Circular RNA circ_0048764 promotes the development of breast cancer by regulating microRNA-1296-5p/tripartite motif containing 14 axis

Abstract

Breast cancer (BC) is one of the leading causes of cancer-related deaths in females. Circular RNA (circRNA), as reported, is involved in the progression of BC. This work focuses on clarifying the biological function of circ_0048764 in BC and its hidden mechanism. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expressions of circ_0048764, microRNA-1296-5p (miR-1296-5p), and tripartite motif containing 14 (TRIM14) in BC tissues and cell lines. Besides, the status of proliferation, migration, invasion and apoptosis of BC cells was probed by cell counting kit-8 (CCK-8), EdU, transwell and flow cytometry assays. Western blot was adopted to examine the level of TRIM14 protein in BC cells. In addition, dual-luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay were conducted to corroborate the targeting relationships between miR-1296-5p and circ_0048764 or TRIM14. It was revealed that circ_0048764 expression was remarkably up-regulated in BC tissues and cells, and circ_0048764 expression was associated with TNM stage and tumor size. Functionally, overexpression of circ_0048764 significantly promoted BC cell proliferative, migrative and invasive abilities and inhibited apoptosis, while circ_0048764 knockdown exerted the opposite effects. Mechanistically, circ_0048764 directly targeted miR-1296-5p and could negatively modulate its expression in BC cells. Besides, miR-1296-5p could reverse the influence of circ_0048764 on BC viability, migration, invasion and apoptosis. Moreover, TRIM14 was confirmed to be a downstream target of miR-1296-5p. Circ_0048764 positively regulated TRIM14 expression in BC cells via targeting miR-1296-5p. Collectively, it is concluded that circ_0048764 promotes the development of BC via modulating the miR-1296-5p/TRIM14 axis.

Keywords: Breast cancer; Trim14; circ_0048764; miR-1296-5p.

Figure 1.

Circ_0048764 is up-regulated in BC tissues and cells (a) the GSE165884 dataset was analyzed by the GEO2R online tool to show the expression changes of circRNAs in BC tissues and normal tissues, with red representing the upregulated circRNAs and blue representing the downregulated circRNAs. (b) A heat map showed the differential expression of circRNAs in BC tissues and normal tissues. (c and d) qRT-PCR was used to detect the expression of circ_0048764 in BC tissues (c) and cell lines (d). Nucleocytoplasmic separation assay was used to detect the subcellular localization of circ_0048764 in SKBR3 and MCF7 cells. **P < 0.01, and ***P < 0.001.

Figure 2.

Circ0048764 promotes BC cells’ proliferation, migration and invasion and inhibits apoptosis (a) pcDNA-NC or pcDNA-circ_0048764 was transfected into SKBR3 cells and si-NC, si-circ0048764-1 and circ_0048764-2 were transfected into MCF7 cells, and the expression of circ_0048764 in SKBR3 and MCF7 cells after transfection was detected by qRT-PCR. (b and c) CCK-8 and EdU assays were used to detect the effects of overexpression or knockdown of circ_0048764 on the proliferation of SKBR3 and MCF7 cells. (d) transwell assay was used to detect the effects of overexpression or knockdown of circ_0048764 on SKBR3 and MCF7 cells’ migration and invasion. (e) flow cytometry was used to detect the effect of overexpression or knockdown of circ_0048764 on the apoptosis of SKBR3 and MCF7 cells. ** P < 0.01, and ***P < 0.001.

Figure 3.

Circ_0048764 adsorbs miR-1296-5p (a) The potential binding site between circ_0048764 and miR-1296-5p was predicted by the Circinteractome database. (b) The interaction between circ_0048764 and miR-1296-5p was detected with a dual-luciferase reporter gene assay with HEK-293 T cells . (c) RIP assay was performed to detect the enrichment of circ_0048764 and miR-1296-5p in the immunoprecipitate of Ago2 group or IgG group. (d) qRT-PCR was used to detect the expression of miR-1296-5p in SKBR3 and MCF7 cells with circ_0048764 overexpression or knockdown. e and f. The expression of miR-1296-5p in BC tissues (e) and cell lines (f) was detected by qRT-PCR. (g) Pearson’s correlation analysis was performed to detect the correlation between circ_0048764 expression and miR-1296-5p expression in BC tissues. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 4.

Circ_0048764 promotes BC cell proliferation, migration and invasion, and inhibits apoptosis by targeting miR-1296-5p (a) pcDNA-NC, pcDNA-circ_0048764 and pcDNA-circ_0048764 + miR-1296-5p mimics were transfected into SKBR3 cells, respectively; si-NC, si-circ_0048764-1 and si-circ_0048764-1 + miR-1296-5p inhibitors were transfected into MCF7 cells, respectively, and the expression of miR-1296-5p was detected by qRT-PCR. (b and c) CCK-8 and EdU assays were used to detect SKBR3 or MCF7 cells’ proliferation after transfection. (d) transwell assay was used to detect SKBR3 or MCF7 cells’ migration and invasion after transfection. (e) flow cytometry was used to detect the apoptosis of SKBR3 or MCF7 cells after transfection. ** P < 0.01, and ***P < 0.001.

Figure 5.

Circ_0048764/miR-1296-5p axis regulates BC progression by regulating the expression of TRIM14 (a)The potential binding site between TRIM14 mRNA 3′ UTR and miR-1296-5p were predicted by targetscan database. (b) dual-luciferase reporter gene assay was used to examine the interaction of TRIM14 mRNA 3’UTR and miR-1296-5p in HEK-293 T cells. (c and d) the levels of TRIM14 mRNA and protein in SKBR3 cells transfected with pcDNA-NC, pcDNA-circ_0048764 and pcDNA-circ_0048764 + miR-1296-5p mimics, or MCF7 cells transfected with si-NC, si-circ_0048764-1 and si-circ_0048764-1 + miR-1296-5p inhibitors were detected by qRT-PCR and western blot assay, respectively. (e) the expression of TRIM14 mRNA in BC tissues and normal tissues was detected by qRT-PCR. (f and g) Pearson’s correlation analysis was used to detect the correlation between TRIM14 mRNA expression and miR-1296-5p expression or circ_0048764 expression in BC tissues, respectively. ** P < 0.01, and ***P < 0.001.