Study On The Function And Mechanism Of MTOR Signaling Pathway In Cell Growth

The mammalian target of rapamycin (mTOR) is a kind of Ser/Thr kinase in mammalian cells. It can recruit and integrate input signals from nutrients, growth factors, energy and environmental stress to regulate cell growth and proliferation via different cellular processes. The mTOR signaling pathway also affects the development of the embryonic stem cells (ESCs) and early embryo, and involved in cancers and metabolism disease. The mTOR signaling pathway has been investigated extensively in human, mouse, rat and cattle, whereas it is not yet studied in goat due to the lack of basic data about genes, gene expression and function. Thus this study uses the human tumor cells as a model to compare with the results from goat to prove that the mTOR signaling pathway plays a critical role in cell proliferation regulation.1 Function and mechanism of mTOR signaling pathway in regulation of human cancer cells growth1.1 Cloning and expression of mTOR and TSC2 genes in human esophageal cancer cells and primary normal esophageal epithelial cellsmTOR and TSC2 are two of the most important protein kinases within mTOR signaling pathway related with regulation of cell growth. mTOR is a central regulator in the pathway, and TSC2 works as a negative regulator to mTOR. mTOR cDNA segment and TSC2 cDNA segment were cloned by RT-PCR from human esophageal cancer cells(SEG-1) and primary normal esophageal epithelial cells (KOB-13). The expression of mRNA and proteins between these two kinds of cells were compared by RT-PCR and Western blot, separately. The results showed that the genes overexpressed in SEG-1 cells.1.2 Effect and mechanism of mTOR inhibition on SEG-1 cells growthSEG-1 cells were treated with RAD001, a specific inhibitor of mTOR and the results showed that SEG-1 cells are sensitive to RAD001 compared to the control group. RAD001 is cytotoxic to SEG-1 cells, and lead to death of the cells and inhibited cell proliferation. The shape of the cells changed and G1/S cell cycle was arrested by mTOR inhibition. RAD001 weakly inhibited phosphorylation of mTOR, and strongly inhibited phosphorylation of its downstream target S6 while the expression of mTOR,S6K1 and S6 genes were inhibited. The inhibitions depended on dosage of the inhibitor. The results showed that RAD001 inhibits activity of mTOR signal pathway. mTOR inhibition induces mTOR and S6 phosphorylation inhibited, and inhibits the expression of the genes.1.3 The relationship between the FAK/IGF-IR and the mTOR signaling pathwayTAE266, a dual inhibitor for FAK and IGF-IR, was used to treat SEG-1 cells. SEG-1 cells were sensitive to TAE266 and TAE226 had lethal function to SEG-1 cells. The cells shape was changed distinctly and cell proliferation was inhibited by the inhibitor with dose-dependant effect. The phosphorylation of mTOR, Akt and S6 was inhibited strongly by FAK/IGF-IR inhibition, and the expression of corresponding genes was inhibited strongly. Inhibition of FAK and IGF-IR lead to low activity of mTOR signal pathway. These data indicated that the activity of FAK may affect the activity of mTOR signal pathway, and FAK signal pathway is possibly the upstream regulatory factor of mTOR signal pathway.2 The function of mTOR signaling pathway in goat fetal fibroblast and its mechanism2.1 Cloning of mTOR signaling pathway related genes in Inner Mongolia Cashmere goat and the analysis of their basal expression patternIn the present study, the 8.6 Kb full length mTOR gene cDNA of Inner Mongolia Cashmere goat was cloned. The partial cDNA segments of S6K1 gene, S6K2 gene, S6 gene and FAK gene were also cloned in the goat. The sequencing analysis was performed and indicated these genes as a highly conserved gene to be very conservative. mTOR gene, S6K1 gene and S6K2 gene were found to be expressed in spleen, kidney, testicle and muscle, showed stronger expression in kidney than in spleen. S6 gene was overexpressed in spleen, testicle and muscle. FAK gene expressed in spleen, testicle and muscle, and expressed thin in testicle. These genes were first cloned and genes expression pattern were first studied in goat, and has laid a bioinformatics foundation for the further study on structure and function.2.2 The function and mechanism of mTOR signaling pathway on regulation of goat fetal fibroblast growthInner Mongolia Cashmere goat fetal fibroblasts (GFb) were treated with CCI-779, a mTOR specific inhibitor. The results indicated that GFb cells were sensitive to CCI-779. CCI-779 inhibits the activity of mTOR signal pathway and cell proliferation, blocks cell cycle, and is cytotoxic to GFb in a dosage dependent manner. GFb cells were treated with CCI-779, cytoskeleton lost normal structure, cell morphology changed. mTOR inhibition leads to the phosphoralation inhibited of S6 while the expression of mTOR,Akt, mTOR, S6kland S6 genes were inhibited. It is possible that CCI-779 can induce apoptosis in GFb cells.In summary, mTOR signaling pathway was first studied and mTOR gene,S6K1 gene,S6K2 gene,S6 gene and FAK gene were cloned firstly. mTOR signaling pathway was proved to be functional in GFb cells and acts as a key regulator to regulate cell growth. By comparing the experimental results on the mTOR signaling pathway from human cancer cells and goat cells, the possible mechanism of mTOR signaling pathway on the regulation of the cell growth and proliferation may involves the regulation of the phosphorilation of its down targets S6K1 and S6 and the expression of related genes after the mTOR is inhibited.