miR-222 regulates proliferation of primary mouse hepatocytes in vitro.

Abstract It is well known that hepatocytes regenerate after liver injury, although it is difficult to reproduce this phenomenon in vitro . The goal of this research was to determine the factors that stimulate proliferation of primary mouse hepatocytes (PMHs) in vitro . We first tested knockdown (KD) of tumor protein 53 (p53) alone as well as partial hepatectomy (PH, performed 72 h prior to PMHs preparation) alone. However, neither intervention stimulated hepatocyte proliferation during the 72-h observation period in vitro . We then tested the combination of p53 KD with PH and found that these interventions together stimulated cell proliferation in vitro . Under these latter conditions we analyzed gene expression of these cells by mRNA sequencing (RNA-seq) and microRNA sequencing (miRNA-seq). TargetScan analysis, which determines the relationship between microRNAs and gene expression, found a relationship between downregulated mmu-mir-222 (miR-222) and upregulated genes such as mitogen-activated protein kinase kinase kinase 2 (Map3k2). To confirm this relationship, we performed miR-222 KD and overexpression (OE) and observed the expected changes in target gene expression. Furthermore, the finding that miR-222 KD or OE stimulates or suppresses, respectively, hepatocyte proliferation is well explained by the association between miR-222 and its target genes, which stimulate growth. Our results suggest that miR-222 is one of the key factors regulating PMH proliferation in vitro .