Figure 4.

LncRNA-H19 acts as an endogenous miRNA let-7 sponge in breast cancer cells. (a) Comparison of the expression of H19 in cytoplasm and in nucleus by RT-qPCR. Data are represented as mean±S.D. ***P<0.001, n=3. (b) Shown was the representative image of the in situ location of H19 transcripts in MDA-MB-231 cells. The scale bar represents 20 μm (left) and 10 μm (right). (c) Immunoprecipitation using anti-AGO2 antibody (lane 3) or IgG (lane 2) followed by western blot analysis using a mouse monoclonal anti-AGO2 (right). Co-IP with rabbit anti-AGO2 antibody or preimmune IgG from extracts of MDA-MB-231 cells. H19 RNA levels in immunoprecipitates were determined by RT-qPCR (left). Data were shown as mean±S.D. from three independent experiments, **P<0.01. (d) The target validation using luciferase reporters; the indicated constructs were each transfected into MDA-MB-231 cells together with negative control miRNA (NC) or let-7 mimics (mlet-7) at a final concentration of 48 nM. Numbers are mean±S.D. (n=3, ***P<0.001). (e) Let-7 sensor (psiCHECK2-let-7 4x) was transfected into MDA-MB-231 cells, together with 0, 20, 40 or 80 ng of sponge plasmid wide type H19 (WT) or mutant H19 (Mut). Numbers are mean±S.D. (n=3, *P<0.05, **P<0.01). (f) Empty vector (EV) or full-length H19 (WT) was transfected into MDA-MB-231 cells. The relative H19 mRNA level was normalized to ACTB and let-7a/7b miRNA levels were normalized against those of U6B. Numbers are mean±S.D. (n=3). (g) The protein levels of let-7 targets DICER and RAS were confirmed by western blot. (h) MDA-MB-231 cells were transfected with siRNA targeting H19 (siH19) or negative control RNA (siNC), H19 expression levels and let-7a/7b miRNA levels were evaluated by RT-qPCR, and (i) the protein levels of let-7 targets were detected by western blot