Circ_0000437 promotes proliferation, invasion, migration and epithelial-mesenchymal transition of breast cancer cells by targeting the let-7b-5p/CTPS1 axis

doi:10.12122/j.issn.1673-4254.2025.08.13

Abstract

Abstract:

Objective To investigate the role of circular RNA circ_0000437 in regulating biological behaviors of breast cancer cells and the molecular mechanism. Methods Breast cancer MCF-7 and MDA-MB-231 cells were transfected with sh-circ_0000437, mimics, inhibitor, si-CTPS1, or their respective negative controls. qRT-PCR was used to detect the expression levels of circ_0000437, let-7b-5p, CTPS1, Notch1, Hes1, and Numb in breast cancer cell lines and tissues. RNase R digestion was used to confirm the circular structure of circ_0000437 and its subcellular localization in the breast cancer cells was determined by cellular distribution analysis. The changes in proliferation, invasion and migration of the transfected cells were assessed using CCK-8 assay, Transwell assay and scratch assay. Dual-luciferase reporter gene and RNA immunoprecipitation assays were employed to validate binding interactions among circ_0000437, let-7b-5p, and CTPS1. The cellular expressions of CTPS1, E-cadherin, N-cadherin, and vimentin proteins were detected with Western blotting. A tumor-bearing mouse model was used to verify the oncogenic mechanism of circ_0000437 and CTPS1. Results Circ_0000437 and CTPS1 were upregulated while let-7b-5p was downregulated in breast cancer tissues and cell lines. Circ_0000437 or CTPS1 knockdown obviously suppressed breast cancer cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT). Overexpression of let-7b-5p produced similar inhibitory effects, whereas inhibition of let-7b-5p significantly enhanced malignant behaviors of the cells. In the tumor-bearing mouse models, circ_0000437 knockdown significantly suppressed tumor growth, but co-transfection of the cells with pcDNA-CTPS1 accelerated tumor growth. Binding sites were identified between circ_0000437 and let-7b-5p and between let-7b-5p and CTPS1, and circ_0000437, let-7b-5p, and CTPS1 showed functional interactions in breast cancer cells. Conclusion Circ_0000437 is upregulated in breast cancer tissues and cells, and its high expression promotes proliferation, invasion, migration and EMT of breast cancer cells through the let-7b-5p/CTPS1 axis.

Key words: breast cancer, epithelial-mesenchymal transition, circular RNA, ceRNA

Siyuan MA, Bochao ZHANG, Chun PU. Circ_0000437 promotes proliferation, invasion, migration and epithelial-mesenchymal transition of breast cancer cells by targeting the let-7b-5p/CTPS1 axis[J]. Journal of Southern Medical University, 2025, 45(8): 1682-1696.

Figures/Tables 12

Tab.1 Primer sequences for qRT-PCR

Gene name	Primer sequence (s)
Circ_0000437	F:5'-AATCCCCGTACGTCCACTAC-3'
	R:5'-AGGGTCATAGAAAGGCAGCA-3'
CORO1C	F:5'-ATGAGGCGGCACATATAC-3'
	R:5'-ATCCCAGGTCACACGAGAAAC-3'
CTPS1	F:5'-CAGTGTGGGCACAATACTCAA-3'
	R:5'-CGCTCATAGTTACCCAGGTCA-3'
Notch1	F:5'-CGAACCCGTGCCAGAA-3'
	R:5'-CAGATGCCCAGTGAAGC-3'
Hes1	F:5'-CAGTGCCTTTGAGAAGCAGG-3'
	R:5'-CAGATAACGGGCAACTTCGG-3'
Numb	F:5'-CACAACTGCCACTGAGCAAG-3'
	R:5'-GTTGCCAGGAGCCACTGAT-3'
GAPDH	F:5'-CATCAAGAAGGTGGTGAAGCAG-3'
	R:5'-GTGTCGCTGTTGAAGTCAGAG-3'
let-7b-5p	5'-GCTGAGGTAGTAGGTTGTGTGGG-3'
U6	5'-CGCTTCGGCACATATAC-3'

Fig.1 Expression levels of circ_0000437 in breast cancer cells and tissues. A: Expression of circ_0000437 in breast cancer cell line. B: Expression of circ_0000437 in datasets GSE165884 and 182471. C: Expression of circ_0000437 in breast cancer tissues. D: Expression of circ_0000437 and linear CORO1C in two breast cancer cell lines treated with RNase R. E: Subcellular localization of circ_0000437 in breast cancer cells. **P<0.01, ***P<0.001.

Fig.2 Effects of circ_0000437 knockdown on proliferation, invasion, migration and EMT of breast cancer cells. A, B: Gene knockout efficiency and fluorescence staining of the cells after lentivirus transfection (Original magnification: ×100). C: CCK-8 assay for assessing the effect of circ_0000437 on cell proliferation. D: Transwell assay for assessing the effect of circ_0000437 on cell invasion (×400). E: Scratch assay for assessing the effect of circ_0000437 on cell migration (×100). F: Western blotting for assessing the effect of circ_0000437 on EMT in breast cancer cells. *P<0.05, **P<0.01, ***P<0.001.

Fig.3 Circ_0000437 has a binding site with let-7b-5p. A: Expression of let-7b-5p in breast cancer in TCGA database. B: The predicted binding diagram and binding sequence of let-7b-5p and circ_0000437. C: Luciferase activity in the cells co-transfected with mimics or mimics-NC and circ_0000437 WT plasmid or MUT plasmid. D: RIP analysis showing enrichment of circ_0000437 and let-7b-5p in the AGO2 part. **P<0.01, ***P<0.001.

Fig.4 Overexpression of let-7b-5p inhibits proliferation, invasion, migration and EMT of breast cancer cells. A, B: Expression of let-7b-5p in breast cancer tissues and cell lines. C: Transfection efficiency of let-7b-5p mimics. D: CCK-8 analysis to evaluate the effects of mimics on cell proliferation. E: Transwell assay to verify the effect of mimics on cell invasion (×400). F: Effect of mimics on cell migration determined using scratch assay (×100). G: Effect of mimics on EMT in breast cancer cells. *P<0.05, **P<0.01, ***P<0.001.

Fig.5 Inhibition of let-7b-5p suppresses proliferation, invasion, migration and EMT of breast cancer cells. A: Transfection efficiency of let-7b-5p inhibitor. B: CCK-8 analysis to evaluate the effects of inhibitor on cell proliferation. C: Transwell assay to verify the effect of inhibitor on cell invasion ability (×400). D: Effect of the inhibitor on cell migration determined using scratch assay (×100). E: Effect of the inhibitor on EMT in breast cancer cells. *P<0.05, **P<0.01, ***P<0.001.

Fig.6 Expression of CTPS1 up-regulated in breast cancer. A: Expression of CTPS1 in breast cancer in TCGA database. B, C: Expression of CTPS1 in breast cancer tissues and cell lines. D: Predicted CTPS1 and let-7b-5p binding sites. E: Mimics or mimics-NC were co-transfected with CTPS1 WT plasmid or MUT plasmid, respectively, and luciferase activity was measured in each group. **P<0.01, ***P<0.001.

Fig.7 CTPS1 knockdown inhibits EMT and regulates the Notch signaling pathway in breast cancer cells. A: Knockdown efficiency after transfection with si-CTPS1. B: Western blotting for assessing the effect of si-CTPS1 on expression of CTPS1 protein in breast cancer cells. C: Western blotting for assessing the effect of CTPS1 on EMT of breast cancer cells. D: qRT-PCR for assessing the effect of si-CTPS1 on the expression of genes related to the Notch signaling pathway. *P<0.05, **P<0.01, ***P<0.001.

Fig.8 CTPS1 knockdown inhibits proliferation, invasion and migration of breast cancer cells. A: CCK-8 assay for evaluating the effect of transfection with si-CTPS1 on cell proliferation. B: Transwell assay for assessing the effect of transfection with si-CTPS1 on cell invasion ability (×400). C: Effect of transfection with si-CTPS1 on cell migration determined using scratch assay (×100). *P<0.05, **P<0.01, ***P<0.001.

Fig.10 Circ_0000437 promotes EMT in breast cancer cells by regulating the let-7b-5p/CTPS1 axis. A: Expression of let-7b-5p in each group (sh-NC+inhibitor-NC, sh-circ_0000437+inhibitor-NC, and sh-circ_0000437+inhibitor) detected by qRT-PCR. B: qRT-PCR for detecting CTPS1 mRNA expression in breast cancer cells in each group. C: Western blotting for detecting the expression of CTPS1 protein in breast cancer cells in each group. D: Western blotting for detecting the expression of EMT-related proteins in breast cancer cells in each group. *P<0.05, **P<0.01, ***P<0.001.

Fig.11 Circ_0000437 promotes proliferation, invasion, and migration of breast cancer cells as a molecular sponge for let-7b-5p. A: CCK-8 assay of the breast cancer cells. B: Transwell assay of the breast cancer cells (×400). C: Scratch assay of the breast cancer cells (×100). *P<0.05, **P<0.01, ***P<0.001.

Fig.12 Construction of the tumor-bearing model in mice. A: Comparison of tumor volume 25 days after cell inoculation. B: Tumor growth curve in the mice. C: Comparison of tumor weight. **P<0.01, ***P<0.001.

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