Studies On The Mechanism Of Mir-101in Regulating Liver Fibrosis

Liver fibrosis is a common outcome of chronic hepatic injuries or diseases. The activation of hepatic stellate cells (HSCs) is the key step of liver fibrosis. HSCs activation and liver fibrosis process are regulated by a variety of cytokines and signaling pathways. Of these, TGF-β1has been indentified as the key cytokine to promote the HSC activation through the Smad2/3pathway. MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules, which are21-25nucleotides in length. They are highly conserved in evolution and widely present in animal and plant cells. Recent studies show that miRNAs can regulate a series of hepatic fibrosis-related gene expression and play a very important role in the development of hepatic fibrosis.Although existing reports explain the mechanism of KLF6raising TGFBR1expression at transcription level in HSC activation process, there is no report about the regulation of TGFβ receptor expression at the transcription level so far. Here, we induced hepatic fibrosis in mouse by repetitive intraperitoneal injections of CCl4and detected marked downregulation of miR-101in the fibrotic liver, while the expression levels of KLF6and TGFBRl increased significantly. qRT-PCR results also revealed that miR-101level in the isolated HSCs dramatically decreased upon CCl4treatment. Meanwhile, TβRI and KLF6mRNA expression in the isolated HSCs significantly increased during the process of CCl4-induced liver fibrogenesis. Luciferase assay and western blot results showed that miR-101dramatically reduced TβRI and KLF6 expression by direct targeting the3’UTR of their mRNA. Lenti-miR-101, that can express miR-101in vivo, was intravenously injected into CCl4-treated mice via tail vein, and greatly reduced CCl4-induced liver fibrosis as demonstrated by HE, Sirius red and α-SMA staining. miR-101expression were greatly upregulated in Lenti-miR-101-infected mice. Meanwhile, hepatic TβRI and KLF6expression were significantly reduced, confirming negative regulation of TβRI and KLF6by miR-101in liver. Nevertheless, blocking hepatic miR-101expression by a chemically modified antisense oligonucleotide in a liver fibrosis model would aggravate fibrosis. Western-blot of HSCs revealed that miR-101markedly suppressed the upregulation of KLF6and TβRI induced by TGF-β1, and consequently reduced phosphorylation of Smad2. Moreover, HSCs transdifferentiation is greatly suppressed by miR-101In conclusion, we have identified the miR-101as a negative regulator of fibrotic TGF-β signaling in liver fibrosis by regulating TβRI production. These findings were further validated in vivo, where restored miR-101expression inhibited the liver fibrogenesis, indicating that miR-101may be a possible therapy for the treatment or reversion of liver fibrosis. Hence, the identification of miR-101and its target genes provide useful insights into mechanisms underlying liver fibrosis.