Abstract
Background
The significant roles of circular RNAs (circRNAs) in different cancers and diseases have been reported. We now focused on the possible role of a newly recognized circRNA, circ_0004674 in triple-negative breast cancer (TNBC), and the related downstream mechanism.
Methods
The expression of circ_0004674 in TNBC tissues and cells was determined followed by analysis of the correlation between circ_0004674 and TNBC patients’ prognosis. The interaction between circ_0004674, miR-377-3p, E2F6, and PNO1 was then identified using bioinformatics analysis combined with FISH, RIP, RNA pull-down, RT-qPCR, and Western blot analysis. Using gain-of-function and loss-of-function methods, we analyzed the effect of circ_0004674, miR-377-3p, E2F6, and PNO1 on TNBC in vivo and in vitro.
Results
Increased circ_0004674 and E2F6 but decreased miR-377-3p were observed in TNBC tissues and MDA-MB-231 TNBC cells, all of which findings were associated with poor prognosis in patients with TNBC. Silencing of circ_0004676 remarkably suppressed the proliferation, cell cycle progression, and migration of TNBC cells in vitro, as well as inhibiting tumorigenesis and metastasis in vivo. Additionally, circ_0004676 served as a sponge of miR-377-3p which bound to the transcription factor E2F6. In the presence of overexpression of circ_0004676, E2F6 expression and its target PNO1 expression were elevated, while miR-377-3p expression was decreased. Interestingly, overexpression of E2F6 could reverse the inhibitory effect on tumor growth caused by downregulation of circ_0004676.
Conclusion
Our study highlighted the carcinogenic effect of circ_0004676 on TNBC through regulation of the miR-377-3p/E2F6/PNO1 axis.
Graphical abstract
1. Circ_0004674 is highly expressed in TNBC tissues and cells.
2. Circ_0004674 upregulates the expression of E2F6 by sponging miR-377-3p.
3. E2F6 upregulates PNO1 by binding to the PNO1 promoter.
4. Circ_0004674 favors TNBC progression by regulating the miR-377-3p/E2F6/PNO1 axis.
5. This study provides a new target for the treatment of TNBC.
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Data availability
The datasets generated/analyzed during the current study are available.
Abbreviations
- ANOVA :
-
analysis of variance
- ATCC :
-
American Type Culture Collection
- BSA :
-
bovine serum albumin
- ceRNA :
-
competing endogenous RNA
- ChIP :
-
chromatin immunoprecipitation
- circRNAs :
-
circular RNAs
- Co-IP :
-
co-immunoprecipitation
- DFS :
-
disease-free survival
- DMEM :
-
Dulbecco’s modified Eagle’s medium
- EDTA :
-
ethylenediaminetetraacetic acid
- EdU :
-
5-ethynyl-2′-deoxyuridine
- EMT :
-
epithelial-mesenchymal transition
- FDR :
-
false discovery rate
- FISH :
-
fluorescence in situ hybridization
- FITC :
-
fluorescein isothiocyanate
- EP :
-
Eppendorf
- GEO :
-
Gene Expression Omnibus
- GFP :
-
green fluorescent protein
- HE :
-
hematoxylin-eosin
- IHC :
-
immunohistochemistry
- lncRNAs :
-
long noncoding RNAs
- MEBM :
-
minimum essential basal medium
- miRNAs :
-
microRNAs
- NC :
-
negative control
- ncRNAs :
-
non-coding RNAs
- OS :
-
overall survival
- PI :
-
propidium iodide
- PPI :
-
protein-protein interaction
- RIP :
-
RNA binding protein immunoprecipitation
- RLU :
-
relative luciferase
- RPMI :
-
Roswell Park Memorial Institute
- RT-qPCR :
-
reverse transcription quantitative polymerase chain reaction
- TNBC :
-
triple-negative breast cancer
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GLS and XLF designed the study. PSZ and XWL collated the data, carried out the data analyses, and produced the initial draft of the manuscript. LH and SFJ contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.
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Our experiment was approved by the Ethics Committee of Zhujiang Hospital of Southern Medical University and conducted in compliance with the Declaration of Helsinki. Informed consent was obtained from all participants prior to the experiment. Animal experiments were performed with approval of the Animal Ethics Committee of Zhujiang Hospital of Southern Medical University.
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Supplementary information
Supplementary Fig. 1
Overexpression of circ_0004674 enhances the proliferation and migration of TNBC cells. A, Sequences of all plasmids used in this study and the circ_0004674 qPCR product. B, RT-qPCR analysis of circ_0004674 expression in MDA-MB-231 cells upon treatment of oe-circ_0004674. C, EdU assay of MDA-MB-231 cell proliferation upon oe-circ_0004674 treatment and colony formation of MDA-MB-231 cells upon treatment of oe-circ_0004674 determined by colony formation assay. D, E, Flow cytometric analysis of MDA-MB-231 cell cycle upon oe-circ_0004674 treatment. E, Migration of MDA-MB-231 cells upon oe-circ_0004674 treatment measured by Transwell assay. F, Invasion of MDA-MB-231 cells upon oe-circ_0004674 treatment measured by Transwell assay. * p < 0.05 vs. oe-NC treatment. The experiment was run in triplicate independently. (JPG 395 kb)
Supplementary Fig. 2
Overexpression of circ_0004674 facilitates the tumorigenesis and metastasis of TNBC cells in vivo. A, Representative macroscopic images of breast tumor upon oe-circ_0004674 treatment. B, Quantification of tumor volume over time upon oe-circ_0004674 treatment. C, Quantification of tumor weight upon oe-circ_0004674 treatment. D, Expression of circ_0004674 in mouse tumor tissues upon oe-circ_0004674 treatment determined by RT-qPCR. E, IHC of Ki67 and CD31 proteins in mouse tumor tissues upon oe-circ_0004674 treatment. F, Quantification of HE staining of lung metastasis in mouse tumor tissues upon oe-circ_0004674 treatment. * p < 0.05 vs. oe-NC treatment. n = 8 for mice upon each treatment. (JPG 510 kb)
Supplementary Fig. 3
Downregulated miR-377-3p expression and upregulated E2F6 and PNO1 expression predict poor prognosis in TNBC patients. A, Correlation of miR-377-3p expression with the DFS and OS of TNBC patients analyzed by the Kaplan-Meier method. B, Correlation of E2F6 expression with the DFS and OS of TNBC patients analyzed by the Kaplan-Meier method. C, Correlation of PNO1 expression with the DFS and OS of TNBC patients analyzed by the Kaplan-Meier method. (JPG 584 kb)
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Shao, G., Fan, X., Zhang, P. et al. Circ_0004676 exacerbates triple-negative breast cancer progression through regulation of the miR-377-3p/E2F6/PNO1 axis. Cell Biol Toxicol 39, 2183–2205 (2023). https://doi.org/10.1007/s10565-022-09704-6
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DOI: https://doi.org/10.1007/s10565-022-09704-6