Biotin pull-down assay of microRNA-25-3p-specific mRNA targets in human hepatic stellate cells

Hepatic stellate cells (HSCs) are considered to be the main source of extracellular matrix in liver fibrosis. During chronic liver injury HSCs become activated and transdifferentiate into pro-fibrotic myofibroblasts. This process is associated with major changes in gene expression as well as intracellular signaling and can potentially be regulated by microRNAs (miRNA). In a recent study we found miRNA-25-3p (miR-25) to be upregulated in serum of children with Cystic Fibrosis (CF) who have no evidence of liver disease, compared to children with CF liver disease and healthy individuals. In this study we investigated the role of miR-25 in HSC biology and on the development of liver disease. LX-2 (Hepatic stellate cells) were transfected with biotynilated miR-25a-3p synthetic duplexes. Total cell lysate was used as control and miRNA coupled with its target mRNA were pulled sown with streptavidin and compared to the control.