Human Liver-specific Expression Of Small Rna-mir-122 Expression Regulation Mechanisms And Functional Studies

miRNAs are a class of noncoding RNAs that are about 22 nucleotides (nt) in length. Several hundred human miRNAs have been cloned and represent 2%-3%of total human genes.They specifically regulate the expression of many protein-coding genes by targeting their messenger RNAs through pairing interactions that direct cleavage or translational repression in a combinatorial fashion. It is estimated that 10%-30%of all protein-coding genes are regulated by miRNAs. miRNAs and their targets seem to constitute remarkably complex regulatory networks since a single miRNA can bind to and regulate many different mRNA targets and, conversely, several different miRNAs can bind to and cooperatively control a single mRNA target (Lewis et al, 2003). Except some intronic microRNAs derived from the intron of known coding genes, the majority of miRNA genes are located in intergenic regions or in antisense orientation to annotated genes (Lagos-Quintana et al,2001; Lau et al,2001; Lee and Ambros,2001;Mourelatos et al,2002). In addition, besides the intronic microRNAs, most non-intronic microRNAs are supposed to be transcribed by RNA polymeraseⅡexcept that some interspersed repetitive sequenced-derived microRNA genes are transcribed by RNA polymeraseⅢ.To dissect these complex networks operated by miRNAs, two aspects would be concerned: one is microRNA function; the other is the regulation of microRNA genes. In the past several years, there are abundant reports on microRNA function in various biological processes and some malignant diseases. Moreover, our knowledge on miRNA biogenesis has been significantly advanced in recent years, which provides good opportunity for the research on the regulation of microRNA genes. Current models for miRNA biogenesis and maturation suggest that compartmentalized stepwise processing of miRNAs takes place first in the nucleus and then in the cytoplasm. The prevailing view is that primary transcripts of miRNAs (pri-miRNAs) are processed in the nucleus by the RNaseⅢenzyme, Drosha, to stem-loop intermediates known as pre-miRNAs (Lee et al.2003). These pre-miRNAs are then transported to the cytoplasm for cleavage by Dicer and maturation to their active forms (Lee et al.2003). In the recent years, increasing evidence shows some non-intronic microRNAs are actually transcribed by RNA polymerase II and can be modulated by some specific transcriptional factors or signaling pathways and execute their functions in different cellular process. In a summary, above evidence indicates that microRNA network is tightly connected with traditional protein-based gene regulatory network. Although RNAs of mature form, pre-miRNAs as well as longer pri-miRNA transcripts have been detected by Northern blot analyses for a handful of miRNAs, the transcription units (TUs) or gene hosts that give rise to the vast majority of miRNAs have not been examined in great detail.mir-122, a liver-specific microRNA, constitutes 70%of the total miRNA population(16,18). Recent evidence shows that mir-122a is involved in multiple hepatocellular processes including HCV replication, cholesterol biosynthesis, fatty-acid synthesis and oxidation. However, how mir-122 is regulated during these processes is largely unkown. Here we identify a remote promoter region critical for the expresion of miR-122 and determine the upstream boundary of the primary transcript of miR-122. Using mutant analysis combined with luciferase report assay, we identify some key cis-elements in miR-122 promoter region and give a list of possible binding transcriptional factors. Amongst them, hepatocyte nuclear factor-4a (HNF-4a) was identified as an indispensable regulator of human mir-122 (hsa-mir-122) gene. Inhibition of HNF-4a expression by PMA, an agonist of MAPK signaling pathway, resulted in down-regulation of mir-122. Deletion, mutagenesis and binding assays revealed that HNF-4a directly regulates human hsa-mir-122 promoter through at least two DR1 (direct repeat separated by one nucleotide) cis-elements. In addition, we show that the coactivator PGC-1αwas capable of stimulating the HNF-4a-dependent transactivation of hsa-mir-122 promoter in a dose-dependent manner. Chromatin Immunoprecipation (ChIP) experiment also shows that HNF4 can bind to hsa-mir-122 promoter region. To explore the mir-122 expression profile under various stress conditions, we perform Northern blot assay to detect the mir-122 expression in Huh7 treated by different stimuli. Our results showed that h2o2, a strong oxidative stress inducer, and Insulin, the pivotal controller in sugar and lipid metabolism hometostais, could both result in significant down-regulation of mir-122 in a dose-dependent manner although the molecular mechanisms are still obscure. We also explore the function of hsa-mir-122 using luciferase reporter gene assay by cotransfect the mir-122 expression plasmid and luciferase reporter gene construct with or without putative mir-122 target gene 3'UTR. Through comparing the luciferase reporter gene expression level in the above two kinds of constructs, we identified several key mir-122 target genes participating sugar or lipid metabolism and cell cycle control etc. Based on the above results, we propose a regulatory model of hsa-mir-122 in different hepatocellular processes under stress conditions to better explain the hsa-mir-122 function and adjustment mechanism, providing more clues for treatment of many hepatocellular malignancies and metabolic diseases.