Introduction Activation and proliferation of myofibroblastic hepatic stellate cells (HSC) is a pivotal event in liver fibrogenesis. MicroRNAs (miRNAs) are implicated in the regulation of a large number of important cellular functions including cell proliferation, differentiation and apoptosis.
Aim To characterise changes in miRNA expression during HSC activation and to investigate the effect of silencing candidate miRNAs on HSC proliferation and apoptosis.
Method Expression of all known miRNAs was determined in quiescent (day 1) and culture-activated (day 10) rat HSC by microarray. Expression of selected, differentially regulated miRNAs was verified by real-time PCR at multiple time-points during culture-activation. Putative target genes of up- and down-regulated miRNA were organised into hierarchical categories based on their gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) classification. Activated HSC were transfected with chemically modified, single stranded nucleic acid miRNA inhibitors by electroporation. HSC proliferation and apoptosis were determined by [3H]-thymidine incorporation and acridine orange staining, respectively.
Results A total of 21 and 17 miRNA were up- or down-regulated >1.5-fold during HSC culture-activation. The level of expression of eight selected miRNAs identified by microarray was confirmed by real-time PCR, with up to 170-fold change in expression observed between day 1 and day 10 (Abstract OP19 Table 1). Multiple GO terms and KEGG pathways were functionally enriched amongst the targets of up- and down-regulated miRNAs. Inhibition of mir-143 in activated myofibroblastic HSC inhibited proliferation by 33.5% (p=0.001) and increased serum-deprivation induced apoptosis by 68.3% (p=0.027).
Conclusion Activation of rat HSC was accompanied by marked up- and down-regulation of multiple miRNAs with potential to influence many cellular functions. Inhibition of mir-143 is pro-apoptotic and anti-proliferative in HSC, suggesting an important pro-fibrotic role for this miRNA in HSC and identifying a potential novel target for anti-fibrotic therapy in the liver.
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