Journal: Scientific Reports

Article Title: An alternative microRNA-mediated post-transcriptional regulation of GADD45A by p53 in human non-small-cell lung cancer cells

doi: 10.1038/s41598-017-07332-3

Figure Lengend Snippet: AGO2 (EIF2C2) is a miR-138 target in human NSCLC cells. miR-138 targets identified using a microarray analysis and bioinformatics in H1299 cells ( a , b ). a Only the top thirteen highest-ranking downregulated genes are listed (for more details, see GEO: GSE69482). ( c ) Schematic representation of miR-138 targets in the 3′ UTR of human AGO2 (top). The positions of miR-138 binding sites correspond to the locations in GenBank accession NM_001164623. The artificial mutant 3′ UTR without the miR-138 binding sites is shown in the bottom image. ( d ) The full length 3′ UTR of AGO2 , containing two miR-138 binding sites, was cloned downstream of a firefly luciferase gene, and the plasmid was named pGL3-AGO2-Full. AGO2 3′ UTR-containing reporter plasmids with either or both miR-138 mutant binding sites were named pGL3-AGO2-Mut1, pGL3-AGO2-Mut2 and pGL3-AGO2-Mut-all, respectively. Dual-luciferase reporter assays were performed to test the interaction between miR-138 and the predicted wild-type AGO2 3′ UTR targeting sequence (pGL3-AGO2-Full) and the mutated targeting sequences (pGL3-AGO2-Mut1, pGL3-AGO2-Mut2, and pGL3-AGO2-Mut-all). A CMV-driven Renilla luciferase construct was co-transfected as a normalization control for firefly luciferase activity. The columns represent the mean normalized relative luciferase activity (RLU) from three independent experiments, with 95% confidence intervals. * P < 0.05 vs . NC by rank-sum test. ( e ) AGO2 mRNA and miR-138 were quantified using real-time PCR (top), and AGO2 protein expression was detected by western blotting in H460 and H1299 cells (bottom). * P < 0.05 and ** P < 0.01 vs. NC by rank-sum test. Full-length blots are in Supplementary information. ( f ) AGO2 mRNA was quantified using real-time PCR (top), and the expression of AGO2 protein was analyzed by western blotting (bottom) in H1299 cells transfected with miR-138, anti-miR-138, AGO2 siRNA, and negative control. * P < 0.05 vs . NC by LSD test. Full-length blots are in Supplementary information. ( g ) AGO2 mRNA was quantified using real-time PCR (top), the AGO2 protein was analyzed by western blotting and the immunoblots were quantified (bottom) in H1299 cells transfected with p53 expression plasmid (pRC/p53) or pRC/CMV control plasmid. * P < 0.05 by LSD test. Full-length blots are in Supplementary information. ( h ) AGO2 mRNA was quantified using real-time PCR (top), the AGO2 protein was analyzed by western blotting, and the immunoblots were quantified (bottom) in H460 cells transfected with p53 siRNA or negative control siRNA. * P < 0.05 by LSD test. Full-length blots are in Supplementary information.

Article Snippet: For in vitro pre-miRNA processing assays, commercial recombinant Dicer enzyme (Takara) and AGO2 protein (OriGene Technologies) were used.

Techniques: Microarray, Binding Assay, Mutagenesis, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Construct, Transfection, Control, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Negative Control

Journal: Scientific Reports

Article Title: An alternative microRNA-mediated post-transcriptional regulation of GADD45A by p53 in human non-small-cell lung cancer cells

doi: 10.1038/s41598-017-07332-3

Figure Lengend Snippet: AGO2 affects miR-130b abundance in human NSCLC cells. The miRNAs in H1299 cells that were significantly changed in both the miR-138 and AGO2 siRNA groups vs . the negative control (top) ( a , b ). The filtered miRNA array data were subjected to unsupervised hierarchical clustering analysis. Upregulated and downregulated miRNAs are represented by red and green colours, respectively, with the common miRNAs marked with red and black arrows, respectively (bottom). ( c ) The effects of AGO2 on the biogenesis of miR-130b. Quantitative RT-PCR assays were performed to examine the effects of AGO2 overexpression, the miR-138 mimic or AGO2 siRNA transfection on miR-130b processing. GAPDH or U6 served as an internal control. * P < 0.05 vs . NC by one-way ANOVA test. ( d ) Processing of pre-miR-130b by recombinant Dicer and AGO2 proteins in vitro . Briefly, 20 pmol of pre-miR-130b was incubated with 2 U of Dicer or 200 ng of Ago2 for 4 h. The processed products were detected using northern blotting with a biotin-labelled anti-miR-130b probe. Lane 1: 22 nt biotin-ssRNA; 2: pre-miR-130b; 3: pre-miR-130b with Dicer; 4: pre-miR-130b with AGO2. Full-length blots are in Supplementary information. ( e ) The sequence and the predicted secondary structure of Ac-pre-miR-130b. The most abundant sequence and secondary structure (http://rna.urmc.rochester.edu/RNAstructureWeb/Servers) are illustrated. ( f ) Dicer cleavage of miR-130b precursors in vitro . Briefly, 20 pmol of miR-130b precursors were incubated with 2 U of Dicer in vitro for 4 h and then a northern blot analysis was performed as in c . Lane 1: 22 nt biotin-ssRNA; 2: pre-miR-130b; 3: Ac-pre-miR-130b. Full-length blots are in Supplementary information. ( g ) AGO2 catalysis promotes the maturation of miR-130b. Pre-miR-130b was incubated with Dicer (2 U) and AGO2 in vitro . Lane 1: 22 nt biotin-ssRNA; 2: pre-miRNA; 3: pre-miRNA with Dicer; 4: pre-miRNA with Dicer and 200 ng AGO2; 5: pre-miRNA with Dicer and 500 ng AGO2. Full-length blots are in Supplementary information. ( h ) The effects of AGO2 on the stabilities of mature miR-130b in H1299 cells. H1299 cells were transfected with AGO2 siRNA or miR-138 mimic for 24 h. Cells were co-transfected with an AGO2 expression plasmid (pCMV-AGO2) for complementation analysis. Cells were then treated with actinomycin D for another 24 h. miRNA expression was determined with qRT-PCR. U6 served as an internal control. miR-130b precursors were prepared by in vitro transcription. * P < 0.05 and **P < 0.01 by LSD test. The data are representative of three independent experiments (mean ± s.d.).

Article Snippet: For in vitro pre-miRNA processing assays, commercial recombinant Dicer enzyme (Takara) and AGO2 protein (OriGene Technologies) were used.

Techniques: Negative Control, Quantitative RT-PCR, Over Expression, Transfection, Control, Recombinant, In Vitro, Incubation, Northern Blot, Sequencing, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: An alternative microRNA-mediated post-transcriptional regulation of GADD45A by p53 in human non-small-cell lung cancer cells

doi: 10.1038/s41598-017-07332-3

Figure Lengend Snippet: The regulatory effect of p53 on miR-130b. ( a ) Sequences and positions of the predicted p53-binding sites in the promoter of the miR-130b gene. The predicted p53-binding sites were incorporated separately into the basic promoter of a firefly luciferase reporter gene (named pGL4-(-4223) and pGL4-(-3540)). ( b – d ) pRC/p53 plasmids were co-transfected with the luciferase reporter plasmid pGL4-(-4223), pGL4-(-3540), or empty pGL4 vector into HeLa ( b ), H1299 ( c ) and H460 ( d ) cells. After 48 h, the luciferase activity was measured. * P < 0.05 vs . the control by one-way ANOVA test. ( e ) Quantitative RT-PCR assays were performed to examine the relative miR-130b expression levels in H1299 and H460 cells. * P < 0.05 by rank-sum test. ( f ) Quantitative RT-PCR assays were performed to examine the relative miR-130b expression levels in H460 cells transfected with anti-miR-138, p53 siRNA, or pCMV-AGO2. * P < 0.05 vs . NC by one-way ANOVA test. ( g ) Quantitative RT-PCR assays were performed to examine the relative miR-130b expression levels in H1299 cells transfected with pRC/p53, miR-138, or AGO2 siRNA. The qRT–PCR expression values were normalized to that of U6. *P < 0.05 and **P < 0.01 vs . the control by one-way ANOVA test. The data are representative of three independent experiments (mean ± s.d.).

Article Snippet: For in vitro pre-miRNA processing assays, commercial recombinant Dicer enzyme (Takara) and AGO2 protein (OriGene Technologies) were used.

Techniques: Binding Assay, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Control, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: An alternative microRNA-mediated post-transcriptional regulation of GADD45A by p53 in human non-small-cell lung cancer cells

doi: 10.1038/s41598-017-07332-3

Figure Lengend Snippet: p53-miR-138-AGO2-miR-130b pathway regulation of GADD45A in human NSCLC cells. Human p53 signalling pathway PCR array analysis. p53 signalling pathway gene expression profiles in H1299 cells treated with AGO2 siRNA, miR-138, or negative control were analyzed using a q-PCR array ( a , b ). a Only the top ten most significantly changed genes are listed (for more details, see GEO: GSE69561). ( c ) The relative expression levels of GADD45A mRNA, AGO2 mRNA, miR-130b and miR-138 were quantified in H460 and H1299 cells using real-time PCR. GAPDH mRNA and U6 snRNA served as internal controls, separately. * P < 0.05 by rank-sum test. ( d ) GADD45A protein was analyzed with western blotting in H1299 cells transfected with miR-138, AGO2 siRNA, negative control or p53 expression plasmid. Full-length blots are in Supplementary information. ( e ) H460 cells were transfected with p53 siRNA, anti-miR-138, miR-130b, pCMV-AGO2, or control plasmid. GADD45A protein was analyzed with western blotting when cells were treated with or without 2 Gy of IR, and then cultured for 8 hours. Full-length blots are in Supplementary information. ( f ) Schematic representation of the miR-130b target in the 3′ UTR of human GADD45A (top). The positions of the miR-138 binding sites correspond to the location of the GenBank accession number NM_001199742. The mutant 3′ UTR without the miR-130b binding sites is shown in the bottom image. ( g ) Dual-luciferase reporter assays were performed to test the interaction between miR-130b and the predicted wild-type GADD45A 3′ UTR targeting sequences (pGL3-GADD45A-wt) and mutated targeting sequences (pGL3-GADD45A-mut). * P < 0.05 by rank-sum test. ( h ) H460 cells transfected with p53 siRNA, anti-miR-138, miR-130b, pCMV-AGO2, or control plasmid were treated with 2 Gy of IR. After 24 h, the cells were stained with Hoechst 33342, and the percentage of cells in G2/M, S, and G0/G1 phase was quantified using an IN Cell Analyzer 2000. ( i ) The relative cell growth of H1299 cells. * P < 0.05 by LSD test. The data are representative of at least three independent experiments (means ± s.d.). ( j ) Schematic model indicating a proposed branch pathway in the p53 regulation of GADD45A by which p53 activates miR-138 to downregulate AGO2 and miR-130b.

Article Snippet: For in vitro pre-miRNA processing assays, commercial recombinant Dicer enzyme (Takara) and AGO2 protein (OriGene Technologies) were used.

Techniques: Gene Expression, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Control, Cell Culture, Binding Assay, Mutagenesis, Luciferase, Staining