The ability of the liver to regenerate provides a unique system to study the in vivo regulation of cell proliferation and gene expression. This remarkable process allows recovery from a number of disease states, including surgical and chemical injuries, and is dependent on the reentry of normally quiescent cells into replication. MicroRNAs are recently discovered ~ 22 nt non-coding RNAs that are known to be critical effectors of gene regulation via mRNA expression. The main objective of this research project is to define the role of microRNAs in the regulation of gene expression in regenerating rat liver after 70% partial hepatectomy (PH). Our hypothesis is that microRNAs are required for liver regeneration and primarily responsible for the observed uncoupling of protein and transcript expression after PH. The first specific aim is designed to investigate whether mina biogenesis is essential for liver regeneration. This will be done by specific knockdown of critical components of Dicer in mina processing and Ago2 in the RNA Induced Silencing Complex (RISC). Knockdown of Dicer or Ago2 will be carried out in vivo using siRNAs, antisense oligonucleotides or plasmid-based expression-systems. mina levels will be quantitated via microarray analysis in rats after PH. The second specific aim will characterize the association of miRNAs with polysomes during liver regeneration and the mechanism(s) responsible for their polysome distribution. Based on the hypothesis that miRNAs follow mRNAs, we will compare global mRNA and mina profiles in the different polysome and non-polysome fractions. Bioinformatics will be used to correlate miRNAs with their predicted mRNA targets. We will examine in detail the expression and modulation of c-myc and p53 mRNA activity and other verified target mRNAs. The third specific aim is designed to identify miRNAs during liver regeneration that are directly involved in the uncoupling of mRNA and protein levels. We will profile miRNAs associated with liver regeneration via microarray analysis and determine the mechanism by which miRNAs levels are modulated. Using bioinformatics, we will select putative mRNA targets of uncoupling by miRNAs during liver regeneration. Finally, we will establish proof of function for selected miRNAs via their knockdown by 2'-O-methyoxyethyl phosphorothioate antisense `antagomir'oligonucleotides, and determine the effect on protein expression. The long-term goal of the research project is to formulate a model of posttranscriptional regulation of mRNA by microRNAs in liver regeneration. The proposed studies will provide important and new information in our understanding of cell growth and the control of gene expression in the liver's ability to recover from injury. PROJECT NARRATIVE: The liver constitutes one of the few, normally quiescent tissues in the adult animal that has the capacity to regenerate in response to cell loss through physical, infectious or hepatotoxic injury. The best-characterized model of liver regeneration is the 70% partial hepatectomy (PH) in rat. In the lobes that remain intact, the majority of hepatic cells rapidly reenter the growth cycle and begin to replicate. Restoration of liver mass is governed primarily by functional rather than anatomical factors, and occurs by growth of the remnant tissue. The compensatory regrowth of the liver after PH is a precise, highly regulated process which exhibits very defined temporal patterns of gene expression. This remarkable physiologic model results from an orchestrated balance of biological controls, which maintain normal liver function and architecture as the full-size of the organ is reconstituted. This grant proposal is based on three specific aims intended to address the hypothesis and elucidate the role of microRNAs in the regulation of gene expression in regenerating liver. These remarkable small RNA molecules have only recently been discovered;and it is now apparent that they are key players in development and growth of tissues. The proposed studies will provide important information for understanding the control of normal cell growth in vivo. Our goal is to understand the role that microRNAs play in the regeneration of liver after injury. Ultimately, the results may provide the basis for novel therapies designed to promote liver regeneration and accelerate recovery from disease states.
