Blockade of miR-3614 Maturation by IGF2BP3 Increases TRIM25 Expression and Promotes Breast Cancer Cell Proliferation
Description
Supplementary information Fig. S1 The data from The Cancer Genome Atlas (TCGA) database showing increased expression of TRIM25 and IGF2BP3 was found in BC samples compared with hyperplastic (non-tumor) breast tissue. (A,B) The levels of TRIM25 and IGF2BP3 mRNA was assayed, the data from TCGA by deep sequencing. (C) Correlation analyses of TRIM25 mRNA and IGF2BP3 mRNA in human BC specimens. (D) The level of TRIM25 mRNA was assayed in ER+ and ER- breast cancer (p>0.05, Student's t-test). Fig. S2 HuR-depleted did not affect the expression of TRIM25 in BC cells. (A,B) The levels of HuR and TRIM25 were measured by qRT-PCR analysis after transfection of Ctrl or HuR siRNAs. (C) Forty-eight hours after transfection of HuR siRNAs, the level of HuR and TRIM25 and loading control β-actin were tested by western blot (**P < 0.01, ***P < 0.001 Student's t-test). Fig. S3 The expression of miR-3614-3p was analyzed by qRT-PCR after transfection with pre-miR-3614, miR-3614-3p inhibitor or controls in BC cells. (*P < 0.05, ***P < 0.001, ANOVA analysis) Fig. S4 Up-regulation of miR-3614 was achieved through lentiviral transduction and inhibited TRIM25 expression. (A) Assessment of lentivirus infection by observing the expression of GFP under the fluorescent microscopy. Magnification ×40, Scale bar, 50 μm. (B) qRT–PCR analysis of miR-3614-3p level after lentivirus transduction. (C) The expression of TRIM25 was analyzed by western blot after lentivirus transduction. (**P < 0.01, Student's t-test) Fig. S5 The expression of IGF2BP3 in BC tumors. (A) qRT-PCR was performed to examine IGF2BP3 mRNA expression in BC tissues versus non-tumor tissues and a sample expression comparison. (B) Representative images of IHC of IGF2BP3 in the non-tumor tissues and BC tissues microarray. Magnification ×40. Scale bar, 50 μm. (C) IGF2BP3 mRNA and protein levels in BC cell lines (MCF-7, MDA-MB-231, HCC-1937 and MDA-MB-453) and normal human mammary gland cell line (HBL-100) were quantified by qRT-PCR and Western blot. (*P < 0.05, **P < 0.01, Student's t-test) Fig. S6 Down-regulation of IGF2BP3 was achieved through lentiviral transduction. (A) Assessment of lentivirus infection by observing the expression of GFP under the fluorescent microscopy. Magnification ×40, Scale bar, 50 μm. (B) qRT–PCR analysis of IGF2BP3 level after lentivirus transduction. (C) The expression of IGF2BP3 and TRIM25 was analyzed by western blot after lentivirus transduction. (**P < 0.01, ***P < 0.001 Student's t-test).