A Study Of MiR-122 Regulating HBV

The emerging picture that small yet versatile RNA molecules can regulate gene expressions has at attributed as the most important advance in biology for decades。From the very beginning, computational methods have been utilized as indispensable tools, and many discoveries have been obtained through combination of experimental and computational approaches. Many technology are performed in miRNA research.Recently it has been recognized that miRNAs comprise one of the abundant gene families in multicellular species, and their regulatory functions in various biological processes are widely spread which post-transcriptionally regulate gene expression in plants and animals.An increasing body of research show that animal miRNAs play fundamental roles in cell growth ,development and differentiation,recent data suggest that miRNAs are aberrantly expressed in many human cancers and that they may play significant roles as on cogenes or tumour suppressors.MiR-122 is an abundant liver specific miRNA ,implicated in fatty acid and cholesterol metabolism.With the biological and computational prospecting we obtain a perspective that there is a miR-122 target sequence in HBV 1689nt-1711nt. For the further reserch of miR-122 , the eukaryotic expression vector of PEGFP-c1-hcr was constructed at first. The genomic hcr sequence surrounding miR-122 was amplified by PCR .The PCR product was cloned into plasmid vector pEGFP。The fusion protein expression in HepG2 cell was evaluated by the expression of GFP .Total RNA was extracted and the expression of micR-122 was detected by RT-PCR and TA clone. The miR-122 expression recombinant plasmid of PEGFP-c1- hcr was successfully constructed. For researching the miR-122 regulating HBV by targeting HBx,the positive miR-15 luciferase reporter plasmids was constructed. For luciferase reporter experiments a 3'UTR ,segment of 546bp of the 3'UTRof the BCL2 gene was amplified by PCR from HepG2 cells genomic DNA and inserted into the pGL3-control vector ,using the XbaⅠsite immediately downstream from the stopcodon of luciferase.We also constructed luciferase reporter plasmids inserted miR-122 target sequence -HBx at the same site. Luciferase activity was assayed .miR-122 with HBx protein expression vector was cotransfected into the HEK293 cells . HBx protein expression was detected by western blot analysis.The perspective was demonstrated that miRNA-122 negatively regulat HBV by targeting HBx sequence. Then,cotransfection of miR-122with HBV resulted in HBV replication down regulated.