Expression profile of circular RNA in LPS-treated murine peritoneal macrophages
Objective 
To analyze the expression profile of circular RNA (circRNA) in LPS-treated murine peritoneal macrophages (MPMs) and to investigate the effects of mmu_circ_0000790 (circ790) on the secretion of pro-inflammatory cytokines in MPMs following LPS stimulation. 
 
 
Methods 
MPMs were isolated from C57BL/6 male mice and then stimulated with (LPS treatment group) or without (blank control group) 1 mg/L of LPS for 12 hours. Supernatants of cell culture and cell pellets were collected from each group. Enzyme linked immunosorbent assay (ELISA) was used to measure the changes in the secretion of IL-1β, IL-6 and TNF-α in the supernatants. Expression profile of circRNA in macrophages from the two groups (n=3) was analyze by circRNA microarray. Some differentially expressed circRNAs were validated by real-time PCR (RT-PCR). Moreover, RT-PCR was also performed to detect the expression of circ790 in MPMs after LPS stimulation. Small interfering RNA (siRNA) oligo specific for circ790 was designed and synthesized. Then the synthesized siRNA oligo and normal control (NC) oligo were transfected into MPMs by Lipofectamine™2000. The transfected MPMs were treated with LPS. ELISA was used to detect the levels of IL-1β, IL-6 and TNF-α in the supernatants. Arraystar′s home-made microRNA target prediction software was used to predict circ790/microRNA. 
 
 
Results 
The concentrations of IL-1β , IL-6 and TNF-α in the LPS treatment group were significantly higher than those in the blank control group (P<0.01). These results indicated that the inflammatory model of MPMs was successfully constructed. Statistical differences in 34 differentially expressed circRNAs were found between the two groups (P<0.05). Among them, 12 circRNAs were up-regulated and the other 22 circRNAs were down-regulated in the LPS treatment group. RT-PCR results were generally consistent with the microarray data. The expression of circ790 was increased by (1.94±0.15), (3.18±0.13) and (4.21±0.22) folds as compared with that of the blank control group at 3, 6 and 12 h after LPS stimulation (P<0.05). After transfecting the MPMs with circ790-siRNA, the secretion of IL-6 was significantly decreased (P<0.05) without notable influence on the secretion of IL-1β and TNF-α. circ790 could function as a microRNA sponge to regulate the gene expression. 
 
 
Conclusion 
These data show a significantly altered circRNA expression profile in the LPS-treated MPMs. circ790 may be involved in the regulation of IL-6 secreted by macrophages. 
 
 
Key words: 
Circular RNA; LPS; Macrophage; Inflammatory response