Effects Of Simulated Microgravity On Transcriptome Gene Expression And Cell Adhesion Of Human Gastric Mucosa GES-1 Epithelial Cells

Objective: China’s manned spaceflight project has entered the third stage,and astronauts will stay longer in space.It is of great significance to study the impact of weightlessness on the digestive system.In this study,we used Illumina High-throughput sequencing technology to perform transcriptome gene sequencing on human gastric mucosal epithelial GES-1 cells before and after RCCS simulated microgravity treatment.Then,Gene sequence was annotated and compared.The differentially expressed genes(DEGs)were screened by DESeq2 software.Functional enrichment analysis of DEGs was performed according to Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database,so as to analyze the expression of DEGs before and after simulated microgravity exposure and perform functional analysis.The interaction network analysis of DEGs was carried out according to the STRING database,and the key genes in the gene interaction network were screened out.The effect of simulated microgravity on cell ultrastructure and cell adhesion was further discussed,and aimed to provide theoretical basis for the management of gastric mucosal stress injury under weightless environment.Methods: Human gastric mucosa epithelium GES-1 cells were studied,and the cells of3-10 generations in logarithmic growth stage were selected,and then the simulated microgravity group(SMG)and the normal gravity group(NG)were randomly set.Two groups of cells were collected on day 1 and 7 of cell culture.Illumina second-generation sequencing platform was used for transcriptome sequencing to screen DEGs.The DEGs were screened by DESeq2,and the GO function enrichment analysis,KEGG pathway enrichment analysis and gene interaction network analysis were performed.The ultrastructure of the cells at day 7 was observed under transmission electron microscope.5794 differentially expressed genes at 7 days were assessed by stratification,and GO and KEGG enrichment analysis were performed.The expression of cell adhesion molecules was verified by RT-PCR.Results: The analysis data showed that,compared with NG group,there were 1285 DEGs in SMG group after 1 day of RCCS culture,among which 421 were up-regulated and864 were down-regulated;After 7 days of RCCS culture,there were 5794 different genes,among which 3,391 were up-regulated and 2,403 were down-regulated.One-day GO functional enrichment analysis showed that the simulated microgravity was related to extracellular matrix organization,extracellular structure organization,regulation of vasculature development,biological adhesion,system development,cell adhesion,negative regulation of multicellular organismal process,positive regulation of multicellular organismal process,anatomical structure morphogenesis,regulation of intracellular signal transduction;The GO function enrichment analysis of the gene at 7 days showed that the simulated microgravity was related to pattern specification process,nervous system development,regulation of neuron differentiation,animal organ morphogenesis,enzyme linked receptor protein signaling pathway,tissue morphogenesis,morphogenesis of an epithelium,biological adhesion,regulation of anatomical structure morphogenesis,cell adhesion.The simulated microgravity at both day 1 and day 7 was related to biological adhesion and cell adhesion function.One-day KEGG pathway analysis showed that simulated microgravity was involved in 5 pathways,including Neuroactive ligand-receptor interaction,MAPK signaling pathway,AGE-RAGE signaling pathway in diabetic complications,ECM-receptor interaction,Protein digestion and absorption.Seven days of KEGG pathway analysis showed that simulated microgravity was associated with 32 pathways,including Pathways in cancer,MAPK signaling pathway,TGF-beta signaling pathway,TNF signaling pathway,AMPK signaling pathway and Focal adhesion and so on.The simulated microgravity at both day 1 and day 7 was related to the MAPK signaling pathway.The interaction network analysis of 1 day showed that the simulated microgravity was closely related to CXCL8,CXCL2,IL6,COL1A1,COL5A1,MXRA8,ALB and IGFBP.Gene interaction network analysis at 7 d showed that simulated microgravity was closely related to CXCL8,FGA,FGG,CCL5,ITGAM and FGB.5794 DEGs at 7 days were analyzed by stratification according to the set range of FC values,and GO and KEGG enrichment analysis were performed.GO analysis of each subgroup of genes showed that the simulated microgravity was related to cell adhesion,mitochondria and endoplasmic reticulum.The down-regulated KEGG pathway analysis showed that simulated microgravity was related to focal adhesion,TGF-β signaling pathway and AMPK signaling pathway.The up-regulated gene KEGG pathway analysis showed that simulated microgravity was related to cytokinecytokine receptor interactions,TNF signaling pathway and MAPK signaling pathway.The ultrastructure of the cells was observed under transmission electron microscopy.In cultured cells for 7 d,simulated microgravity resulted in nuclear shrinkage,ER swelling and mitochondrial pyknosis.The expression of CDH1 gene was relatively decreased,while the expression of CTNNB1 gene was relatively up-regulated.Conclusion: The RCCS simulated microgravity has important impacts on the transcriptome gene expression,cell adhesion function and ultrastructure of human gastric mucosal GES-1 cells,and related mechanisms and protective measures need to be studied in depth.