miR-23a-3p/SIX1 regulates glucose uptake and proliferation through GLUT3 in head and neck squamous cell carcinomas

Abstract

SIX1 overexpression has been reported in several cancers. However, its involvement in head and neck squamous cell carcinoma (HNSCC) remains unclear. In this study we investigated the clinical significance and biological roles of SIX1 in HNSCC. SIX1 expression was upregulated in HNSCC and correlated with TNM stage and nodal metastasis. Analysis of TCGA dataset demonstrated that high SIX1 expression correlated with poor patient prognosis. Overexpression of SIX1 in the Fadu cell line upregulated cell proliferation, colony formation, glucose uptake and ATP production. In contrast, SIX1 depletion in the Detroit562 cell line downregulated cell proliferation, colony formation, glucose uptake and ATP production. We analyzed a series of genes involved in glucose metabolism and found that SIX1 overexpression upregulated GLUT3, an important glucose transporter, at both mRNA and protein levels. Using the TRANSFAC database, we found that SIX1 had potential binding sites on the GLUT3 promoter, which was validated by chromatin immunoprecipitation (ChIP) assays. Next, we focused on miR-23a-3p, which could target SIX1 in HNSCC cells. The miR-23a-3p mimic downregulated SIX1 expression while the miR-23a-3p inhibitor upregulated SIX1 expression. The binding of miR-23a-3p to the 3'-UTR of SIX1 was confirmed using the luciferase reporter assay. Analysis of TCGA dataset showed a negative correlation between the miR-23a-3p and SIX1. Furthermore, the miR-23a-3p mimic inhibited cell proliferation, ATP production and glucose uptake, which could be rescued by transfection with the SIX1 plasmid. In summary, our study demonstrated that SIX1 facilitated HNSCC cell growth through regulation of GLUT3 and glucose uptake. miR-23a-3p targeted the SIX1/GLUT3 axis and suppressed glucose uptake and proliferation in HNSCC.

Keywords: GLUT3; SIX1; glucose metabolism; head and neck squamous cell carcinoma; miR-23a-3p.

Figure 1

Expression of SIX1 in HNSCC. A. Negative SIX1 expression in normal laryngeal squamous epithelium. B. Negative SIX1 expression in a case of normal oral epithelial tissue. C. Positive nuclear and cytoplasmic SIX1 expression in a case of laryngeal squamous cell carcinoma. D. Positive nuclear SIX1 expression a case of oral squamous cell carcinoma. E. Negative SIX1 expression in a case of laryngeal squamous cell carcinoma. F. Negative SIX1 expression in a case of oral squamous cell carcinoma. G. Western blot analysis of SIX1 protein expression in 10 cases of HNSCC tissues and their adjacent normal tissues. H. External datasets from TCGA showed that overexpression of SIX1 correlated with poor overall survival of HNSCC.

Figure 2

SIX1 regulates glucose metabolism and ATP production in HNSCC cells. A. SIX1 protein expression in a 4 HNSCC cell lines. B and C. Efficiencies of SIX1 plasmid transfection in FaDu cell line siRNA knockdown in Detroit562 cell line. D. MTT assay showed that SIX1 overexpression promoted proliferation rate in FaDu cell line. SIX1 depletion inhibited proliferation rate in Detroit562 cell line. E. Colony formation assay demonstrated that SIX1 overexpression upregulated colony number in FaDu cell line while SIX1 depletion downregulated colony number in Detroit562 cell line. F. 2-NBDG glucose uptake assay demonstrated that SIX1 overexpression upregulated glucose uptake in FaDu cell line. SIX1 depletion showed the opposite effect in Detroit562 cell line. G. SIX1 overexpression increased ATP production in FaDu cells while SIX1 depletion decreased ATP production in Detroit562 cells. * indicates p<0.05.

Figure 3

SIX1 regulates GLUT3 in HNSCC. A. Western blot showed regulation of SIX1 on GLUT family proteins. B. Real-time PCR shows the effects of SIX1 on GLUT3. C. Prediction of binding sites and sequences by TRANSFAC database analysis. D. ChIP assay showed that SIX1could bind to the GLUT3 promoter. * indicates p<0.05.

Figure 4

miR-23a-3p targets and downregulates SIX1 in HNSCC. A and B. miR-23-3p regulates SIX1 mRNA and protein expression in FaDu cells. C. Prediction of SIX1 as a target of miR-23a-3p using Targetscan analysis. D. miR-23a-3p mimic suppressed the luciferase reporter activity of wild-type reporter, while no significant change was observed in that of mutant reporter. E. TCGA data showed that there was a significant negative correlation between miR-23a and SIX1 expression using linear regression. * indicates p<0.05.

Figure 5

miR-23a-3p regulates glucose uptake via SIX1 in HNSCC cells. A. Western blot demonstrated that miR-23a-3p mimic suppressed GLUT3 protein, which was partly restored by SIX1 in FaDu cells. B and C. 2NBDG glucose uptake and ATP production assays showed that miR-23a-3p mimic suppressed glucose uptake, which could be restored by SIX1 overexpression. D. MTT assay showed that miR-23a-3p decreased proliferation of FaDu cells, which was restored by SIX1 overexpression. * indicates p<0.05.