The ability to suppress macrophage-mediated inflammation in orbital fat stem cells is controlled by miR-671-5p

Lien, Gi-Shih; Liu, Jen-Fang; Chien, Ming-Hsien; Hsu, Wei-Tse; Chang, Tzu-Hao; Ku, Chia-Chi; Ji, Andrea Tung-Qian; Tan, Peng; Hsieh, Ting-Lieh; Lee, Liang-Ming; Ho, Jennifer H

To determine whether miR-671-5p in OFSCs response to activated macrophages, we compared the level of miR-671-5p in OFSCs under normal conditions and noncontact cultured with LPS-activated macrophages. We showed that miR-671-5p expression was significantly downregulated in OFSCs when co-cultured with activated macrophages ( Figure 5A ). Furthermore, we identified the direct targets of miR-671-5p by transfection of miR-671-5p inhibitor into OFSCs. As shown in Figure 5A , miR-671-5p inhibitor successfully reduced the miR-671-5p expression in OFSCs both under normal conditions and co-culture with activated macrophages. Inhibition of miR-671-5p in OFSCs resulted in increasing the mRNA level of sTNFR type II and IL-1RA, but not IDO and IL-10 ( Figure 5B) . Notably, the binding sequences of miR-671-5p to sTNFR type II ( Figure 5C ) and IL-1RA ( Figure 5D ) demonstrated that the binding affinities between miR-671-5p and these two targets were strong, which is evidenced by a low minimal free energy and a high miRanda alignment score ( Figure 5C,D) . OFSCs expressed the low protein level of sTNFR type II and no IL-1RA protein could be detectable in OFSCs ( Figure 5E ). However, protein levels of both sTNFR type II and IL-1RA in OFSCs were increased after inhibiting miR-671-5p, which confirmed that cytokine levels in response to miR-671-5p were in line with the mRNA expression values ( Figure 5E ). The above findings confirmed that sTNFR type II and IL-1RA were direct targets of miR-671-5p.