H19 elevates PDK1 expression in a HIF-1α-dependent manner. (a, b) MDA-MB-231 and MCF-7 cells expressing either NTC or shH19 were cultured under hypoxic condition for 12 h. HIF-1α mRNA and protein expression levels were detected by RT-qPCR (up) and western blotting (down). (c) MDA-MB-231 cells were cultured under normoxic or hypoxic conditions for 12 h. The mRNA level of H19 in cytoplasm and nucleus were detected by RT-qPCR. (d) Diagram represents the let-7 putative binding site on 3′UTR of HIF1A, this site was inserted to the cloning site of psiCHECK2 vector. (e) The psi-HIF1A-FL, MRE, Mut and psi-let-7 4x, HIF-1α and let-7a, let-7b vectors were co-transfected into MDA-MB-231 cells and the regulation of HIF-1α by let-7a and let-7b was studied by luciferase assay. (f) HIF-1α expression was measured in MDA-MB-231 cells with let-7 mimics (mlet-7) and let-7 inhibitors (ilet-7). DICER is a target of let-7 and used as a positive control. (g) MDA-MB-231 cells were transfected with let-7 sensor (psi-HIF1A-MRE) together with 0, 20, 40 and 80 ng of wild-type H19 (WT) or mutant H19 (Mut) plasmids. Dual-luciferase reporter activity was analyzed. (h) HIF-1α expression was detected in siH19 and siH19 plus let-7 inhibitor compared with negative control in MDA-MB-231 cells. DICER is a target of let-7 and used as a positive control. (i, j) MDA-MB-231 and MCF-7 cells expressing either NTC or shHIF1A were infected with vector expressing H19 and control vector. PDK1 and HIF-1α expression was detected by western blot. Data shown are mean±s.d. (n=3), *P<0.05, **P<0.01 and ***P<0.001, respectively.