The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

Abstract

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

Keywords: LEF1; SGK1; circRAB3IP; eIF4A3; enzalutamide resistance.

Figure 1

(A) Analysis of circRNA microarray data (GSE118959) from EnzR PCa cells and RNA-seq data of 25 pairs of matched tumor and normal for PRAD samples by Josh et al. (B) Schematic diagram of the genomic location and splicing pattern of circRAB3IP. The divergent and convergent primers were designed to detect circRAB3IP and RAB3IP, respectively. (C) PCR was performed to detect the existence of circRAB3IP from cDNA and gDNA in C4-2-EnzR cells using divergent and convergent primers, respectively. (D) qRT-PCR was conducted to determine the abundances of circRAB3IP and linear RAB3IP mRNA in PCa cells treated with RNase R at the indicated time points. (E) The RNA expression levels of circRAB3IP and RAB3IP were detected by qRT-PCR after treatment with actinomycin D at the indicated time points. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001 compared with the controls. N.S., not significant compared with the controls.

Figure 2

(A) The proliferation ability of C4-2-EnzR cells was evaluated by MTT assay after knocking down circRAB3IP. (B) The proliferation ability of LNCaP-EnzR cells was evaluated by MTT assay after knocking down circRAB3IP. (C) The proliferation ability of C4-2 cells was evaluated by MTT assay after overexpressing circRAB3IP. (D) The proliferation ability of LNCaP cells was evaluated by MTT assay after overexpressing circRAB3IP. (E) C4-2-EnzR cells were treated with different concentrations of Enz as indicated after knocking down circRAB3IP #1, and cell viability was evaluated by CCK-8 assay. (F) LNCaP-EnzR cells were treated with different concentrations of Enz as indicated after knocking down circRAB3IP #1, and cell viability was evaluated by CCK-8 assay. (G) C4-2 cells were treated with different concentrations of Enz as indicated after overexpressing circRAB3IP, and cell viability was evaluated by CCK-8 assay. (H) LNCaP cells were treated with different concentrations of Enz as indicated after overexpressing circRAB3IP, and cell viability was evaluated by CCK-8 assay. (I) C4-2 cells were treated with/without 1μM Enz after overexpressing circRAB3IP or not (PWPI), and cell viability was evaluated by CCK-8 assay. (J) LNCaP cells were treated with/without 2μM Enz after overexpressing circRAB3IP or not (PWPI), and cell viability was evaluated by CCK-8 assay. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared with the controls.

Figure 3

(A) Nuclear mass separation assays indicated the location of circRAB3IP, RAB3IP, U6 and GAPDH in C4-2-EnzR and LNCaP-EnzR cells. (B) RNA-FISH indicated the location of circRAB3IP in C4-2-EnzR and LNCaP-EnzR cells. (C) AGO2-RIP assays were performed using an antibody against AGO2 on extracts and detected by qRT-PCR assays in C4-2-EnzR and LNCaP-EnzR cells. (D) circRIP assays were performed in C4-2-EnzR cells using a circRAB3IP-specific probe, and the enrichment of miRNAs was detected by PCR. (E) A pull-down assay for biotin-labeled miRNA was used to evaluate the binding properties between miR-133a-3p/miR-133b and circRAB3IP in C4-2-EnzR cells. (F) A dual luciferase reporter assay was used to prove the binding properties between miR-133a-3p/miR-133b and circRAB3IP. (G) C4-2 cells were treated with different concentrations of Enz as indicated after overexpressing circRAB3IP-Mut or not (PWPI), and cell viability was evaluated by CCK-8 assay. (H) LNCaP cells were treated with different concentrations of Enz as indicated after overexpressing circRAB3IP-Mut or not (PWPI), and cell viability was evaluated by CCK-8 assay. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the controls. N.S., not significant compared with the controls.

Figure 4

(A) The protein levels of SGK1 were detected by WB after treatment with miR-133a-3p mimics in C4-2-EnzR and LNCaP-EnzR cells. (B) The protein levels of SGK1 were detected by WB after treatment with miR-133a-3p inhibitors in C4-2 and LNCaP cells. (C) The upregulation of SGK1 in C4-2-EnzR cells transfected with miR-133a-3p inhibitors was reversed by knocking down circRAB3IP, as detected by WB. (D) The downregulation of SGK1 in C4-2 cells transfected with miR-133a-3p mimics was reversed by overexpression of circRAB3IP, as detected by WB. (E) C4-2-EnzR cells were treated with different concentrations of Enz as indicated after overexpressing circRAB3IP, knocking down SGK1 or their combination, and cell viability was evaluated by CCK-8 assay. (F) LNCaP-EnzR cells were treated with different concentrations of Enz as indicated after overexpressing circRAB3IP, knocking down SGK1 or their combination, and cell viability was evaluated by CCK-8 assay. (G) LNCaP cells were treated with different concentrations of Enz as indicated after overexpressing SGK1, knocking down circRAB3IP or their combination, and cell viability was evaluated by CCK-8 assay. (H) C4-2 cells were treated with different concentrations of Enz as indicated after overexpressing SGK1, knocking down circRAB3IP or both in combination, and cell viability was evaluated by CCK-8 assay. (I) C4-2-EnzR cells were treated with different concentrations of Enz, GSK650394 or their combination as indicated, and cell viability was evaluated by CCK-8 assay. (J) LNCaP-EnzR cells were treated with different concentrations of Enz, GSK650394 or their combination as indicated, and cell viability was evaluated by CCK-8 assay. (K) Mice bearing C4-2-EnzR xenografts were treated with control, Enz, GSK650394 or their combination for 24 days, and tumor volumes were measured every 4 days. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the controls. N.S., not significant compared with the controls.

Figure 5

(A) The protein levels of eIF4A3 were detected by WB in PCa cells. (B) The expression of circRAB3IP was detected by RT-PCR after knocking down eIF4A3 in C4-2-EnzR and LNCaP-EnzR cells. (C) The expression of circRAB3IP was detected by RT-PCR after overexpressing eIF4A3 in C4-2 and LNCaP cells. (D) The putative binding sites of eIF4A3 in the upstream and downstream regions of the circRAB3IP pre-mRNA were predicted with the CircInteractome database. (E) RIP assay confirmed that eIF4A3 could directly bind to the RAB3IP pre-mRNA in C4-2-EnzR cells. (F) The protein levels of LEF1 and PAX2 were detected by WB in PCa cells. (G) The expression of circRAB3IP was detected by RT-PCR after knocking down LEF1 in C4-2-EnzR and LNCaP-EnzR cells. (H) The expression of circRAB3IP was detected by RT-PCR after overexpressing LEF1 in C4-2 and LNCaP cells. (I) Schematic illustration of three putative binding sites of LEF1 on the RAB3IP promoter is shown. (J) ChIP assays were performed to determine which putative LEF1 binding site the RAB3IP promoter was bound to in C4-2-EnzR cells. (K) Luciferase reporter assays were performed to determine whether LEF1 could promote the luciferase activity of the wild-type RAB3IP promoter. (L) The schematics of the mechanism which circRAB3IP might sponge miR-133a-3p and miR-133b to regulate SGK1 expression, leading to Enz resistance in PCa cells. Data are presented as mean ± SEM. *P < 0.05 compared with the controls. N.S., not significant compared with the controls.