MCS formation
Fig. 1 and Fig. 2 show changes over time in the morphology and number of SNU-790 and SNU-80 cells cultured under simulated microgravity and normal gravity (1G) conditions. Adherent SNU-790 cells were identified as elongated spindle epithelial cells. Over time, adherent SNU-790 cells became densely cultured. SNU-790 cells formed MCSs after 24 h of incubation under simulated microgravity, but not under 1G (Fig. 1). Meanwhile, adherent SNU-80 cells were observed as polygonal epithelial cells with large, round nuclei. These cells reached confluence over time. The formation of MCSs was observed in SNU-80 cells after 24-h incubation under simulated microgravity, with the number of MCSs increasing over time. However, no MCSs were observed in SNU-80 cells cultured under 1G.
Altered gene expression in SNU-790 cells
Differentially expressed gene (DEG) analysis in SNU-790 cells revealed 1,014 DEGs under simulated microgravity compared with those under 1G conditions. Of these, 306 were upregulated and 708 were downregulated (Fig. 3). Moreover, heatmap and principal component analysis (PCA) results showed that SNU-790 cultured in simulated microgravity exhibited transcriptional profiles distinct from those of cells cultured under 1G (Fig. 3).
Fig. 4 and Table 1 summarize the results of KEGG pathway and Gene Ontology (GO) enrichment analyses conducted on SNU-790 cells cultured under simulated microgravity conditions. The DEGs identified in SNU-790 cells were associated with 44 KEGG pathways. Among the top 20 pathways analyzed, systemic lupus erythematosus (ID:05322), alcoholism (ID:05034), neutrophil extracellular trap formation (ID:04613), necroptosis (ID:04217), shigellosis (ID:05131), and viral carcinogenesis (ID:05203) were observed to express more histone-cluster-related genes than other genes. Moreover, a total of 32 GO biological process (GO:BP) terms, 3 GO molecular function (GO:MF) terms, and 5 GO cellular component (GO:CC) terms were enriched. Protein-DNA complex subunit organization (GO:BP, GO:0071824), chromatin remodeling (GO:BP, GO:0006338), protein heterodimerization activity (GO:MF, GO:0046982), and nucleosome (GO:CC, GO:0000786), as well as similar terms were related to the overexpression of genes related to the histone cluster. Meanwhile, analysis of DEGs in SNU-790 cells cultured under simulated microgravity conditions revealed an overexpression of genes associated with histone clusters: HIST1H3G (fold change [FC] 3.286, adjusted P-value [adj. P] < 0.001), HIST1H3B (FC 3.066, adj. P < 0.001), HISTH2AB (FC 2.555, adj. P < 0.001), HIST1H1B (FC 2.382, adj. P < 0.001), HIST1H2AI (FC 2.140, adj. P < 0.001), and HIST1H1E (FC 2.072, adj. P < 0.001; Table 2).
KEGG pathway enrichment analysis of SNU-790 cells under simulated microgravity revealed that most genes related to cancer-associated microRNAs (ID:05206) were downregulated. Additionally, terms related to gene silencing, such as gene silencing by miRNA (GO:BP, GO:0035195) and posttranscriptional gene silencing (GO:BP, GO:0035194), were associated with low expression of microRNA-related genes. The top genes downregulated in the simulated microgravity culture of SNU-790 were MIR4798 (FC −3.437, adj. P < 0.001), MIR548K (FC −3.234, adj. P < 0.001), MIR4762 (FC −3.217, adj. P < 0.001), MIR548O2 (FC −3.111, adj. P < 0.001), MIR1284 (FC −3.066, adj. P < 0.001), MIR4524A (FC −2.925, adj. P < 0.001), MIRLET7A2 (FC −2.835, adj. P < 0.001), MIR4668 (FC −2.672, adj.P < 0.001), MIR4742 (FC -2.613, adj.P < 0.001), MIR4441 (FC -2.607, adj.P < 0.001), and MIR4500 (FC -2.597, adj.P < 0.001).
Altered gene expression in SNU-80 cells
SNU-80 cell analysis revealed 96 DEGs under simulated microgravity compared with those under 1G. Of these, 41 were upregulated and 55 were downregulated (Fig. 3). The heatmap and PCA showed that SNU-80 cultured in simulated microgravity had transcriptional profiles distinct from those of cells cultured in 1G (Fig. 3).
Fig. 5 and Table 3 summarize the results of the KEGG pathway enrichment and GO enrichment analyses of SNU-80 cells under simulated microgravity. A total of 15 KEGG pathways showed remarkable results in the enrichment analysis using the SNU-80 DEG set. The HIF-1 signaling pathway (ID 04066, P = 0.001, FDR = 0.159) was analyzed, and genes related to response to hypoxic conditions, including EDN1 (FC −1.979, adj. P < 0.001), PDK1 (FC −1.844, adj. P < 0.001), ENO2 (FC −1.628, adj. P < 0.001), and HK2 (FC −1.552, adj. P < 0.001), were downregulated. Moreover, DEGs related to the response to hypoxia were observed in pathways such as central carbon metabolism in cancer (ID 05230, P = 0.008, adj. P = 0.217), lipid and atherosclerosis (ID 05417, P = 0.008, adj. P = 0.217), and metabolic pathways (ID 01100, P = 0.008, adj. P = 0.217). A total of 164 GO:BP terms, 3 GO molecular function (GO:MF) terms, and 5 GO cellular component (GO:CC) terms were analyzed. A total of six GO:BP terms were associated with the response to hypoxic conditions, including response to hypoxia (GO:BP, GO:0001666, adj. P < 0.001), response to decreased oxygen levels (GO:BP, GO:0036293, adj. P < 0.001), and response to oxygen levels (GO:BP, GO:0070482, adj. P < 0.001).
Table 4 summarizes the top 17 up- and 20 down-regulated mRNAs from microarray analysis of SNU-80 cells in simulated microgravity culture. CA9 (FC −2.211, adj. P < 0.001), BNIP3 (FC −2.130, adj. P < 0.001), NPPB (FC −2.017, adj. P < 0.001), EDN1 (FC −1.979, adj. P < 0.001), PDK1 (FC −1.844, adj. P < 0.001), VLDLR (FC −1.792, adj. P < 0.001), and NDRG1 (FC −1.677, adj. P < 0.001) were downregulated under simulated microgravity in SNU-80 cells, and were related to the cellular response to hypoxia.