Anti-cancer role of curcumin in prostate cancer cells via regulation of m6A-modified circ0030568-FMR1 signaling pathway

Abstract

Background: Prostate cancer (PCa) is the most prevalent adult malignancies worldwide and studies have shown that circular RNAs (circRNAs) play critical roles in the development and progression of PCa. As the most abundant modification, N6-methyladenosine (m6A) modification functions in regulating circRNAs expression and has been shown to regulate PCa progression. However, the biological relevance of m6A modification of circRNAs in PCa remains unclear. In addition, curcumin is reported to inhibit a variety of cancer cells while the biological functions in PCa have not yet been fully elucidated. Thus, our study aims to investigate whether curcumin can suppress PCa progression through the m6A-modified circRNAs.

Methods: By conducting m6A methylation immunoprecipitation combined with quantitative real-time polymerase chain reaction (MeRIP-qPCR) assay, cell counting kit-8 (CCK-8) assay and wound healing assay, increased m6A modification on circ0030568 was detected and upregulated circ0030568 was also observed in different PCa cells lines, which promotes proliferation and migration of PCa cells.

Results: More importantly, the results confirmed that curcumin could suppress the proliferation and migration of PCa cells lines by inhibiting METTL3-modified circ0030568. Mechanistically, m6A reader YTHDF2 elevated the stability of circ0030568 via m6A modification and curcumin could suppress PCa progression by inhibiting YTHDF2 mediated circ0030568 stability.

Conclusions: Taken together, circ0030568 may act as a promising biomarker and an attractive target for PCa treatment and curcumin's inhibition of m6A-modified circ0030568 may be a potential mechanism of its anti-PCa.

Keywords: Curcumin; FMR1; N6-methyladenosine (m6A); circ0030568; prostate cancer (PCa).

Figure 1

Characterization of circ0030568 in PCa cells. (A) Heat map showing m6A modification levels of circular RNAs differentially expressed in WPMY-1, PC3 and DU145 cells. (B) MeRIP-qPCR analysis for the m6A modification levels of indicated circular RNAs. (C) qRT-PCR analysis for the expression of indicated circular RNAs. (D) Volcano plot of GSE179321 constructed using log2(fold change values) and adjusted P value. Red and blue points in the plot represent the up-regulated and down-regulated circRNAs in normal or prostate cancer tissues. Grey points represent circRNAs with no significant changes. (E) qRT-PCR analysis of the relative expression of circ0030568 in normal or prostate cancer tissues from GSE179321 database. (F) CircBase database analysis of structural characteristics of circ0030568. (G) UCSC database analysis of characterization of circ0030568. (H) Circ0030568 and linear ABCC4 mRNA levels were detected by qRT-PCR in PC3 and DU145 cells treated with or without RNase R. The relative RNA levels were normalized to those measured in the mock group. (I) Circ0030568 and ABCC4 mRNA were abundant in the cytoplasm of PC3 and DU145 cells. Actin and U6 were used as positive controls in the cytoplasm and nucleus, respectively. (J-K) RNA-FISH for circ0030568. Nuclei were stained with DAPI; scale bar: 10 µm. **, P<0.01; ***, P<0.001. Data represent mean ± SEM. N=3. PCa, prostate cancer; qRT-PCR, quantitative real-time polymerase chain reaction; UCSC, University of California Santa Cruz; DAPI, 4’,6-diamidino-2-phenylindole; SEM, standard error of mean.

Figure 2

Circ0030568 promotes PCa cells proliferation and migration. (A,B) qRT-PCR analysis of circ0030568 and ABCC4 expression was performed for PC3 cells transfected with negative control (scramble or vector), sicirc0030568, or circ0030568 OE plasmids. Relative circ0030568 and ABCC4 expression was normalized to Actin expression. (C,D) qRT-PCR analysis of circ0030568 expression and ABCC4 was performed for DU145 cells transfected with negative control (scramble or vector), sicirc0030568, or circ0030568 OE plasmids. Relative circ0030568 and ABCC4 expression was normalized to Actin expression. (E,F) CCK8 assays were performed to assess the cellular proliferation in different treatment groups. (G,H) Wound healing assays were performed to explore the effects of circ0030568 on the migration of PC-3 and DU145 cells transfected with negative control, sicirc0030568, or circ0030568 OE plasmids. Scale bar, 50 µm. *, P<0.05; **, P<0.01; ***, P<0.001; ns means no significance. Data represent mean ± SEM. N=3. PCa, prostate cancer; qRT-PCR, quantitative real-time polymerase chain reaction; OE, overexpression; CCK8, cell counting kit-8; SEM, standard error of mean.

Figure 3

Curcumin inhibits PCa cells proliferation and migration by downregulating circ0030568. (A) The chemical structure formula of curcumin. (B) qRT-PCR analysis of circ0030568 expression in PC3 cells treated with different concentrations of curcumin (1, 10, 20, and 50 µM). (C) qRT-PCR analysis of circ0030568 expression in DU145 cells treated with different concentrations of curcumin (1, 10, 20, and 50 µM). (D-E) The expression level of circRNAs was measured by RT-qPCR analysis in PC3 and DU145 cells after treatment with 10 µM curcumin for 24 h. (F-G) The CCK-8 assays were performed to determine the viability of PC3 and DU145 cells treated with 10 µM curcumin for different time. (H-I) The migration of PC3 and DU145 cells treated with or without 10 µM curcumin was tested by the cell scratch wound assay. Scale bar, 50 µm. Experiments were performed in triplicate. **, P<0.01p ***, P<0.001. Error bars indicate SEM. N=3. PCa, prostate cancer; qRT-PCR, quantitative real-time polymerase chain reaction; CCK-8, cell counting kit-8; SEM, standard error of mean.

Figure 4

METTL3 mediates m6A modification of circ0030568. (A,B) Predicted m6A site in circ0030568 from results of a sequence-based N6-methyladenosine (m6A) modification site predictor (SRAMP) and RMBase. (C) The expression pattern of YTHDF2 were analyzed in 498 PCa tissues and 52 normal controls (TCGA database). (D,E) RIP assays showing the association of METTL3 and METTL14 with circ0030568 in PC3 and DU145 cells. Relative enrichment representing RNA levels associated with METTL3 and METTL14 relative to an input control. IgG antibody served as a control. (F,G) The circ0030568-protein complex pulled down by circ0030568 junction probe with protein extracts from PC3 and DU145 cells. Immunoblot analysis of METTL3 after pull-down assay showing its specific association with circ0030568. “+” means control probe or circ0030568 probe treatment. “−” means no control probe or circ0030568 probe treatment. (H) Western blotting analysis for the expression of METTL3 in PC3 and DU145 cells. (I,J) Western blotting and qRT-PCR analysis for the interference efficiency of METTL3 in PC3 cells transfected with negative control (scramble) or siMETTL3 #1-3. (L,M) Western blotting and qRT-PCR analysis for the interference efficiency of METTL3 in DU145 cells transfected with negative control (scramble) or siMETTL3 #1-3. qRT-PCR analysis for the expression of circ0030568 in PC3 (K) and DU145 (N) cells transfected with METTL3 siRNA. ***, P<0.001. Data represent mean ± SEM. N=3. RMBase, RNA modification database; PCa, prostate cancer; SRAMP, sequence-based RNA adenosine methylation site predictor; TCGA, The Cancer Genome Atlas; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of mean.

Figure 5

YTHDF2 upregulates circ0030568 expression by increasing its stability. (A) The expression pattern of YTHDF2 were analyzed in 498 PCa tissues and 52 normal controls (TCGA database). (B) The structure of YTHDF2 domain. (C,D) RIP assays showing the association of YTHDF2 with circ0030568 in PC3 and DU145 cells. Relative enrichment representing RNA levels associated with YTHDF2 relative to an input control. IgG antibody served as a control. “+” means control probe or circ0030568 probe treatment. “−” means no control probe or circ0030568 probe treatment. (E) Western blotting analysis for the expression of YTHDF2 in PC3 and DU145 cells. (F,G) Western blotting analysis for the expression of YTHDF2 in PC3 cells transfected with negative control (scramble or vector), siYTHDF2 #1-3, or oeYTHDF2 #1-3. (H) qRT-PCR analysis of circ0030568 expression was performed for PC3 cells transfected with negative control (scramble or vector), siYTHDF2, or oeYTHDF2. (I,J) Western blotting analysis for the expression of YTHDF2 in DU145 cells transfected with negative control (scramble or vector), siYTHDF2, or oeYTHDF2. (K) qRT-PCR analysis of circ0030568 expression was performed for DU145 cells in different treatment groups. (L) Western blotting analysis for the expression of YTHDF2 in PC3 and cells in DU145 cells treated with curcumin. (M) qRT-PCR analysis for the expression of circ0030568 in PC3 and DU145 cells treated with curcumin combined with YTHDF2 siRNA or overexpression. *, P<0.05; ***, P<0.001. Data are represented as mean ± SEM. N=3. PCa, prostate cancer; TCGA, The Cancer Genome Atlas; RIP, RNA immunoprecipitation; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of mean.

Figure 6

Curcumin inhibits PCa progression via regulation of m6A-modified circ0030568-FMR1 signaling pathway. (A) AGO2-RIP experiments show the enrichment of circ0030568 the AGO2 antibody. (B) The exploration of circ0030568’s protein coding potential using the circRNADb database. (C) Prediction of RNA-protein interaction of circ0030568 with FMR1 using the catRAPID algorithm. (D) RIP assays showing the association of FMR1 with circ0030568 in PC3 and DU145 cells. Relative enrichment representing RNA levels associated with FMR1 relative to an input control. IgG antibody served as a control. (E) Western blotting analysis for the expression of FMR1 in PC3 and DU145 cells. (F) Western blotting analysis for the expression of FMR1 in PC3 cells transfected with negative control (scramble or vector), sicirc0030568, or oecirc0030568. (G) qRT-PCR analysis of FMR1 expression was performed for PC3 cells transfected with negative control (scramble or vector), sicirc0030568, or oecirc0030568. (H) Western blotting analysis for the expression of FMR1 in DU145 cells transfected with negative control (scramble or vector), sicirc0030568, or oecirc0030568. (I) qRT-PCR analysis of FMR1 expression was performed for DU145 cells in different treatment groups. (J) Western blotting analysis for the expression of FMR1 in PC3 and cells in DU145 cells treated with curcumin. (K) qRT-PCR analysis for the expression of FMR1 in PC3 and DU145 cells treated with curcumin combined with circ0030568 siRNA or overexpression. *, P<0.05; ***, P<0.001; ns means no significance. Data represent mean ± SEM. N=3. PCa, prostate cancer; RIP, RNA immunoprecipitation; circRNADb, circRNA database; catRAPID, an algorithm to estimate the binding propensity of protein-RNA pairs; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of mean.

Figure 7

Proposed model of curcumin inhibiting PCa progression via regulation of m6A-modified circ0030568-FMR1 pathway. PCa, prostate cancer.