Fig 1.
Gene expression profiling of mouse liver regeneration after PHx.
(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 Gapdh 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.
Fig 2.
(A) LncPHx2 gene structure and genomic location. (B) qPCR analysis of LncPHx2 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 Fig 1C. (C) Northern blot of LncPHx2 in mouse livers. Lane 1, RNA from mouse liver treated with PBS. Lane2, RNA from mouse liver treated with ASO specifically targeting LncPHx2. Lane 3, RNA from mouse liver collected at 48 hours after PHx surgery. (D) qPCR analysis of LncPHx2 levels in nine mouse tissues. LncPHx2 level was normalized to total RNA amount measured by Ribogreen (Life Technologies). LncPHx2 level in mouse liver was set as 1. (E) LncPHx2 single molecule RNA in situ hybridization in livers of mice subjected to sham or PHx surgery. Upper panels: Light microscopy images. In situ signal of LncPHx2 is red, as indicated by arrows. Liver sections were counter-stained by H&E. Lower panels: Fluorescent microscopy images. In situ signal of LncPHx2 is red, as indicated by arrows. The nucleus was stained by DAPI.
Fig 3.
Depletion of LncPHx2 promotes hepatocyte proliferation in liver regeneration.
(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 LncPHx2 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 Fig 1C. (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 Fig 1C. (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.
Fig 4.
Genome-wide gene expression profiling of LncPHx2 regulated genes.
RNA-seq analysis of liver RNAs from mice treated with PBS or LncPHx2_ASO1 and subjected to Sham or PHx surgery. n = 3 for each group. (A) Pie charts of differentially expressed genes at 48 hours after PHx surgery compared to sham surgery in PBS-treated mice (left panel) and in LncPHx2_ASO1-treated mice (right panel). (B) Venn diagram comparing differentially expressed genes at 48 hours after PHx in PBS- and LncPHx2_ASO1-treated mice. Left panel: Upregulated genes. Right panel: Downregulated genes. (C) Pie charts of differentially expressed genes in LncPHx2_ASO1-treated mice compared to PBS-treated mice at 48 hours after sham (left panel) and PHx surgery (right panel). (D) Venn diagrams of differentially expressed genes in LncPHx2_ASO1-treated mouse livers under either sham or PHx conditions compared to PBS-treated mouse livers under the same conditions. Left panel: Upregulated genes. Right panel: Downregulated genes. (E) KEGG pathway analysis of genes upregulated in regenerating livers from LncPHx2_ASO1-treated mice compared to PBS-treated mice at 48 hours after PHx. * Pathway with FDR<0.05. (F) RNA-seq results of representative genes in each treatment condition indicated. mRNA levels in PBS-treated mice with sham surgery were set as 1. Ratios and statistics were done using cuffdiff. * p<0.01; **p < 0.001; ***p < 0.0001
Fig 5.
Identify LncPHx2 RNA-interactome.
(A) qPCR analysis of LncPHx2 recovery in RNA samples from LncPHx2 RNA-interactome experiment. Odd pool: pool of odd numbered probes bind to LncPHx2 RNA. Even pool: pool of even numbered probes bind to LncPHx2 RNA. LacZ: control probes bind to LacZ mRNA. (B) Upper panel: LncPHx2 RNA-interacting motif identified from 415 LncPHx2 interacting sites using MEME de novo motif search tool. Lower panel: LncPHx2 RNA-interacting motif in LncPHx2, Mcm2, Mcm3 and Mcm7 transcripts. (C) LncPHx2 RNA-interacting motif enrichment in differentially expressed genes in regenerating liver upon LncPHx2-depletion. Motif-search for the LncPHx2 RNA-interacting motif was performed on 300 upregulated, 291 downregulated and 100 sets of 300 randomly sampled unchanged gene transcript sequences in LncPHx2-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.