Figure 3. lncRNA-ES3 suppressed miR-34c-5p expression by direct interaction. (A) Schematic representation of the putative binding sites between lncRNA-ES3 and miR-34c-5p, and the mutant sites in Mut-lncRNA-ES3 reporter were underlined. (B) qRT-PCR showed the expression of lncRNA-ES3 in HA-VSMCs cultured with NG, OC, and HG. (C) HA-VSMCs was transfected with miRNA NC and miR-34c-5p mimics and harvested for the examination of lncRNA-ES3 by qRT-PCR. (D) The inhibitory efficiency of shRNAs targeting lncRNA-ES3 was verified by qRT-PCR. (E) HA-VSMCs were transfected with shRNA NC and shRNA-ES3-2, and the expression of miR-34c-5p was detected by qRT-PCR. (F) The WT-lncRNA-ES3 3’UTR and the Mut-lncRNA-ES3 3’UTR reporters were co-transfected with miR-34c-5p mimics or control oligos into HA-VSMCs. Forty-eight hours after transfection, luciferase activities were measured. (G) The expression of lncRNA-ES3 was detected by qRT-PCR after biotin-labeled miR-34c-5p pull-down assay. (H) RIP and qRT-PCR assays were performed to explore the binding efficiency of miR-34c-5p and lncRNA-ES3 to Ago2 protein in HA-VSMCs. The data are expressed as mean ± SD, n=3, *p<0.05, **p<0.005, ***p<0.0005. NG: normal glucose; OC: osmolarity control; HG: high glucose; NC: negative control.