Effect of hsa_circ_0001573 on biological behaviors of breast cancer cells and its molecular mechanism

doi:10.19401/j.cnki.1007-3639.2023.04.004

Abstract

Abstract:

Background and purpose: Circular RNA (CircRNA) is a type of non-coding RNA with a closed circular structure, which has important potential in gene expression regulation. CircRNA is closely related to the occurrence and development of tumor. This study aimed to explore the influence of hsa_circ_0001573 on the biological behavior of breast cancer cells and its mechanism. Methods: Breast cancer tissues and paracancerous tissues of 4 patients surgically removed at the First Affiliated Hospital of Chongqing Medical University from January 2020 to January 2021 were collected, sequenced and analyzed by RNA microarray analysis. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was used to detect the relative expression of hsa_circ_0001573 in breast cancer tissues and paracancerous tissues of 40 cases, as well as normal human breast cancer epithelial cell MCF-10A and breast cancer cells (MCF-7 and SK-BR-3), and the location and expression were further examined by fluorescence in situ hybridization (FISH) assay. The interference vector was transfected into breast cancer cells. Cell counting kit-8 (CCK-8), EdU, wound healing, clonal formation, Hoechst 33342, TUNEL, flow cytometry and transwell assays were adopted to determine cell migration, invasion, proliferation and apoptosis. Western blot was employed to measure the expressions of CCND1, CCND2 and CDK4. The effect of hsa_circ_0001573 on the growth of xenograft tumors was observed in nude mice. RNA pulldown assay was performed to identify hsa_circ_0001573-associated proteins. Subcellular localization of hsa_circ_0001573 and GNB4 was clarified by FISH and immunofluorescence (FISH-IF), to explore potential molecular mechanisms. Results: The expression level of hsa_circ_0001573 was high in breast cancer (P<0.001) and breast cancer cells (P<0.001). Downregulation of hsa_circ_0001573 could inhibit the proliferation, migration and invasion of breast cancer cells, induce cell apoptosis leading to cell cycle arrest in G₁ phase, and obviously decrease the expressions of CCND1, CCND2 and CDK4. The results of in vivo experiments showed that knockdown of hsa_circ_0001573 inhibited the growth of xenograft tumors. RNA pulldown experiment showed that hsa_circ_0001573 combined with GNB4 protein. FISH-IF indicated that hsa_circ_0001573 was co-localized with GNB4 in the nucleus and interacted with GNB4 to promote the expression of c-myc. Conclusion: CircRNA hsa_circ_0001573 is highly expressed in breast cancer. Knockdown of hsa_circ_0001573 could regulate cell proliferation, invasion, apoptosis and cell cycle arrest, and inhibit the growth of xenograft tumors in vivo, thus providing a new target for breast cancer treatment.

Key words: CircRNA, Breast cancer, Hsa_circ_0001573, Cell proliferation, Apoptosis, Xenograft tumors, GNB4

CLC Number:

R737.9

CHEN Hong, CHEN Junxia. Effect of hsa_circ_0001573 on biological behaviors of breast cancer cells and its molecular mechanism[J]. China Oncology, 2023, 33(4): 342-353.

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Clinicopathological features	All cases	Relative expression of hsa_circ_0001573		Chi-square	P value
Clinicopathological features	All cases	Low (n=15)	High (n=25)	Chi-square	P value
Age/year				2.373	0.123
<55	26	12	14
≥55	14	3	11
Grade				3.175	0.075
Ⅱ	12	7	5
Ⅲ	28	8	20
T stage				5.184	0.023
T₁	15	9	6
T_2-3	25	6	19
N stage				7.111	0.008
N₀	24	13	11
N_1-3	16	2	14
TNM stage				6.222	0.013
Ⅰ	12	8	4
Ⅱ/Ⅲ	28	7	21