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. 2021 May 11;12:2699. doi: 10.1038/s41467-021-23052-9

Fig. 5. Dysregulation of FOXO3a-miRNAs axis conferred Herceptin resistance.

Fig. 5

a The expression levels of miR-128-3p and miR-30a-5p in SKBR3, pool2, BT474, or HR20 cells were measured by qRT-PCR, ****p < 0.0001. b Pool2 or HR20 cells were transfected with miR-128-3p or/and miR-30a-5p mimics. The expression of IRS1, p-FOXO3a, FOXO3a, and β-actin was examined by western blot assays. c Pool2 cells were transfected with miRNA mimics in combination with pLEX-IRS1 followed by Herceptin treatment at indicated concentrations for 72 h (left). SKBR3 cells were transfected miRNA inhibitors in combination with IRS1 shRNA, and then treated with 80 ng/ml rhIGF2 along with Herceptin (right). Cell viability was evaluated by MTS assays. Pool2 Mimics: **p = 0.0022, ***p = 0.0003, ****p < 0.0001, Mimics+pLEX-IRS1: ***p = 0.0001, ****p < 0.0001, SKBR3 Inhibitors+IGF2: **p = 0.0072, **p = 0.0035, ****p < 0.0001, Inhibitors+IGF2 + sh-IRS1: **p = 0.0079, ***p = 0.0002, ****p < 0.0001. d The expression levels of miR-193a-5p in SKBR3, pool2, BT474, and HR20 cells were detected by qRT-PCR, ****p < 0.0001. e Pool2 or HR20 cells were transfected with a miR-193a-5p mimic. SKBR3 or BT474 were transfected with a miR-193a-5p inhibitor. IGF2 levels in the CM were measured by ELISA. f Pool2 or HR20 cells were treated with vehicle (Mock) or WAY-600 (1 μM, WAY). The expression of p-Akt(S473), Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, and β-actin was examined by western blot assays (left); the expression levels of miR-128-3p, miR-30a-5p and miR-193a-5p were detected by qRT-PCR (right), ****p < 0.0001. g Pool2 or HR20 cells were treated with WAY-600 for 24 h. The enrichment of FOXO3a at miR-193a-5p promoter was examined by ChIP-qPCR assays, ****p < 0.0001. n = 3 biological independent samples (a, c–e, g). Data are presented as mean values ± SEM (a, c–e, g). Statistical significance was determined by a two-tailed Student’s t-test (a, c–e, g). Data show a representative of three independent experiments (b, f). All data are provided in the Source Data.