. *P < 0.05

Figure 6

CircABCC4 regulates prostate cancer progression by modulating miR‐1182/FOXP4 signalling. A, B, Overexpression of circABCC4 abrogated the effect of the miR‐1182 mimic on FOXP4 expression in PC3 and DU145 cells, whereas mutation of the binding site in circABCC4 abolished this trend. C, Correlation between circABCC4 and FOXP4 expression in prostate cancer tissues. D, Protein levels of FOXP4 at the indicated cell lines. E, Cell proliferation was measured by CCK8 assay. F, G, Cell migration and invasion was evaluated by transwell assay of the indicated cell lines. *P < 0.05