microRNAについて - tokudai-sanfujinka.jp

1 microrna RNA(non-coding RNA 23,000 . RNA:20,000 . 18-25 RNA. 1/3 RNA . RNA 1800 . miR-122, miR-200 let-7.. RNA (RNA interference). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Fire A, Mello C 1998 Nature 2006 . (C elegans) unc-22 . unc-22 . 742 . 742 . RNA 742 dsRNA RNA mRNA RNase Dicer RNA siRNA(small intefering RNA) . siRNA RISC (RNA-induced silencing complex) . mRNA miRNA, siRNA siRNA miRNA miRNA miRNA 6-8 .. miRNA 100-1000 . (Target Scan .. Dicer, Drosha, and Outcomes in Patients with Ovarian Cancer Merritt. WM, Sood AK, N Eng J Med 2008. Dicer, Drosha mRNA Dicer mRNA.))

2 RNA miRNA miR-124a is required for hippocampal axogenesis and retinal cone survival through Lhx2 suppression. Sanuki R, Furukawa T Nat Neurosci 2011. miR-124a miRNA 25-48%.. miR124a .. C miRNA miR-122 . HCV RNA miR-122 . miR-122 HCV RNA . SPC3649 miR-122 5 miR-122 Therapeutic silencing of microrna -122 in patients with chronic hepatitis C virus infection Robert E, Henrik O Science 2010. HCV C . SPC3649 : high dose (5mg/kg), low dose(1mg/kg) 2 . 12 HCV RBA .. miR-122 . Silencing of miR-122 by SPC3649 in chimpanzees with chronic hepatitis C virus infection. (A) Analysis of HCV RNA. levels in HCV-infected chimpanzees during the study.

3 The HCV titers are shown as genomic equivalents (GE) for the high-dose animals (4x0513, blue triangles; 4x0514, magenta diamonds) and low-dose animals (4x0267, orange squares;. 4x0358, red dots) in serum (GE/ml, solid lines) and liver (GE/. g liver RNA, dashed lines). The placebo and active treatment periods are indicated below. (B) Northern blot analysis of RNA. from chimpanzee liver biopsies using LNA-modified probes detecting free and sequestered miR-122 (upper panel) and SPC3649 (lower panel). The first two lanes are positive controls for free miR-122 and preformed miR-122:SPC3649. heteroduplexes, respectively.

4 (C) Detection of sequence variants in the miR-122 seed sites (boxed) by deep sequencing of the HCV 5 NCR from the high-dose animals at four time points: baseline, end of treatment, viral rebound, and end of the follow-up period. (D) The two miR-122 seed sites (boxed) in the HCV 5 NCR are conserved in all HCV. genotypes and subtypes (see Supporting Online Material for details). Treatment of HCV-infected chimpanzees with SPC3649 was well tolerated. (A) Plasma trough levels of SPC3649. (B and C) Alanine aminotransferase (ALT) levels (B) and creatinine levels (C) in HCV-infected chimpanzees during the study. (D to G) Photomicrographs of hematoxylin and eosin.

5 Stained sections from biopsies of a normal chimpanzee liver (D) and animal 4x0513 at week 4 (E), week 19 (F), and week 25 (G), respectively. A SPC3649 8 . B ALT(GPT) . C . EMT (epithelial mesenchymal transition). E-cadherin . vimentin,fibronectin, ZEB1(E-cadherin ,ZEB2 retrovirus miR-200c miR-200c expression induces an EMT phenotype in UMUC3 bladder cancer cell line. A, phase- contrast microscopy of UMUC3 cells transduced with a miR-200c containing retrovirus (UMUC3/200c) or with an empty, control, retroviral construct (UMUC3-E). Bar, 100 m. B, measurement of in vitro cell migration by "wound- healing" assay. Representative pictures for same single spot.)

6 The experiment was done twice in triplicate experiments. Bar, 400 m. C, quantification of miR-200c in empty virus . transduced (UMUC3-E) and miR-200c-transduced (UMUC3/200c) cells. Levels of miR-200c in UMUC5 cells are plotted for comparison. D, measurement by real-time RT-PCR of the epithelial and mesenchymal markers E-cadherin, ZEB1, and ZEB2 in empty virus transduced cells and miR-200c-transduced UMUC3 cells. E, confocal microscopy analysis of the UMUC3 series costained for ZEB1 or ZEB2 (red pixels) and nuclear DNA (green pixels). Note the down- regulation of nuclear ZEB1 and ZEB2 in UMUC3. clones expressing the miR-200c.

7 F, measurement of ERRFI-1 and E-cadherin protein levels by Western blot. Actin reprobing served as internal (loading) control. Bottom, absorbance relative values expressed as ratios between actin (internal control) and ERRFI-1. Note the up-regulation of E- cadherin and down-regulation of ERRFI-1 protein levels on miR-200c expression. miR200c . ERRFI-1(ErbB receptor inhibitor-1) . EGFR . C225(Cetuximab) p-MAPK . miR-200c expression reverses EGFR resistance in UMUC3 cells. A, confocal microscopy analysis of the UMUC3 series costained for ERRFI-1 (red pixels) and EGFR (green pixels) and a DNA dye, showing the nucleus (blue pixels).

8 Note the yellow pixels (left) as a result of red and green pixels colocalization. B, immunoblot of autophosphorylated EGFR, total EGFR, and total ERRFI-1 in cells transduced with miR-200c from the experiment above. In this experiment, actin served as the internal control. C, top, immunoblot of pMAPK, total MAPK, and total EGFR of the UMUC3 series. Cells grown in 2% serum- supplemented MEM were left untreated or treated with increased concentrations of cetuximab (C225) for 3 h. Bottom, absorbance relative values expressed as ratios between EGFR. (internal control) and pMAPK. D, cell proliferation measurement of the UMUC3 series using radioactive thymidine incorporation.

9 Each experiment was done in at least two different triplicates. miR-200 miR200 Tublin( ZEB1 ErbB receptor inhibitor-1 -catenin Cyclin D1 E-cadherin EGFR)