The functional influence of the RNA transcript complementary to pri-miR-122 for the expression levels of pri-miR-122 and miR-122.

<p>(<b>A</b>) RNA extracted from Huh-7 cells infected with various lenti-ADARs was prepared for assessment of the levels of <b>endogenous</b> miR-122, with that of si-GFP infected cells (set as 1). (<b>B</b>) RNA extracted from HepG2 cells transfected with either the wild-type or the mutant pri-miR-122 containing A-to-G at position −7 was processed for qRT–PCR. (<b>C</b>) The protein lysates extracted from Huh-7 (left) and HepG2 (right) cells infected with various lentiviruses were processed for the Western blotting analysis. (<b>D</b>) RNA extracted from Huh-7 cells infected with either lenti-si-GFP or lenti-si-AS-122, which targets the RNA transcript complementary to pri-miR-122, was processed for qRT–PCR for the antisense transcript (comp-AS-RNA) (left), the pri-miR-122 (middle), and miR-122 (right). (<b>E</b>) The HepG2 cells transfected with the control vector or the plasmid construct expressing comp-AS-RNA were processed for qRT–PCR, for examining the levels comp-AS-RNA (left), pri-miR-122 (middle), and miR-122 (right). The results were shown as the average of three experiments (mean ± SD), and the <i>P</i> values were calculated by <i>t</i> test (*<i>P</i><0.05; ***<i>P</i><0.001).</p

Antisense transcripts exist for pri-miR-214 and pri-miR-122, with A-to-I/(G) RNA editing at specific nucleotide residues.

<p>Schematic illustration of the gene structures for the RNA transcripts complementary to (<b>A</b>) pri-miR-214 and (<b>B</b>) pri-miR-122. (<b>C</b>) The representative results for sequencing of the strand-specific RT–PCR products from HCC with putative editing events in the sense (<b>S</b>) and antisense (<b>AS</b>) transcripts of pri-miR-214 and pri-miR-122, focusing on the regions covering the sites with potential editing events with the editing marked with red asterisks.</p

Specific editing of the RNA transcripts related with pri-miR-214 and pri-miR-122 in Huh-7 cells infected with lenti-ADAR2 lentiviruses.

<p>(<b>A</b>) The cells were uninfected (mock), infected with lenti-sh-luc (negative control), or infected with the lenti-ADAR1S, ADAR1L, and ADAR2 individually. The cell lysates were processed for Western blotting by probing with Abs as indicated at the bottom of each panel. The HRM results for miR-214 (<b>B</b>) and miR-122 (<b>C</b>). The raw data for the relative signals are shown in the upper panel; and the difference of the relative signal by comparing with the “no editing” standard are shown in the lower panel. The stem loop structures for pre-miR-214 (<b>D</b>) and pre-miR-122 (<b>E</b>) are illustrated schematically, with the nucleotides corresponding to mature miRs labeled with green. The positions of the nucleotides changed by overexpression of ADAR2 are marked in red, with the numbers showing their positions relative to the first nucleotide of mature miRs (as position no. 1). The summary results of the nucleotide changes in precursors of these two miRNAs are shown at the right panel, by sequencing of 100 clones from TA cloning of the RT–PCR products amplified by the RNA from lenti-ADAR2 infected cells.</p

ADAR2 edits the specific nucleotide residues at the precursor and complementary antisense RNA transcripts of pri-miR-214 and pri-miR-122.

<p>The Huh-7 cells were transfected with the plasmid constructs expressing either the sense (<b>S</b>) or the complementary antisense (<b>AS</b>) transcripts of pri-miR-214 (<b>A</b>) and pri-miR-122 (<b>B</b>), and then infected with lenti-ADARs as indicated. The RNA was extracted from each cell preparation for RT–PCR and subsequent direct sequencing analysis. The results shown here are representative, focusing on the regions covering the residues identified as the major editing sites (as revealed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081922#pone-0081922-g002" target="_blank">Figure 2</a>), including −37 and −28 for pri-miR-214 and −7 and +57 for pri-miR-122 (marked with arrows). The nucleotide residues showing editing events are marked with red asterisks.</p

ADAR2-mediated editing on the RNA transcript complementary to pri-miR-214 decreases the levels of pri-miR-214 and the miR-214.

<p>(<b>A</b>) The RNA extracted from Huh-7 cells infected with individual lenti-ADARs as indicated was used for assessment of the levels of <b>endogenous</b> miR-214 (left), pri-miR-214 (middle), and complementary antisense transcript of miR-214 (comp-AS-RNA) (right). (<b>B</b>) The individual lenti-ADARs were infected to Huh-7 cells, which were already transfected with the plasmids <b>exogenously</b> expressing either the pri-miR-214 or the complementary antisense transcript (comp-AS-RNA) as indicated. The RNA was extracted for quantification of the levels of both transcripts accordingly. (<b>C</b>) The Huh-7 cells were transfected with the plasmid expressing the wild-type comp-AS-RNA, or the ones introducing either the single mutation as A(−37)G or double mutations as A(−37/−28)G. The levels of antisense transcript were then determined by qRT–PCR. (<b>D</b>) The Huh-7 cells were not infected (mock), infected with control lenti-si-GFP (si-GFP), or infected with lenti-si-AS-214, which targets the RNA transcript complementary to pri-miR-214, for evaluating the effects on the expression levels of comp-AS-RNA (left), pri-miR-214 (middle), and miR-214 (right). (<b>E</b>) The Huh-7 cells were transfected with the control vector or the plasmid expressing the comp-AS-RNA, for evaluating the effect on the levels of comp-AS-RNA (left), pri-miR-214 (middle), and mature miR-214 (right). (<b>F</b>) The protein lysates extracted from Huh-7 cells uninfected or infected with lenti-si-Luc or lenti-pri-miR214 were analyzed by Western blotting analysis (left). The Huh-7 cells transfected with pGL3-vector or pGL3-Rab15-3′UTR reporter constructs were infected with lenti-si-GFP or lenti-pri-miR214. The effect on reporter activity was illustrated relative to that of pGL3-vector-transfected cells infected with lenti-si-GFP (middle). The protein lysates extracted from Huh-7 cells either uninfected or infected with various lentiviruses as described were processed for Western blotting, probing with antibodies against Rab15, ADAR1, ADAR2, and β-actin (right).</p

SVR₁₂ according to baseline patient characteristics and on-treatment HCV viral decline.

SVR12 according to baseline patient characteristics and on-treatment HCV viral decline.</p

Baseline patient characteristics.

Baseline patient characteristics.</p

ADAR2-Mediated Editing of miR-214 and miR-122 Precursor and Antisense RNA Transcripts in Liver Cancers

<div><p>A growing list of microRNAs (miRNAs) show aberrant expression patterns in hepatocellular carcinoma (HCC), but the regulatory mechanisms largely remain unclear. RNA editing catalyzed by members of the adenosine deaminase acting on the RNA (ADAR) family could target the miRNA precursors and affect the biogenesis process. Therefore, we investigate whether RNA editing could be one mechanism contributing to the deregulation of specific miRNAs in HCC. By overexpression of individual ADARs in hepatoma cells, RNA editing on the precursors of 16 miRNAs frequently deregulated in HCC was screened by a sensitive high-resolution melting platform. The results identified RNA precursors of miR-214 and miR-122 as potential targets edited by ADAR2. A subset of HCC showing elevated ADAR2 verified the major editings identified in ARAR2 overexpressed hepatoma cells, either with A-to-I or U-to-C changes. The unusual U-to-C editing at specific residues was demonstrated as being attributed to the A-to-I editing on the RNA transcripts complementary to the pri-miRNAs. The editing event caused a decrease of the RNA transcript complementary to pri-miR-214, which led to the decrease of pri-miR-214 and miR-214 and resulted in the increased protein level of its novel target gene Rab15. In conclusion, the current study discovered ADAR2-mediated editing of the complementary antisense transcripts as a novel mechanism for regulating the biogenesis of specific miRNAs during hepatocarcinogenesis.</p></div

Long-term events of groups according to their hepatitis activities.

<p>Data are expressed as median (25th, 75th quartile) or number (percentage) as indicated.</p>†<p>P<.001,</p>*<p>P<.05 compared with its baseline level.</p>‡<p>P for trend.</p

The ADAR2-mediated editing at specific nucleotide residues in the RNA transcripts related with pri-miR-214 and pri-miR-122 also occurred in the clinical HCC specimens.

<p>The representative sequencing results for miR-214 (<b>A</b>) and miR-122 (<b>B</b>), with the RT–PCR products from four pairs of HCC tissues as indicated. The nucleotide residues with changes in the tumorous (T) tissues, in comparison with those in the corresponding nontumorous (NT) tissues, are marked with red arrows. The positions relative to that of the mature miRNAs are indicated above the arrows. (<b>C</b>) The relative expression levels of ADAR2 mRNA in these HCC tissues were determined by qPCR analysis, with the quantitative results shown as relative to the level of the NT part of patient no. 8. (<b>D</b>) The protein expression of ADAR2 and ADAR1 in these four pairs of HCC tissues was examined by Western blotting.</p