Volume 24, Issue 10, Pages 1806-1822 (October 2016) Role of HCP5-miR-139-RUNX1 Feedback Loop in Regulating Malignant Behavior of Glioma Cells  Hao Teng, Ping Wang, Yixue Xue, Xiaobai Liu, Jun Ma, Heng Cai, Zhuo Xi, Zhen Li, Yunhui Liu  Molecular Therapy  Volume 24, Issue 10, Pages 1806-1822 (October 2016) DOI: 10.1038/mt.2016.103 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Expression of histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) in human glioma tissues and cell lines, and the effects of HCP5 on glioma cells. (a) The expression HCP5 in normal brain tissues (NBTs), Grade I–II glioma tissues, Grade III and Grade IV glioma tissues. Error bars represent as the mean ± SD (n = 5, each group). **P < 0.01, ▴▴P < 0.01, #P < 0.05. (b) The expression of HCP5 in human normal astrocytes and glioblastoma multiforme (GBM) cell lines (U87 and U251). Error bars represent as the mean ± SD (n = 5, each group). **P < 0.01. (c) Effect of HCP5 Knockdown on cell proliferation of U87 and U251 cells. (d) Effect of HCP5 Knockdown on cell migration and invasion of U87 and U251 cells. (e) Effect of HCP5 Knockdown on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05. Scale bars represent 20 μm. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 MicroRNA-139 (miR-139) was down-regulated by histocompatibility leukocyte antigen (HLA) complex P5 (HCP5). (a) Relative expression of miR-139 after cells transfected with the expression of HCP5 changed. Error bars represent as the mean ± SD (n =5, each group). *P < 0.05. (b) Relative luciferase activity was performed by dual-luciferase reporter assay. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05. (c) Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect HCP5 in the sample pulled down by biotinylated miR-139. (n = 5, each group). **P < 0.01. (d) QRT-PCR was used to detect miR-139 in the sample pulled down by biotinylated HCP5 probe. (n = 5, each group). **P < 0.01. (e) MiR-139 expression in normal brain tissues (NBTs), Grade I–II glioma tissues, Grade III and Grade IV glioma tissues. Error bars represent as the mean ± SD (n = 5, each group). **P < 0.01, ▴▴P < 0.01, #P < 0.05. (f) MiR-139 expression in human normal astrocytes and GBM cell lines (U87 and U251). Error bars represent as the mean ± SD (n = 5, each group). **P < 0.01. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 The effects of microRNA-139 (miR-139) on glioma cell lines. (a) Effect of miR-139 on cell proliferation of U87 and U251 cells. (b) Effect of miR-139 on cell migration and invasion of U87 and U251 cells. (c) Effect of miR-139 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, #P < 0.05. Scale bars represent 20 μm. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 MicroRNA-139 (miR-139) inhibited the expression of Runt-related transcription factor 1 (RUNX1) by targeting its 3′-untranslated region (3′-UTR). (a) Western blot analysis for RUNX1 in U87 and U251 cells, after cells transfected with the expression of miR-139 changed. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, #P < 0.05. (b) Relative luciferase activity was performed by dual-luciferase reporter assay. Data represent mean ± SD (n = 5, each). *P < 0.05. (c) The expression of RUNX1 in normal brain tissues (NBTs), Grade I–II glioma tissues, Grade III and Grade IV glioma tissues. Error bars represent as the mean ± SD (n = 5, each group). ***P < 0.001, **P < 0.01, ▴▴▴ P < 0.001,▴▴ P < 0.01, #P < 0.05. (d) RUNX1 expression in human normal astrocytes and glioblastoma multiforme (GBM) cell lines (U87 and U251). Error bars represent as the mean ± SD (n = 5, each group). ***P < 0.001. (e) The protein expression of RUNX1 in human glioma tissues. Error bars represent as the mean ± SD (n = 5, each group). ***P < 0.001, **P < 0.01, ▴▴▴ P < 0.001,▴▴ P < 0.01, #P < 0.05. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 The effects of Runt-related transcription factor 1 (RUNX1) on glioma cell lines. (a) Effect of RUNX1 on cell proliferation of U87 and U251 cells. (b) Effect of RUNX1 on cell migration and invasion of U87 and U251 cells. (c) Effect of RUNX1 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, #P < 0.05. Scale bars represent 20 μm. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 MicroRNA-139 (miR-139) mediated the tumor-suppressive effects of histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) knockdown on glioma cell lines. (a) Cell Counting Kit-8 (CCK8) assay to evaluate the effect of HCP5 and miR-139 on cell proliferation in U87 and U251 cells. (b) Transwell assay to evaluate the effect of HCP5 and miR-139 on cell migration and invasion of U87 and U251 cells. (c) Flow cytometry analysis to evaluate the effect of HCP5 and miR-139 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, **P < 0.01, #P < 0.05. Scale bars represent 20 μm. (d) Western blot analysis for Runt-related transcription factor 1 (RUNX1) in U87 and U251 cells with the expression of HCP5 changed. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, #P < 0.05. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 MicroRNA-139 (miR-139) mediated the tumor-suppressive effects of histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) knockdown on glioma cell lines. (a) Cell Counting Kit-8 (CCK8) assay to evaluate the effect of HCP5 and miR-139 on cell proliferation in U87 and U251 cells. (b) Transwell assay to evaluate the effect of HCP5 and miR-139 on cell migration and invasion of U87 and U251 cells. (c) Flow cytometry analysis to evaluate the effect of HCP5 and miR-139 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, **P < 0.01, #P < 0.05. Scale bars represent 20 μm. (d) Western blot analysis for Runt-related transcription factor 1 (RUNX1) in U87 and U251 cells with the expression of HCP5 changed. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, #P < 0.05. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Runt-related transcription factor 1 (RUNX1) promoted the expression of astrocyte elevated gene-1 (AEG-1) and bound to the promoters of AEG-1. (a) Western blot analysis for AEG-1in U87 and U251 cells with the expression of RUNX1 changed. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, #P < 0.05. (b) RUNX1 on the promoter activity of AEG-1 in U87 and U251 cell lines. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05. (c) RUNX1 bound to the promoter of AEG-1 in U87 and U251 cell lines. Schematic representation of the human AEG-1 promoter region 3,000 bp upstream of the transcription start site (transcription start site (TSS), designated as +1). Polymerase chain reaction (PCR) was conducted with the resulting precipitated DNA. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Runt-related transcription factor 1 (RUNX1) mediated tumor-suppressive effects of microRNA-139(miR-139). (a) Cell Counting Kit-8 (CCK8) assay to evaluate the effect of miR-139 and RUNX1 on cell proliferation in U87 and U251 cells. (b) Transwell assay to evaluate the effect of miR-139 and RUNX1 on cell migration and invasion of U87 and U251 cells. (c) Flow cytometry analysis to evaluate the effect of miR-139 and RUNX1 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, ▴P < 0.05, #P < 0.05. Scale bars represent 20 μm. (d) Western blot analysis for astrocyte elevated gene-1 (AEG-1) in U87 and U251 cells with the expression of miR-139 and RUNX1 changed. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, #P < 0.05, ▴P < 0.05, &P < 0.05. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Runt-related transcription factor 1 (RUNX1) mediated tumor-suppressive effects of microRNA-139(miR-139). (a) Cell Counting Kit-8 (CCK8) assay to evaluate the effect of miR-139 and RUNX1 on cell proliferation in U87 and U251 cells. (b) Transwell assay to evaluate the effect of miR-139 and RUNX1 on cell migration and invasion of U87 and U251 cells. (c) Flow cytometry analysis to evaluate the effect of miR-139 and RUNX1 on cell apoptosis of U87 and U251 cells. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, ▴P < 0.05, #P < 0.05. Scale bars represent 20 μm. (d) Western blot analysis for astrocyte elevated gene-1 (AEG-1) in U87 and U251 cells with the expression of miR-139 and RUNX1 changed. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, #P < 0.05, ▴P < 0.05, &P < 0.05. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 9 Runt-related transcription factor 1 (RUNX1) feedback promoted histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) expression by binding to the promoters of HCP5. (a) Relative expression of HCP5 in U87 and U251 cells with the expression of RUNX1 changed. Error bars represent as the mean ± SD (n = 5, each group). *P < 0.05, #P < 0.05. (b) RUNX1 on the promoter activity of HCP5 in U87 and U251 cell lines. Error bars represent as the means ± SD (n = 5, each). *P < 0.05. (c) RUNX1 bound to the promoter of HCP5 in U87 and U251 cell lines. Schematic representation of the human HCP5 promoter region 3,000 bp upstream of the transcription start site (transcription start site (TSS), designated as +1). Polymerase chain reaction (PCR) was conducted with the resulting precipitated DNA. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 10 In vivo tumor xenografts study. The stable expressing cells were used for the in vivo study. (a) The nude mice carrying tumors from respective groups were shown. (b) The sample tumor from representative groups was shown. (c) Tumor growth curves in nude mice. Tumor volume was calculated every five days after injection. (d) Tumors were harvested on day 30 and weighed. (e) The survival curves of nude mice injected into the right striatum. Error bars represent as the means ± SD (n = 6, each group). *P < 0.05, #P < 0.05,▴P < 0.01. Molecular Therapy 2016 24, 1806-1822DOI: (10.1038/mt.2016.103) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions