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. 2019 Jul 1;11:5983–6001. doi: 10.2147/CMAR.S207084

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© 2019 Zhu et al.

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Linc00460 competitively binds to miR-489-5p to increase FGF7 expression and activates downstream AKT signaling. (A) Predicted miR-489-5p-binding site on the 3ʹUTR of FGF7 mRNA. (B-C) Luciferase reporter plasmids containing FGF-7 3ʹUTR or empty control were co-transfected with miR-489-5p mimics, miR-489-5p inhibitors, or their respective scrambled oligonucleotide control into MCF-7 cells and MDA-MB-231 cells. Firefly luciferase reporter activities determined were normalized with Renilla luciferase reporter activities. Data are presented as mean ± SD, analyzed using independent samples t-test. *P<0.05. (D–E) Protein and mRNA levels of FGF7 in MCF-7 and MDA-MB-231 cells transfected miR-489-5p mimics, miR-489-5p inhibitor, or their respective scrambled oligonucleotide control, were analyzed by Western blot and qRT-PCR, respectively. Data are presented as mean ± SD, analyzed using independent samples t-test. *P<0.05. (F–G) Expression levels of FGF7 in MCF-7 and MDA-MB-231 cells transfected with the Linc00460-expressing plasmid, Linc00460 shRNAs or respective empty vectors were determined by qRT-PCR. Data are presented as mean ± SD, analyzed using independent samples t-test. **P<0.01. (H–I) Protein levels of FGF7, and phosphorylated and total-AKT in MCF-7 and MDA-MB-231 cells stably transfected with Linc00460-expressing plasmid, Linc00460 shRNAs or respective empty vectors were determined by Western blot. (J) Pearson’s correlation was performed to analyze the correlations between the levels of Linc00460 and FGF7 in 87 breast cancer tissues from GSE6532 dataset (R=0.3261, P<0.05). (K) AGO2 protein levels of co-immunoprecipitated products were measured by Western blot in MCF-7-pSin-460oe cells or –vector cells. (L–M) RIP-qRT-PCR assays were used to examine the FGF7 and miR-489-5p levels in associated with AGO2 upon Linc00460 overexpression in MCF-7 cells. Data are present as mean ± SD from three independent experiments. *P<0.05; **P<0.01 (Student’s t-test). (N) AGO2 protein levels of co-immunoprecipitated products were measured by Western blot in MDA-MB-231-pSin-460oe cells or –vector cells. (O–P) RIP-qRT-PCR assays were used to examine FGF7 and miR-489-5p levels in associated with AGO2 after Linc00460 overexpression in MDA-MB-231 cells. Data are present as mean ± SD from three independent experiments. *P<0.05; **P<0.01 (Student’s t-test).