LncRNA ODRUL Contributes to Osteosarcoma Progression through the miR- 3182/MMP2 Axis  Kun-Peng Zhu, Xiao-Long Ma, Chun-Lin Zhang  Molecular Therapy  Volume 25, Issue 10, Pages 2383-2393 (October 2017) DOI: 10.1016/j.ymthe.2017.06.027 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 ODRUL Was Upregulated in the Osteosarcoma Tissues and Cell Lines, Correlated with Lung Metastasis and Worse Prognosis (A) Expression level of ODRUL in five human OS cell lines and normal osteoblast cell line hFOB1.19. (B) Expression level of ODRUL in 80 pairs of OS and paracancerous tissues. (C) Expression level of ODRUL in OS tissues of lung metastasis and lung non-metastasis groups at early stage. (D) OS patients with higher expression of ODRUL had a shorter overall survival time than those with lower expression. Data are presented as mean ± SEM. *p < 0.05. Molecular Therapy 2017 25, 2383-2393DOI: (10.1016/j.ymthe.2017.06.027) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 ODRUL Promoted OS Cell Proliferation, Migration, Invasion, and Tumor Growth In Vitro and In Vivo (A) qRT-PCR analysis of the effect on knockdown or overexpression of ODRUL by shRNA or vector transfection. (B) CCK-8 assays were performed to examine the cell proliferation rate of MG63 and 143B cells after knockdown or overexpression of ODRUL. (C) Clone formation assays were performed to examine cell vitality after transfection. (D) Transwell assays were performed to identify the capacity of cell invasion after transfection. (E) Wound healing assays were performed to examine the capacity of cell migration after transfection. (F) General conditions and in vivo imaging of nude mice in the four groups when exposed to the same treatment. (G) The nude mice were sacrificed in the seventh week. Tumors that formed in the ODRUL group grew much faster, compared with those in the ODRUL-NC group, and the volumes of transplanted tumors and weights of nude mice were smaller in the sh-ODRUL group when compared with the sh-NC group. Data are presented as mean ± SEM. *p < 0.05. Molecular Therapy 2017 25, 2383-2393DOI: (10.1016/j.ymthe.2017.06.027) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 ODRUL Was Predominantly Localized in the Cytoplasm and Regulated Posttranscriptional Expression of miR-3182 and MMP2 (A) Subcellular localization of ODRUL by RNA-FISH in the MG63 and 143B cells. Nuclei are stained blue (DAPI), and ODRUL is stained red. (B) Nuclear and cytoplasmic fractions assay further validated the subcellular localization of ODRUL. (C) ODRUL regulated the mRNA level of MMP2 expression. (D) ODRUL regulated the protein level of MMP2 expression. (E) miRNA and mRNA microarrays were used to screen differentially expressed miRNAs and mRNAs associated with ODRUL in the paired sh-ODRUL and sh-NC MG63 cells. (F) qRT-PCR was performed to study the interaction of the miRNA expression levels with ODRUL overexpression or knockdown, and only miR-3182 was consistently negative with the expression of ODRUL. (G) qRT-PCR was performed to study the interaction between the ODRUL expression levels with miR-3182 overexpression or knockdown. (H) The miR-3182 response element (MRE) between the sequence of ODRUL and MMP2 by bioinformatics analysis. (I) miR-3182 negatively regulated the mRNA level of MMP2 expression. (J) miR-3182 negatively regulated the protein level of MMP2 expression. Data are presented as mean ± SEM. *p < 0.05. Molecular Therapy 2017 25, 2383-2393DOI: (10.1016/j.ymthe.2017.06.027) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 ODRUL Was Directly Targeted by miR-3182, Further Regulating the Expression of MMP2 through Competitively Binding with miR-3182 (A) The mRNA expression of MMP2 was examined in the MG63 and 143B cells co-transfected with miR-3182 mimics or inhibitor, ODRUL vector or sh-ODRUL, or their NCs. (B) The protein expression of MMP2 was examined in the same cells previously described. (C) Transwell assays were performed to observe the biological behaviors of OS cells co-transfected with miR-3182 mimics or inhibitor, sh-ODRUL or ODRUL vector, and their NCs. (D) WT and MUT sequences designed for the ODRUL and MMP2 according to their binding sites with miR-3182. (E) ODRUL luciferase activity assays. Wild-type or mutant ODRUL was co-transfected with miR-3182 mimics or miR-3182 inhibitor, respectively. Mir-3182 mimics repressed, but miR-3182 inhibitor enhanced, the luciferase activity of the WT-ODRUL reporter, including the wild-type sequence of ODRUL. There was no obvious change of the luciferase activity for the MUT-ODRUL reporter, which contained mutant ODRUL sequence. (F) MMP2 3′ UTR luciferase activity assays. Overexpression of ODRUL rescued the luciferase activity, which was repressed by the transfection with miR-3182 mimics in the WT-MMP2 but not in the MUT-MMP2 reporter. (G) Downregulation of ODRUL with shRNA reversed the luciferase activity, which was enhanced by the transfection with miR-3182 inhibitor in the WT-MMP2 but not in MUT-MMP2 reporter. The normalized luciferase activity in the control group was set to 1. Data are presented as mean ± SEM. *p < 0.05. Molecular Therapy 2017 25, 2383-2393DOI: (10.1016/j.ymthe.2017.06.027) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 miR-3182 Expression Was Inversely Correlated with ODRUL and Poor Prognosis (A) Expression level of miR-3182 in five human OS cell lines and normal osteoblast cell line hFOB1.19. (B) Expression level of miR-3182 in 80 pairs of OS and paracancerous tissues. (C) The relative expression levels of ODRUL and miR-3182 in five human OS cell lines and normal osteoblast cell line hFOB1.19. (D) The relative expression levels of ODRUL and miR-3182 in 80 pairs of OS and paracancerous tissues. (E) Expression level of miR-3182 in OS tissues of lung metastasis and lung non-metastasis groups at early stage. (F) OS patients with lower expression of miR-3182 had a shorter overall survival time than those with higher expression. Data are presented as mean ± SEM. *p < 0.05. Molecular Therapy 2017 25, 2383-2393DOI: (10.1016/j.ymthe.2017.06.027) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 6 miR-3182 Suppressed OS Cell Proliferation, Migration, Invasion, and Tumor Growth In Vitro and In Vivo (A) CCK-8 assays were performed to examine the cell proliferation rate of cells transfected with miR-3182 mimics or inhibitor. (B) Clone formation assays were performed to examine the vitality of cells transfected with miR-3182 mimics or inhibitor. (C) Transwell assays were performed to identify the capacity of cell invasion after miRNA transfection. (D) Wound healing assays were performed to examine the capacity of cell migration after miRNA transfection. (E) General conditions and in vivo imaging of nude mice in the miR-3182-mimics and mimics-NC transfected groups when exposed to the same treatment. (F) The nude mice were sacrificed in the seventh week. Tumors formed in the miR-3182 mimics group grew more slowly, compared with the mimics-NC group, and the volumes of transplanted tumors were smaller in the miR-3182 mimics group when compared with those in the mimics-NC group. Data are presented as mean ± SEM. *p < 0.05. Molecular Therapy 2017 25, 2383-2393DOI: (10.1016/j.ymthe.2017.06.027) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions