luciferase reporter pmir report  (Addgene inc)

Journal: Molecular Cancer

Article Title: The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells

doi: 10.1186/1476-4598-12-92

Figure Lengend Snippet: Regulation of MAP7 expression by miR-16 at both the transcript and protein levels. (A, D and G) Relative quantification RT-PCR analysis of miR-16 levels in A549 (A), Hela (D) and MCF-7 (G) cells treated with pre-miR-16, pre-miR-control, anti-miR-16 or anti-miR-control. The results presented are the mean ± SD of three independent experiments (*** p < 0.001). (B, E and H) Relative quantification RT-PCR analysis of MAP7 mRNA levels in A549 (B) , Hela (E) and MCF-7 (H) cells treated with pre-miR-16, pre-miR-control, anti-miR-16 or anti-miR-control. The results presented are the mean ± SD of three independent experiments (*** p < 0.001). (C, F and I) Western blot analysis of MAP7 protein levels in A549 (C) , Hela (F) and MCF-7 (I) cells treated with pre-miR-16, pre-miR-control, anti-miR-16 or anti-miR-control. Left panel: representative image; right panel: quantitative analysis (*** p < 0.001).

Article Snippet: The PCR products were inserted into the p-MIR-report plasmid (Ambion).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Molecular Cancer

Article Title: The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells

doi: 10.1186/1476-4598-12-92

Figure Lengend Snippet: The role of miR-16 targeting MAP7 in the regulation of proliferation, apoptosis and cell cycle progression. (A and B) Efficient overexpression or knockdown of MAP7 expression. For knockdown of MAP7, siRNA against MAP7 and a scrambled control siRNA were transfected into A549 cells. For overexpression of MAP7, MAP7 overexpressing plasmid and an empty plasmid were transfected into A549 cells. Cells were harvested at 24 h post-transfection. MAP7 mRNA and protein levels were assessed by relative quantification RT-PCR (A) and Western blotting (B) . Left panel: quantitative analysis; right panel: representative image. (C) MTT cell viability assay at 12, 24, 36, and 48 h after transfection of A549 cells with equal doses of pre-miR-control, pre-miR-16, anti-miR-control or anti-miR-16. (D) MTT cell viability assay at 12, 24, 36, and 48 h after transfection of A549 cells with equal doses of control siRNA or siRNA against MAP7 or equal doses of empty plasmid or MAP7 overexpressing plasmid. (E) MTT cell viability assay at 12, 24, 36, and 48 h after transfection of A549 cells with equal doses of pre-miR-control, pre-miR-16, or pre-miR-16 along with the MAP7 overexpressing plasmid. (F) A549 cells transfected with equal doses of pre-miR-control, pre-miR-16, control siRNA or siRNA against MAP7 were labeled with FITC-Annexin V/PI, and serum deprivation-induced apoptosis was measured by flow cytometry. (G) Quantification of the apoptotic cells in panel F. (H) A549 cells were transfected with equal doses of scrambled ncRNA or pre-miR-16 or equal doses of control siRNA or siRNA against MAP7. Cell cycle profiles were analyzed using flow cytometry. Shown in the panel are histograms of cell numbers (y axis) against DNA content (x axis) determined by measuring fluorescence intensity. (I) Quantification of the percentages of cells in the G0/G1, S, and G2/M phases in panel (H) (mean ± SD; ** p < 0.01; *** p < 0.001).

Article Snippet: The PCR products were inserted into the p-MIR-report plasmid (Ambion).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Viability Assay, Labeling, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: miR-203 Suppresses the Proliferation and Migration and Promotes the Apoptosis of Lung Cancer Cells by Targeting SRC

doi: 10.1371/journal.pone.0105570

Figure Lengend Snippet: ( A ) Schematic description of the hypothetical duplexes formed by the interactions between the binding sites in the SRC 3′-UTR (top) and miR-203 (bottom). The predicted free energy value of each hybrid is indicated. The seed recognition sites are denoted, and all nucleotides in these regions are highly conserved across species, including human, mouse, and rat. ( B ) Quantitative RT-PCR analysis of the expression levels of miR-203 (in the form of the miRNA/U6 ratio) in six pairs of CL and NL samples. ( C ) Pearson's correlation scatter plot of the fold change of the levels of miR-203 and SRC protein in human lung cancer tissues. ( D ) Pearson's correlation scatter plot of the fold change of the levels of miR-203 and SRC mRNA in human lung cancer tissues. * P<0.05; ** P<0.01.

Article Snippet: The PCR products were inserted into the p-MIR-reporter plasmid (Ambion), and the insertion was confirmed as correct by sequencing.

Techniques: Binding Assay, Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: miR-203 Suppresses the Proliferation and Migration and Promotes the Apoptosis of Lung Cancer Cells by Targeting SRC

doi: 10.1371/journal.pone.0105570

Figure Lengend Snippet: ( A ) Quantitative RT-PCR analysis of the miR-203 levels in A549 cells treated with pre-miR-control or pre-miR-203 and in HLF cells treated with anti-miR-control or anti-miR-203. ( B and C ) Western blot analysis of SRC protein levels in A549 cells treated with pre-miR-control or pre-miR-203 and in HLF cells treated with anti-miR-control or anti-miR-203. B: representative image; C: quantitative analysis. ( D ) Quantitative RT-PCR analysis of SRC mRNA levels in A549 cells treated with pre-miR-control or pre-miR-203 and in HLF cells treated with anti-miR-control or anti-miR-203. ( E ) Firefly luciferase reporters containing wild-type (WT) or mutant (MUT) miR-203 binding sites in the SRC 3′-UTR were co-transfected into A549 cells with pre-miR-control or pre-miR-203 and into HLF cells with anti-miR-control or anti-miR-203. Twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit. The results are calculated as the ratio of firefly luciferase activity in the pre-miR-203- or anti-miR-203-transfected cells normalized to the control cells. * P<0.05; ** P<0.01.

Article Snippet: The PCR products were inserted into the p-MIR-reporter plasmid (Ambion), and the insertion was confirmed as correct by sequencing.

Techniques: Quantitative RT-PCR, Western Blot, Luciferase, Mutagenesis, Binding Assay, Transfection, Activity Assay

Journal: PLoS ONE

Article Title: miR-203 Suppresses the Proliferation and Migration and Promotes the Apoptosis of Lung Cancer Cells by Targeting SRC

doi: 10.1371/journal.pone.0105570

Figure Lengend Snippet: ( A and B ) Western blotting analysis of the protein levels of SRC, Ras-GTP, total Ras, phosphorylated ERK1/2, and ERK1 in A549 cells transfected with control siRNA, SRC siRNA, control vector, or SRC vector. A: representative image; B: quantitative analysis. ( C ) Quantitative RT-PCR analysis of the SRC mRNA levels in A549 cells treated with control siRNA, SRC siRNA, control vector, or SRC vector. ( D and E ) Western blotting analysis of the protein levels of SRC, Ras-GTP, total Ras, phosphorylated ERK1/2, and ERK1 in A549 cells transfected with pre-miR-control or pre-miR-203. D: representative image; E: quantitative analysis. * P<0.05; ** P<0.01.

Article Snippet: The PCR products were inserted into the p-MIR-reporter plasmid (Ambion), and the insertion was confirmed as correct by sequencing.

Techniques: Western Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR