circROBO1 promotes prostate cancer growth and enzalutamide resistance via accelerating glycolysis

Abstract

Background and aim: As non-coding RNAs, circular RNAs (circRNAs) contribute to the progression of malignancies by regulating various biological processes. In prostate cancer, however, there is still a lack of understanding regarding the potential molecular pathways and roles of circRNAs. Methods: Loss-off function experiments were performed to investigate the potential biological function of circRNA in the progression of prostate cancer. Western blot, qRT-PCR, and IHC assay were used to examine the expression level of different genes or circRNAs. Further molecular biology experiments were conducted to uncover the molecular mechanism underlying circRNA in prostate cancer using dual luciferase reporter and RNA immunoprecipitation (RIP) assays. Results: A novel circRNA (hsa_circ_0124696, named circROBO1) was identified as a significantly upregulated circRNA in both prostate cancer cells and tissues. Suppression of circROBO1 significantly attenuated the proliferation of prostate cancer cells. In addition, we found that the knockdown of circROBO1 remarkably increased the sensitivity of prostate cancer to enzalutamide treatment. A deceleration in glycolysis rate was observed after inhibition of circROBO1, which could suppress prostate cancer growth and overcome resistance to enzalutamide. Our results revealed that circROBO1 promotes prostate cancer growth and enzalutamide resistance via accelerating glycolysis. Conclusion: Our study identified the biological role of the circROBO1-miR-556-5p-PGK1 axis in the growth and enzalutamide resistance of prostate cancer, which is the potential therapeutic target of prostate cancer.

Keywords: PGK1; circROBO1; circular RNAs; enzalutamide resistance; prostate cancer.

Figure 1

circROBO1 is upregulated in prostate cancer cells and tissues. (A) Comparison of the expression level of circROBO1 in normal WPMY1 cells and prostate cancer cells. (B) The expression level of the circROBO1 expression level in ten pairs of prostate cancer tissues and adjacent normal tissues, detected by qPCR analysis. (C) RNase R assays were used to examine circROBO1's circular feature in C4-2B and 22RV1 prostate cancer cell line. (D) Actinomycin D treated assays showed that circular ROBO1 transcripts were more stable than linear ROBO1 transcripts in C4-2B and 22RV1 prostate cancer cell line.

Figure 2

circROBO1 promotes the glycolysis and enzalutamide resistance of prostate cancer cells. (A) Short hairpin RNA (shRNA) targeting the back-splice sequence of circRNA were designed to investigate the function of circROBO1 in prostate cancer. (B) The efficacy of shRNA was determined in C4-2B and 22RV1 prostate cancer cell lines. (C) The proliferation of cells was evaluated using the CCK-8 assay in C4-2B and 22RV1 prostate cancer cell line. (D) The colony-formation ability of cells was evaluated using the Colony-formation assay in C4-2B and 22RV1 prostate cancer cell line. (E) A graph showing the statistical data for the colony-formation test. (F-G) Knockdown of circROBO1 significantly increased the sensitivity of C4-2B and 22RV1 prostate cancer cells to enzalutamide treatment. (H-I) circROBO1-induced glucose uptake and lactate production was found to accelerate glycolysis in C4-2B and 22RV1 prostate cancer cells.

Figure 3

circROBO1 acts as a sponge of miR-556-5p in prostate cancer. (A) The predicted binding sites for miR-556-5p within circROBO1. (B) Comparison of the expression level of miR-556-5p in normal WPMY1 cells and prostate cancer cells. (C) The expression level of the miR-556-5p expression level in ten pairs of prostate cancer tissues and adjacent normal tissues, detected by qPCR analysis. (D-E) RT-qPCR analysis was performed on mRNA expression of 18S, ACTB, circROBO1 and ROBO1 in the nuclear and cytoplasmic fractions. (F-G) Dual luciferase reporter assay of C4-2B and 22RV1 prostate cancer cell lines transfected with miR-556-5p mimics and circROBO1 wild-type or mutant-type luciferase vectors. (H-I) The ms2-related RIP assay was performed by transfecting the ms2-circROBO1 plasmid, the ms2-circROBO1-mutant plasmid, or the Rluc-NC plasmid.

Figure 4

Glycolysis regulating enzyme PGK1 is the downstream target of miR-556-5p in prostate cancer. (A) The downstream target of miR-556-5p, PGK1 mRNA, was predicted by TargetScan online. (B) Comparison of the expression level of PGK1 in normal WPMY1 cells and prostate cancer cells. (C) The expression level of the PGK1 expression level in ten pairs of prostate cancer tissues and adjacent normal tissues, detected by qPCR analysis. (D-E) Enrichment of circROBO1, PGK1 and miR-556-5p on AGO2 in C4-2B and 22RV1 prostate cancer cell lines assessed by RIP assay. (F-G) Dual luciferase reporter assay of C4-2B and 22RV1 prostate cancer cell lines transfected with miR-556-5p mimics and PGK1 mRNA 3'-UTR wild-type or mutant-type luciferase vectors. (F) Enrichment of PGK1 mRNA to AGO2 was significantly increased after the suppression of circROBO1 in C4-2B and 22RV1 prostate cancer cell lines.

Figure 5

circROBO1 promotes prostate cancer enzalutamide resistance and glycolysis through circROBO1-miR-556-5p-PGK1 axis. (A-B) The cell proliferation rate of the C4-2B and 22RV1 prostate cancer cell lines was decreased after the inhibition of circROBO1, which was reversed after the transfection of miR-556-5p mimics. (C-D) The enzalutamide resistance was also reversed by the transfection of miR-556-5p mimics when circROBO1 was inhibited in C4-2B and 22RV1 prostate cancer cells. (E-F) The glycolysis activity of C4-2B and 22RV1 prostate cancer cells was decreased after silencing of circROBO1, which was also reversed after supplement of miR-556-5p mimics. (G) Western blot assay revealed that PGK1 expression was decreased after the suppression of circROBO1 in C4-2B and 22RV1 prostate cancer cell lines. (H) Western blot assay revealed that the decrease of PGK1 was rescued via the supplement of miR-335-5p mimics in C4-2B prostate cancer cells, which further determined the circROBO1-miR-556-5p-PGK1 axis in prostate cancer.