Fig. 5

Characterizations of lncRNA RP11-390F4.3 using different in vitro and in vivo metastatic assays. a Immunofluorescence staining shows the nuclear localization of lncRNA RP11-390F4.3. Cell nuclei were stained by DAPI. b Measurement of the copy number of lncRNA RP11-390F4.3 in FADU cells (normoxia vs. hypoxia). Titration standard curve was used for measurement of the copy number of lncRNA RP11-390F4.3 per 500,000 cells. The red point represents the qRT-PCR value from a standard sample of 500,000 FADU cells under hypoxia. c Knockdown of lncRNA RP11-390F4.3 significantly decreased the induction of various HIF-1α target genes using qRT-PCR analysis. Knockdown using the scrambled control siRNA was used as a control. N, normoxia; H, hypoxia. The asterisk (*) indicated statistical significance (P<0.05) between experimental and control clones (n=3). d qChIRP assays show the significantly decreased lncRNA RP11-390F4.3 binding to the promoter regions of EMT regulators and Glut1 gene in hypoxic FADU cells under knockdown of lncRNA RP11-390F4.3 vs. the hypoxic control knockdown cells. e Heatmap analysis of the EMT transcription regulator genes induced by lncRNA RP11-390F4.3 or METTL4 overexpression using RNA-seq datasets. f Western blot analysis shows that knockdown of lncRNA RP11-390F4.3 abolished the EMT phenotypes induced by METTL4 overexpression in FADU and BFTC909 cells. g Overexpression of lncRNA RP11-390F4.3 in BFTC909 cells significantly increased metastatic lung nodules in mice after injection of these cells into mice. Representative gross anatomy and histology are shown on the left, and measurement of metastatic lung nodules is shown on the right. BFTC control-transfected clone was used as a control. h Knockdown of lncRNA RP11-390F4.3 in BFTC909 cells overexpressing METTL4 significantly decreased the metastatic lung nodules in mice. Representative gross anatomy and histology are shown on the left, and measurement of metastatic lung nodules is shown on the right. BFTC control-transfected clone was used as a control. The asterisk (*) indicates statistical significance (P<0.05) between experimental and control groups