Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Novel MicroRNA-455-3p and its Protective Effects Against Abnormal APP Processing and Amyloid Beta Toxicity in Alzheimer’s Disease

doi: 10.1016/j.bbadis.2019.06.006

Figure Lengend Snippet: (A) The two putative binding sites of miR-455-3p at the 3′-UTR regions of a wild-type APP gene in humans and mice. Region A (522-528) had 7 nucleotide binding sites, and Region B (3139-3145) had 6 nucleotide binding sites with the seed sequence of miR-455-3p in both the human and mouse wild-type APP gene (shown in green). (B) Luciferase reporter assay for miR-455-3p binding with the APP gene. Normalized luciferase activity (Firefly/Renilla) in neuroblastoma cells co-transfected with the APP 3’UTR clone (HmiT009578-MT06) and miR-455-3p mimics. Firefly/Renilla luciferase activity of the APP 3’UTR clone was significantly decrease by the miR-455-3p. (C) Representative images of neuroblastoma cells morphology after 24 hr of miR-455-3p mimics and inhibitor transfection (10× magnification). Quantitative measurement of miR-455-3p (D), and APP fold-change (E), in cells at 24 hr post mimics and inhibitor transfection. (F) Representative images of Annexin V and PI staining of cells at 24 hr post-transfection of miR-455-3p mimics and inhibitor. White arrow represents the viable (green) and dead (red) cells. Percentage (%) of apoptotic cells (G), and viable cells populations (H), after 24 hr of miR-455-3p mimics and inhibitor transfection. (*P<0.05) (**P<0.01) (***P<0.001)

Article Snippet: MiR-455-3p expression vector MiR-455-3p expression vector (pRP[Exp]-U6>hsa-miR-455-3p-CAG-EGFP) was purchased from VectorBuilder (Cyagen Biosciences, Santa Clara, CA, EISA).

Techniques: Binding Assay, Sequencing, Luciferase, Reporter Assay, Activity Assay, Transfection, Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Novel MicroRNA-455-3p and its Protective Effects Against Abnormal APP Processing and Amyloid Beta Toxicity in Alzheimer’s Disease

doi: 10.1016/j.bbadis.2019.06.006

Figure Lengend Snippet: (A) Representative images for transfection of the miR-455-3p expression vector (GFP), the miR-control (GFP) vector, and the mutant APP cDNA in neuroblastoma cells (10× magnification). Bright field images and green fluorescence detected in the same field at 24 hr post-transfection (10× magnification). (B) Representative images of Annexin V and Propidium Iodide (PI) stained neuroblastoma cells transfected with the miR-control vector, the miR-455-3p vector, and the mutant APP cDNA. The white arrow indicates the populations of viable (green) cells and dead (red) cells. (C) Percentage (%) of an apoptotic cell population after 24 hr of the miR-455-3p vector, the miR-control vector, and the mutant APP cDNA transfecting cells. (D) Percentage (%) of a viable cell population after 24 hr of the miR-control vector, the miR-455-3p vector and the mutant APP cDNA transfecting cells. (E) Quantitative measurement of miR-455-3p fold change expression after 24 hr of the miR-455-3p vector and the mutant APP cDNA transfecting cells. (F) Quantitative measurement of APP mRNA folds change expression after 24 hr of the miR-455-3p vector and the mutant APP cDNA transfecting cells. (G) Western blot for APP (6E10), the C-terminal fragment of APP (C99) and B-actin proteins in cells transfected with the miR-control vector, the miR-455-3p vector, mutant APP cDNA and co-transfected cells. Quantitative measurement of (H) APP (6E10) and C-terminal fragments of APP C99 (I), and C83 (J) proteins levels by densitometry in the mutant APP cDNA and the miR-455-3p vector transfecting cells. (*P<0.05) (**P<0.01) (***P<0.001)

Article Snippet: MiR-455-3p expression vector MiR-455-3p expression vector (pRP[Exp]-U6>hsa-miR-455-3p-CAG-EGFP) was purchased from VectorBuilder (Cyagen Biosciences, Santa Clara, CA, EISA).

Techniques: Transfection, Expressing, Plasmid Preparation, Mutagenesis, Fluorescence, Staining, Western Blot

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Novel MicroRNA-455-3p and its Protective Effects Against Abnormal APP Processing and Amyloid Beta Toxicity in Alzheimer’s Disease

doi: 10.1016/j.bbadis.2019.06.006

Figure Lengend Snippet: (A) Representative immunostaining images of neuroblastoma cells transfected with the miR-control vector, the miR-455-3p vector, the mutant APP cDNA, the mutant APP and miR-455-3p co-transfected. Cells were stain with the APP (6E10) antibody producing red fluorescence (20× magnification). Green fluorescence showed the miR-control and miR-455-3p vector expression and the nucleus of the cell stained with DAPI (blue). Fluorescence intensity (red) of the mutant APP was reduce in mutant APP and miR-455-3p co-transfected cells. (B) Quantitative measurement of fluorescence intensity of the APP (6E10) protein in mutant APP, miR-455-3p, and miR-control vector transfected cells. (C) ELISA analysis for amyloid (1-40) and (1-42) levels in neuroblastoma cells. Quantitative detection of the (i) human amyloid-β(1-40) and (ii) amyloid-β(1-42) peptide level in mutant APP cDNA and miR-455-3p vector transfected cells. (*P<0.05)

Article Snippet: MiR-455-3p expression vector MiR-455-3p expression vector (pRP[Exp]-U6>hsa-miR-455-3p-CAG-EGFP) was purchased from VectorBuilder (Cyagen Biosciences, Santa Clara, CA, EISA).

Techniques: Immunostaining, Transfection, Plasmid Preparation, Mutagenesis, Staining, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Novel MicroRNA-455-3p and its Protective Effects Against Abnormal APP Processing and Amyloid Beta Toxicity in Alzheimer’s Disease

doi: 10.1016/j.bbadis.2019.06.006

Figure Lengend Snippet: (A) Representative western blot images for PGC1α, NRF1, NRF2, TFAM, and beta-actin proteins levels in 1) Neuroblastoma cells transfected with the miR-control vector, 2) Neuroblastoma cells transfected with the miR-455-3p expression vector, 3) Neuroblastoma cells transfected with the mutant APP cDNA, 4) Neuroblastoma cells co-transfected with the miR-455-3p and mutant APP cDNA, and 5) Neuroblastoma cells co-transfected with the mutant APP and miR-control vector. Quantitative measurement of the levels of (B) the PGC1α protein (C) NRF1, (D) NRF2, and (E) TFAM using densitometry in 1) Cells transfected with the miR-control vector, 2) Cells transfected with the miR-455-3p expression vector, 3) Cells transfected with mutant APP cDNA, 4) Cells co-transfected with the miR-455-3p and mutant APP cDNA, and 5) Cells co-transfected with the mutant APP and miR-control vector. (F) Representative western blot images for synaptophysin, PSD95, and beta-actin proteins levels in 1) Cells transfected with the miR-control vector, 2) Cells transfected with the miR-455-3p expression vector, 3) Cells transfected with the mutant APP cDNA, 4) Cells co-transfected with the miR-455-3p and mutant APP cDNA, and 5) Cells co-transfected with the mutant APP and miR-control vector. Quantitative measurement of the levels of (G) the synaptophysin protein and (H) PSD95 using densitometry in same groups of cells. (*P<0.05) (**P<0.01)

Article Snippet: MiR-455-3p expression vector MiR-455-3p expression vector (pRP[Exp]-U6>hsa-miR-455-3p-CAG-EGFP) was purchased from VectorBuilder (Cyagen Biosciences, Santa Clara, CA, EISA).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Mutagenesis

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Novel MicroRNA-455-3p and its Protective Effects Against Abnormal APP Processing and Amyloid Beta Toxicity in Alzheimer’s Disease

doi: 10.1016/j.bbadis.2019.06.006

Figure Lengend Snippet: (A) Representative western blot images for DRP1, FIS1, OPA1, Mfn1, Mfn2 and beta-actin proteins levels in: 1) Neuroblastoma cells transfected with the miR-control vector, 2) Neuroblastoma cells transfected with the miR-455-3p expression vector, 3) Neuroblastoma cells transfected with the mutant APP cDNA, 4) Neuroblastoma cells co-transfected with the miR-455-3p and mutant APP cDNA, and 5) Neuroblastoma cells co-transfected with the mutant APP and miR-control vector. Quantitative measurement of the levels of (B) the DRP1, (C) FIS1, (D) OPA1, (E) Mfn1 and (F) Mfn2 proteins using densitometry in the same groups of cells. (G) Representative TEM images of Neuroblastoma cells transfected with the miR-control vector, the miR-455-3p vector, mutant APP cDNA, co-transfected with mutant APP and miR-455-3p, and co-transfected with mutant APP and miR-control, showing mitochondrial organization (600 nm magnification). (H) Quantification of the number of mitochondria in the Neuroblastoma cells transfected with miR-control vector, the miR-455-3p vector, mutant APP cDNA, co-transfected with mutant APP and miR-455-3p, and co-transfected with mutant APP and miR-control. (I) Quantification of the size of mitochondria (μm) in the neuroblastoma cells transfected with miR-control vector, the miR-455-3p vector, mutant APP cDNA, co-transfected with mutant APP and miR-455-3p, and co-transfected with mutant APP and miR-control. (*P<0.05) (**P<0.01).

Article Snippet: MiR-455-3p expression vector MiR-455-3p expression vector (pRP[Exp]-U6>hsa-miR-455-3p-CAG-EGFP) was purchased from VectorBuilder (Cyagen Biosciences, Santa Clara, CA, EISA).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Mutagenesis