miR-223 is repressed and correlates with inferior clinical features in mantle cell lymphoma through targeting SOX11  Keshu Zhou, Xiaoyan Feng, Yanying Wang, Yanyan Liu, Long Tian, Wenli Zuo, Shuhua Yi, Xudong Wei, Yongping Song, Lugui Qiu  Experimental Hematology  Volume 58, Pages 27-34.e1 (February 2018) DOI: 10.1016/j.exphem.2017.10.005 Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

Figure 1 miR-223 is downregulated in MCL patients. (a) qRT-PCR was performed on isolated CD19+ lymphocytes from PBMCs of 21 treatment-naive MCL patients and 20 healthy donors. Results are shown as the relative expression level compared with RNU48 and as mean ± SD. (b) Statistical significance was determined by nonparametric test. ****p < 0.0001. Experimental Hematology 2018 58, 27-34.e1DOI: (10.1016/j.exphem.2017.10.005) Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

Figure 2 miR-223 predicts poor clinical outcome in MCL. OS was analyzed for MCL patients according to expression of miR-223. Survival analysis was performed via Kaplan–Meier survival analysis, with differences between curves analyzed by log–rank test. Experimental Hematology 2018 58, 27-34.e1DOI: (10.1016/j.exphem.2017.10.005) Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

Figure 3 miR-223 overexpression reduces cell proliferation while boosting apoptosis. (a) Ectopic expression of miR-223 plasmid was constructed and transduced into the Granta519 cell line. The expression level of miR-223 was determined by qRT-PCR and is shown as relative expression to empty vector (“Vector”). (b) Cell viability was determined using the CCK-8 method after incubation for the indicated time periods and is shown as absorbance values. (c) Percentages of each subset in different cell cycle phases based on propidium iodide staining. (d) Apoptosis as detected by Annexin V staining. Error bars represent the SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. Experimental Hematology 2018 58, 27-34.e1DOI: (10.1016/j.exphem.2017.10.005) Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

Figure 4 miR-223 targets SOX11 directly. (a) Seed sequences of miR-223 and wild-type/mutated 3′-UTR SOX11. Complement bases are highlighted in red. (b) Protein level of SOX11 of miR-223-overexpressing or control Grant519 cells. (c) Luciferase assay performed by cotransfecting HEK293T cells with the indicated combination of plasmids. Data are shown as the mean ± SD of three independent experiments. 3′-UTR NC is represented as the empty plasmid control of 3′-UTR of SOX11. (d) Correlation of miR-223 and SOX11 on 21 MCL patients. Data are expressed as relative expression levels to internal control RNU48. Experimental Hematology 2018 58, 27-34.e1DOI: (10.1016/j.exphem.2017.10.005) Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

Figure E1 Plasmids construction. (a) Plasmid diagram of miR-223 overexpression. (b) Plasmid diagram for luciferase assay. Experimental Hematology 2018 58, 27-34.e1DOI: (10.1016/j.exphem.2017.10.005) Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

Figure E2 Transfection efficiencies determined by fluorescence microscope, left panel represented bright field, while right panel was obtained from fluorescence field. Experimental Hematology 2018 58, 27-34.e1DOI: (10.1016/j.exphem.2017.10.005) Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions