Partial Hepatectomy Induced Long Noncoding RNA Inhibits Hepatocyte Proliferation during Liver Regeneration

<div><p>Liver regeneration after partial hepatectomy (PHx) is a complex and well-orchestrated biological process in which synchronized cell proliferation is induced in response to the loss of liver mass. To define long noncoding RNAs (lncRNAs) that participate in the regulation of liver regeneration, we performed microarray analysis and identified more than 400 lncRNAs exhibiting significantly altered expression. Of these, one lncRNA, <i>LncPHx2</i> (Long noncoding RNA induced by PHx 2), was highly upregulated during liver regeneration. Depletion of <i>LncPHx2</i> during liver regeneration using antisense oligonucleotides led to a transient increase in hepatocyte proliferation and more rapid liver regeneration. Gene expression analysis showed that <i>LncPHx2</i> depletion resulted in upregulation of mRNAs encoding proteins known to promote cell proliferation, including MCM components, DNA polymerases, histone proteins, and transcription factors. <i>LncPHx2</i> interacts with the mRNAs of MCM components, making it a candidate to regulate the expression of MCMs and other genes post-transcriptionally. Collectively, our data demonstrate that LncPHx2 is a key lncRNA that participates in a negative feedback loop modulating hepatocyte proliferation through RNA-RNA interactions.</p></div

Suppressing transthyretin production in mice, monkeys and humans using 2nd-Generation antisense oligonucleotides

<p>Transthyretin amyloidosis (ATTR amyloidosis) is a rare disease that results from the deposition of misfolded transthyretin (TTR) protein from the plasma into tissues as amyloid fibrils, leading to polyneuropathy and cardiomyopathy. IONIS-TTR_Rx (ISIS 420915) is a 2nd-Generation 2′-<i>O</i>-(2-methoxyethyl) modified “2′-MOE” antisense oligonucleotide (ASO) that targets the TTR RNA transcript and reduces the levels of the TTR transcript through an RNaseH1 mechanism of action, leading to reductions in both mutant and wild-type TTR protein. The activity of IONIS-TTR_Rx to decrease TTR protein levels was studied in transgenic mice bearing the Ile84Ser human TTR mutant, in cynomolgus monkeys and in healthy human volunteers. Robust (>80%) reductions of plasma TTR protein were obtained in all three species treated with IONIS-TTR_Rx, which in mice and monkeys was associated with substantial reductions in hepatic TTR RNA levels. These effects were dose-dependent and lasted for weeks post-dosing. In a Phase 1 healthy volunteer study, treatment with IONIS-TTR_Rx for four weeks was well tolerated without any remarkable safety issues. TTR protein reductions up to 96% in plasma were observed. These nonclinical and clinical results support the ongoing Phase 3 development of IONIS-TTR_Rx in patients with ATTR amyloidosis.</p

Gene expression profiling of mouse liver regeneration after PHx.

<p>(A) Differentially expressed mRNAs and putative lncRNAs were clustered based on their expression pattern during liver regeneration after PHx. Normalized average probe intensity was plotted over the time course of liver regeneration. Cluster 1 contains 401 mRNA and 30 lncRNA transcripts. Cluster 2 contains 471 mRNA and 91 lncRNA transcripts. Cluster 3 contains 610 mRNA and 110 lncRNA transcripts. Cluster 4 contains 385 mRNA and 46 lncRNA transcripts. Cluster 5 contains 146 mRNA and 17 lncRNA transcripts. Cluster 6 contains 410 mRNA and 73 lncRNA transcripts. Cluster 7 contains 254 mRNA and 37 lncRNA transcripts. Cluster 8 contains 330 mRNA and 17 lncRNA transcripts. Cluster 9 contains 646 mRNA and 44 lncRNA transcripts. Sham: liver RNAs of mice subjected to sham surgery. PHx: liver RNAs of mice subjected to PHx surgery. n = 5 for each time point. (B) Pathway analysis of the eight gene clusters using David KEGG pathway tools. Cluster 5 has no enriched pathway (not shown). * Pathway with FDR<0.05. (C) qPCR analysis of the levels of lncRNA transcripts. The mRNA level of the housekeeping gene <i>Gapdh</i> and total RNA amount determined by Ribogreen staining (Life Technology) were used as controls. The RNA levels in livers of mice subjected to sham surgery were set as 1. n = 5. Statistical analysis was performed using the Student's t test. * p<0.05; **p < 0.01; ***p < 0.001. (D) LncRNA expression in nine mouse tissues collected from Encode RNA-seq database. FPKM of lncRNA expression in each tissue was plotted against the mean value of each row. Red indicates higher expression. Blue indicates lower expression.</p

Characterization of <i>LncPHx2</i>.

<p>(A) <i>LncPHx2</i> gene structure and genomic location. (B) qPCR analysis of <i>LncPHx2</i> expression after PHx. The RNA levels in the livers at time 0 after sham surgery were set as 1. Data were quantified and statistically analysed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132798#pone.0132798.g001" target="_blank">Fig 1C</a>. (C) Northern blot of <i>LncPHx2</i> in mouse livers. Lane 1, RNA from mouse liver treated with PBS. Lane2, RNA from mouse liver treated with ASO specifically targeting <i>LncPHx2</i>. Lane 3, RNA from mouse liver collected at 48 hours after PHx surgery. (D) qPCR analysis of <i>LncPHx2</i> levels in nine mouse tissues. <i>LncPHx2</i> level was normalized to total RNA amount measured by Ribogreen (Life Technologies). <i>LncPHx2</i> level in mouse liver was set as 1. (E) <i>LncPHx2</i> single molecule RNA <i>in situ</i> hybridization in livers of mice subjected to sham or PHx surgery. Upper panels: Light microscopy images. <i>In situ</i> signal of <i>LncPHx2</i> is red, as indicated by arrows. Liver sections were counter-stained by H&E. Lower panels: Fluorescent microscopy images. <i>In situ</i> signal of <i>LncPHx2</i> is red, as indicated by arrows. The nucleus was stained by DAPI.</p

Identify <i>LncPHx2</i> RNA-interactome.

<p>(A) qPCR analysis of <i>LncPHx2</i> recovery in RNA samples from <i>LncPHx2</i> RNA-interactome experiment. Odd pool: pool of odd numbered probes bind to <i>LncPHx2</i> RNA. Even pool: pool of even numbered probes bind to <i>LncPHx2</i> RNA. LacZ: control probes bind to LacZ mRNA. (B) Upper panel: <i>LncPHx2</i> RNA-interacting motif identified from 415 <i>LncPHx2</i> interacting sites using MEME de novo motif search tool. Lower panel: <i>LncPHx2</i> RNA-interacting motif in <i>LncPHx2</i>, <i>Mcm2</i>, <i>Mcm3</i> and <i>Mcm7</i> transcripts. (C) <i>LncPHx2</i> RNA-interacting motif enrichment in differentially expressed genes in regenerating liver upon <i>LncPHx2</i>-depletion. Motif-search for the <i>LncPHx2</i> RNA-interacting motif was performed on 300 upregulated, 291 downregulated and 100 sets of 300 randomly sampled unchanged gene transcript sequences in <i>LncPHx2</i>-depleted regenerating liver using MAST with default parameters (e-value cutoff 100, maximum p-value for motif match = 0.0001). Student’s t-test was performed on log-transformed e-scores from the 3 groups.</p

Depletion of <i>LncPHx2</i> promotes hepatocyte proliferation in liver regeneration.

<p>(A) Experimental procedure. Mice were given two doses of PBS or LncPHx2_ASO1 at 50 mg/kg (mpk) (indicated by green arrows) before PHx (indicated by red arrow). Animals were sacrificed at eight time points after PHx (indicated by blue arrows). n = 3 for 0, 24, 36, 48, 60, 72, and 168 hour time points. n = 4 for 336 hour time point. (B) qPCR analysis of <i>LncPHx2</i> in livers of mice treated with LncPHx2_ASO1 or PBS. The RNA levels in the PBS-treated livers at time 0 after PHx were set as 1. Data were quantified and statistically analysed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132798#pone.0132798.g001" target="_blank">Fig 1C</a>. (C) Liver to body weight ratios measured at indicated time points after PHx. (D) Left panel: Representative images of Ki67 staining and BrdU labelling of livers from mice treated with LncPHx2_ASO1 or PBS at 36 hours after PHx. Right panel: Histogram and statistics of Ki67 staining and BrdU labelling of mouse livers after PHx at indicated time points after PHx. (E) qPCR analysis of cell-cycle marker gene expression in mouse livers at indicated time points after PHx. mRNA levels in the PBS-treated livers at time 0 after PHx were set as 1. Data were quantified and statistically analysed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132798#pone.0132798.g001" target="_blank">Fig 1C</a>. (F) Liver aspartate transaminase (AST) levels (top panel) and alanine transaminase (ALT) levels (bottom panel) in mouse plasma measured at indicated time points after PHx.</p