Research On S100A6 Expression In Non-functional Pituitary Adenomas And Its Biological Function In TtT/GF Cell Line

Objectives:We aim to investigate S100A6 protein expression level and sub-cellular location in non-functional pituitary adenomas(NFPAs)tissues and normal pituitary gland;The correlation will be analyzed between S100A6 protein and clinical characteristics of NFPAs patients,such as tumor invasion;S100A6 protein expression in serum samples will also be detected;Mouse-derived pituitary adenoma cell line(TtT / GF)are studied for exploring the role of S100A6 protein on the proliferation,migration,and invasion from the cell functional level.Through the above experiments,we aim to determine the clinical significance of S100A6 protein expression in NFPAs and its biological function on the pituitary adenoma cell.This study is to explore new invasion markers or potential molecular therapeutic targets involved NFPAs.Methods:Immunohistochemistry was used to detect the expression and distribution of S100A6 protein in NFPAs and normal pituitary samples.ELISA served to detect the expression of S100A6 protein in the serum of patients with NFPAs and its clinical significance.Standard cell culture methods were used in order to culture the pituitary adenoma cell line TtT / GF,and the expression of S100A6 was down-regulated by siRNA transfection.Western Blot was used in order to detect the expression of S100A6 and ?-catenin in multiple samples.Edu labeling and colony formation assays detect cell proliferation after transfection.Transwell chambers were utilized to detect migration and invasion of pituitary adenoma cell.Statistical software was used to analyze the correlation between S100A6 expression and clinical characteristics and differences in different experimental groups.Results:1.Immunohistochemistry showed that the expression of S100A6 protein in normal pituitaries was weak positive and mainly distributed in the cell nucleus;while in NFPAs tissue were scattered among the nucleus,cytoplasm and extracellular,and mainly in the cytoplasm or extracellular fluid.Immunohistochemistry identified a total of 31 samples with high expression and 39 samples with low expression.Statistical analysis showed that S100A6 expression was not significantly related to age,sex,tumor size,and clinical symptoms of NFPAs;while the invasiveness of NFPAs was significantly related to high expression of S100A6.2.ELISA showed that there was no significant difference in expression of S100A6 between NFPAs group and healthy group;Whereas it was significantly higher in invasive group compared to non-invasive group.3.Western Blot results showed that S100A6 expression was higher in NFPAs compared with normal pituitary gland.The expression of S100A6 and ?-catenin in the SiRNA1 group was significantly reduced compared with the NC-SiRNA group and Blank group.4.Edu showed that EDU-labeled cells in the SiRNA1 group were significantly reduced than NC-SiRNA group and the Blank group.5.Colony formation experiments showed that the number of colony formation in SiRNA-1 group was significantly less than NC-SiRNA and Blank group.6.The cells number that migrated longitudinally into the Transwell chamber and passed through the Matrigel are both markedly reduced in SiRNA-1 group compared with the other control group.Conclusion:1.S100A6 protein was over-expression in NFPAs compared to normal pituitaries.Localization of S100A6 expression scattered among the nucleus,the cytoplasm and extracellular,mainly in the cytoplasm or extracellular fluid.The invasiveness of NFPAs was significantly related to high expression of S100A6.2.The expression of S100A6 protein in serum of patients with NFPAs was not significantly different from that in healthy human serum;Whereas its expression in serum was significantly higher in invasive NFPAs compared to non-invasive NFPAs.3.The down-regulation of S100A6 mediated by SiRNA inhibited the invasion,proliferation and migration of pituitary adenoma cells;Down-regulation of S100A6 mediated by siRNA transduction induced the expression of ?-catenin protein.4.The above studies suggested that over-expression of S100A6 may play an important part in the development of NFPAs.Down-regulation of S100A6 may be as a potential therapeutic target in the future.S100A6 protein is markedly correlated with NFPAs invasiveness,which may be an effective serum marker to predict NFPAs invasiveness in the future.