Knockdown of Secernin 1 inhibit cell invasion and migration by activating the TGF-β/Smad3 pathway in oral squamous cell carcinomas

Abstract

Secernin-1 (SCRN1) is a regulator of exocytosis in mast cells. Recently, SCRN1 was reported to be correlated with the prognosis of colorectal cancer and gastric cancer, but its functional effects on oral squamous cell carcinoma (OSCC) remain unclear. Our aim was to explore the expression pattern and the migration and invasion effects of the newly identified SCRN1 in OSCC. Western blotting (WB) was performed to measure SCRN1 expression in human OSCC tissue samples and OSCC cell lines. The effects of SCRN1 on OSCC cell proliferation, invasion and migration were analyzed by cell counting kit-8 and Transwell assays. The expression levels of TGF-β, Smad3 and phosphorylated Smad3 (p-Smad3) were measured by WB. The secretion of matrix metalloproteinase (MMP)-2 and MMP-9 was determined by the enzyme-linked immunosorbent assay. The expression of SCRN1 was significantly elevated in OSCC tissues and cell lines. SCRN1 knockdown reduced the expression of TGF-β and p-Smad3 in OSCC cells. TGF-β stimulation promoted proliferation, invasion and migration and enhanced the expression of p-Smad3 and the secretion of MMP9 in SCRN1-knockdown OSCC cell lines. Our study demonstrated that SCRN1 is upregulated in OSCC. Further analyses demonstrated that SCRN1 promotes the proliferation, invasion and migration of OSCC cells via TGF-β/Smad3 signaling.

Figure 1

SCRN1 expression is significantly increased in OSCC tissues and cell lines. (A) Normal tissue was negative for SCRN1 staining (× 500). (B) Weak expression of SCRN1 was defined as low expression in OSCC (× 500). (C) Strong expression of SCRN1 with cytoplasmic and membranous staining was defined as high expression in OSCC (× 500). (D, E) The expression levels of SCRN1 were measured in 15 paired OSCC tissues and adjacent normal mucosa by WB. The data are presented as the mean ± SD. ***p < 0.001. (F, G) The expression levels of SCRN1 in the HSC3, HSC6, SCC15, SCC25, UM1 and UM2 cell lines were examined by WB. Significant differences were determined using t test. The data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 2

Knockdown of SCRN1 suppresses proliferation, invasion and migration in OSCC cell lines. (A) The protein expression levels of SCRN1 in HSC3 cell transfected with three short-hairpin RNAs, namely, shRNA-a, shRNA-b and shRNA-c, were evaluated by WB. (B) The protein expression levels of SCRN1 in SCC15 cell transfected with three short-hairpin RNAs, namely, shRNA-a, shRNA-b and shRNA-c, were evaluated by WB. (C) The mRNA expression levels of SCRN1 in HSC3 and SCC15 cells transfected with three short-hairpin RNAs, namely, shRNA-a, shRNA-b and shRNA-c, were evaluated by qPCR. (D) SCRN1 knockdown suppressed HSC3 and SCC15 cell proliferation, as determined by the CCK-8 assay. (E, F) Transwell migration assays were performed to assess and quantify the migration of SCRN1-knockdown and control HSC3 and SCC15 cells. (G-H) Transwell migration assays were performed to assess and quantify the invasion of SCRN1-knockdown HSC3 and SCC15 cells and control cells. The data are presented as the mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 3

SCRN1 promotes cell invasion and migration by activating the TGF-β/Smad3 pathway. (A) The protein expression levels of TGF-β, Smad3 and p-Smad3 in HSC3 and SCC15 cells were evaluated by WB. (B) The levels of TGF-β and p-Smad3 in SCRN1-knockdown HSC3 and SCC15 cells and control cells were determined by WB. The ratios of p-Smad3 to the total levels were calculated based on the densities of the respective bands. The data are presented as the mean ± SD of three independent experiments. **p < 0.01. (C) After 48 h of treatment with TGF-β, SCRN1-knockdown HSC3 and SCC15 cell proliferation was assessed by the CCK-8 assay. Compared with the HSC3-shRNA group (Green), TGF-β stimulation promoted the proliferation of OSCC cell lines in the HSC3-shRNA + TGF-β group (Red). (D) After 48 h of treatment with TGF-β, Transwell invasion and migration assays were performed to assess and quantify migration in SCRN1-knockdown HSC3 and SCC15 cells and control cells. (E) After 48 h of treatment with TGF-β, Transwell invasion and migration assays were performed to assess and quantify invasion in SCRN1-knockdown HSC3 and SCC15 cells and control cells. (F) After 48 h of treatment with TGF-β, the levels of p-Smad3 in HSC3 and SCC15 cells were determined by WB. The ratios of p-Smad3 to the total levels were calculated based on the densities of the respective bands. The data are presented as the mean ± SD of three independent experiments. **p < 0.01.

Figure 4

SCRN1 enhances MMP-9 exocytosis by activating the TGF-β/Smad3 pathway. (A) qPCR revealed that no significant difference in MMP-2/9 mRNA between control cells and SCNR1-knockdown cells. The data are presented as the mean ± SD, P > 0.05 and ns = no significant difference. (B) MMP-2 and MMP-9 protein secretion from SCRN1 knockdown SCC15 and HSC3 cells as determined by ELISA. The data are presented as the mean ± SD of three independent experiments. **p < 0.01. (C) Gelatinase activity as determined by zymogram densitometric analysis. The data are presented as the mean ± SD of three independent experiments. (D) After 48 h of treatment with TGF-β, MMP-2 and MMP-9 protein from SCRN1-knockdown SCC15 and HSC3 cells was detected by ELISA. The data are presented as the mean ± SD of three independent experiments. **p < 0.01.