SIRT6 inhibits metastasis by suppressing SNAIL expression in nasopharyngeal carcinoma cells

Abstract

Nasopharyngeal carcinoma (NPC) is a head and neck cancer with severe local invasion and early distant metastasis. SIRT6 serves as a critical modulator of the development and metastasis of multiple types of cancer; however, the roles and underlying mechanisms of SIRT6 in regulating NPC metastasis remain largely unknown. Here, the expression of SIRT6 in high metastatic 5-8F cells and low metastatic 6-10B cells was analyzed. SIRT6 expression was found to be negatively associated with the metastatic capability of NPC cells. Moreover, we identified that SIRT6 inhibited NPC cell metastasis through suppression of SNAIL expression. Mechanistically, we demonstrated that SIRT6 interacted with transcription factor p65 (NF-kB subunit) and deacetylated histone H3 lysine 9 (H3K9) and lysine 56 (H3K56) at the promoter of SNAIL, leading to reduced transcription of SNAIL. In summary, SIRT6 functions as a metastasis suppressor in NPC cells through epigenetic regulation of SNAIL gene expression.

Keywords: Nasopharyngeal carcinoma; SIRT6; SNAIL; metastasis; p65.

Figure 1

Comparison of SIRT6 expression in NPC cell lines. (A) qPCR analysis of SIRT6 expression in 5-8F and 6-10B cells. (B) Western blot analysis of SIRT6, SNAIL and E-cadherin protein levels in 5-8F and 6-10B cells. (C and D) qPCR (C) and western blot (D) analysis of SIRT6 expression in 5-8F-GFP and 5-8F-SIRT6 cells. (E and F) qPCR (E) and western blot (F) analysis of SIRT6 expression in 6-10B-shGFP and 6-10B-shSIRT6 cells. Data are shown as mean ± SEM (n=3-4 per group). *, P<0.05 by t test. For gel source data, see Supplementary Figure 1.

Figure 2

SIRT6 suppresses the migration and invasion of NPC cells. (A-D) In 5-8F cells that stably overexpressed with GFP or SIRT6, cell motility was analyzed using a wound healing assay (Magnification: 40×; Scale bars: 100 µm) (A) and the percentage of wound closure was quantified (B); migration and invasion abilities were evaluated by migration assay and Matrigel invasion assay, respectively (Magnification: 100×; Scale bars: 50 µm) (C); the percentage of invaded or migrated cells (% of GFP group) was quantified (D). (E-H) In control (shGFP) or stable SIRT6 knockdown (shSIRT6) 6-10B cells, cell motility was measured using a wound healing assay (Magnification: 40×; Scale bars: 100 µm) (E) and the percentage of wound closure was quantified (F); invasion and migration abilities were measured by Matrigel invasion assay and migration assay, respectively (Magnification: 100×; Scale bars: 50 µm) (G); the percentage of invaded or migrated cells (% of shGFP group) was quantified (H). Data are shown as mean ± SEM (n=4 for each group). *, P<0.05 by t test.

Figure 3

SIRT6 inhibits NPC cell metastasis by suppression of SNAIL expression. (A) qPCR analysis of SNAIL expression in 5-8F and 6-10B cells. (B) Western blot analysis of SNAIL and E-cadherin protein levels in stable 5-8F cell lines with GFP or SIRT6 overexpression and stable 6-10B cell lines with control or SIRT6 knockdown. (C-F) In control (shGFP) and stable SIRT6 knockdown (shSIRT6) 6-10B cells transfected with siSCR or siSNAIL, SIRT6 and SNAIL protein levels were analyzed by western blot (C); invasion and migration abilities were measured by Matrigel invasion assay and migration assay, respectively (Magnification: 100×; Scale bars: 50 µm) (D); the percentage of invaded (E) or migrated (F) cells (% of shGFP siSCR group) was quantified. Data are shown as mean ± SEM (n=3-4 per group). *, P<0.05 by t test. For gel source data, see Supplementary Figure 2.

Figure 4

SIRT6 interacts with p65 and deacetylates H3K9 and H3K56 at the promoter of Snail1. (A) Co-IP assay to test the potential interaction between SIRT6 and p65 was performed by transfection of corresponding constructs into 6-10B cells. IP, immunoprecipitation; IB, immunoblot. (B) The sequence of the Snail1 promoter region (-500) is presented. The p65 DNA binding sites were predicted by program PROMO 3.0. (C and D) ChIP assay of the binding of p65 (C) and SIRT6 (D) to the promoter regions of the Snail1 gene in 6-10B cells using IgG as a negative control for chromatin pull-down. (E) The effect of SIRT6 overexpression on histone acetylation in the region 2 of Snail1 promoter was evaluated using ChIP in 6-10B cells transfected with GFP or SIRT6 expression constructs. Data are shown as mean ± SEM (n=3 for each group). *, P<0.05 by t test. For gel source data, see Supplementary Figure 3.