956 MIR29 TARGETS THE POTENT PROFIBROGENIC GROWTH FACTORS SUCH AS IGF-I AS WELL AS TWO MEMBERS OF THE VEGF/PDGF FAMILY

Background and Aim: HSC have been taken centre stage of liver fibrogenesis due to their remarkable synthesis of extracellular matrix proteins and secretion of profibrogenic mediators.MicroRNAs (miRNAs) are small noncoding RNAs, that posttranscriptionally inhibit gene expression by targeting the untranslated region (UTR) of various transcripts.Recently, we have shown that miR-29a and miR-29b reduce collagen expression in HSC.In the present study, we now investigated the role of miR-29 during the expression of profibrogenic growth factors.Methods: miR-29 expression was analysed by Real Time PCR in HSC after stimulation with Hepatocyte Growth Factor (HGF) and in rat liver after four weeks bile-duct obstruction (N = 22 BDL and N = 13 sham).In order to identify fibrogenic mediators as putative targets of miR-29a and miR-29b, different databases were screened.The putative targets were subsequently analysed by reporter assays.For this purpose the psiCheck vector providing endogenous normalisation of the luciferase reporter activity was used and putative miR-29 binding sites of the identified UTRs were cloned downstream to the luciferase reporter gene.Ago-miR-29, mimicking the miR-29a, were transfected into HSC and synthesis of identified targets were determined.Results: miR-29a and miR-29b were downregulated in rat livers suffering from cholestasis induced experimental fibrosis.Searching of different databases revealed that transcripts of IGF-I and members of the PDGF/VEGF family are putative miR-29 target sequences.Transfection of Ago-miR-29 did not affect reporter expression of PDGF-B and PDGF-B receptor.However, reporter assays demonstrate functional miR-29 binding to the 3 -UTR of the PDGF-C, IGF-I and VEGF-A mRNA but not to the corresponding mutated sequences taken as negative controls.Furthermore, we were able to show that miR-29 was markedly upregulated after stimulation of HSC with the antifibrogenic mediator HGF.Conclusion: miR-29, that is downregulated during experimental fibrosis, but upregulated by HGF is postulated to be involved in regulation of extracellular matrix synthesis as well as in secretion of the profibrogenic mediators IGF-I, VEGF-A and PDGF-C.Therefore, miR-29, are antifibrogenic miRNAs not only by targeting collagen biosynthesis but also by interfering with the profibrogenic cell communication.