1. Derepression of co-silenced tumor suppressor genes by nanoparticle-loaded circular ssDNA reduces tumor malignancy
by Jing Meng, Shuang Chen, Jing-xia Han, Qiang Tan, Xiao-rui Wang, Hong-zhi Wang, Wei-long Zhong, Yuan Qin, Kai-liang Qiao, Chao Zhang, Wan-feng Gao, Yue-yang Lei, Hui-juan Liu, Yan-rong Liu, Hong-gang Zhou, Tao Sun, and Cheng Yang
Sci Transl Med
Volume 10(442):eaao6321
May 23, 2018
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

2. Fig. 1 Co-silencing of KLF17, CDH1, and LASS2 expression correlated with escalated tumor malignancy.
Co-silencing of KLF17, CDH1, and LASS2 expression correlated with escalated tumor malignancy. (A) Cluster analysis based on differences in median survival time of patients with different expression levels of 51 tumor suppressor genes between breast, lung, and ovarian cancer in Kaplan-Meier plotter (14). (B) Identification of five tumor suppressor genes with relatively large differences in median survival time. (C) Survival time of hepatocellular carcinoma patients with low or high expression of KLF17, CDH1, and LASS2. (D) Percentages of clinical stage, pathological grade, (E) metastasis, and (F) AFP in groups of hepatocellular carcinoma patients with low (−) or high (+) expression of KLF17, CDH1, and LASS2.
Jing Meng et al., Sci Transl Med 2018;10:eaao6321
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

3. Fig. 2 Increasing KLF17, CDH1, and LASS2 expression reduced malignant progression and promoted apoptosis of tumor cells.
Increasing KLF17, CDH1, and LASS2 expression reduced malignant progression and promoted apoptosis of tumor cells. (A) Western blot analysis of KLF17, CDH1, and LASS2 expression in HeLa cells transfected with pcDNA3.1-KLF17, pcDNA3.1-CDH1, or pcDNA3.1-LASS2, respectively. (B) Cell proliferation, (C) migration (scale bars, 50 μm), (D) invasion (scale bars, 100 μm), (E) colony formation (scale bars, 2 mm), and (F) apoptosis of HeLa cells transfected with pcDNA3.1-KLF17, pcDNA3.1-CDH1, and pcDNA3.1-LASS2 were measured. FITC, fluorescein isothiocyanate. All data are represented as means ± SEM versus control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P <
Jing Meng et al., Sci Transl Med 2018;10:eaao6321
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

4. Fig. 3 Nanoparticle-loaded CSSD-9 served as a miR-9 sponge that increased tumor suppressor gene expression and inhibited tumor proliferation and metastasis.
Nanoparticle-loaded CSSD-9 served as a miR-9 sponge that increased tumor suppressor gene expression and inhibited tumor proliferation and metastasis. (A) Motifs from miRNAs targeting KLF17, CDH1, and LASS2 found by a database search. (B) KLF17, CDH1, and LASS2 protein levels assessed by Western blot analysis after transfection with miR-9 for 48 hours. (C) Several miR-9 sponges (linear RNA-9, circR-9, CSSD-9-s, and CSSD-9) were designed. (D) Stability of engineered miRNA sponges after exonuclease VII (Exo VII), T5 exonuclease (T5 Exo), RNase R, medium with serum, or transfection reagent treatment. (E) Representative scanning electron microscopy images of nanoparticles. (F) qRT-PCR detection of miR-9 expression in HeLa cells after transfection of nanoparticles with different miR-9 sponges for different time points. miR-9 expression was normalized by reference gene U6 small nuclear RNA expression. (G) Western blot analysis of KLF17, CDH1, and LASS2 proteins in HeLa cells transfected with nanoparticles with different miR-9 sponges. (H) Scanning electron microscopy images of phenotypic changes observed 48 hours after HeLa cells were transfected by CSSD-9 nanoparticles. The red arrows indicate apoptotic vesicles. Scale bars, 10 μm (left) and 5 μm (right). (I) Cell proliferation, (J) migration, (K) invasion, (L) colony formation, and (M) apoptosis were detected in HeLa cells transfected with CSSD-9 nanoparticles at doses of 0.25, 0.5, and 1 μg per 105 cells. All data are represented as means ± SEM. *P < 0.05, **P < 0.01, **** P <
Jing Meng et al., Sci Transl Med 2018;10:eaao6321
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

5. Fig. 4 Gene expression analysis revealed key biological functions influenced by CSSD-9.
Gene expression analysis revealed key biological functions influenced by CSSD-9. (A) Workflow of the whole-genome gene expression chip analysis of cells treated with CSSD-9. (B) Up- and down-regulated biological processes and molecular functions in HeLa cells 48 hours after CSSD-9 treatment. Up-regulated biological processes including immune and inflammatory response and apoptotic signaling pathway are shown in green. Down-regulated biological processes including migration, invasion, proliferation, angiogenesis, and growth factors are shown in red. IκB, inhibitor of nuclear factor κB; NF-κB, nuclear factor κB. (C) Up-regulated genes in HeLa cells after CSSD-9 treatment were associated with inflammatory response, immune response, apoptosis, and chemokines/chemokine receptors. (D) Down-regulated genes in HeLa cells after CSSD-9 treatment were associated with migration, invasion, angiogenesis, extracellular matrix, growth factors, and chemokines/chemokine receptors. (E) Inferred up-regulated protein-protein interaction networks based on differentially expressed genes after CSSD-9 treatment were enriched for apoptosis and inflammatory response. (F) Inferred down-regulated protein-protein interaction networks based on differentially expressed genes after CSSD-9 treatment were enriched for invasion, migration, and proliferation.
Jing Meng et al., Sci Transl Med 2018;10:eaao6321
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

6. Fig. 5 CSSD-9 sensitivity against multiple tumor cells.
CSSD-9 sensitivity against multiple tumor cells. (A) Proliferation of HeLa, SiHa, A549, H1299, and HepG2 cells was measured at 48 hours after transfection with CSSD-9. (B) Gelatin zymography demonstrating repressed MMP2 and MMP9 activity in HeLa, SiHa, A549, H1299, and HepG2 cells after CSSD-9 treatment. (C) Cell invasion was determined by a Matrigel-coated transwell assay. The cells that crossed the Matrigel-coated filter were fixed, stained, and counted. (D) Migration of HeLa, SiHa, A549, H1299, and HepG2 cells was detected at 24 and 48 hours after transfection with CSSD-9. Data were normalized to 0 hours. (E) Effect of CSSD-9 on apoptosis of tumor cells analyzed by flow cytometry. Harvested cells were stained with annexin V and propidium iodide to determine the percentage of early and late apoptotic cells and viable cells. All data are represented as means ± SEM. **P < 0.01, ****P <
Jing Meng et al., Sci Transl Med 2018;10:eaao6321
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

7. Fig. 6 Antitumor effect on tumor growth and pulmonary metastasis of CSSD-9 in vivo.
Antitumor effect on tumor growth and pulmonary metastasis of CSSD-9 in vivo. (A) HeLa tumor volumes after CSSD-9 treatment at different doses. When tumors reached 100 to 200 mm3 in size, mice were treated with 10 and 20 μg of CSSD-9 via I.T. or I.V. injection. Tumor sizes were measured every 3 days, and tumor volumes were calculated. (B) SiHa, A549, and HepG2 tumor images and growth curves after CSSD-9 treatment by I.V. and I.T. injection. (C) Visible metastatic nodules on the surface of lungs in the control and CSSD-9–treated mice (top). Representative figures of hematoxylin and eosin (H&E) staining were performed on serial sections of metastatic tumors in the lungs. Scale bars, 50 μm. (D) Number of metastatic lung nodules was quantified in the control and CSSD-9 treatment groups. (E) miR-9 expression in the control and CSSD-9 groups was detected by qRT-PCR after lysis of tumor tissues and RNA extraction. (F) mRNA expression of KLF17, CDH1, and LASS2 in control and CSSD-9 treatment groups was detected by qRT-PCR. (G) Expression of KLF17, CDH1, and LASS2 was confirmed with Western blot analysis after lysis of tumor tissues. (H) Left: In vivo fluorescence imaging of a BALB/c nude mouse that received I.V. injection of A549-GFP cells and treated with CSSD-9 by I.V. injection. Right: CTCs were detected by flow cytometry. (I) Infiltration of CD8+ T cell (CD3+CD8+) and IFN-γ+ cell percentage in the tumor tissues after CSSD-9 treatment by I.T. or I.V. injection. (J) Percentages of MDSCs in tumor tissues, bone marrow (BM), and spleen after CSSD-9 treatment. All data are represented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, **** P <
Jing Meng et al., Sci Transl Med 2018;10:eaao6321
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

8. Fig. 7 CSSD-9 specifically inhibited tumors with high miR-9 expression in the PDX model.
CSSD-9 specifically inhibited tumors with high miR-9 expression in the PDX model. (A) Information on clinicopathological features of patients with different tumors. (B) Schematic of primary tumor treatment and I.T. injection of CSSD-9 in the PDX model. (C) Representative images of positive and negative KLF17, CDH1, and LASS2 expression and miR-9 expression in different hepatocellular carcinoma tissues detected by IHC and FISH. (D) Linear regression analysis indicated a significant negative correlation between miR-9 and the expression of KLF17, CDH1, and LASS2 in tumor tissues of patients. (E) Tumor growth inhibition rate of CSSD-9 was positively correlated with miR-9 and negatively correlated with the expression of KLF17, CDH1, and LASS2.
Jing Meng et al., Sci Transl Med 2018;10:eaao6321
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works