The Effects Of Simulated Microgravity On M Tor Signaling Pathway In BL6-10 Cells

Previous studies have shown that microgravity has significant impacts on the physiological status of cells in organisms,but its molecular mechanisms behind them are still elusive.The mammalian target of rapamycin(mTOR)signaling pathway is one of the many pathways that involved in the process but little studies have been made.The upstream of the mTOR pathway is linked with other signaling pathways,and the downstream involves protein synthesis,apoptosis and autophagy.In this study,the simulated microgravity model and drug control model were established to investigate the effect of different molecular activities on mTOR pathway on adhesion,proliferation,migration and apoptosis.By investigating the changes of mTOR signaling pathway,we elucidate the molecular mechanism that simulates the influence of microgravity on cell physiological status.In this study,BL6-10 melanoma cells were used as the research object.A threedimensional biaxial gyroscope was used to establish a simulated microgravity model and the drug control was established using the mTOR inhibitor rapamycin and the CNF1(Cytotoxic Necrotizing Factor 1),which can up-regulate the mTOR activity.Immunofluorescence staining was used to observe the effects of microgravity and the changes of cell morphology and adhesion on micrrofilaments,Tubulin,Vinculin and Actin The expression of cytoskeleton and adhesion-related protein ?-tubulin,?-tubulin,FAK,vincullin,Rho A and SUN2 were detected by Western blotting.The changes of cell growth were determined by measuring the number of cells at different time points.MTS was used to explore the proliferation of cells.The cell cycle arrest was detected by flow cytometry.In vitro and in vivo migration experiments to explore changes in tumor cell migration ability.Western blotting was used to quantitatively analyze the expression of Cyclin B1,CD44,Met and p-Met.The apoptosis of the two models was detected by flow cytometry,and the changes of nuclear material and nuclear cytoskeleton were observed by immunofluorescence staining.Western blotting was used to detect the expression of apoptosis-related signaling pathway and DNA repair pathway.Finally,Western blotting was used to detect the expression of mTOR signaling pathway related molecules,and associated with the above phenomenon.The results showed that compared with the normal gravity group,morphological changes of the cells were observed under simulated microgravity and mTOR inhibitor rapamycin,and the distribution of microfilaments,microtubules and actin was abnormal.The distribution of focal adhesion occurred A certain disorder.The changes caused by simulated microgravity have some recovery in the treatment of CNF1.The expression of certain cytoskeletal proteins and adherent proteins is adaptive.Under simulated microgravity and rapamycin treatment,the rate of cell proliferation slowed down,CNF1 partially restored this effect,but failed to return to the normal gravity level.Cell cycle arrest occurred in both the simulated microgravity group and the rapamycin treated group,but the period of arrest was not the same.CNF1-treated group failed to completely repair the cell cycle arrest.Migration of tumor cells decreased after simulated microgravity and mTOR inhibitor treatment,and recovered after treatment with CNF1.Some of the molecular weight associated with metastasis is down-regulated in the effects of microgravity and rapamycin,but some molecules are adaptive.Similarly,mimicking microgravity and the mTOR inhibitor rapamycin both resulted in disruption of the nuclear cytoskeleton,apoptosis of melanoma cells,and decreased expression of nuclear scaffold proteins.The above results show that mTOR and Rho A activity are down-regulated under simulated microgravity,which affect the expression of downstream molecules and inhibit the related proteins synthesis,resulting in disorganization of cytoskeleton,focal adhesions and nuclear scaffolds,the changes of cell morphology and adhesion,and activating the apoptosisrelated signaling molecule expression,and lead to the occurrence of apoptosis.All above effects are consistent with the inhibition of mTOR activity,demonstrating that mTOR signaling pathway plays an important role in the influence of microgravity on cells.