FOXK1, Regulated by miR-365-3p, Promotes Cell Growth and EMT Indicates Unfavorable Prognosis in Breast Cancer

Abstract

Background: Forkhead box K1 (FOXK1) is members of the FOX transcription factor family. Previous work has found out that FOXK1 promotes cell proliferation, migration and invasion in several cancers, such as gastric cancer, glioma cancer and lung cancer; however, the exact role of FOXK1 in breast cancer is still poorly known.

Methods: Here, the association between FOXK1 expression and the clinicopathological characteristics of patients with breast cancer was identified. To further decipher the functional roles of FOXK1, it was overexpressed or knocked down in MCF-7, MDA-MB-231 and MCF-10A cells. Cell Counting Kit-8, colony formation and cell cycle assays were performed to examine the proliferation of breast cancer cells. Moreover, wound-healing and Transwell invasion analyses were carried out to explore the effect of FOXK1 on breast cancer cell migration and invasion.

Results: Our findings discovered that FOXK1 promotes cell proliferation, migration and invasion in breast cancer. In addition, consistent with the previous report, FOXK1 also facilitates EMT in breast cancer. TargetScan was used to predict up-stream of FOXK1, indicating that miR-365-3p could regulate FOXK1 expression in breast cancer.

Conclusion: The findings of the present study demonstrated that miR-365-3p-FOXK1 axis plays a key role in breast cancer.

Keywords: EMT; FOXK1; breast cancer; miR-365-3p; migration.

Figure 1

FOXK1 is up-regulated in breast cancer tissues and cells. (A) The expression of FOXK1 in breast cancer tissues and adjacent normal tissue samples as detected by RT-qPCR and Western blotting. *p<0.05 vs Adjacent normal tissues. (B) The expression of FOXK1 in breast cancer cells as detected by RT-qPCR and Western blotting. *p<0.05 vs MCF-10A. (C) The survival curve was analyzed on GEPIA website (gepia.cancer-pku.cn).

Figure 2

FOXK1 promotes the proliferation of breast cancer cells. (A) FOXK1 was overexpressed or knocked down in MCF-7 and MDA-MB-231 cells, the expression of FOXK1 was established using RT-qPCR and Western blotting. *p<0.05 vs vector or scramble siRNA (SCR). (B) CCK-8 analysis of FOXK1-transfected MCF-7 and MDA-MB-231 and control cells. *p<0.05 vs vector or SCR. (C) Colony formation analysis of FOXK1-transfected MCF-7 and MDA-MB-231 and control cells. *p<0.05 vs vector or SCR. (D) After transfection, cell cycle assay was performed. *p<0.05 vs vector or SCR.

Figure 3

FOXK1 improves the invasive ability of breast cancer cells. (A) Transwell invasion assay in FOXK1-transfected MDA-MB-231 and control cells. *p<0.05 vs vector or SCR. (B) Wound-healing assay in FOXK1-transfected MDA-MB-231 and control cells. *p<0.05 vs vector or SCR. (C) The effect of FOXK1 on MMP2/9 secretion was identified by MMP2/9 ELISA kit. *p<0.05 vs vector or SCR.

Figure 4

FOXK1 regulates EMT in breast cancer. (A and B). FOXK1 was overexpressed or knocked down in MCF-10A or MDA-MB-231 cells, respectively. The expression of EMT markers was established using RT-qPCR and Western blotting. *p<0.05 vs vector or SCR. (C and D). The effect of FOXK1 on slug and snail expression was identified by RT-qPCR and Western blotting. *p<0.05 vs vector or SCR.

Figure 5

FOXK1 is a target of miR-365-3p in breast cancer. (A) FOXK1 is predicted to be a potential target of miR-365-3p. (B) The luciferase reporter gene plasmids containing the wild type (WT) and mutant type (MT) of FOXK1 3ʹ-UTR were generated. (C) Dual-luciferase reporter assay revealed that the luciferase activity was decreased in MCF-7 cells co-transfected with miR-365-3p mimics and WT 3ʹ-UTR of FOXK1 reporter plasmid, but unaltered in cells co-transfected with miR-365-3p mimics and MT FOXK1 3ʹ-UTR plasmid. *p<0.05 vs miR-NC. (D) RT-qPCR and Western blotting analyses were conducted to examine the effect of miR-365-3p on the expression of FOXK1. *p<0.05 vs miR-NC.

Figure 6

Up-reComment: Dear TS, please replace fig 6 with attached fig 6gulation of miR-365-3p reverses the effects of FOXK1 on proliferation and invasion in breast cancer cells. (A) MDA-MB-231 cells were co-transfected with FLAG-FOXK1 plasmid or/and miR-365-3p mimic or co-transfecting siFOXK1 or/and miR-365-3p inhibitor, the expression of FOXK1 was identified using RT-qPCR and Western blotting analyses. *p<0.05 vs vector or SCR. (B) After transfection, CCK-8 assay was carried out to determine cell proliferation. *p<0.05 vs vector or SCR. (C) After transfection, Transwell assay was performed to determine cell invasion. *p<0.05 vs vector or SCR.