Study About Growth Inhibition Of HUVEC-C After Simulated Microgravity

Space microgravity causes cardiovascular dysfunction, which has been shown to be related to dysregulation of vascular endothelial cells. However, it is unclear about how microgravity-induced damage response adapts, what effects recover, and what regulatory mechanism is involved after astronants return from space to earth (1 g). In the present study, rotary cell culture system (RCCS-1) is utilized to perform simulate microgravity experiments in human umbilical vein endothelial cells (HUVEC-C). The biological effects of simulated microgravity and its subsequent effects after simulated microgravity are compared to investigate the characteristics of biological changes. The regulatory roles of miRNAs and their target genes in these processes are also discussed to reveal the possible mechanism in microgravity-induced biological effects.In this study, we co-cultured HUVEC-C cells and microcarriers for 24h before simulated microgravity so as to achieve complete combination of cells with microcarriers. The results showed that rearrangement of stress fibers occurred at 6h and recovered at 24h and 48h after simulated microgravity, indicating that simulated microgravity induced cytoskeleton remodeling. After recovery for 24h from simulated microgravity, cytoskeleton changed withactindepolymerization, and returned to normal again at 48h from simulated microgravity. Analysis of cell cycle showed that G2 arrest was observed at 48h after simulated microgravity, and disappeared after cells were cultured in 1g condition for 48h. SA-β-gal staining results showed that simulated microgravity promoted cell senescence. After recovery from simulated microgravity, cell growth of HUVEC-C was significantly inhibited. The adhesion ability decreased at the early recovery time (2h,4h), and gradually recovered. The apoptotic cell rate increased at later recovery time (72h and 96h) after simulated microgravity. These findings suggested that cell growth inhibition might contribute to the decrease in early cell adhesion ability, cytoskeleton remodeling and later apoptosis. To further analyze whether miRNA were involved in the regulation of biological effects after simulated microgravity, we examined the expression levels of three miRNAs (miR-1, miR-22 and miR-34a) under and after simulated microgravity. The results showed that simulated microgravity increased the expression levels of miRNAs (miR-1, miR-22 and miR-34a) with different expression pattern at different timepoints after simulated microgravity. Based on qRT-PCR and western blot analysis, we confirmed that miR-22 and miR-34a could play a role in cell growth inhibition through their target genes SIRT1 and BCL2, respectively. In addition, PTEN and Apaf-1 might be involved in apoptosis through PTEN-PI3K-Akt and P53-Apaf-1 pathway.Our study provides important values for evaluating the health states of the astronants after the return to earch and evaluating the risk of spaceflight.