HOTAIR is a target of miR-331-3P or miR-124 and controls miR-331-3p target. (A) The 11 putative miRNA binding sites in the HOTAIR sequence. The HOTAIR cDNA containing the putative miRNAs recognition sites was cloned downstream of the luciferase gene and named RLuc-HOTAIR. (B) Left: The luciferase reporter plasmid (RLuc-HOTAIR) was co-transfected into HEK-293 T cells with the 11 various miRNA-coding plasmids. Right: the luciferase reporter plasmid containing wild-type or mutant HOTAIR was co-transfected into HEK-293 T cells with miR-331-3p in parallel with an empty plasmid vector. Luciferase activity was determined using the dual luciferase assay and shown as the relative luciferase activity normalized to renilla activity. Histogram indicates the values of luciferase measured 48 h after transfection. (C) The 3’-UTR of HER2 was fused to the luciferase coding region (RLuc-HER2 3’-UTR) and transfected in HEK293T cells with miR-331-3p to confirm HER2 is the target of miR-331-3p. RLuc-HER2 3’-UTR and miR-331-3p constructs were co-transfected into HEK293T cells with plasmids expressing HOTAIR (pCDNA/HOTAIR) or with a control vector to verify the ceRNA activity of HOTAIR. rno-miRNA-344 was used as a negative control. Histogram indicates the values of luciferase measured 48 h after transfection. (D) RIP with mouse monoclonal anti-Ago2, preimmune IgG or 10% input from BGC 823-cell extracts. RNA levels in immunoprecipitates were determined by qRT-PCR. Top: levels of HOTAIR, miRNA331-3P and FOS RNA were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates. Bottom: relative RNA levels of U1 snRNA in SNRNP70 relative to IgG immunoprecipitates. Numbers are mean ± s.d. (n = 3). (E) Western blot analysis of HER2 protein level following treatment of BGC-823 cells with si-HOTAIR or pCDNA/HOTAIR, and SGC-7901 cells with pCDNA/HOTAIR. GAPDH was used as control. *P < 0.05, **P < 0.01 and N.S. not significant.