Long non-coding RNA HOTAIR is targeted and regulated by miR-141 in human cancer cells - PubMed (original) (raw)

Ago2 mediates HOTAIR silencing by miR-141. A, Ago2 siRNA reduced Ago2 expression. 786-O cells were transiently transfected with siRNA control or one of two Ago2 siRNAs for 72 h, and Western blot analysis was performed. B, Ago2 knockdown reduces endogenous HOTAIR expression by miR-141. 786-O cells were co-transfected with 30 n

m

control siRNA or Ago2 siRNA and 30 n

m

miR negative control or miR-141. HOTAIR and miR-141 expression at 72 h after the transfection was analyzed by real-time PCR and normalized to that of the control. C, Ago2 siRNA knockdown reduces the effect of miR-141 on overexpressed HOTAIR. 786-O cells were co-transfected with 30 n

m

control siRNA or Ago2 siRNA, 30 n

m

miR negative control or miR-141, and a control plasmid or HOTAIR expression plasmid. HOTAIR and miR-141 expression at 72 h of the transfection was analyzed by real-time PCR and normalized to that of the control. D, 786-O cells in 6-well plates were co-transfected with 30 n

m

miR negative control or miR-141, 100 ng of control plasmid or HOTAIR expression plasmid, and 250 ng of FLAG-tagged Ago2 expression plasmid for 48 h, and total cell lysates were immunoprecipitated with FLAG antibody. Immunoprecipitated RNA was analyzed by real-time PCR and was normalized to that of the control. Bottom, Western blot of immunoprecipitated Ago2 probed by anti-FLAG antibody. 1, pcDNA3.1(+) and pre-miR negative control; 2, pcDNA3.1(+) and pre-miR-141; 3, pcDNA3.1(+)-HOTAIR and pre-miR negative control; 4, pcDNA3.1(+)-HOTAIR and pre-miR-141. FLAG-tagged Ago2 expression plasmid was transfected in 1–4. E, 786-O cells in 6-well plates were co-transfected with 30 n

m

miR negative control or miR-141, 100 ng of control plasmid, and 250 ng of FLAG-tagged Ago2 expression plasmid for 48 h, and total cell lysates were immunoprecipitated with FLAG antibody. In vitro transcribed HOTAIR was incubated with the immunocomplex at 4 °C for 3 h. Immunoprecipitated RNA was analyzed by real-time PCR and was normalized to that of the control. Bottom, Western blot of immunoprecipitated Ago2 probed by anti-FLAG antibody. 1, pre-miR negative control; 2, pre-miR-141; 3, purified HOTAIR and pre-miR negative control; 4, purified HOTAIR and pre-miR-141. FLAG-tagged Ago2 expression plasmid was transfected in 1–4. F, Ago2 immunocomplex cleaves HOTAIR. 786-O cells in 10-cm dishes were co-transfected with 30 n

m

miR negative control or miR-141 and 1 μg of control plasmid or FLAG-tagged Ago2 expression plasmid for 48 h, and total cell lysates were immunoprecipitated with FLAG antibody. DIG-labeled in vitro transcribed HOTAIR was incubated with immunoprecipitated Ago2 complex. After Northern blotting, DIG was probed with a DIG nucleic acid detection kit. 1, control plasmid and pre-miR negative control; 2, FLAG-tagged Ago2 expression plasmid and pre-miR negative control; 3, FLAG-tagged Ago2 expression plasmid and pre-miR-141. Error bars, S.D.