Research Paper Volume 12, Issue 3 pp 2974—2991

Figure 5. TUSC8 functions as molecular sponge for miR-190b-5p in breast cancer cells. (A) The subcellular location score of TUSC8 in IncLocator prediction database. (B) The relative TUSC8 expression levels in the cytoplasm and nucleus of MCF-7 cells. (C) Complementary sequence between miR-190b-5p and wild type (wt) TUSC8. The putative binding sites of miR-190b-5p were mutated in mutant (mut) TUSC8. MCF-7 cells that were co-transfected with miR-190b-5p mimics and wt or mut TUSC8 vectors were measured for luciferase activity. (D) miR-190b-5p was highly enriched in the sample pulled down with biotinylated wt TUSC8 rather than mut TUSC8 by RNA pull-down assay. (E, F) AGO2-RIP assay was performed in SK-BR-3 cell lysates, followed by qRT-PCR to detect TUSC8 and miR-190b-5p association with AGO2. The results indicated that TUSC8, miR-190b-5p and AGO2 formed a complex in SK-BR-3 cells. (G) Knock-down of TUSC8 significantly elevated the miR-190b-5p expression level in MCF-7 and HCC1937 cell lines. (H) Over-expression of TUSC8 significantly reduced the miR-190b-5p expression level in SK-BR-3 and MDA-MB-435 cell lines. The asterisks (*, **) indicate a significant difference (p < 0.05, p < 0.01) respectively.