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. 2017 Oct 26;9:387–398. doi: 10.1016/j.omtn.2017.10.016

Figure 4.

Figure 4

The Suppression of Breast Tumor Growth and Metastasis by Shrimp miR-34 In Vivo

(A) A flow chart of the in vivo experiments. Tumor growth and metastasis were examined weekly. (B) Tumor growth curves in mice subjected to different treatments. (C) Sizes of the solid tumors in mice subjected to different treatments. Tumors were harvested 6 weeks after injection. (D) The expression level of miR-34 in the blood of mice. At 6 weeks after injection, the blood of mice subjected to different treatments was analyzed by real-time qPCR to evaluate miR-34 expression. (E) The detection of miR-34 expression in solid tumors of mice subjected to various treatments by real-time qPCR analysis. (F) The expression levels of miR-34 target genes in solid tumors. (G) The expression levels of miR-34 target gene-encoding proteins in solid tumors. Protein levels were detected by western blot analysis. Tubulin was used as a control. (H) Immunohistochemical analysis of miR-34 target gene-encoding proteins in solid tumors. The brown and blue colors represent the proteins and nuclei, respectively. The proteins for the detection are indicated on the left. Scale bar, 100 μm. (I) The loading of shrimp miR-34 onto human Ago2 complex in tumor cells derived from mice. Human Ago2 complex of tumor cells treated with shrimp mR-34 or physiological saline was immunoprecipitated, followed by western blot analysis (bottom). Shrimp miR-34 in human Ago2 complex was detected by real-time qPCR (top). (J) The metastasis of breast cancer cells in mice. The capacity of breast cancer cell metastasis in mice subjected to different treatments was examined weekly after injection. Images showing cancer cell metastasis to the lungs of mice at 4 weeks after injection are shown. (K) Representative images displaying lung tumor nodules in mice subjected to various treatments. (L) Detection of miR-34 expression level in the blood of mice subjected to different treatments using real-time qPCR. Statistically significant differences between treatments are indicated using asterisks (error bar, SD; *p < 0.05 and **p < 0.01).