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The 3′UTR of the pseudogene CYP4Z2P promotes tumor angiogenesis in breast cancer by acting as a ceRNA for CYP4Z1

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A Correction to this article was published on 26 October 2019

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Abstract

Pseudogenes are now known to regulate their protein-coding counterparts. Additionally, disturbances of 3′UTRs could increase the risk of cancer susceptibility by acting as modulators of gene expression. The aim of this study was to investigate the roles of the pseudogene CYP4Z2P-3′UTR and functional gene CYP4Z1-3′UTR in breast cancer angiogenesis process. The levels of CYP4Z2P- and CYP4Z1-3′UTR and miRNA of interests were measured in 22 cancerous tissues paired with non-cancerous samples by qRT-PCR. The effects of CYP4Z2P- and CYP4Z1-3′UTR were studied by overexpression and RNA interference approaches in vitro and ex vivo. Insights of the mechanism of competitive endogenous RNAs were gained from bioinformatic analysis, luciferase assays, and western blot. The positive CYP4Z2P/CYP4Z1 interaction and negative interaction between predicted miRNAs and CYP4Z2P or CYP4Z1 were identified via qRT-PCR assay and bivariate correlation analysis. CYP4Z2P- and CYP4Z1-3′UTR share several miRNA-binding sites, including miR-211, miR-125a-3p, miR-197, miR-1226, and miR-204. The CYP4Z2P- and CYP4Z1-3′UTRs arrest the interference caused by of these miRNAs, resulting in increased translation of CYP4Z1. Moreover, ectopic expression of the CYP4Z2P- and CYP4Z1-3′UTRs exhibit tumor angiogenesis-promoting properties in breast cancer collectively by inducing the phosphorylation of ERK1/2 and PI3K/Akt. Co-transfection with Dicer siRNA reversed the CYP4Z2P 3′UTR-mediated changes. Additionally, PI3K or ERK inhibitors reversed CYP4Z2P- and CYP4Z1-3′UTR-mediated changes in VEGF-A expression. Increased CYP4Z2P- and CYP4Z1-3′UTR expression promotes tumor angiogenesis in breast cancer partly via miRNA-dependent activation of PI3K/Akt and ERK1/2. The CYP4Z2P- and CYP4Z1-3′UTRs could thus be used as combinatorial miRNA inhibitors.

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Change history

  • 26 October 2019

    After publication of this article [1], it came to our attention that there was an error in Fig. 4b and 4c. In Fig.4b, the migration images of vector groups were incorrect based on this error, the authors re-constructed the migration experiments of Fig. 4b, and the consistent results were obtained. In Fig. 4c, the tube formation image of the Z2P-UTR-siRNA&Z1-UTR group (MCF-7) of Fig. 7d was accidentally misused in control group (MCF-7) and vector group (MCF-7) of Fig. 4c. The corrected Fig. 4 is provided below. This error did not impact the conclusions of the article. The authors apologize for this error.

  • 26 October 2019

    After publication of this article [1], it came to our attention that there was an error in Fig. 4b and 4c. In Fig.4b, the migration images of vector groups were incorrect based on this error, the authors re-constructed the migration experiments of Fig. 4b, and the consistent results were obtained. In Fig. 4c, the tube formation image of the Z2P-UTR-siRNA&Z1-UTR group (MCF-7) of Fig. 7d was accidentally misused in control group (MCF-7) and vector group (MCF-7) of Fig. 4c. The corrected Fig. 4 is provided below. This error did not impact the conclusions of the article. The authors apologize for this error.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China [Grants 81372331] (to Tao Xi), Major Drug Discovery of Science and Technology Major Projects [Grants 2009ZX09103-652] and the project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

Conflict of interest

The authors declare that they have no conflict of interests.

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    Authors and Affiliations

    1. School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China

      Lufeng Zheng, Xiaoman Li, Yi Gu, Xiaobo Lv & Tao Xi

    2. Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China

      Lufeng Zheng, Xiaoman Li, Yi Gu, Xiaobo Lv & Tao Xi

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    1. Lufeng Zheng
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    Correspondence to Tao Xi.

    Additional information

    Lufeng Zheng and Xiaoman Li contributed equally to this study.

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    Fig. S1

    CYP4Z2P and CYP4Z1 expression were regulated by miR-211, miR-1226, miR-125a, miR-197 and miR-204. (A) miR-211, miR-1226, miR-125a, miR-197 and miR-204 level were lower in breast cancer tissues than in mammary gland tissues. Twenty-two pairs of breast tumors with adjacent mammary gland epithelial tissues were analyzed by qRT-PCR. (B) Correlation analysis of the expression of CYP4Z2P, CYP4Z1 and miR-211, miR-1226, miR-125a, miR-197, miR-204 in breast cancer tissues. The expression of CYP4Z2P and CYP4Z1 is inversely correlated with miRNAs level. (C) Luciferase reporter assay after transfection. Fluc activity in cells was measured and normalized to β-galactosidase activity. miR-211, miR-1226, miR-125a, miR-197 and miR-204 could repress the luciferase signals of wild type not the mutant CYP4Z2P- and CYP4Z1-3’UTR reporters in 293T cells. Values are mean ± SD. *P<0.05, **P < 0.01 (n = 3, one-way ANOVA) (TIFF 8073 kb)

    Fig. S2

    Z1-UTR overexpression promotes tumor-induced angiogenesis in CAM of the chick embryo and rat aortic ring assays. (A) Different CM was gently placed on the CAM. The eggs were incubated for 48 h and photographed. Blood vessel density was quantified by counting the number of branching blood vessels. (B) Each rat aortic ring was placed in a collagen pre-coated 96-well plate, different CM was added to the wells. On day 6, the rings were analyzed by phase-contrast microscopy and microvessel outgrowths were quantified and photographed. Values are mean ± SD. *P<0.05, **P < 0.01 (n = 4, Student’s t-test) (TIFF 6100 kb)

    Fig. S3

    miR-211, miR-1226, miR-125a, miR-197 and miR-204 can repress tumor-induced angiogenesis. The methods were described as above in Fig. 4 and Fig. S2 (TIFF 15104 kb)

    Fig. S4

    miRNAs level could be regulated by siRNA-Dicer but not CYP4Z2P- and CYP4Z1-3’UTR. (A) The knockdown efficiency of siRNA-Dicer was examined by qRT-PCR. (B) MCF-7 cells were transfected with siRNA-Dicer to block the miRNA biogenesis pathway. Knocking down Dicer decreased levels of the miRNAs tested. (C) Levels of miRNAs were determined in MCF-7 cells transfected with Z2P-UTR or Z1-UTR by qRT-PCR. Little difference could be detected. (D) miRNA expression was analyzed in cells transfected with different miRNAs or a control oligo. Transfection with miRNA mimics increased mature miRNA levels. Values are mean ± SD. **P < 0.01 (n = 3, one-way ANOVA) (TIFF 9871 kb)

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    Zheng, L., Li, X., Gu, Y. et al. The 3′UTR of the pseudogene CYP4Z2P promotes tumor angiogenesis in breast cancer by acting as a ceRNA for CYP4Z1. Breast Cancer Res Treat 150, 105–118 (2015). https://doi.org/10.1007/s10549-015-3298-2

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    • DOI: https://doi.org/10.1007/s10549-015-3298-2

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