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. 2023 Mar 5;19(5):1543-1563.
doi: 10.7150/ijbs.77133. eCollection 2023.

M6A-modified circRBM33 promotes prostate cancer progression via PDHA1-mediated mitochondrial respiration regulation and presents a potential target for ARSI therapy

Chuanfan Zhong  1 , Zining Long  1 , Taowei Yang  1 , Shuo Wang  1 , Weibo Zhong  1 , Feng Hu  2 , Jeremy Yuen-Chun Teoh  3 , Jianming Lu  1   4 , Xiangming Mao  1
Affiliations

M6A-modified circRBM33 promotes prostate cancer progression via PDHA1-mediated mitochondrial respiration regulation and presents a potential target for ARSI therapy

Chuanfan Zhong et al. Int J Biol Sci. .

Abstract

N6-Methyladenosine (m6A) is the most prevalent RNA modification in various types of RNA, including circular RNAs (circRNAs). Mounting evidence has shown that circRNAs may play critical roles in diverse malignancies. However, the biological relevance of m6A modification of circRNAs in prostate cancer (PCa) remains unclear and needs to be elucidated. Our data showed that circRBM33 was m6A-modified and was more highly expressed in PCa cells than in normal cells/tissues. The in vitro and in vivo experiments showed that downregulation/upregulation of circRBM33 inhibited/promoted tumour growth and invasion, respectively. Decreasing m6A levels rescued the tumour-promoting effect of circRBM33. Additionally, once modified by m6A, circRBM33 interacts with FMR1 by forming a binary complex that sustains the mRNA stability of PDHA1, a downstream target gene. Suppressed/overexpressed circRBM33 lowered/enhanced the ATP production, the acetyl-CoA levels and the NADH/NAD+ ratio. Moreover, depletion of circRBM33 significantly increased the response sensitivity to androgen receptor signalling inhibitor (ARSI) therapy, including enzalutamide and darolutamide, in prostate tumours. Our study suggested that the m6A-mediated circRBM33-FMR1 complex can activate mitochondrial metabolism by stabilizing PDHA1 mRNA, which promotes PCa progression, and can attenuate circRBM33 increased ARSI effectiveness in PCa treatment. This newly discovered circRNA may serve as a potential therapeutic target for PCa.

Keywords: ARSI therapy; N6-methyladenosine; Prostate cancer; circRBM33; mitochondrial respiration.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
CircRBM33 is m6A-modified and predicts a poor prognosis in PCa. (A) The Venn diagram shows the only overlapping circRNA among the BCR-related circRNAs in PCa, circRNAs with FPKM > 0.5, and circRNAs in the MeRIP analysis. (B) Sanger sequencing confirms the back-splicing site of circRBM33. (C) The motif analysis predicts the potential m6A-modified sites in circRBM33. (D) The MeRIP assay examines the is m6A-modified status of circRBM33 (E) Divergent and convergent primers amplification assays confirms the derivation of circRBM33. (F) The RNase R assay confirms the stability of circRBM33 and linearRBM33. (G) The Actinomycin D assay detects the stability disparity between circRBM33 and linearRBM33. (H) The Kaplan-Meier survival curves display the prognosis value of circRBM33 in PCa.
Figure 2
Figure 2
CircRBM33 promotes PCa's proliferation and invasion in vitro. (A) qRT-PCR confirms circRBM33 expression in five PCa cell lines (LNCaP, C4-2, 22Rv1, PC-3, and DU145), as well as RWPE-1. (B) qRT-PCR confirms the transfection efficiency of overexpressing circRBM33 in C4-2 and PC-3 cell lines. (C) qRT-PCR confirms the transfection efficiency of silencing circRBM33 (shC1 and shC2) in 22Rv1 and DU145 cell lines. (D) The CCK-8 assay measures the cell viability in circRBM33-silenced and negative control PCa cells. (E) The plate colony formation assay detects the colony formation ability in circRBM33-silenced and negative control PCa cells. (F) The Transwell assay determines the invasiveness discrepancy between circRBM33-silenced and negative control PCa cells. (G) The CCK-8 assay measures the cell viability in circRBM33-upregulated and negative control PCa cells. (H) The plate colony formation assay detects the colony formation ability in circRBM33-overexpressed and negative control PCa cells. (I) The Transwell assay determines the invasiveness discrepancy between circRBM33-overexpressed and negative control PCa cells.
Figure 3
Figure 3
CircRBM33 interacts with FMR1 in m6A-mediated manner. (A) FISH assays show the subcellular localization of circRBM33 in PCa cells (PC-3 and C4-2) with 18S acting as a positive control. (B) The nuclear and cytoplasmic extraction experiments show the nucleus/cytoplasm proportion of circRBM33. (C) The protein that overlapped between the catRAPID database and the CircInteractome database. (D) qRT-PCR confirms the enrichment of the ChIRP probe designed to circRBM33. (E) WB confirms the pulldown of FMR1 by the ChIRP probe. (F) The RIP experiments show the enrichment of circRBM33 by FMR1. (G) A schematic representation of the potential m6A-modified sites in circRBM33. (H) Wb confirms the transfection efficiency of shRNA to METTL3. (I) MeRIP assay confirms the enrichment of circRBM33 by m6A in METTL3-knockdown and control groups. (J) FMR1-RIP assay detects the enrichment of circRBM33 by FMR1 in METTL3-knockdown and control groups. (K) D) qRT-PCR confirms the enrichment of the ChIRP probe designed to circRBM33 in METTL3-knockdown groups. (L) WB confirms the pulldown of FMR1 by the ChIRP probe under the downregulation of METTL3.
Figure 4
Figure 4
FMR1 promotes PCa aggressiveness and relates to a poor prognosis in PCa. (A) WB confirms the transfection efficiency of siRNA targeting FMR1 in 22Rv1 and DU145 cell lines. (B) CCK-8 assays demonstrate the proliferation changes after FMR1 downregulation in 22Rv1 and DU145 cell lines. (C) Plate colony formation assays exhibit FMR1-silenced PCa cells' ability to form colonies. (D) Transwell experiments determine invasiveness differences in FMR1-silenced PCa cells with si-NC as a negative control. (E) KM survival analysis validated FMR1's prognostic value in PCa.
Figure 5
Figure 5
CircRBM33 significantly increases mitochondrial respiration in PCa cells. (A) KEGG pathway analysis of FMR1-RIP sequencing. (B) qRT-PCR determined the RNA expression levels of the key molecules involved in pyruvate metabolism, the pentose phosphate pathway, glycolysis/gluconeogenesis, and the citrate cycle in PCa cells when circRBM33 was upregulated or downregulated. (C) WB detects the PDHA1 expression differences in PCa cells after circRBM33 is overexpressed or silenced. (D-E) Detection of the changes in ATP production and acetyl-CoA levels in circRBM33-silenced and circRBM33-overexpressed cells compared to negative control cells. (F) Detection of the variation in the NAD+/NADH ratio in circRBM33-silenced and circRBM33-overexpressed cells compared to their own negative control cells. (G) Seahorse experiments detect the OCR in circRBM33-silenced and circRBM33-overexpressed cells in comparison to their own negative control cells.
Figure 6
Figure 6
Downregulating FMR1 impairs circRBM33-mediated aggressive phenotypes in PCa cells. (A) WB validates the influence of FMR1 knockdown on PDHA expression in circRBM33-overexpressed cells (22Rv1 and C4-2). (B) The CCK-8 assay examines the influence of FMR1 silencing on cell growth in circRBM33-overexpressed cells. (C) The influence of FMR1 downregulation on colony formation ability was examined in circRBM33-overexpressed cells. (D) The Transwell assay determines the impact of FMR1 silencing on invasiveness in circRBM33-overexpressed cells. (E) Detection of the NAD+/NADH ratio changes in the circRBM33-overexpressed cells when FMR1 is downregulated. (F-G) Detection of ATP and acetyl-CoA production changes in circRBM33-overexpressed cells when FMR1 is downregulated. (H) Seahorse experiments detects the basal and maximal OCR in the circRBM33-overexpressed cells when FMR1 was downregulated.
Figure 7
Figure 7
CircRBM33-FMR1 Complex sustains PDHA1 mRNA stability. (A) The PDHA1 and FMR1 expression correlation analysis in TCGA database. (B) Using the Integrative Genomics Viewer (IGV) visualizes the FMR1 binding regions of PDHA1. (C) The distribution of the potential FMR1 binding sites in PDHA1. (D) FMR1-RIP assays confirm the enrichments of PDHA1. (E) WB confirmed the transfection efficiency of downregulating or overexpressing FMR1 in PCa cell lines. (F) The variation in PDHA1 stability when FMR1 is upregulated or downregulated. (G) Actinomycin D assay detects the mRNA stability of PDHA1 when circRBM33 is upregulated or downregulated.
Figure 8
Figure 8
CircRBM33 promotes the PCa tumor growth in vivo. (A) The influence of overexpressing circRBM33 on subcutaneous PCa tumor growth in nude mice. (B) The impact of circRBM33 knockdown on subcutaneous PCa tumor growth in nude mice. (C) The IHC assay detects the expression changes in Ki67, FMR1 and PDHA1 in circRBM33-overexpressed PCa subcutaneous tumors. (D) The IHC assay detects the expression changes in Ki67, FMR1 and PDHA1 in circRBM33-silenced PCa subcutaneous tumors.
Figure 9
Figure 9
Knockdown of circRBM33 enhances tumor sensitivity to ARSI therapy in PCa. (A) The effects of ARSIs (Enzalutamide and Darolutamide) on circRBM33-depleted 22Rv1 cells in comparison to the negative control. (B) The effects of ARSIs (Enzalutamide and Darolutamide) on circRBM33-silenced C4-2 cells in comparison to the negative control. (C) The workflow shows the subcutaneous tumor mouse model construction based on ARSIs (enzalutamide and darolutamide) oral administration. (D) The comparison of ARSIs' influence on the growth of circRBM33-downregulated tumors.
Figure 10
Figure 10
The schematic graph presents the potential molecular mechanism of circRBM33 regarding tumor aggressiveness modulation and ARSI therapy sensitivity in prostate cancer.
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