Accumulating studies have shown the important role of circular RNAs (circRNAs) in the oncogenesis and metastasis of various cancers. We previously reported that circACTN4 could bind with FUBP1 to promote tumorigenesis and the development of breast cancer (BC) by increasing the expression of MYC. However, its exact molecular mechanism and biological function have not been fully elucidated.

Here, Circular RNA microarray analysis was conducted in 3 pairs of BC and paracancerous tissues. The expression of circACTN4 in BC cells and tissues was detected via reverse transcription‒quantitative PCR (RT‒qPCR). Cell Counting Kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), transwell migration, and invasion assays were performed to further detect the biological functions of circACTN4 in BC cells. Xenograft models were used to investigate the in vivo role of circACTN4. Fluorescence in situ hybridization, Chromatin immunoprecipitation (ChIP)‒qPCR, coimmunoprecipitation, fluorometric, western blot, and rescue experiments were performed to explore the mechanism of circACTN4.

Our results revealed that circACTN4 was highly expressed in BC cells and tissues. The upregulated expression of circACTN4 was significantly related to the T stage and TNM stage and poor prognosis of patients with BC. circACTN4 was located primarily in the nucleus of BC cells. Upregulation of circACTN4 significantly increased the proliferation, invasion, and growth of BC cells, whereas the downregulation of circACTN4 exerted the opposite effects and induced G1/S cell cycle arrest. Mechanistically, we showed that circACTN4 could upregulate the expression of MYC and that MYC might interact with TIP60 histone acetyltransferase to increase the recruitment of TIP60 to MYC target genes and histone H4 acetylation (AcH4), thus promoting the progression of the breast cancer cell cycle and tumorigenesis.

Taken together, our findings reveal for the first time a new mechanism by which circACTN4 could promote oncogenesis and the development of BC by increasing the AcH4 of MYC target genes via TIP60. Therefore, circACTN4 could be a novel target for BC diagnosis and remedy.

To investigate the regulatory mechanism of long non-coding RNA (lncRNA) ENST00000579933 (hereinafter referred to as lncRNA ENST 933) on the development of breast cancer (BC).

The expression level of lncRNA ENST 933 in BC tissues as well as in BC cell lines (MCF-7, MDA-MB-231, MDA-MB-453) was detected by fluorescence-based real-time quantitative polymerase chain reaction RT-qPCR. The effects of lncRNA ENST 933 and miR-588 on the proliferation, migration, invasion and cell cycle in BC were determined using CCK-8 assay, clone formation assay, EdU, Transwell assay, flow cytometry, Western blotting and in vivo transplantation tumor assay. Finally, dual luciferase reporter gene, RNA immunoprecipitation assay, RT-qPCR, Western blotting and rescue assay were used to evaluate the interactions among lncRNA ENST 933, miR-588, and eukaryotic initiation factor 6 (EIF6).

The expression of lncRNA ENST 933 was up-regulated in both BC tissues and BC cell lines (P < 0.05). Overexpression of lncRNA ENST 933 could enhance the proliferation, migration and invasion abilities of BC cells and promote the growth of transplanted tumors, while knockdown of lncRNA ENST 933 resulted in inhibited development of BC and arrested cell cycle (P < 0.05). The expression of miR-588 was obviously down-regulated in BC tissues, and its overexpression inhibited cell proliferation, migration and invasion abilities in the BC cells (P < 0.05). Moreover, the results of rescue assay indicated that lncRNA ENST 933 promoted the expression of target gene EIF6 by competitively binding with miR-588. Western blotting revealed that miR-588 decreased the phosphorylation of AKT and mTOR (P < 0.05).

lncRNA ENST 933 promotes the progression of BC by modulating the miR-588/EIF6 axis.

To explore the effects of the interaction between circular RNA hsa_circ_0000231 and HnRNPK on the proliferation, migration and apoptosis in breast cancer.

Microarray analysis was used to investigate the expression profile of circRNAs in 4 pairs of breast cancer tissues and corresponding adjacent normal tissues. A total of 35 breast cancer tissue specimens were collected from the patients admitted in the First Affiliated Hospital of Chongqing Medical University from September 2019 to January 2021. qRT-PCR was carried out to detect the relative expression of hsa_circ_0000231, and fluorescence in situ hybridization (FISH) assay was performed to observe its location and expression in the cells. Breast cancer MCF-7 and SK-BR-3 cells were transfected with the interference vector of hsa_circ_0000231 respectively. Then cell wound healing assay, CCK-8 assay, EdU cell proliferation assay, clone formation experiment, hoechst33342 staining, TUNEL, flow cytometry and Transwell assay were adopted to determine cell migration, invasion, proliferation and apoptosis, and Western blotting was employed to measure the expression of CCND2, CCND1 and CDK4 after the knockdown. The effect of hsa_circ_0000231 on the growth of transplanted tumors was observed in nude mice. RNA pulldown assay was performed to identify hsa_circ_0000231-associated proteins, and FISH-immunofluorescence (IF) assay were employed to clarify the subcellular localization of hsa_circ_0000231 and HnRNPK. qRT-PCR and Western blotting were conducted to detect the expression of c-Myc.

Circular RNA hsa_circ_0000231 was highly expressed in breast cancer tissues (P<0.001) and breast cancer cells (P<0.01). Down-regulation of hsa_circ_0000231 inhibited the proliferation, migration and invasion of breast cancer cells, induced cell apoptosis, led to cell cycle arrest in G₁ phase, and obviously decreased the expression of CCND2, CCND1 and CDK4. The results of in vivo experiments showed that knockdown of hsa_circ_0000231 inhibited the growth of tumor xenograft. HnRNPK was co-localized in the nucleus with hsa_circ_0000231, and interacted with hsa_circ_0000231 to promote c-Myc expression.

Knockdown of hsa_circ_0000231 could suppress the proliferation, migration and invasion of breast cancer cells, induce cell apoptosis and cell cycle arrest, and inhibit tumor growth in vivo. The interaction of hsa_circ_0000231 with HnRNPK enhances the expression of c-Myc, and thus promotes the occurrence and development of breast cancer.

To investigate the expression of circular RNA circACTN4 (also called hsa_circ_0050900) in breast cancer (BC) and its effect on the proliferation, invasion and metastasis of BC cells.

The circRNA expression profiles of 4 pairs of BC tissues and adjacent tissues were analyzed by circRNA chip analysis, and the relative expression level of circACTN4 was detected by qRT-PCR. After BC cell lines MCF-7/MDA-MB-231 were transfected with circACTN4 overexpression plasmid and control plasmid, respectively, the effects of circACTN4 on cell proliferation, migration and invasion were tested by Cell Counting Kit-8 (CCK-8), EdU assay, colony formation, scratching assay, and Transwell migration and invasion assay. Western blotting was executed to determine the protein expression of CCND1, CCND2 and CDK4. The effects of circACTN4 on the growth and metastasis of transplanted tumors were also observed in nude mice.

The expression of circACTN4 was significantly higher in BC tissues and cells than in the paired adjacent tissues and normal breast epithelial cells (P<0.01). Up-regulation of circACTN4 enhanced the proliferation, migration and invasion of BC cells (P<0.05), and facilitated the expression of CCND1, CCND2 and CDK4 (P<0.01). The results of in vivo experiments also showed that up-regulation of circACTN4 promoted the growth and metastasis of transplanted tumors, with the tumor microvascular density and the number of lung metastatic nodules significantly increased in the circACTN4 group, as compared with the control group (P<0.05).

circACTN4 is highly expressed in BC tissues and cells. It can promote the proliferation, migration and invasion of BC cells, and facilitate the growth and metastasis of transplanted tumors in vivo.