The novel circFKBP8/miR-432-5p/E2F7 cascade functions as a regulatory network in breast cancer

Abstract

Background: Circular RNAs (circRNAs) are capable of affecting breast cancer (BC) development. However, the role and underneath mechanism of circFKBP8 (also known as hsa_circ_0000915) in BC remain largely unknown.

Methods: Expression analyses were performed using quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC) assays. Effects on cell functional phenotypes were determined by assessing cell proliferation, migratory capacity, invasion, and stemness in vitro. The relationship between microRNA (miR)-432-5p and circFKBP8 or E2F transcription factor 7 (E2F7) was examined by RNA pull-down, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assays. Xenograft assays were used to identify the function of circFKBP8 in vivo.

Results: CircFKBP8 was presented at high levels in BC tissues and cells. High circFKBP8 expression was associated with worse overall survival in BC patients. CircFKBP8 suppression inhibited BC cell proliferation, migratory capacity, invasion and stemness in vitro. CircFKBP8 suppression blocked xenograft tumor growth in vivo. Mechanistically, circFKBP8 functioned as a miR-432-5p sponge to modulate E2F7 expression. CircFKBP8 modulated BC cell malignant behaviors by miR-432-5p, and miR-432-5p affected these cell phenotypes through E2F7.

Conclusion: Our observations prove that circFKBP8 promotes BC malignant phenotypes through the miR-432-5p/E2F7 cascade, offering a promising therapeutic and prognostic target for BC.

Keywords: Breast cancer; E2F7; circFKBP8; miR-432-5p.

Fig. 1

CircFKBP8 was highly expressed in BC. (A) CircFKBP8 expression was detected by qRT-PCR in BC tissues (n = 70) and adjacent non-cancer tissues (NC) (n = 70); (B) circFKBP8 expression in BC patients with no metastasis and distant metastasis; (C) The prognostic value of circFKBP8 by survival curve analysis; (D) circFKBP8 expression in BC cells (MDA-MB-231, MDA-MB-453, MDA-MB-468, and BT549) and human normal breast cells MCF-10 A; (E) The expression of circFKBP8 and GAPDH was detected after treatment with or without RNase R in BC cells. ** P < 0.01, *** P < 0.001

Fig. 2

CircFKBP8 inhibition suppressed BC cell proliferation, migration, invasion, and stemness. (B) The expression of circFKBP8 was detected after transfected with sh-NC, sh-circFKBP8#1, sh-circFKBP8#2, and sh-circFKBP8#3 in BC cells; (B-D) CCK-8 assay, EDU assay and colony formation assay were used to detect cell proliferation; (E-F) Transwell migration and invasion assays were performed to assess cell migration and invasion ability; (G) Sphere formation assay was used to evaluate cell stemness; (H) The protein levels of KI-67, MMP2 and Nanog in BC cells were detected by western blot assays. ** P < 0.01, *** P < 0.001

Fig. 3

CircFKBP8 bound to miR-432-5p in BC. (A) The nuclear and cytoplasmic fractions of circFKBP8 were detected by qRT-PCR in BC cells; (B) Prediction of downstream target miRNAs for circFKBP8 binding by bioinformatics websites (starbase, circBank, and circatlas); (C) The enrichment of miR-490-3p or miR-432-5p was detected by qRT-PCR after pull-down assay with oligo probe or circFKBP8 probe; (D) The expression of miR-432-5p was assessed after transfection with miR-432-5p or miR-NC. (E) Schematic diagram of binding sites between miR-432-5p and circFKBP8; (F) The luciferase activity was detected using dual-luciferase reporter assay after co-transfection with miR-NC or miR-432-5p and circFKBP8-WT or circFKBP8-MUT; (G-H) The enrichment of miR-432-5p and circFKBP8 was evaluated after immunoprecipitation with Ago2 in RIP assay; (I-J) miR-432-5p expression was detected in BC tissues and cells. ** P < 0.01, *** P < 0.001

Fig. 4

MiR-432-5p inhibition rescued the effect of circFKBP8 knockdown on BC cell malignant behaviors. (A-H) MDA-MB-231 and MDA-MB-468 cells were transfected with sh-NC + inh-NC, sh-circFKBP8#1 + inh-NC, or sh-circFKBP8#1 + inh-miR-432-5p; (A) miR-432-5p expression was detected by qRT-PCR in MDA-MB-231 and MDA-MB-468 cells; (B-D) CCK8 assay, EDU assay and colony formation assay were used to assess cell proliferation; (E-G) Transwell assay and tube formation assay were performed to assess cell migration, invasion ability and stemness; (H) Western blot assay was performed to assess the protein levels of KI-67, MMP2 and Nanog. ***P < 0.001

Fig. 5

E2F7 was a direct target of miR-432-5p. (A) The binding sites was predicted by starbase between miR-432-5p and E2F7; (B) Luciferase activity was detected in dual-luciferase assay; (C) The protein expression of E2F7 was detected in different groups; (D) UALCAN was used to analyze E2F7 expression in BC tissues in TCGA database; (E-G) The mRNA (E) and protein (F-G) levels of E2F7 in BC tissues were analyzed by qRT-PCR and western blot/IHC assays; (H) Western blot was used for protein analysis of E2F7 in BC cells; (I) After BC cells were transfected with sh-NC + inh-NC, sh-circFKBP8#1 + inh-NC, or sh-circFKBP8#1 + inh-miR-432-5p, the protein level of E2F7 was detected. ** P < 0.01, *** P < 0.001

Fig. 6

E2F7 promoted BC cell proliferation, migration, invasion, and stemness. MDA-MB-231 and MDA-MB-468 cells were transfected with si-NC or si-E2F7. (A) The protein level of E2F7 was detected after knockdown of E2F7 in BC cells; (B-G) CCK-8 assay, EDU assay, colony formation assay, transwell migration and invasion assay, and sphere formation assay were used to detect cell proliferation, migration and invasion ability and cell stemness; (H) Western blot assay was used to detect the protein levels of KI-67, MMP2 and Nanog in BC cells. *** P < 0.001

Fig. 7

MiR-432-5p inhibited BC cells malignant behaviors by negatively regulating E2F7 expression. (A-H) MDA-MB-231 and MDA-MB-468 cells were transfected with miR-NC + vector, miR-432-5p + vector or miR-432-5p + E2F7; (A) Western blot analysis was used to evaluate the protein level of E2F7 in BC cells; (B-F) The proliferation, migration invasion, and stemness of BC cells were detected by CCK8 assay, EDU assay, colony formation assay, transwell migration and invasion assay, and sphere formation assay, respectively; (G) The KI-67, MMP2 and Nanog protein levels were detected in MDA-MB-231 and MDA-MB-468 cells by western blot assays. ***P < 0.001

Fig. 8

CircFKBP8 knockdown inhibited BC tumor growth in vivo. Xenograft model of sh-NC or sh-circFKBP8#1 group was established in mice. (A-B) Tumor volume and Tumor weight were detected in Xenograft assay; (C) The levels of KI-67, E2F7, MMP2 and Nanog was assessed by IHC staining in different groups; (D) The expression of circFKBP8, miR-432-5p and E2F7 was detected by qRT-PCR in tumors from different groups; (E) The protein levels of KI-67, E2F7, MMP2 and Nanog were evaluated by western blot assays in tumors form different groups. ** P < 0.01, *** P < 0.001