Up-regulation of miR-326 regulates pro-inflammatory cytokines targeting TLR-4 in buffalo granulosa cells

Abstract

A genome-wide profiling of microRNA (miRNA) in endotoxin tolerant buffalo granulosa cells identified miR-326 amongst top-10 upregulated miRNAs. In this study, we have elucidated the role of miR-326 in granulosa cells in vitro. In-silico analysis revealed that miR-326 have binding site for 3′UTR of TLR-4 (Toll-like receptor 4). Transfection experiments showed that there was a significant inhibition of TLR-4 when miR-326 mimic was transfected to buffalo granulosa cells. Furthermore, when miR-326 transfected granulosa cells were exposed to LPS, followed by expression analysis of TLR-4 complex genes (TLR-4 and MyD88) and pro-inflammatory cytokines, we found a decreased expression of both TLR-4 complex and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β). We also found that the expression of anti-inflammatory (IL-10) gene was upregulated. To the best of our knowledge, this is the first study that showed the regulation of TLR-4 using miR-326. The present findings on regulation of TLR-4 are important and would help in understanding innate immunity regulation under different patho-physiology.