The Role Of STIM In Cell Apoptosis And Cell Proliferation Of HNSCC

Objective: STIM1 is an endoplasmic reticulum(ER) Ca2+ sensor that triggers the store-operated Ca2+ entry(SOCE). Calcium signal was an important second signal in cellular metabolism. In recent years, many studies have demonstrated that intracellular Ca2+ signaling implicated in various cancer progression and carcinogenesis, such as proliferation, apoptosis, migration and metastasis. As we all known, reisting cell death and sustaining proliferative signaling are important factors that contribute to tumor progression. The clinical relevance of STIM1 has been highlighted in multiple cancers, but the molecular mechanism by which STIM1 promotes cancer progression in head and neck squamous cell cancer(HNSCC) remains unclear. In this study, we detected the STIM1 expression in HNSCC tumor tissues and design the experiment to demonstrate its role in HNSCC cell apoptosis and proliferation in vitro and in vivo, hoping to further understand the relationship among calcium signal, STIM1 and HNSCC. The results will provide a new insight to mechanism investigation and molecular therapy of HNSCC.Method: 1. A total of 56 HNSCC formalin-fixed paraffin-embedded tumor samples were randomly collected from Tianjin Medical University Cancer Institute & Hospital during 2009 to 2010. Simultaneously, we reserved the neighbouring normal mucosa tissues from the same patients as the control. IHC was used to detect the expression of STIM1. The overall survival and clinical data were collected. At the same time, we collected 8 surgical fresh tissues for western blot to dectect STIM1 expression. Statistics was determined using Student’s t-test using SPSS18.0. Statistical significance was determined as P<0.05. Univariate survival analysis was determined using Kaplan-Meier and time series test(log-rank test). 2. Our study chose TSCCA and Hep2 cell lines to transfect STIM1-si RNA and Tb3.1 to transfect STIM1 plasmid. We used RT-PCR and western blot to verify the transfection efficiency. FACS and Hoechst33258 were used to detect3. apoptosis rate. FACS was used to detect cell cycle distribution. Western blot was used to detect the changes of apoptotic and proliferative relevant protein expression. 4. To study the possible mechanisms of STIM1 to resist apoptosis in HNSCC cells, we used fluo-4/AM fluorescent calcium probe to detect the calcium distribution changes. 5. TSCCA cell xenograft model was used to verify the in vitro experiments. HE staining was used to observe nuclear morphology and IHC was used to detect the expression change of caspase3, caspase12, BAX, Bcl2, P21 and cyclin D1.Result: 1. STIM1 expression was significantly higher in tumor tissues(6.286±0.259) compared with the adjacent normal tissues(2.393±0.226). There were significant differences between the two groups. STIM1 expression level was Western blot showed the same results. The STIM1 expression level was related with tumor size and pathology grade. The patients 5-year survival rate with STIM1-positive(25.9%)was obviously lower than that of patient with STIM1-negative(48.3%, P =0.032). 2. In TSCCA and Hep2 cell lines, STIM1 was down-regulated after transfection of STIM1-si RNA; in the contrary, STIM1 was up-regulated after transfection of GV144-STIM1. FACS and Hoechst33258 staining results revealed that apoptosis rate in STIM1-si RNA group was higher than the control group. The cell cycle distribution was blocked at G0/G1 phase after STIM1 was knockdown. In Tb3.1 cell line, apoptosis rate was declined after transfected with GV144-STIM1. Western blot showed that caspase3, caspase12, BAX and P21 was up-regulated, meanwhile, Bcl2 and cyclin D1 was down-regulated significantly after STIM1 was down-regulated. 3. In TSCCA and Hep2 cell lines, calcium influx was decreased, that is to say, SOCE was decreased once STIM1 was knockdown which triggered ER stress. 4. The tumor weight and volume growth curve suggested that, si RNA-STIM1 group was significantly suppressed compared with the PBS treated xenograft tumors.5. HE staining showed less mitotic nuclei phenotype and atypia cells in si RNA-STIM1 group. IHC showed intensive brown staining signal of caspase3, caspase12, BAX and P21 in the cytoplasm.Conclusion: 1. The expression of STIM1 was much higher in HNSCC tumor tissues compared with normal tissues. 2. The STIM1 expression was associated with cell apoptosis and cell cycle distribution in HNSCC cell lines. STIM1 participated in SOCE and regulate cell apoptosis through ER stress pathway by targeting caspase12. 3. The result of experiment in vitro was in correspondence with that of experiment in vivo. STIM1 could be potential therapeutic markers for treating HNSCC through targeting SOCE dependent processes.