MicroRNA-101 Inhibits Growth, Proliferation and Migration and Induces Apoptosis of Breast Cancer Cells by Targeting Sex-Determining Region Y-Box 2 Cell.

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MicroRNA-101 Inhibits Growth, Proliferation and Migration and Induces Apoptosis of Breast Cancer Cells by Targeting Sex-Determining Region Y-Box 2 Cell Physiol Biochem 2017;43:717–732 - DOI:10.1159/000481445 Fig. 1. miR-101 was significantly downregulated in BC tissues and cell lines. (A) By qRT-PCR, miR-101 was expressed at lower levels in 64 BC samples than paired carcinoma-adjacent samples. (B) By qRT-PCR, miR-101 was downregulated in the BC cell lines MCF7, MDA-MB-231 and SK-BR-3 compared with normal breast cell line MCF-10A. *P<0.05. **P<0.01. The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

MicroRNA-101 Inhibits Growth, Proliferation and Migration and Induces Apoptosis of Breast Cancer Cells by Targeting Sex-Determining Region Y-Box 2 Cell Physiol Biochem 2017;43:717–732 - DOI:10.1159/000481445 Fig. 2. miR-101 upregulation inhibits BC cell growth, proliferation and migration, and induces apoptosis. (A) By qRT-PCR, miR-101 was upregulated by miR-101 mimics in MCF-7 and MDA-MB-231 cells. (B) The effects of miR-101 mimics on the viability of BC cells was determined using the MTT assay. (C) MCF-7 and MDA-MB-231 cells were transfected with miR-101 mimics or negative control and subjected to a colony formation assay. The number of colonies was quantitatively analyzed. (D) The ability of cells to invade through Matrigel was decreased in the high miR-101 expression groups compared with control groups. (E) The wound healing rates of the high miR-101 expression groups were lower than control groups. (F) miR-101 overexpression induced apoptosis of BC cells. *P<0.05. **P<0.01. The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

MicroRNA-101 Inhibits Growth, Proliferation and Migration and Induces Apoptosis of Breast Cancer Cells by Targeting Sex-Determining Region Y-Box 2 Cell Physiol Biochem 2017;43:717–732 - DOI:10.1159/000481445 Fig. 3. miR-101 inhibitors increase BC cell growth, proliferation and migration, and suppress apoptosis. (A) By qRT-PCR, miR-101 was downregulated by miR-101 inhibitors in HBL-100 cells. (B) The effects of miR-101 inhibitors on the viability of BC cells was determined using the MTT assay. (C) HBL-100 cells were transfected with miR-101 inhibitors or negative control and subjected to a colony formation assay. The number of colonies was quantitatively analyzed. (D) The ability of cells to invade through Matrigel was increased in the low miR-101 expression groups compared with control groups. (E) The wound healing rates of the low miR-101 expression groups were faster than control groups. (F) miR-101 downregulation suppressed apoptosis of BC cells. **P<0.01. The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

MicroRNA-101 Inhibits Growth, Proliferation and Migration and Induces Apoptosis of Breast Cancer Cells by Targeting Sex-Determining Region Y-Box 2 Cell Physiol Biochem 2017;43:717–732 - DOI:10.1159/000481445 Fig. 4. SOX2 is a direct target of miR-101 in BC. (A) Sequences of the miR-101 binding site within the SOX2 3’UTR and its mutated version are as shown. (B) miR-101 overexpression suppressed SOX2 expression in MCF-7 and MDA-MB-231 cells and miR-101 inhibitors increased SOX2 expression in HBL-100 cells. (C) Relative Luciferase activity was repressed by adding the miR-101 binding site from the SOX2 3’UTR to the reporter construct. **P<0.01 The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

MicroRNA-101 Inhibits Growth, Proliferation and Migration and Induces Apoptosis of Breast Cancer Cells by Targeting Sex-Determining Region Y-Box 2 Cell Physiol Biochem 2017;43:717–732 - DOI:10.1159/000481445 Fig. 5. miR-101 overexpression suppresses the growth of subcutaneous transplanted tumors and induces apoptosis. (A) Subcutaneous tumor volumes of the miR-101 overexpression groups were smaller than control groups in the xenograft tumorigenicity assay. (B) Tumor volumes were measured every 4 days for 32 days. The results demonstrated that miR-101 upregulation inhibited tumor growth in vivo. (C) SOX2 expression in transplanted tumors. (D) Apoptosis in transplanted tumors was determined by TUNEL assay. miR-101 upregulation induced apoptosis in vivo. **P<0.01. The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

MicroRNA-101 Inhibits Growth, Proliferation and Migration and Induces Apoptosis of Breast Cancer Cells by Targeting Sex-Determining Region Y-Box 2 Cell Physiol Biochem 2017;43:717–732 - DOI:10.1159/000481445 Fig. 6. SOX2 is overexpressed in BC tissues and cell lines. (A) Positive SOX2 expression was detected in the nuclei of paraffinsectioned BC tissues. Strong positive SOX2 expression is shown in case1 and weak positive expression is shown in case2. Western blotting was performed in BC tissue samples and paired adjacent tissues. Four cases of strong positive SOX2 expression in BC tumor tissues are as shown. (B) At both the protein and mRNA levels, SOX2 was upregulated in BC cells. (C) In BC tissues, the expression of miR-101 and SOX2 were negative correlated. **P<0.01. The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

MicroRNA-101 Inhibits Growth, Proliferation and Migration and Induces Apoptosis of Breast Cancer Cells by Targeting Sex-Determining Region Y-Box 2 Cell Physiol Biochem 2017;43:717–732 - DOI:10.1159/000481445 Fig. 7. SOX2 knockdown inhibits BC cell growth, proliferation and migration, and induces apoptosis. (A) SOX2 was silenced by shRNA in MCF-7 and MDA-MB-231 cells. (B) The influence of SOX2 downregulation on the viability of BC cells was determined using the MTT assay. (C) BC cells were transfected with sh-SOX2 or sh-NC and then subjected to a colony formation assay. The number of colonies in the sh-SOX2 group was significantly lower than the sh-NC group. (D) The ability of cells to invade through Matrigel was decreased in the sh-SOX2 groups compared with the control groups. (E). The effect of SOX2-knockdown on cell migration were determined using the wound healing assay. The wound healing rates of sh-SOX2 groups were lower than control groups. (F). SOX2 silencing induced apoptosis of BC cells. *P<0.05. **P<0.01. The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

MicroRNA-101 Inhibits Growth, Proliferation and Migration and Induces Apoptosis of Breast Cancer Cells by Targeting Sex-Determining Region Y-Box 2 Cell Physiol Biochem 2017;43:717–732 - DOI:10.1159/000481445 Fig. 8. miR-101 inhibits BC cell malignant phenotypes and induces apoptosis by directly targeting SOX2. (A) MTT assays were performed to show the viability of BC cells. (B). The relative proliferation of BC cells was assessed using colony formation assays. (C) Transwell assays were performed to show the ability of BC cells to invade through Matrigel. (D) Wound healing assays showed the migratory ability of BC cells. (E) BC apoptosis rates were detected by flow cytometry. (F) miR-101 overexpression and SOX2 down-regulation can both regulate the levels of Bcl2, Bax and cleaved caspase3. SOX2 overexpression can mitigate the changes in Bcl2, Bax and cleaved caspase 3 levels induced by miR-101 upregulation. *P<0.05, **P<0.01. The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0