Figure 4. lncRNA GAS5 sponged miR-221 through both directly targeting and Ago2-dependent manner. (A) qPCR results showed that GAS5 expression level in nucleus was higher than that in the cytoplasm; (B) FISH results indicated that GAS5 was chiefly located in the cytoplasm; (C) Direct binding sites between GAS5 and miR-221 were revealed; (D) Binding relationship between GAS5 and miR-221 was detected by luciferase reporter assay with wild or mutant type of luciferase reporter plasmids of GAS5; (E) There is negative correlation between lncRNA GAS5 and miR-221 in T2D with DN tissues (N=30). (F, G) miR-221 expression was detected by qPCR. MCs were transfected with LV-GAS5 and sh-GAS5; (H, I) RIP assay was performed, which used input from cell lysate, IgG, or anti-Ago2. The expression levels of GAS5 and miR-221 were measured by qPCR.