The pluripotency factor LIN28 is targeted by neuronal microRNAs participating in myofibroblastic differentiation

Background and Aim: Activated hepatic stellate cells (HSC), which transdifferentiate into myofibroblastic cells, are the main producers of extracellular components upon chronic liver disease. Myofibroblastic transition is associated with an altered profile of profibrogenic growth factors, but also of neuronal signal transducers. MicroRNAs are small non-coding RNAs that regulate gene expression by translational inhibition. Therefore, we studied the role of microRNAs known to be involved in neuronal differentiation during HSC transdifferentiation. Methods: RNA was extracted by the Trizol method from primary rat HSC cultured for up to 10 days. miRNA expression profiling was performed by microarray analyses of miRNA extracted from HSC during myofibroblastic differentiation. Further polyadenylation combined reverse transcription was carried out and neuronal miR-9, miR-125b and miR-128 were quantified by Real-Time PCR. Putative targets were searched using various algorithms and a novel immunoprecipitation assay of the Argonaute 2 protein. Potential miRNA binding sites of the 3'-UTR were verified by luciferase reporter analyses. Results: During myofibroblastic HSC differentiation an upregulation for miR-9, miR-125b and miR-128 was observed. In silico target prediction identified putative binding sites of the neuronal miRNAs in transcripts encoding for several pluripotency factors such as LIN28. To evaluate the targets functional reporter assays were performed by cloning the wild type in comparison to corresponding mutated sequences with two point mutations in the binding consensus. We identified for all three neuronal miRNAs the potential target LIN28 which triggers pluripotency in embryonic stem cells. Conclusion: In conclusion, the neuronal microRNAs miR-9, miR-125b and miR-128 are highly upregulated during HSC transdifferentiation and are suggested to play an important role in myofibroblastic transition and expression of neuronal features by targeting LIN28 mRNA in HSC.