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Figure 3 | Breast Cancer Research

Figure 3

From: MicroRNA-18a inhibits hypoxia-inducible factor 1α activity and lung metastasis in basal breast cancers

Figure 3

Effects of ectopic microRNA-18a expression on primary tumor growth and spontaneous lung metastasis. (A) Growth curves of primary tumors generated from lung metastatic and green fluorescent protein microRNA-18a cells (LM-miR-18a and LM-GFP, respectively). The results are presented as mean ± SE (n = 10). *P < 0.05 by Kruskal-Wallis analysis of variance followed by Dunn’s multiple-comparisons test. (B) Wet weights of primary tumors that were surgically removed at either 4 weeks or 6 weeks postinoculation. *P < 0.05 by Kruskal-Wallis analysis of variance followed by Dunn’s multiple-comparisons test. (C) Expression levels of miR-18a in end-stage primary tumors. Total RNA was prepared from primary tumor tissues harvested 6 weeks after inoculation and subjected to quantitative PCR (qPCR) analysis for miRNA expression. The results are presented as mean ± SD (n = 3). *P < 0.05 by Student’s t-test. (D) Spontaneous lung metastasis from orthotopic sites. Metastatic foci of red fluorescent protein (RFP)–expressing cells on the dorsal surface of the left lung lobe were imaged and counted using a fluorescence microscope at × 10 magnification. *P < 0.05 by Mann-Whitney U test (n = 8). The presence of tumor cells in the lungs was visualized by hematoxylin and eosin (H&E) staining of formalin-fixed lung sections (10 μM) and quantified by qPCR using the human-specific primers of Alu repeats (hAlu-qPCR). *P < 0.05 by Mann-Whitney U test (n = 8). (E) Growth curves of primary tumors and images of lung metastasis generated from MDA-MB-231 sublines with low miR-18a activity (MB231-18aIN) and control cells expressing mCherry only (MB231-C). Tumor volumes are presented as mean ± SE (n = 14). Metastatic foci of mCherry-expressing cells on the dorsal surface of the left lung lobe were imaged using a fluorescence microscope at × 10 magnification. The presence of tumor cells in the lungs was quantified by hAlu-qPCR. *P < 0.05 by Mann-Whitney U test (n = 7). n, the number of experimental repeats.

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