Journal: PLoS ONE

Article Title: MicroRNA-608 and MicroRNA-34a Regulate Chordoma Malignancy by Targeting EGFR, Bcl-xL and MET

doi: 10.1371/journal.pone.0091546

Figure Lengend Snippet: Small RNAs were extracted from chordoma UCH1 and UCH2 cells and control fibroblasts. miRNA levels were measured using qRT-PCR relative to control U6B snRNA.

Article Snippet: From 100 ng of cDNA template, quantitative real-time PCR analyses for 26 miRNAs (miR-335, miR-202, miR-29a, miR-20a*, miR-34a, miR-148a, miR-23b, miR-23b, miR-10b, miR-204, miR-221, miR-297, miR-328, miR-421, miR-663, miR-7, miR-134, miR-196, mIR-582-3p, miR-885-5p, miR-768-5p, miR-582-5p, miR-887, miR-363-miR-22, miR-768-3p, and mir-608) and U6B control were performed using miRNA specific primer assays and U6B-specific primers according to the manufacturer's protocol (Qiagen).

Techniques: Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: MicroRNA-608 and MicroRNA-34a Regulate Chordoma Malignancy by Targeting EGFR, Bcl-xL and MET

doi: 10.1371/journal.pone.0091546

Figure Lengend Snippet: A) Predicted binding sequences of miR-608 in the 3′UTR sequences of EGFR and Bcl-xL mRNA; B) Predicted binding sequences of miR-34a in the 3′UTR sequence of MET mRNA; C), D) UCH1 and C24 cells were transfected with pre-miR-608 (C) or pre-miR-34a (D) or control pre-miR for 48 hrs. Cell lysates were immunoblotted for EGFR or Bcl-xL (C) or MET (D), The results show that the miRNAs significantly inhibited these predicted target proteins in chordoma cells; E), F) UCH1 cells were transfected with pre-miR-608, pre-miR-34a or pre-miR-con and then with either EGFR 3′UTR, Bcl-xL 3′UTR, MET 3′UTR or control reporter plasmids together with β-Galactosidase (β-Gal) plasmid, and 3′UTR reporter activity was measured by a luciferase assay and normalized to β-Gal activity. The results show that miR-608 expression down-regulates EGFR and Bcl-xL luciferase activities (E) and that miR-34a expression repressed MET luciferase activity (F) in UCH1 cells. (* P<0.05)

Article Snippet: From 100 ng of cDNA template, quantitative real-time PCR analyses for 26 miRNAs (miR-335, miR-202, miR-29a, miR-20a*, miR-34a, miR-148a, miR-23b, miR-23b, miR-10b, miR-204, miR-221, miR-297, miR-328, miR-421, miR-663, miR-7, miR-134, miR-196, mIR-582-3p, miR-885-5p, miR-768-5p, miR-582-5p, miR-887, miR-363-miR-22, miR-768-3p, and mir-608) and U6B control were performed using miRNA specific primer assays and U6B-specific primers according to the manufacturer's protocol (Qiagen).

Techniques: Binding Assay, Sequencing, Transfection, Control, Plasmid Preparation, Activity Assay, Luciferase, Expressing