Article Figures & Data
Figures
- Fig. 1.
Hierarchical clustering analysis of miRNA expression (light gray, overexpressed genes; dark gray underexpressed genes). miRNAs were isolated from actively translated RNA population in HCT-116 (wt-p53) and HCT-116 (null-p53) cells using gel fractionation. The level of miRNAs were quantified with miRNA array analysis. The expression of miRNAs were analyzed using Feature Extraction Software. The signal of each probe was set as its median intensity. The threshold for reliable probe signals was set at 1,500. Clustering analysis was done using CLUSTER 3.0/TreeView software (23).
- Fig. 2.
A, effect of p53 siRNA knockdown on the expression of wt-p53 via Western immunoblot analysis. HCT-116 (wt-p53) cells were transfected with siRNA mismatch control and wt-p53-specific siRNA and the expression levels of wt-p53 were quantified via Western immunoblot analysis [lane 1, control; lane 2, mismatch control; lane 3, Lamin A/C positive control; lane 4, 100 nmol/L siRNA; lane 5, 400 nmol/L siRNA; lane 6, HCT-116 (null-p53)]. B, real-time qRT-PCR analysis of has-miR-26a expression [n = 3; lane 1, control; lane 2, mismatch control; lane 3, Lamin A/C positive control; lane 4, 100 nmol/L siRNA; lane 5, 400 nmol/L siRNA; lane 6, HCT-116 (null-p53)]. 5S rRNA was used as internal standard for expression normalization.
- Fig. 3.
A, hierarchical clustering analysis of steady-state total mRNAs expression profile between HCT-116 (wt-p53; lane 1) and HCT-116 (null-p53; lane 2) cells via microarray expression analysis (red, overexpressed genes; blue, underexpressed genes). B, hierarchical clustering analysis of actively translated mRNAs expression profile between HCT-116 (wt-p53; lane 1) and HCT-116 (null-p53; lane 2) cells (red, overexpressed genes; blue, underexpressed genes).
- Fig. 5.
A, effect of 5-FU treatment on the expression of p53 via Western immunoblot analysis [lane 1, control HCT-116 (wt-p53); lane 2, HCT-116 (wt-p53) cells treated with 10 μmol/L 5-FU for 24 hours; lane 3, control HCT-116 (null-p53); lane 4, HCT-116 (null-p53) cells treated with 10 μmol/L 5-FU for 24 hours]. B, expression of wt-p53 mRNA from HCT-116 (wt-p53) control cells and cells treated with 10 μmol/L 5-FU for 24 hours analyzed by real-time qRT-PCR analysis (n = 3). The expression of housekeeping gene GAPDH was used as internal control. C, effect of 5-FU treatment on hsa-miR-26a expression analyzed by real-time miRNA qRT-PCR (n = 3) and 5S rRNA was used as internal standard. The ratio of target miRNA and 5S rRNA was used to calculate the relative expression.
- Fig. 6.
Schematic illustration of p53 regulatory pathways. Wt-p53 not only acts as a typical transcription factor to regulate a number of cellular mRNAs at the transcriptional level, but also regulates gene expression at posttranscriptional and translational events via its RNA-binding function. In addition, wt-p53 regulates a number of noncoding miRNAs at the transcriptional level thereby influences certain cellular mRNAs translation through its mediated miRNAs. Wt-p53 also influences apoptosis pathway via accumulation to the mitochondria.
Tables
- Table 1.
Expression analysis of miRNAs using MIRCHIP2 array in HCT-116 (wt-p53) and HCT-116 (null-p53) cells
Up-regulated miRNA Fold-change Down-regulated miRNA Fold change Hsa-miR-30a-5p +32.56 Hsa-miR-15b −126.34 Hsa-miR-181b +11.87 Hsa-miR-27a −60.76 Hsa-miR-372 +6.57 Hsa-miR-200c −58.30 Hsa-let-7g +4.93 Hsa-miR-191 −56.39 Hsa-miR-26a +2.27 Hsa-miR-30c −50.12 Hsa-let-7b +2.26 Hsa-miR-25 −49.24 Hsa-miR-296 +2.26 Hsa-miR-107 −48.55 Hsa-miR-30a-3p +2.00 Hsa-miR-339 −46.52 Hsa-miR-21 +1.89 Hsa-miR-125a −39.97 Hsa-miR-132 +1.58 Hsa-miR-27b −32.84 Hsa-miR-181a +1.34 Hsa-miR-23a −28.88 Hsa-miR-320 +1.05 Hsa-miR-10a −3.51 - Table 2.
Prediction of putative p53-binding sites of miRNA promoters
miRNA Site position (upstream miRNA) Gap Sequence Site score Shorter gap sites Hsa-let-7b 828 0 AGCCATGTCT…CTTCTTGTCT 87.56 Hsa-mir-26a-1 3,108 2 CAGCAAGACT…GGGCAAGAGC 86.96 Hsa-mir-26a-2 3,024 0 GCCCTTGCCC…CTGCTTGTCT 86.30 Hsa-mir-372 3,661 3 CGCCATGTTG…AGGCTAGTCT 84.81 Hsa-let-7b 3,628 1 TCGCATGCCT…TGTCTTGCTG 83.73 Hsa-mir-181b-1 1,420 0 AGCCAAGCTT…TGGCATGACT 82.44 Hsa-mir-200c 2,183 2 AGACAAGGAG…GAGCAAGGGT 81.59 Hsa-mir-26a-2 174 3 CAGCATGTTG…AGTCAAGTTC 80.06 Longer gap sites Hsa-mir-200c 3,807 11 ATACAAGCCG…AGGCAAGTCC 89.80 Hsa-mir-181b-1 1,470 7 AAACATGTCC…CAACTTGCCT 89.00 Hsa-mir-181b-1 4,594 6 GAACTAGCCC…GGCCATGTTT 88.70 Hsa-mir-26a-2 4,840 13 AAGCAAGCAC…GAGCAAGACT 87.77 Hsa-let-7g 1,620 11 AGGCTTGCCT…CAGCAAGCGC 86.69 Hsa-mir-26a-2 4,066 12 CAGCTTGCTT…TGCCATGCCC 85.20 Hsa-mir-26a-2 4,070 8 TTGCTTGCCC…TGCCATGCCC 84.06 Hsa-mir-26a-1 4,496 6 ACGCAAGTCC…TCCCATGTCC 83.60 Hsa-mir-26a-1 4,496 12 ACGCAAGTCC…GTCCTTGCTT 83.29 Hsa-mir-26a-2 4,846 7 GCACAAGATC…GAGCAAGACT 83.12 Hsa-mir-27a 2,060 11 CCTCATGCCT…GAGCTTGGTT 81.88 Hsa-let-7b 2,624 10 GGGCATGGGG…TAGCATGCCG 80.81 Hsa-mir-26a-2 4,863 12 GAGCAAGACT…TGTCTAGTCT 80.66 Hsa-mir-26a-1 1,437 9 TGCCTTGTTC…GGGCATGCAG 80.52 Hsa-mir-26a-1 4,512 4 TCCCATGTCC…TTTCTTGCTT 80.33 Hsa-mir-181b-2 1,330 6 AAACATGAAT…TGACATGCTG 80.28 - Table 3.
Expression analysis of steady-state total mRNA transcripts in HCT-116 (wt-p53) and HCT-116 (null-p53) cells
Genbank accession no. Gene ID Fold change Biological function Increased genes NM_000474 TWIST +34.30 Cell differentiation; chromosome organization and biogenesis NM_001225 CASP4 +20.22 Apoptosis; proteolysis and peptidolysis NM_012427 KLK5 +11.13 Epidermis development; proteolysis and peptidolysis NM_004172 SLC1A3 +11.07 l-Glutamate transport; dicarboxylic acid transport NM_005930 MGEA6 +10.92 RNA processing D16350 SAH +10.07 Metabolism; regulation of blood pressure NM_004864 PLAB +9.75 Cell-cell signaling; signal transduction NM_025048 FLJ22684 +9.46 Neuropeptide signaling pathway AB029015 PLCL2 +9.11 Intracellular signaling cascade; lipid metabolism NM_016135 TEL2 +9.00 Organogenesis; regulation of transcription NM_006017 PROML1 +8.88 Visual perception NM_002923 RGS2 +8.54 Cell cycle; G-protein signaling pathway; signal transduction M23419 EIF5A;TNNI3 +7.09 Protein biosynthesis; translational initiation NM_003633 ENC1 +6.99 Development; neurogenesis AK023349 Nup43 +4.97 Intracellular protein transport NM_005596 NFIB +4.89 DNA replication; regulation of transcription, DNA dependent NM_012198 GCA +3.96 Membrane fusion NM_001654 ARAF1 +3.92 Cell growth and/or maintenance; intracellular signaling cascade NM_001387 DPYSL3 +3.88 Neurogenesis; signal transduction NM_000043 TNFRSF6 +3.80 Apoptosis; immune response; protein assembly; signal transduction NM_000389 CDKN1A +3.55 Cell cycle; apoptosis; cell proliferation; regulation of CDK activity BC007613 CRMP1 +3.14 Neurogenesis; nucleobase, nucleoside, nucleotide and nucleic acid metabolism AK057343 ZNF131 +3.11 Regulation of transcription, DNA-dependent NM_052966 C1orf24 +3.08 Protein folding Decreased genes NM_014178 HSPC156 −21.83 Vesicle-mediated transport NM_000582 SPP1 −19.53 T-helper 1 type immune response; antiapoptosis; cell-cell signaling NM_001147 ANGPT2 −13.04 Angiogenesis; cell growth and/or maintenance; signal transduction NM_003121 SPIB −11.76 Regulation of transcription from Pol II promoter BC010398 PMPCB −7.41 Proteolysis and peptidolysis NM_000310 PPT1 −5.26 Neurogenesis; protein modification; visual perception NM_000465 BARD1 −4.59 Protein ubiquitination NM_016611 KCNK4 −4.29 Ion transport; potassium ion transport NM_001901 CTGF −3.50 DNA metabolism; cell adhesion; cell growth; cell motility NM_003925 MBD4 −3.34 Base-excision repair NM_005627 SGK −3.09 Apoptosis; phosphorylation; response to stress; sodium ion transport NM_005834 TIMM17B −3.06 Protein-mitochondrial targeting NM_013961 NRG1 −3.06 Cell differentiation; embryonic development; neurogenesis - Table 4.
Expression analysis of actively translated mRNA transcripts in HCT-116 (wt-p53) and HCT-116 (null-p53) cells
Genbank accession no. Gene ID Fold change Biological function Increased genes NM_016292 TRAP1 +19.01 Protein folding NM_014474 ASML3B +13.51 Carbohydrate metabolism NM_001654 ARAF1 +12.79 Cell growth and/or maintenance; intracellular signaling cascade NM_000178 GSS +11.20 Amino acid metabolism; glutathione biosynthesis; neurogenesis NM_001225 CASP4 +8.83 Apoptosis; proteolysis and peptidolysis NM_001402 EEF1A1 +8.44 Protein biosynthesis; regulation of translation NM_000918 P4HB +7.86 Electron transport NM_004335 BST2 +7.28 Cell proliferation; cell-cell signaling; development; immune response NM_006743 RBM3 +6.85 RNA processing NM_004046 ATP5A1 +6.83 ATP synthesis–coupled proton transport NM_000546 TP53 +6.58 Apoptosis; DNA recombination; DNA repair; cell cycle; transcription NM_004317 ASNA1 +6.54 Anion transport; response to arsenate NM_004127 GPS1 +6.19 c-Jun-NH2-kinase cascade; cell cycle; inactivation of mitogen-activated protein kinase NM_000291 PGK1 +6.13 Glycolysis NM_003752 EIF3S8 +6.12 Protein biosynthesis; regulation of translational initiation NM_005500 SAE1 +5.85 Protein ubiquitination NM_000107 DDB2 +5.70 Nucleotide-excision repair AK024835 CNN2 +5.68 Cytoskeleton organization; smooth muscle contraction NM_003915 CPNE1 +5.62 Lipid metabolism; vesicle-mediated transport NM_006374 STK25 +5.53 Phosphorylation; response to oxidative stress; signal transduction NM_017916 FLJ20643 +5.44 Metabolism NM_006400 DCTN2 +5.35 Cell proliferation; microtubule-based process; mitosis NM_000474 TWIST +5.19 Cell differentiation; chromosome organization and biogenesis NM_005030 PLK +5.09 Mitosis; protein amino acid phosphorylation; regulation of cell cycle BC033103 INPP5E +5.04 Proteolysis and peptidolysis NM_032272 MAF1 +4.91 Regulation of transcription, DNA-dependent NM_001970 EIF5A +4.85 Protein biosynthesis; translational initiation NM_006201 PCTK1 +4.73 Protein amino acid phosphorylation; regulation of cell cycle NM_005654 NR2F1 +4.66 Regulation of transcription, DNA-dependent; signal transduction NM_003633 ENC1 +4.62 Development; neurogenesis NM_016016 CGI-69 +4.60 Transport NM_000967 RPL3 +4.58 Protein biosynthesis NM_016645 NEUGRIN +4.56 Neuron differentiation NM_018658 KCNJ16 +4.38 Ion transport; potassium ion transport NM_004864 PLAB +4.33 Cell-cell signaling; signal transduction NM_003624 RANBP3 +4.29 Small GTPase-mediated signal transduction NM_004559 NSEP1 +4.28 Regulation of transcription; response to pest/pathogen/parasite NM_012427 KLK5 +4.14 Epidermis development; proteolysis and peptidolysis NM_001085 SERPINA3 +4.12 Inflammatory response; regulation of lipid metabolism NM_021734 SLC25A19 +4.12 Deoxynucleotide transport NM_000175 GPI +4.10 Carbohydrate metabolism; gluconeogenesis; glycolysis; hemostasis NM_001908 CTSB +4.10 Proteolysis and peptidolysis NM_002923 RGS2 +4.01 Cell cycle; G-protein signaling pathway; signal transduction NM_000687 AHCY +3.95 One-carbon compound metabolism NM_000190 HMBS +3.94 Heme biosynthesis NM_013388 PREB +3.93 Regulation of transcription, DNA dependent NM_005410 SEPP1 +3.92 Response to oxidative stress NM_015679 CLONE24922 +3.87 RNA processing NM_021008 DEAF1 +3.82 Regulation of transcription, DNA dependent AK000822 DKFZP564M182 +3.75 Protein biosynthesis NM_000182 HADHA +3.74 Fatty acid metabolism NM_002611 PDK2 +3.70 Glucose metabolism; signal transduction NM_005802 TP53BPL +3.68 Protein ubiquitination AF016266 TNFRSF10B +3.59 Caspase activation; electron transport; induction of apoptosis NM_001536 HRMT1L2 +3.58 Signal transduction; defense response; methylation NM_133455 LOC129080 +3.52 Phosphate transport NM_001640 APEH +3.51 Proteolysis and peptidolysis NM_001416 EIF4A1 +3.50 Protein biosynthesis NM_006388 HTATIP +3.33 Regulation of transcription, DNA-dependent; transcription NM_006112 PPIE +3.29 Protein folding NM_000308 PPGB +3.28 Intracellular protein transport; proteolysis and peptidolysis NM_002046 GAPD +3.24 Glucose metabolism; glycolysis NM_001130 AES +3.24 Wnt receptor signaling pathway; development; transcription Decreased genes NM_014178 HSPC156 −27.25 Vesicle-mediated transport NM_006533 MIA −15.34 Cell proliferation NM_006993 NPM3 −7.58 Protein folding NM_005554 KRT6A −6.62 Ectoderm development NM_002274 KRT13 −5.75 Epidermis development NM_003125 SPRR1B −4.81 Epidermis development NM_000117 EMD −4.76 Muscle contraction; muscle development NM_006072 SCYA26 −4.69 Cell-cell signaling; immune response; signal transduction BC014000 LOC115509 −4.18 Regulation of transcription, DNA dependent NM_024710 FLJ23469 −3.88 Metabolism NM_002906 RDX −3.47 Cytoskeletal anchoring
Volume 12, Issue 7
