Blockade of miR-3614 Maturation by IGF2BP3 Increases TRIM25 Expression and Promotes Breast Cancer Cell Proliferation

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).补充资料 图S1 来自癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库的数据显示，与增生性（非肿瘤性）乳腺组织相比，乳腺癌（Breast Cancer, BC）样本中TRIM25和IGF2BP3的表达水平显著升高。(A、B) 采用深度测序获取的TCGA数据集，检测了TRIM25与IGF2BP3的mRNA水平。(C) 对人类乳腺癌标本中TRIM25 mRNA与IGF2BP3 mRNA进行相关性分析。(D) 检测了雌激素受体阳性（ER+）与雌激素受体阴性（ER-）乳腺癌组织中TRIM25的mRNA水平（p>0.05，学生t检验）。 图S2 敲低HuR并不会影响乳腺癌细胞中TRIM25的表达。(A、B) 转染对照（Ctrl）或HuR小干扰RNA（small interfering RNA, siRNA）后，采用实时荧光定量反转录PCR（quantitative real-time reverse transcription PCR, qRT-PCR）检测HuR与TRIM25的表达水平。(C) 转染HuR siRNA 48小时后，采用蛋白质印迹法检测HuR、TRIM25及内参蛋白β-肌动蛋白的表达水平（**P < 0.01，***P < 0.001，学生t检验）。 图S3 在乳腺癌细胞中转染pre-miR-3614、miR-3614-3p抑制剂或对照试剂后，采用qRT-PCR分析miR-3614-3p的表达水平（*P < 0.05，***P < 0.001，方差分析（Analysis of Variance, ANOVA））。 图S4 过表达miR-3614可通过慢病毒转染实现，并可抑制TRIM25的表达。(A) 通过荧光显微镜观察绿色荧光蛋白（Green Fluorescent Protein, GFP）的表达以评估慢病毒感染效率，放大倍数×40，比例尺50 μm。(B) 慢病毒转染后，采用qRT-PCR分析miR-3614-3p的表达水平。(C) 慢病毒转染后，采用蛋白质印迹法分析TRIM25的表达水平（**P < 0.01，学生t检验）。 图S5 乳腺癌组织中IGF2BP3的表达情况。(A) 采用qRT-PCR检测乳腺癌组织与非肿瘤组织中IGF2BP3的mRNA表达水平，并展示样本表达对比。(B) 非肿瘤组织与乳腺癌组织芯片中IGF2BP3的免疫组织化学（Immunohistochemistry, IHC）代表性图像，放大倍数×40，比例尺50 μm。(C) 采用qRT-PCR与蛋白质印迹法定量检测乳腺癌细胞系（MCF-7、MDA-MB-231、HCC-1937及MDA-MB-453）与正常人类乳腺细胞系（HBL-100）中IGF2BP3的mRNA与蛋白水平（*P < 0.05，**P < 0.01，学生t检验）。 图S6 敲低IGF2BP3可通过慢病毒转染实现。(A) 通过荧光显微镜观察GFP的表达以评估慢病毒感染效率，放大倍数×40，比例尺50 μm。(B) 慢病毒转染后，采用qRT-PCR分析IGF2BP3的表达水平。(C) 慢病毒转染后，采用蛋白质印迹法分析IGF2BP3与TRIM25的表达水平（**P < 0.01，***P < 0.001，学生t检验）。