Fig. 4

ACTA2-AS1 functioned as a ceRNA in sponging miR-4428. A FISH analysis of ACTA2-AS1 subcellular localization in TPC1 cells. ACTA2-AS1 probes are stained in green and nuclei are stained in blue. Scale bar, 20 µm and magnification, 400 × . B Total RNA was extracted from the nucleus and cytoplasm following which the ACTA2-AS1 level was assessed using qRT-PCR. C A RNA immunoprecipitation (RIP) assay was performed with an anti-AGO2 antibody to assess the enrichment of ACTA2-AS1 in AGO2-immunoprecipitated complexes in TPC1 cells. IgG served as a negative control; 10% of the total RNA was used as input. D MiRNA expression in ACTA2-AS1-overexpressing TPC1 cells was assessed by qRT-PCR. E Sequence analysis revealed that ACTA2-AS1 harbors a complementary miR-4428 target site. F pGL3-ACTA2-AS1-wt or pGL3-ACTA2-AS1-mut and miR-4428 were co-transfected into TPC1 cells and then luciferase activity was measured. G pGL3-ACTA2-AS1-wt and miR-4428 or miR-4428-mut were co-transfected into TPC1 cells and then luciferase activity was measured. H RNA pull-down was carried out with biotin-labeled miR-4428 to assess the enrichment of ACTA2-AS1 in miR-4428-immunoprecipitated complexes in TPC1 cells. **p < 0.01