Identification of a novel TGF-β-miR-122-fibronectin 1/serum response factor signaling cascade and its implication in hepatic fibrogenesis.

Transforming growth factor-β (TGF-β) is a potent cytokine that promotes the development of fibrogenic cells, stimulates the expression of fibrosis-related genes, and consequently results in hepatic fibrogenesis. The involvement of miRNAs in this process remains largely unknown. We showed that miR-122 was substantially expressed in hepatic stellate cells (HSCs) and fibroblasts, the major sources of fibrogenic cells in liver tissues. Notably, exposure to TGF-β led to significant downregulation of miR-122. Furthermore, reintroduction of miR-122 suppressed TGF-β-induced expression of fibrosis-related genes, including alpha smooth muscle actin (α-SMA), fibronectin 1 (FN1) and α1 type I collagen (COL1A1), in HSCs and fibroblasts. Subsequent mechanism investigations revealed that miR-122 directly inhibited FN1 expression by binding to its 3'-untranslated region and indirectly reduced the transcription of α-SMA and COL1A1 by inhibiting the expression of serum response factor (SRF), a key transcription factor that mediated the activation of fibrogenic cells. Further in vivo studies disclosed that intravenous injection of miR-122-expressing lentivirus successfully increased miR-122 level and reduced the amount of collagen fibrils, FN1 and SRF in the livers of CCl4-treated mice. These findings disclose a novel TGF-β-miR-122-FN1/SRF signaling cascade and its implication in hepatic fibrogenesis, and suggest miR-122 as a promising molecular target for anti-fibrosis therapy.

2 analyses, total RNA from cells was extracted using the TRIzol reagent, subjected to DNase I digestion (1U/μL, Fermentas) at 37°C for 30 min and then to heat inactivation in DNase I at 65°C for 10 min.
The expression levels of miR-122 and the reference gene U6 were quantified using the TaqMan MicroRNA Assay kit (Applied Biosystems, Foster City, CA, USA). qPCR analyses for the expression of pri-miR-122, -SMA, COL1A1, FN1 and the reference gene GAPDH were performed using Power SYBR ® Green PCR Master Mix (Applied Biosystems). The temperature cycle profile for the qPCR reactions was 95 °C for 1 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
When Power SYBR ® Green PCR Master Mix was used, melting curve analysis was performed to verify the specificity of the PCR product immediately after amplification, as follows: heating to 95 °C for 20 s, cooling to 60 °C for 20 s, followed by a temperature increase to 95°C with a transition rate of 0.11°C /s and the continuous detection of fluorescence.
All qPCR reactions were performed on a LightCycler 480 (Roche Diagnostics, Germany), and were run in triplicate. The cycle threshold (Ct) values did not differ by more than 0.5 among the triplicate runs. The level of target genes was normalized to the levels of the internal control genes to yield a 2 -△△Ct value. Sequences for primers are listed in Supplementary Table 1.

Plasmids
The details for plasmid construction are described below. The lentivirus vector 3 pCDH-miR-122 was generated by cloning the genomic fragment (621 bp) that encompasses the mouse miR-122 precursor and its 5'-and 3'-flanking sequences into the XbaI and BamHI sites of pCDH-CMV-MCS-EF1-copGFP (System Biosciences, Mountain View, CA, USA).
To verify the miR-122-targeted 3'UTR, a pGL3cm-FN1-3'UTR-WT was created by inserting the full length 3'UTR (1090 bp) of human FN1 into the EcoRI and XbaI sites downstream of the stop codon of firefly luciferase in pGL3cm [1], which was previously produced based on the pGL3-control (Promega). The pGL3cm-FN1-3'UTR-MUT plasmid, which carried the mutated sequence in the complementary site for the seed region of miR-122, was generated by fusion PCR based on the pGL3cm-FN1-3'UTR-WT vector.
All constructs were confirmed by direct sequencing, and all primer sequences used for cloning are listed in Supplementary Table 1.

Luciferase reporter assay
To verify the miR-122-targeted 3'UTR, 293T cells grown in a 48-well plate were co-transfected with 10 ng of firefly luciferase reporter, 20 ng of pRL-TK (Promega) and 20 nM of NC or miR-122 duplex. pRL-TK, which expresses Renilla luciferase, was used to correct the differences in both transfection and harvest efficiencies. Fortyeight hours post-transfection, cells were harvested and subjected to the luciferase assay as previously described [1].

Analysis of transaminase activity
Serum samples were collected from mice and stored at -80°C. The alanine transaminase (ALT) was tested using the serum transaminase test kit (Jiancheng, Nanjing, China) following the supplier's instruction.