HOXC6-Mediated miR-188-5p Expression Induces Cell Migration through the Inhibition of the Tumor Suppressor FOXN2

Abstract

Homeobox C6 (HOXC6) is a transcription factor that plays a role in the malignant progression of various cancers. However, the roles of HOXC6 and its regulatory mechanism remain unclear. In this study, we used microRNA (miRNA) regulatory networks to identify key regulatory interactions responsible for HOXC6-mediated cancer progression. In microarray profiling of miRNAs, the levels of miRNAs such as hsa-miR-188-5p, hsa-miR-8063, and hsa-miR-8064 were significantly increased in HOXC6-overexpressing cells. Higher positive expression rates of HOXC6 and miR-188-5p were observed in malignant cancer. We also found that HOXC6 significantly upregulated miR-188-5p expression. The underlying function of HOXC6-mediated miR-188-5p expression was predicted through TargetScan and the MiRNA Database. Overexpression of mir-188-5p inhibited the expression of forkhead box N2 (FOXN2), a tumor suppressor gene. Furthermore, in the luciferase assay, miR-188-5p bound to the 3'-UTR of FOXN2 and was mainly responsible for the dysregulation of FOXN2 expression. Silencing FOXN2 induced cell migration, and the effect of FOXN2 silencing was enhanced when the HOXC6/miR-188-5p axis was induced. These results suggest that HOXC6/miR-188-5p may induce malignant progression in cancer by inhibiting the activation of the FOXN2 signaling pathway.

Keywords: FOXN2; HOXC6; cell migration; miR-188-5p.

Figure 1

Identification of differentially expressed miRNAs. (A) The Affymetrix microRNA microarray revealed differential miRNA expression profiles in FaDu cells vs. HOXC6-expressing FaDu cells. (B) Differentially expressed microRNAs in a scatter plot analysis. (C) Bar plot of significantly different miRNAs. The bar plot shows the most strongly deregulated miRNAs in comparison between the FaDu/HOXC6 cells and the control cells.

Figure 2

Validation of identified miRNAs using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Total RNA was isolated from HOXC6-overexpressing FaDu cells. (A,B) FaDu cells were transfected with pcDNA3-HOXC6. Forty-eight hours after transfection, the cells were collected to measure HOXC6 gene and protein expression. (C) Expression of selected miRNAs (miR-188-5p, miR-1281, miR-8063, and miR-8064) analyzed by qRT-PCR in FaDu/HOXC6 cells, and U6 was used for normalization. (D,E) Kaplan–Meier curves showing overall survival based on HOXC6 or miR-188-5p levels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for RT-PCR. Relative HOXC6 mRNA expression was determined by qRT-PCR, using GAPDH as an internal control. β-actin was used as loading control for Western blot. MicroRNA expression was determined by qRT-PCR, with U6 spliceosomal RNA (U6 snRNA) serving as an internal control. The data are presented the mean ± SD of three replicates. * p < 0.05.

Figure 3

HOXC6/miR-188-5p modulates the expression of FOXN2. FaDu cells were transfected with pcDNA3-HOXC6 or pcDNA3 vector for 48 h, and total RNA and protein were isolated. (A,B) HOXC6 or miR-188-5p expression in FaDu/HOXC6 cells was analyzed by Western blotting or quantitative RT-PCR. (C,D) Expression of FOXN2 in HOXC6- or miR-188-5p mimic-expressing FaDu cells. Actin was used as the internal control. The qRT-PCR data are presented as the mean ± SD from three independent experiments. * p < 0.05. (E,F) Effects of miR-188-5p on FOXN2 expression. FaDu cells were transfected with pSuper basic vector or pSuper/miR-188-5p for 48 h. miR-188-5p and FOXN2 mRNA expression levels were quantified by qRT-PCR analysis. (G) Total proteins were subjected to Western blotting analysis using FOXN2 antibodies. GAPDH was used as a loading control for RT-PCR. The relative level of expression of FOXN2 mRNA normalized to GAPDH was examined by qRT-PCR. mir-188-5p expression were determined by qRT-PCR, using U6 snRNA as an internal control. * p < 0.05 vs. the control.

Figure 4

Effects of HOXC6 or miR-188-5p on FOXN2 expression in HEK293 cells. Cells were transfected with pcDNA3-HOXC6 for 48 h. (A,B) HOXC6 or miR-188-5p was analyzed by Western blotting or quantitative RT-PCR in FaDu/HOXC6 cells. (C–E) Expression of FOXN2 mRNA and protein in HOXC6- or miR-188-5p mimic-expressing HEK293 cells. The qRT-PCR data are presented as the mean ± SD from three independent experiments. * p < 0.05. (F,G) HEK293 cells were transfected with pSuper/miR-188-5p for 48 h. miR-188-5p and FOXN2 mRNA expression levels were quantified by qRT-PCR analysis. * p < 0.05 vs. control. (H) Total proteins were subjected to Western blotting analysis using FOXN2 antibody. β-actin was used as loading control for Western blot. FOXN2 mRNA and mir-188-5p expression were determined by qRT-PCR, with GAPDH and U6 snRNA serving as an internal control, respectively. Moreover, GAPDH was used as an internal for RT-PCR.

Figure 5

miR-188 downregulates FOXN2 expression by targeting the 3′UTR of FOXN2. (A) Schematic of predicted miR-188 binding sites in the FOXN2-3′UTR. The putative binding site of FOXN2 is the red color sequence. (B) Luciferase activity of reporters expressing the wild-type 3′-UTRs of FOXN2 in FaDu cells cotransfected with the miR-188-5p inhibitor and pSuper/miR-188-5p or miR-188-5p mimic as indicated. Luciferase activity was assessed 24 h after transfection. (C) Luciferase activity of reporters with the 3′-UTR of FOXN2 in HEK293 cells cotransfected with the miR-188-5p inhibitor or miR-188-5p mimic oligonucleotides. The data are shown as the mean ± SD of at least three independent experiments. * p < 0.05.

Figure 6

miR-188-5p regulates cell migration in FaDu cells. (A) Cell migration in FaDu cells transfected with miR-188-5p mimics and inhibitor was evaluated via wound healing assays. (B) Effect of FOXN2 in inhibiting cell migration. FaDu cells transfected with siFOXN2 for 24 h or 48 h. A monolayer of confluent FaDu cells was wounded with a sterile pipette tip. Wound closure was observed by phase-contrast microscopy and photographed at 0, 24 h, and 48 h. The graph presents the wound closure rate (%). The data are shown as the mean ± SD of at least three independent experiments. * p < 0.05.