Fig 1.
Overexpression of STIM1 in HNSCC tumor tissue was closely related to tumor size but not to neck node metastasis.
STIM1 expression was detected by immunohistochemistry using monoclonal mouse anti-STIM1 antibody. STIM1 immunoreactivity was visible in the cytoplasm but not in the cell nucleus or on the membranes. (A) Paraffin-embedded sections of tongue squamous carcinoma tissue showed overexpression of STIM1 (Yellow staining). (B) Epiglottis squamous carcinoma tissue showed overexpression of STIM1 (Yellow staining). (C) Paired adjacent normal tissue of tongue showed relatively lower expression level of STIM1. (D) Paired adjacent normal tissue of epiglottis showed relatively lower expression level of STIM1. (E) Protein levels of STIM1 in 56 pairs of HNSCC cancer samples were analyzed by immunoblotting. Results from eight typical samples are shown here. (F) Quantitative analyses of STIM1 immunoblotting. The mean grays were collected through Image J software analysis. STIM1 expression in tumor tissue compared with GAPDH was significantly higher than in adjacent normal tissue. *p<0.05. (G) Association between STIM1 expression and tumor size in HNSCC tissues (n = 56). The value of linear relevance was 0.9034 between them. STIM1 expression in tumor tissue showed higher expression level than adjacent normal tissue in 50 cases (89%). (H) STIM1 expression in tumor tissue had no statistical difference between neck lymph node metastasis positive and negative groups (p>0.05).
Fig 2.
Change STIM1 expression or function influence SOCE in HNSCC cell lines.
(A)Protein level of STIM1 showed different expression in five available HNSCC cell lines. TSCCA and Hep2 were the highest but lowest in Tb3.1. (B) Results of STIM1 expression in mRNA showed higher expression level in TSCCA and Hep2 but lower in Tb3.1. (C) Downregulated STIM1 in TSCCA and Hep2 by transfected with STIM1-siRNA and upregulated STIM1 expression in Tb3.1 by transfecting with GV-144 STIM1. The transfection efficiency was confirmed by WB. (D) Above transfection efficiency was further confirmed by RT-PCR detection* p< 0.05. (E) TG-induced SOCE in TSCCA. SOCE was found in normal group but not in STIM1-siRNA and added SKF96365 groups. (F) TG-induced SOCE in Hep2. SOCE was found in normal group but not in STIM1-siRNA and added SKF96365 groups. (G) TG-induced SOCE in Tb3.1. SOCE was found in normal group and GV144-STIM1 groups but not in GV144-STIM1+SKF96365 groups. (H) Mean number of calcium response peaks, which mainly reflect cell SOCE ability, was significantly decreased in STIM1 siRNA and add SKF96365 groups in TSCCA and Hep2 but significantly increased in GV144 STIM1 group for Tb3.1. *p< 0.05 versus other two groups.
Fig 3.
STIM1 expression influences the HNSCC cell proliferation.
The extracellular calcium (EC) concentration were 0, 2 and 4 mmol/L respectively. (A)TSCCA cell proliferation during 3 days.* p<0.05 versus 0 mmol/L EC. (B)TSCCA STIM1-siRNA cell proliferation during 3 days. * p<0.05 versus 0 mmol/L EC. (C) Hep2 cell proliferation during 3 days.* p<0.05 versus 0 mmol/L EC. (D) Hep2 STIM1-siRNA proliferation during 3 days.* p<0.05 versus 0 mmol/L EC. (E) Tb3.1 cell proliferation during 3 days.* p<0.05 versus 0 mmol/L EC. (F) Tb3.1 STIM1-siRNA proliferation during 3 days.* p <0.05 versus 0 mmol/L EC.
Fig 4.
STIM1 expression influences the HNSCC cell phases.
(A) FACS measurements showed that downregulated STIM1 expression arrested more cells in G0/G1 stage for TSCCA and Hep2. (B) Data analysis about Fig 4A. *p<0.05 (C) WB detection of CyclinD1 and P21. Results showed that CyclinD1 decreased in STIM-siRNA groups than in normal, whereas P21 increased in STIM1-siRNA groups for TSCCA and Hep2.
Fig 5.
STIM1 expression closely related to HNSCC cell apoptosis and participated in TG-induced ER stress.
(A) Transfected with STIM1-siRNA in TSCCA and Hep2 could significantly increase cell apoptosis compared with control groups by FACS detection. (B)Transfection with GV144 STIM1 in Tb3.1 significantly decrease cell apoptosis proportion compared with control groups by FACS detection. (C) Data analysis of A and B. * P< 0.05. (D) 10mmol/L TG was added in all groups to induce ER stress. BAX, cleaved caspase3, and caspase12, Bcl2 were detected using WB detection. (E) Cleaved caspase3, caspase12, and BAX were notably increased in STIM1-siRNA groups than in normal for TSCCA. Caspase3 and Bcl2 expression had no difference among the two groups. (F) Cleaved caspase3, caspase12, and BAX were notably increased in STIM1-siRNA groups than normal for Hep2. Caspase3 and Bcl2 expression had no difference among the two groups. (G) Cleaved caspase3, caspase12, and BAX were notably decreased in GV144-STIM1 groups than in normal for Tb3.1. Caspase3 and Bcl2 expression had no difference among the two groups.
Fig 6.
Downregulated STIM1 expression in vivo TSCCA xenograft could inhibit tumor growth.
(A) Establish mouse oral cancer model by subcutaneous injection TSCCA or TSCCA with STIM1-siRNA. Images of TSCCA SI and TSCCA (STIM1-siRNA) SI-treated xenograft tumors. (B) Growth curves of xenograft tumors. *P< 0.05 (C) Tumor weight of xenograft tumors *P<0.05. (D) IHC staining of STIM1 and caspase12 in two groups. Yellow staining for STIM1 and caspase12.