The Role And Mechanism Of TPPP3 In Obesity And Lung Cancer

Tubulin Polymerization Promoting Proteins (TPPPs) are a protein family, which can promote tubulin monomer binding and make microtubule stable. In 2006, TPPPs were found and studied by a Hungarian research group as a new series of proteins. It includes three members (TPPP1/P25, TPPP2/P18 and TPPP3). For human beings, they are located on chromosome 5,16 and 14, respectively. Except the N-terminal is lost in TPPP1/P25 protein, the three subfamilies have strong homology. TPPP1 and TPPP3 can bind together with tubulin and induce microtubule gathering. TPPP1 is the most powerful protein to cause microtubule gathering while TPPP2 lacks this capacity.This study established the hypothalamic-pituitary-adrenal cDNA gene expression profiling by cDNA and DNA microarray hybridization. A group of new genes were cloned, of which TPPP3 was a novel gene cloned in the pituitary tissue by us in 2000, and was named as brain specific protein. It was also found in C. elegans in 2000 by Taiwanese scientists.At present, the data about TPPP family were heavily focused on how TPPP1 induced and regulated tubulin aggregation. However the studies on TPPP3 function are still scarce. In our previous work, we found a high expression of TPPP3 in adipose and lung tissue by Northern blotting.. Therefore, we conducted this study in two parts to explore the role and the mechanism of TPPP3 gene on obesity and lung cancer development respectively.Part1:Study on the mechanism of tppp3 gene related to obesityObjective:TPPP3-/- mice were fed a high-fat diet to investigate the impact of TPPP3 deficiency on the process of obesity so as to explore the possible molecular mechanism of obesity in TPPP3-/- mice with high fat diet.Methods:TPPP3-/- mice (TPPP3-KO) and its wild type control mice (WT) were fed with a high fat diet and a normal diet for 12 weeks. Food intake was measured every day and weight gain was determined per week. Body fat in mice is scanned and analyzed by Micro-PET/CT. Intraperitoneal glucose tolerance, insulin tolerance, blood pressure, blood lipids and blood insulin were measured respectively. Liver morphological changes were detected microscopically with H&E and Oil Red 0 staining. Inflammatory cytokines were detected on adipose tissue by Real-time PCR. The gene levels of enzymes related to lipid metabolism in liver were also examined by Real-time PCR.Results:1) Compared with the WT mice, TPPP3-/-mice have a significantly higher weight after the high fat diet for 8-12 weeks.2) Whole body fat measured by Micro-PET/CT was increased significantly in TPPP3-/-mice.3) A large number of lipid droplets were accumulated in the liver tissues of TPPP3-/- mice.4) glucose tolerance tests revealed a significant deterioration in glucose metabolism of TPPP33-/- mice than WT mice.5) The concentration of plasma insulin was higher than WT mice.6) Inflammatory factors from visceral adipose tissue of TPPP3-/- mice, such as MCP-1, F4/80, CD68, TNF-α, IL-1β and so on, were higher than those of WT mice. And then, genes involved in adipogenesis and metabolism were altered in TPPP3-/- mice, such as SCD-1, ACC, FDS and Insig-1.Conclusions:TPPP3 is most likely an anti-obesity factor. (Proinflammatory cytokines released by adipose tissue in generating the chronic inflammatory profile associated with visceral obesity).Part 2:The role and mechanisms of TPPP3 on development of Lung cancerObjective:To clarify the biological roles and mechanisms of TPPP3 in the process of lung cancer.Methods:The expression levels of TPPP3 and overall survival of post-operation were detected in the lung cancer tissues array by Immunohistochemistry. Stable TPPP3-knockdown cell lines were established for study of cell function in vitro and subcutaneous xenograft model in vivo. The expression of proteins related to proliferation and apoptosis were detected by Western blot.Results:1) The expression of TPPP3 increased significantly in the tumor tissues. TPPP3 levels in the lung cancer was an independent risk factors for overall survival of lung cancer by Cox regression.2) TPPP3-knockdown cell lines were successfully constructed.3) By CCK-8, Live cell Imaging System, Transwell assay, Flow cytometry assay and Tumor xenograft assay, knockdown of TPPP3 inhibited cell proliferation, migration, cell cycle distribution and tumorigenesis.4) The proteins related to proliferation and anti-apoptosis (P-Stat3, P-AKT, Survivin, Bcl-2) can be influenced after TPPP3 knockdown.Conclusions:TPPP3 maybe a factor for regulating tumor cell proliferation.