Microarray Profiling and Bioinformatic Analysis of Circular RNAs in RAW264.7 Macrophages Under Simulated Microgravity

Abstract

Macrophages play a vital role in the innate immune system. Lots of studies have showed that spaceflight can change the function of macrophage and induce the immune disorder, however, the mechanisms remain to be illustrated. circular RNAs (circRNAs) can act as miRNA sponges and play an important role in regulating gene expression, here we tested the hypothesis that simulated microgravity can induce the differential expression of circRNAs in RAW 264.7 macrophages under simulated microgravity. Arraystar Mouse circRNA Array (8x15K, Arraystar) was used to identify the circRNA expression profiles in RAW 264.7 macrophages from Control(Con) and simulated microgravity(SM) groups, the cells in SM group were treated by clinostat for 72 hours. Total RNA was quantified using the NanoDrop ND‐1000. The sample preparation and microarray hybridization were performed based on the Arraystar's standard protocols, Agilent Feature Extraction software was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering with a threshold of fold change ≥1.5 and P<0.05. Quantitative RT‐PCR was performed in the Real‐time PCR System (Applied Biosystems) using SYBR Green qPCR master mix(Arraystar). Bioinformatic analyses including gene ontology (GO) and KEGG pathway were applied to predict the potential functions of circRNAs. The results from circRNA microarrays revealed that there were 60 differentially expressed circRNAs in SM group compared with Con group, in which 34 circRNAs were found to be up‐regulated and 26 circRNAs were down‐regulated. Furthermore, the up‐regulated expression of circR003780, circR015248 and circR015947 were verified by qRT‐PCR. GO analysis indicated that the top20 up‐regulated enriched GO terms included biological process “positive regulation of chronic inflammatory response”. KEGG analysis showed that the top 30 pathway enrichment included Wnt and mTOR signaling pathway, protein processing in endoplasmic reticulum, apoptosis, et al. These results indicated that simulated microgravity can change the circRNAs expression pattern in RAW 264.7 macrophages, the differentially‐expressed circRNAs may participate in the function changes of macrophage in spaceflight.Support or Funding InformationFunded by National Basic Research Programs of China, No.2014CB744404This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.