Study On Mechanism Of Exogenous S100A8/A9Induced Esophageal Squamous Cancer Cells EC9706Apoptosis

Background and ObjectiveEsophageal squamous cell carcinoma (ESCC) is a common digestive tract malignant tumor with high incidence and mortality rate in our country, especially in Hanan province. The occurrence of ESCC is come from interactions among polygene, multi-factor and multistep. At the molecular level, the variations of multiple genes and proteins were related with ESCC. But its exact pathogenesis is not yet clear that seriously hindered the early prevention and treatment of ESCC. Therefore, to explore the mechanism of ESCC is particularly important to effectively prevent ESCC occur and reduce the ESCC incidence and mortality.Esophagitis is probably one of ESCC occurs precancerous conditions, S100A8and S100A9express in a wide variety of tumor cells and immune cells, with similar function of inflammatory cytokines, and their regulation mechanisms were very complex. S100A8/A9, a heterodimer with proteins S100A8and S100A9formed in a calcium-dependent manner, play variety of biological functions in intracellular and extracellular. S100A8/A9expression were up-regulation in many tumors, playing dual roles in anti and pro-tumor responses. While in esophageal squamous cancer cells, the expression of S100A8/A9is reduced or ansent. The distinct expression patterns between ESCC and other tumor suggested that the roles in ESCC occurrence and development were different from other tumors.Under the premise of determining EC9706cells do not express S100A8and S100A9, This study is focuses on exploring whether EC9706cells express receptors RAGE (receptor of advanced glycation end) and TLR4(Toll-like receptor4), whether exogenous S100A8/A9binding with endogenous receptor RAGE or TLR4, as well as exogenous S100A8/A9effects on cell apoptosis and the molecular mechanism of apoptosis, to provide experimental basis for further research the development of ESCC, and to offer an new gene therapy strategy to ESCC on prevention and treatMethods1. Cultured human esophageal squamous cancer cells EC9706, the cells were divided into three groups:(1) Blank group:cells transfected with only GenJetTM.(2) NC group:cells transfected with pcDNA3.1empty plasmid.(3) Experimental group:cells transfected with S100A8and S100A9eukaryotic transfection pcDNA3.1/Myc-his and pcDNA3.1-Flag. Transfected cells were placed in carbon dioxide incubator and cultured for subsequent experiments.2. In order to count transfection efficiency,24h after transient transfection S100A8and S100A9protein were checked with their antibodies respectively, and observed under fluorescence microscope.3. Using optical microscope, the cell growth of NC group and experimental group were observed24h,48h and72h after transfection.4. The effects of S100A8/A9on cell apoptosis were checked with TUNEL method.5. After cellular immunofluorescence staining, confocal microscope was used to detect the colocalization of S100A8/A9with RAGE and TLR4receptor.6.24h,48h and72h after transient transfection, Q RT-PCR were used to check the expression levels of s100a8, s100a9, Erkl/2, Nf-κb p65and downstream target genes of the Bcl-2and p53mRNA.7. Western blot was used to detect the proteins expression of S100A8, S100A9, ERK1, ERK2, NF-κB P65and downstream target Bcl-2and P53at24h,48h and72h after transient transfection.Results1. The expressions of S100A8and S100A9were absent in human esophageal squamous carcinoma EC9706cells.2. The transfection efficiency was65%.3. The number of cells in experimental group gradually reduce as the transfection time, the experimental cell loss of about60%, the NC group cell loss of about10%.4. Compared with NC group, the number of apoptosis cell in transfection group was significant increased in a time-dependent (P<0.05)5. Confocal scanning electron microscopy positioning results showed that in Blank group, NC group and experimental group RAGE and TLR4are expressed in the all cell, S100A8/A9and RAGE or TLR4displayed a strong colocalization in experimental group.6. Q-RTPCR assays showed that S100a8and s100a9only expressed in transfection group. Compared with the Blank group and NC group, the expression of Erkl/2, Nf-κbp65and p53mRNA in experimental group was obviously raised (P<0.05), while the expression of Bcl-2mRNA in experimental group was down-regulation (P<0.05).7. Western blot assays showed that S100A8and S100A9protein only expressed in experimental group. Compared with the Blank group and NC group, the expression of ERK1-. ERK2, NF-κB P65and P53proteins in experimental group were significantly increase(P<0.05), While the expression of Bcl-2protein is obviously down-regulated with the increase of transfection time(P<0.05). Conclusions1. The expressions of S100A8and S100A9were absent in EC9706cells, but express RAGE and TLR4receptors. Exogenous S100A8/A9proteins can be combined with the two receptors.2. Exogenous S100A8and S100A9induce EC9706cell apoptosis. The mechanism may be through the activate of ERK1/2and NF-κB signaling pathways, up-regulation P53expression and down-regulation Bcl-2expression, then induce the cell apoptosis in ESCC.