. 2017 Nov 1;8(61):102783-102800.
eCollection 2017 Nov 28.
Loss of p53-inducible Long Non-Coding RNA
LINC01021 Increases Chemosensitivity
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Loss of p53-inducible Long Non-Coding RNA
LINC01021 Increases Chemosensitivity
Free PMC article
We have previously identified the long non-coding RNA
LINC01021 as a direct p53 target (Hünten et al. Mol Cell Proteomics. 2015; 14:2609-2629). Here, we show that LINC01021 is up-regulated in colorectal cancer (CRC) cell lines upon various p53-activating treatments. The LINC01021 promoter and the p53 binding site lie within a MER61C LTR, which originated from insertion of endogenous retrovirus 1 (ERV1) sequences. Deletion of this MER61C element by a CRISPR/Cas9 approach, as well as siRNA-mediated knockdown of LINC01021 RNA significantly enhanced the sensitivity of the CRC cell line HCT116 towards the chemotherapeutic drugs doxorubicin and 5-FU, suggesting that LINC01021 is an integral part of the p53-mediated response to DNA damage. Inactivation of LINC01021 and also its ectopic expression did not affect p53 protein expression and transcriptional activity, implying that LINC01021 does not feedback to p53. Furthermore, in CRC patient samples LINC01021 expression positively correlated with a wild-type p53-associated gene expression signature. LINC01021 expression was increased in primary colorectal tumors and displayed a bimodal distribution that was particularly pronounced in the mesenchymal CMS4 consensus molecular subtype of CRCs. CMS4 tumors with low LINC01021 expression were associated with poor patient survival. Our results suggest that the genomic redistribution of ERV1-derived p53 response elements and generation of novel p53-inducible lncRNA-encoding genes was selected for during primate evolution as integral part of the cellular response to various forms of genotoxic stress.
LINC01021; chemosensitivity; colorectal cancer; p53; tumor suppression.
Conflict of interest statement
CONFLICTS OF INTEREST The authors declare no conflicts of interest.
Figure 1. Identification of ERV1 LTR elements within promoters of p53-inducible lncRNAs
(A) Heatmap visualization depicting p53 binding events ranked by read number in a 4 kbp window up- and down-stream of high-confidence p53 ChIP-Seq peaks identified with MACS2 peak calling after ectopic expression of p53 or vector control in SW480 cells . P53-bound regions were filtered for regions intersecting with genomic locations of the indicated ERV1-derived LTR sequences. Genomic coordinates for 1839 LTR sequences were obtained from the UCSC genome browser. The resulting heatmap visualization depicts p53 binding ranked by read number in a 4 kbp window up- and downstream of the LTR center. (B) Genomic DNA sequences associated with p53-bound LTRs (n=231) were subjected to MEME analysis. The resulting p53 DNA binding motif and statistical significance (E-value) are indicated. (C) Scatter plot representation of RNA-Seq data obtained and adapted from our NGS analysis previously published in Hünten et al.  showing differential lncRNA expression after induction of p53 in SW480 cells. Red lines denote 1.5-fold expression change cut-offs. LncRNAs with ≥ 1.5-fold upregulation and RPKM > 0.5 in at least one condition are shown in green, LncRNAs with ≥ 1.5-fold downregulation and RPKM > 0.5 in at least one condition shown in red. Orange dots indicate lncRNAs with p53 binding within +/- 20 kbp of the TSS. LncRNAs with ERV1-associated TSSs are highlighted in red, additional lncRNAs without ERV1-associated TSSs in black. RP3-326I13.1/PINCR, RP3-510D11.2/Lnc34a, TP53TG1, and NEAT1/ AP000944.1 have been functionally characterized recently [19, 48, 56, 57].
Figure 2. Analysis of p53-binding and retroviral elements within the
LINC01021 promoter region
(A) RNA-Seq and ChIP-Seq results were obtained after induction of ectopic p53 in SW480 cells and adapted from our previous NGS analysis published in Hünten et al. . Genomic coordinates of repetitive DNA elements were obtained by analyzing the DNA sequence of the indicated genomic interval using the Dfam database . The MER61C element harboring the p53 binding site associated with the LINC01021 promoter is indicated in red. Additional MER61 DNA sequences are shown in black, other repetitive elements in grey. Lower part: Schematic representation of the LINC01021 genomic locus and its annotated transcript variants. The length of spliced transcripts is indicated. (B) Alignment of the LINC01021 promoter sequence analyzed in luciferase reporter assays and the MER61C profile hidden Markov model (HMM) obtained from the Dfam database. The p53 binding site is given in red. Pairwise sequence alignment was performed with Promoterwise  and edited with the BioEdit software. Only the proximal promoter sequence with sequence homology to MER61C is shown. The nucleotide positions within the reporter fragment used for luciferase reporter assays are indicated. (C) A 412 bp fragment of the LINC01021 promoter harboring the p53 binding site or a mutated version was cloned upstream of the luciferase coding sequence. The proximal promoter sequence with sequence homology to MER61C is high-lighted in blue. Base exchanges in the p53 binding site of the mutated promoter fragment are highlighted in red. RKO p53-/- cells were transfected with the indicated reporter constructs and subjected to dual reporter assays 48 hours post transfection. Renilla activity was used for normalization. Results are represented as mean +/- s.d. ( n=3).
Figure 3. Comparative analysis of lncRNA regulation by p53 in CRC cell lines
(A) Expression of selected lncRNAs was determined by qPCR analyses after induction of ectopic p53 by addition of doxyxcycline (DOX) in SW480 cells for 24 hours and normalized to the expression in untreated cells and to GAPDH. (B-E) Expression of selected lncRNAs was determined by qPCR analyses after treatment of HCT116 p53+/+ and p53-/- cell lines with 5-FU, doxorubicin (DOXO), etoposide, Nutlin-3 or, as a control, DMSO for 24 hours, and normalized to the expression in untreated cells and to GAPDH. (F)
LINC01021 expression was analyzed by qPCR in isogenic p53+/+ and p53-/- HCT116, RKO and SW48 cell lines and normalized to the expression in p53+/+ cells and to GAPDH. (A-F) Results are represented as mean +/- s.d. ( n=3).
Figure 4. Characterization of
LINC01021 by CRISPR/Cas9-mediated deletion of the promoter elements, isoform-specific qPCR and subcellular fractionation
(A) Position of guide RNAs (g1 and g2) used to delete portions of the LINC01021 promoter. Genomic DNA from single cell HCT116 clones was analyzed by PCR with the indicated primers (red). (B) Expression of LINC01021 was determined by qPCR after treatment with Nutlin-3 for 24 hours. CDKN1A served as a positive control for p53-mediated target gene induction. (C) Expression of LINC01021 isoforms was verified by qPCR with isoform-specific primers in parental HCT116 cells and LINC01021 KO clones. Expression was normalized to untreated, parental HCT116 cells and GAPDH. (A-C) Results are represented as mean +/- s.d. ( n=3). (D) The subcellular localization of LINC01021 splice variants was determined in HCT116 cells by qPCR analyses after fractionation of cytoplasmic and nuclear RNA as described . NEAT1_1 and pri-miR-34a were used as positive controls for nuclear RNAs, GAPDH was used as a positive control for cytoplasmic RNA localization.
Figure 5. Loss of
LINC01021 sensitizes HCT116 cells to 5-FU
(A) Parental HCT116, HCT116 p53-/- cells and LINC0121 wt or KO clones were treated with DMSO or 5-FU at a final concentration of 10 μg/ml for 24 hours. After staining of DNA with propidium iodide samples were subjected to FACS analysis. (B) HCT116 cells were transfected with 40 nM siRNA pools consisting of 30 different siRNAs that target all LINC01021 splice variants (siPOOLs) or control siPOOLs. 24 hours after transfection, 5-FU or DMSO was added. After 24 hours apoptosis was determined by FACS detection of Annexin-V positive cells. Left: representative dot plots. Right: quantification of biological replicates. Results are represented as mean +/- s.d. (n=3). (C) HCT116 cells were seeded at 1x10 4 cells per E-plate well and transfected with 40 nM final siPOOL concentration. 24 hours after transfection, 5-FU or DMSO was added at the indicated final concentration. Cellular impedance was measured for 48 hours. For cell counting, cells from the same transfection were seeded at 1x10 4 cells per 96-well and viable cells were enumerated 48 hours after addition of 5-FU at the indicated concentrations or DMSO using trypan blue exclusion. Cell numbers are displayed as mean +/- s.d. ( n=3).
LINC01021 inactivation sensitizes to doxorubicin
(A) Apoptosis was determined by FACS analysis of Annexin-V/PI stained cells after treatment of LINC01021 wt and KO clones with DMSO or doxorubicin (DOXO) for 40 hours. Left: representative FACS plots. Right: Quantification. (B) Colony formation assays of LINC01021 wt and KO clones. Cells were seeded, cultivated for 24 hours and treated with DMSO or DOXO for the indicated periods. After removal of DOXO cells were incubated for 2 additional days in fresh medium before fixation and crystal violet staining. (C) Cellular impedance was determined after seeding 1x10 4 cells per E-plate well and treatment with DMSO or DOXO after 25 hours. Values represent mean +/- s.d. ( n=3).
Figure 7. Modulating
LINC01021 expression does not affect p53 activation
(A) Western blot analysis of p53 and p21 expression after treatment of LINC01021 wt and KO HCT116 cell lines with DMSO or 5-FU for 12 hours. (B) Western blot analysis of p53 and p21 protein expression after treatment of LINC01021 wt and KO HCT116 cell lines with DMSO or doxorubicin (DOXO) for 12 (left) or 24 hours (right). (C) Western Blot analysis of p53 and p21 expression after treatment of SW48 p53+/+ expressing different LINC01021 isoforms with DMSO or 5-FU for the indicated time periods. Ectopic LINC01021 expression was induced by addition of doxycycline (DOX) for 32 hours (pre-treatment) before addition of 5-FU.
Figure 8. Assocation of
LINC01021 expression with wild-type p53 associated expression signatures and clinical outcome of CRC subtypes
(A) Genes were preranked by expression correlation coefficient (Pearson r) with LINC01021 in descending order from left (positive correlation) to right (negative correlation) based on publically available TCGA datasets from human colorectal tumors  and analyzed by GSEA. LINC01021 expression values were obtained from . Pos. corr.: positive correlation, neg. corr.: negative correlation. NES: normalized enrichment score. (B) Heatmap depicting the hierarchically clustered correlation matrix of pairwise expression correlation coefficients (Pearson r) between previously described direct p53 protein-coding target genes  and LINC01021 based on TCGA COAD RNA expression data from 134 patient samples. (C) Averaged expression of ten p53 target genes selected in (B) based on normalized expression z-scores was calculated for each patient sample and associated with p53 mutational status. (D)
LINC01021 RNA expression levels within normal mucosa and primary tumors samples from CRC patients. Horizontal bars indicate mean +/- s.e.m. (E) Box plots showing LINC01021 RNA expression levels in patient samples associated with the different CRC consensus molecular subtypes (CMS) . Association of TCGA patient samples with CMS categories was obtained from the Colorectal Cancer Subtyping Consortium (CRCSC) at www.synapse.org. Horizontal bars indicate mean +/- s.e.m. (F) Kaplan-Meier plots showing overall survival of patients with primary CRCs classified as CMS1-3, or CMS4 with either high LINC01021 or low LINC01021 expression levels. P-values were calculated by log-rank (Mantel-Cox) test.
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