Research ArticleCancer

Derepression of co-silenced tumor suppressor genes by nanoparticle-loaded circular ssDNA reduces tumor malignancy

See allHide authors and affiliations

Science Translational Medicine  23 May 2018:
Vol. 10, Issue 442, eaao6321
DOI: 10.1126/scitranslmed.aao6321

Protecting tumor suppressors

Strategies to target inactive tumor suppressor genes in cancer lag behind advances in targeting oncogenes. In new work, Meng et al. developed a therapeutic strategy to simultaneously up-regulate three different tumor suppressor genes that are sometimes co-silenced in different cancers by a microRNA called miR-9. They created a circular single-stranded DNA (CSSD) containing sequences to bind miR-9, thus sequestering it away from its target tumor suppressor genes. Loading the CSSDs onto nanoparticles improved delivery into human tumor cells. The CSSD increased expression of the three tumor suppressor proteins and displayed an antitumor effect in both cell line–based ex vivo models and dozens of patient-derived xenografts in mice.

Abstract

The co-silencing of multiple tumor suppressor genes can lead to escalated malignancy in cancer cells. Given the limited efficacy of anticancer therapies targeting single tumor suppressor genes, we developed small circular single-stranded DNA (CSSD) that can up-regulate the expression of co-silenced tumor suppressor genes by sequestering microRNAs (miRNAs) that negatively regulate these genes. We found that cancer patients with low tumor expression of the tumor suppressor genes KLF17, CDH1, and LASS2 had shortened survival times. The up-regulation of these genes upon transfection of artificial CSSD-9 inhibited tumor proliferation and metastasis and promoted apoptosis in vitro as well as in ex vivo and patient-derived xenograft models. In addition, CSSD is more stable and effective than current miRNA inhibitors, and transfecting CSSDs via nanoparticles substantially improved delivery efficiency. The use of a single CSSD can promote the inhibition of multiple tumor suppressor genes. This study provides evidence for the possibility of using CSSDs as therapeutic miRNA inhibitors to target the co-silencing of multiple tumor suppressor genes.

INTRODUCTION

Epigenetic dysregulation and somatic mutations leading to the silencing of multiple tumor suppressor genes are prevalent in tumors. Traditional antitumor drugs, however, mostly target single oncogenes and not tumor suppressor genes (1). Thus, nucleic acid–based methods to release the repression of tumor suppressor genes may be of clinical therapeutic significance and may help promote the development of new antitumor drugs.

The dysregulation of microRNAs (miRNAs) has been implicated in tumor malignancy, and each miRNA can have multiple target genes (2, 3). Therefore, regulating the expression of even a single miRNA to simultaneously release the co-silencing of multiple tumor suppressor genes may be an effective tumor therapy. To block the functions of oncogenic miRNAs, approaches based on oligonucleotides, small molecules, and miRNA “sponges” have been developed (4, 5). However, the tumor suppression efficiency and the duration of these inhibitors need to be improved (4, 6).

Circular RNAs (circRNAs) are a class of RNAs formed by covalently closed continuous loops; they can be evolutionarily conserved and more stable than linear RNAs in the cytoplasm (7, 8). Recently, circRNAs enriched with miRNA binding sites were observed to act as natural miRNA sponges (7, 9), by which they can regulate gene expression (10, 11). CircRNA MTO1 was found to act as a sponge of miR-9 to suppress hepatocellular carcinoma progression (12), and silencing of the endogenously occurring RNA circHIPK3 has been shown to inhibit human tumor cell growth by sponging multiple miRNAs (13). Because circRNAs are not easily degraded, they may absorb miRNAs more effectively than linear miRNA inhibitors. These observations suggested the potential of engineered circular nucleic acids rich in miRNA binding sites to inhibit miRNA function and prevent tumor growth.

Here, by mimicking circRNA characteristics, we designed an artificial circular single-stranded DNA (CSSD) molecule containing continuous, multiple miR-9 complementary sites that are stable and resistant to degradation. Our results showed that CSSDs effectively released multiple, co-silenced tumor suppressor genes (KLF17, CDH1, and LASS2), up-regulating their expression by absorbing oncogenic miRNA9 and consequently suppressing tumor progression and lung metastasis. To improve delivery efficiency, CSSD-9 was transfected via nanoparticles. Accordingly, this study may provide stable and effective miRNA inhibitors (CSSD) to release co-silenced tumor suppressor genes for further research and potential clinical application.

RESULTS

Co-silencing of KLF17, CDH1, and LASS2 expression is correlated with escalated tumor malignancy

To examine the effect of tumor suppressor genes on patient survival time in different tumors, we searched the Kaplan-Meier plotter database (14) and compared the median survival time linked to 51 tumor suppressor genes in breast, lung, and ovarian cancers by hierarchical cluster analysis (Fig. 1A). We also analyzed which common tumor suppressor genes’ expression was associated with relatively greater median survival time in the three cancers. Of these genes that possibly influenced median survival time, five were observed to overlap across the three cancers (Fig. 1B). Survival curves striated according to expression of either KLF17, CDH1, or LASS2 in breast, lung, and ovarian cancer showed that high expression of any of these tumor suppressor genes associated with longer median survival time (fig. S1). The three genes’ expression was positively correlated in breast cancer (fig. S2). We further tested the association between survival time and the expression of the three proteins in hepatocellular carcinoma tissues and found that simultaneous low expression of KLF17, CDH1, and LASS2 was associated with shorter overall survival in hepatocellular carcinoma patients (Fig. 1C). Moreover, we examined the correlations between KLF17, CDH1, and LASS2 expression and clinical stage or pathological grade in hepatocellular carcinoma cases. The percentages of clinical stages I and II were 50.8 and 63.9% and those of stages III and IV were 49.2 and 36.1% in groups with low and high expression of the three tumor suppressor genes, respectively (P = 0.029). The percentages of pathological grade III were 23.8 and 7.4% in the low and high expression groups, respectively (P = 0.004) (Fig. 1D). The percentages of metastasis were 63.3 and 26.7% (P = 0.002) (Fig. 1E), and the percentages of α-fetoprotein (AFP) higher than 400 ng/ml were 69.2 and 58.8% in the low and high expression groups, respectively (P = 0.010) (Fig. 1F). These findings indicated that the co-silencing of KLF17, CDH1, and LASS2 in hepatocellular carcinoma is strongly correlated with poor prognosis, tumor progression, metastasis, and serum AFP content.

Fig. 1 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.

miR-9 contributes to KLF17, CDH1, and LASS2 co-silencing and malignant tumor progression

We studied the effect of releasing the co-silencing of KLF17, CDH1, and LASS2 in cancer cells overexpressing these genes upon transfection (Fig. 2A). Overexpression of the three genes inhibited cell proliferation (Fig. 2B), migration (Fig. 2C), invasion (Fig. 2D), and colony formation (Fig. 2E) and promoted cell apoptosis (Fig. 2F). The effect was more evident when the three genes were overexpressed simultaneously than when each gene was overexpressed individually.

Fig. 2 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 < 0.0001.

Taking into account the efficiency of inhibiting tumor cells with overexpressed KLF17, CDH1, and LASS2, we screened miRNA databases for common carcinogenic miRNAs that targeted the three genes and verified the inhibitory effects of these miRNAs on the three target genes by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (fig. S3A). Furthermore, we found that 11 miRNA seed sequences shared some sequence similarity with miR-9 (Fig. 3A). We detected the proliferation inhibition caused by these miRNAs in HeLa cells, and the inhibitory effect of miR-9 on proliferation was the most obvious (fig. S3B). TCGA (The Cancer Genome Atlas) database query results also support the notion that miR-9 expression is higher in a variety of tumor tissues than in normal tissues (fig. S4). Bioinformatics analysis by using miRTarBase (15), TargetScan (16), or miRanda (17) revealed that miR-9 has highly conserved binding sites on the 3′ untranslated regions (3′UTRs) of KLF17, CDH1, and LASS2 (fig. S5A). Furthermore, we found that miR-9 suppressed KLF17, CDH1, and LASS2 3′UTR luciferase activity (fig. S5B) and decreased the expression of KLF17, CDH1, and LASS2 (Fig. 3B). Thus, the expression of these three genes can be increased by inhibiting miR-9.

Fig. 3 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 < 0.0001.

Nanoparticle-loaded CSSD-9 serves as a miR-9 sponge that increases tumor suppressor gene expression and inhibits tumor malignancy

To reduce the miR-9 content in tumor cells, we designed several inhibitors based on the sequence of mature miR-9. Linear RNA is an oligonucleotide containing one copy of the miR-9 binding sites; circR-9 and CSSD-9-s contained two binding sites, and CSSD-9 contained four miR-9 binding sites (Fig. 3C). Given that imperfect miRNA bulged sponges are more effective than sponges with a perfect antisense sequence (10, 11, 18), each miRNA binding site included a bulged site. Next, we used exonuclease VII, T5 exonuclease, ribonuclease (RNase) R, medium with serum, or transfection reagent for enzymatic digestion of engineered miRNA sponges. Compared to circR-9, CSSD-9 was more resistant to enzymatic degradation and more stable in medium with serum or transfection reagent (Fig. 3D).

Nanoparticle morphology was observed under a scanning electron microscope (Fig. 3E). CSSD-9 and other miR-9 inhibitors that we designed were transferred into cells on nanoparticles. The effect of CSSD-9 on reducing miR-9 content and increasing KLF17, CDH1, and LASS2 protein expression was the most obvious (Fig. 3, F and G). We observed the HeLa cell phenotype after CSSD-9 treatment using a scanning electron microscope. The cell morphology became spherical from the original spindle shape; cell surface schistose pseudopodia decreased, and apoptotic vesicles appeared (Fig. 3H). We also assessed the ability of CSSD-9 to inhibit tumor cell malignancy and found that CSSD-9 inhibited cell proliferation (Fig. 3I), migration (Fig. 3J), invasion (Fig. 3K), and colony formation (Fig. 3L) and promoted cell apoptosis (Fig. 3M). These effects of CSSD-9 on cell proliferation, migration, invasion, colony formation, and apoptosis were dose-dependent (0, 0.25, 0.5, and 1 μg per 105 cells), with a larger effect achieved at higher doses (Fig. 3).

Whole-genome gene expression chip analysis reveals key biological functions influenced by CSSD-9

To assess the influence of CSSD-9 on key biological functions, we analyzed the whole-genome gene expression in HeLa cells treated with CSSD-9. Gene expression profiling was conducted to assess the effects of CSSD-9 on cancer cells. Bulk RNA was extracted from cells treated with CSSD-9, and the expressed transcripts were analyzed by whole-genome gene expression chip (Fig. 4A). To analyze the expression of chemokine and growth factors in the whole-genome gene expression chip data, we optimized the analysis conditions and chose genes that were differentially expressed by more than twofold for gene ontology (GO) analysis. The results showed that after CSSD-9 treatment, apoptosis, intracellular pH regulation, immune response, inflammatory response, interleukin-18 (IL-18)– and IL-8–related biological processes, and CCR chemokine receptor binding–related molecular function were all up-regulated. By contrast, cell adhesion, invasion, migration, angiogenesis, and positive regulation of IL-4 production–related biological processes and growth factor activity–related molecular function were down-regulated (Fig. 4B). Up-regulated genes were associated with inflammatory response, immune response, apoptosis, chemokines, and chemokine receptors (Fig. 4C), whereas down-regulated genes were associated with migration, invasion, angiogenesis, extracellular matrix, chemokines, chemokine receptors, and growth factor (Fig. 4D). Furthermore, we analyzed protein-protein interactions present among differentially expressed genes (up- or down-regulated) using the STRING database (19). Genes up-regulated upon CSSD-9 treatment were enriched in protein-protein interactions associated with cell apoptosis and inflammatory response (Fig. 4E), whereas down-regulated genes were associated with cell invasion, metastasis, and proliferation (Fig. 4F).

Fig. 4 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.

CSSD-9 inhibits tumors in cells expressing miR-9

To examine the sensitivity of other tumor cells to CSSD-9, we measured miR-9 expression (fig. S6) and mRNA expression of KLF17, CDH1, and LASS2 (fig. S7) in various tumor cell types by qRT-PCR. We selected HeLa, SiHa, A549, H1299, and HepG2 cells to investigate the inhibitory effect of CSSD-9 because these cells had the highest relative expression of miR-9 and the lowest relative expression of the target tumor suppressor genes of all the studied cell lines. CSSD-9 reduced cell proliferation in all of the cell lines 48 hours after transfection (Fig. 5A). CSSD-9 overexpression inhibited the metastasis-promoting activity of matrix metalloproteinase-2 (MMP2) and MMP9 in the cells, as assessed by gelatin zymography (Fig. 5B). Furthermore, CSSD-9 inhibited cell invasion (Fig. 5C) and migration (Fig. 5D) and promoted apoptosis (Fig. 5E). In addition, CSSD-9 was not toxic to HEK293, LO2, HUVEC, or HaCaT cells (fig. S8). We also designed other CSSDs that target carcinogenic miRNAs and validated their inhibitory effect in HeLa cells (fig. S9).

Fig. 5 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 < 0.0001.

To investigate the inhibitory effect of CSSD-9 in vivo, about 1 × 106 HeLa cells were injected subcutaneously in the mid-dorsal region of BALB/c nude mice. When the tumor volume reached 100 to 200 mm3, mice were treated with mixed CSSD-9 (10 μg) and nanoparticles (10 μl) or mixed CSSD-9 (20 μg) and nanoparticles (20 μl) by intratumoral (I.T.) or intravenous (I.V.) injection every 3 days to ascertain the dose dependency of the effect (Fig. 6A). Compared with 10-μg CSSD-9 treatment, the administration of 20 μg of CSSD-9 exhibited more pronounced tumor-inhibiting effects. Therefore, we selected a high dose (20 μg) of CSSD-9 treatment to inhibit tumor detection in vivo. A mixture of 20 μg of CSSD-9 and 20 μl of nanoparticles was injected into SiHa, A549, and HepG2 tumor-bearing mice I.V. or I.T. every 3 days. CSSD-9 decreased the tumor growth in these three models, with the inhibitory effect more apparent in I.T. injection groups than in I.V. injection groups (Fig. 6B). The number of lung metastatic nodules decreased in mice treated with CSSD-9 (two-tailed unpaired Student’s t test or Mann-Whitney test, P < 0.05) (Fig. 6, C and D). We also detected miR-9 and mRNA expression of KLF17, CDH1, and LASS2 in tumor tissues by qRT-PCR. The results showed that CSSD-9 suppressed miR-9 expression (Fig. 6E) and increased KLF17, CDH1, and LASS2 expression (Fig. 6F). KLF17, CDH1, and LASS2 protein expression was also increased in the CSSD-9 treatment group as shown by Western blot analysis (Fig. 6G). The mRNA expression of some chemokines, chemokine receptors, and growth factors in tumor tissues was detected by qRT-PCR in CSSD-9 treatment groups. The mRNA expression variation of CXCR5, IL33, CCL18, FGF11, and VEGFB in tissues was consistent with that of the whole-genome gene expression chip data: The expression of CXCR5 increased, whereas IL33, CCL18, FGF11, and VEGFB decreased in CSSD-9–treated tumor tissues (fig. S10).

Fig. 6 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 < 0.0001.

We next investigated the effect of the nanoparticles delivered with CSSD-9 on normal cells in the animal models. We evaluated the complete blood count, liver function, and kidney function in normal mice (normal group), tumor-bearing mice (control group), and mice treated with different doses of I.T. or I.V. injection of CSSD-9 (that is, I.T.-20 μg, I.T.-10 μg, I.V.-20 μg, and I.V.-10 μg groups). Compared with normal mice, the percentage of neutrophils increased [one-way analysis of variance (ANOVA), P < 0.0001], but there was no significant statistical difference between monocytes, lymphocytes, and eosinophils in tumor-bearing mice and normal mice. The percentage of basophils was close to 0. After CSSD-9 treatment, there was no statistical difference in neutrophils, monocytes, lymphocytes, eosinophils, and basophils (fig. S11, A and B). We also performed alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), total protein (TP), albumin (ALB), creatinine (CREA), and urea (UREA) tests to evaluate the effect of CSSD-9 on liver and kidney functions. Compared with normal mice, the ALT, ALP, TP, and ALB concentrations in tumor-bearing mice decreased, whereas the AST and TBIL concentrations increased. After CSSD-9 treatment, these indexes reverted to near-normal; however, these changes were not statistically significant. In addition, there was no marked difference in CREA and UREA concentrations in all groups (fig. S11, C and D). The results showed that CSSD-9 exhibits little effect on complete blood count, liver function, and kidney function in mice.

To investigate the in vivo effects of CSSD-9 on metastasis, an experimental metastasis assay was used to compare lung metastatic and circulating tumor cells (CTCs) in mice injected with A549 cells, stably labeled by green fluorescent protein (GFP). Six weeks after injection, mice in the control group showed marked pulmonary metastasis by in vivo fluorescence imaging. The CTCs were analyzed by flow cytometry, and the results showed that CTC was higher in the control group than in the CSSD-9 group (Fig. 6H). The number of lung metastatic nodules decreased in mice treated with CSSD-9 (two-tailed unpaired Student’s t test, P < 0.05) (fig. S12).

Furthermore, we examined immune cell development and function after CSSD-9 transfection in vivo. We evaluated the infiltration of CD8+ T cells and interferon-γ–positive (IFN-γ+) cells in the tumor tissues after CSSD-9 transfection by I.T. or I.V. injection using flow cytometry. The percentages of infiltrating CD8+ T cells and IFN-γ+ cells in the tumor tissues increased after CSSD-9 treatment, and the increase in 20-μg CSSD-9 treatment group was more obvious (Fig. 6I). To detect the reversal of immunosuppression and the target cells besides tumor cells, we measured the percentage of myeloid-derived suppressor cells (MDSCs) in tumor, bone marrow, and spleen. The percentage of MDSCs decreased after CSSD-9 treatment (Fig. 6J).

CSSD-9 specifically inhibits tumors with high miR-9 expression in a patient-derived tumor xenograft model

We obtained 43 different primary tumor fragments (hepatocellular carcinoma, lung cancer, cervical cancer, ovarian cancer, and breast cancer) from 61 patients and established 43 patient-derived tumor xenograft (PDX) models, whose clinical and pathological features are shown in Fig. 7A. A schematic of the experiments including treatment of fresh surgical tumor tissue, passaging in vivo, and I.T. injection of CSSD-9 in the PDX model is shown in Fig. 7B. We detected KLF17, CDH1, LASS2, and miR-9 expression in these tumors by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) (Fig. 7C). Statistical analysis showed that the expression of KLF17, CDH1, and LASS2 was negatively correlated with miR-9 in these tumor tissues (Fig. 7D). To investigate the inhibitory activity of CSSD-9 on the growth of patient tumors, mice were implanted with different tumor tissues and assigned to control and CSSD-9 groups. We analyzed the relationship between the tumor suppressor rate of CSSD-9 and expression of miR-9 and KLF17, CDH1, and LASS2 in patient tumors. The results showed that tumor inhibitory rate was positively correlated with miR-9 expression but negatively correlated with KLF17, CDH1, and LASS2 expression (Fig. 7E). PDX model results suggested a potential clinical therapeutic value of CSSD-9, which can be viewed as preclinical susceptibility evaluation before the use of CSSD therapy.

Fig. 7 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.

DISCUSSION

Co-silencing multiple oncogenes or releasing the suppression of tumor suppressor genes may have more potential in tumor therapy than traditional targeting antitumor drugs. Here, we analyzed the expression of more than 50 tumor suppressor genes and the associated survival time in breast, lung, and ovarian cancers in the Kaplan-Meier plotter database (14). The survival time of patients with low tumor expression of KLF17, CDH1, and LASS2 was shorter than that of patients with high expression of these tumor suppressor genes. Subsequently, the result was verified in hepatocellular carcinoma tissue microarray. In clinical case analysis and cell experiments in vitro, co-silencing of KLF17, CDH1, and LASS2 was related to tumor malignancy, and their overexpression inhibited tumor malignancy. Therefore, drugs that improve the expression of these genes may serve as potential antitumor agents. The existing antitumor drugs have difficulty in regulating the expression of multiple genes simultaneously, and excessive inhibition by targeted drugs may also cause side effects, such as nausea, skin ulcers, and abnormal blood pressure.

Each miRNA can regulate multiple target genes in different signaling pathways. Therefore, suppressing miRNAs to temporarily and indirectly regulate multiple tumor-related genes may be advantageous in cancer therapy. We analyzed a series of common miRNAs targeting KLF17, CDH1, and LASS2, and their seed sequences shared similar sequences with miR-9. Therefore, searching for inhibitors of miR-9 that enhance the expression of tumor suppressor genes by absorbing miR-9 is essential. It was reported that increased miR-9 expression predicted worse overall survival and promoted tumor malignancy in various carcinomas (2024). The current miRNA inhibitors, expression vectors, and chemically modified antisense oligonucleotides may provide a transient depression of miRNAs in cells (4), or the effective dose in vivo is high (6).

Here, CSSD-9 was designed as a miR-9 sponge mimicking circRNA. A large number of binding sites increase the likelihood of reaching maximal miRNA sequestration; however, such a number may also increase the formation of secondary structures (18). Taking this into account, we designed four miRNA binding sites in each CSSD-9, with each binding site including a bulged site. The imperfect miRNA sponges were attributed to the impediment of quick turnover of the sponge by endonucleolytic cleavage, which prolonged the sequestering effects of the miRNA (25, 26). The results showed that CSSD-9 was more efficient and stable than other linear or cyclic miRNA sponges. In tumor cells, CSSD-9 still produced a stable inhibitory effect on miR-9 that lasted up to 96 hours. This finding suggested that the clinical application of CSSD will reduce the frequency of medications and increase persistence and effectiveness.

In vitro experiments showed that CSSD-9 transfected in cells competitively bound and inhibited miR-9 activity, thereby increasing the expression of miR-9–targeted tumor suppressor genes—KLF17, CDH1, and LASS2—in cancer. KLF17, CDH1, and LASS2 were correlated with tumor malignancy and apoptosis through the regulation of essential signaling pathways (2729). CSSD-9–induced KLF17 overexpression decreased cancer metastasis by down-regulating the inhibition of DNA binding 1 to promote E-cadherin expression, which inhibits tumor migration and invasion through epithelial-mesenchymal transition–associated processes (27, 30, 31). CSSD-9 also directly absorbed miR-9, which maintained the levels of E-cadherin by prolonging the half-life of targeted mRNA (28). In addition, it enhanced the expression of miR-9 target gene LASS2 (also called tumor metastasis suppressor gene 1), which can inhibit Bcl2 and survivin expression (29, 32) to promote apoptosis and decrease MMP2 and MMP9 activities to inhibit epithelial-mesenchymal transition (33). Whole-genome gene expression chip analysis was conducted to assess the effects of CSSD-9 on cancer cells. The differentially expressed genes were subjected to GO analysis. The results showed that after CSSD-9 treatment, cell adhesion, invasion, migration, and angiogenesis-related biological processes were down-regulated, whereas apoptosis-related biological processes were up-regulated. CSSD-9 was found to enhance immune and inflammatory responses. It was reported that exogenous circRNA can induce cellular immune response (34), so we detected the infiltration of CD8+ T cells in tumor tissues and MDSCs in tumor, bone marrow, and spleen after CSSD-9 treatment. CSSD-9 increased infiltration of CD8+ T cells and IFN-γ+ cell percentages in the tumor tissues and inhibited the MDSCs. This indicated that CSSD-9 also increased the immune response in vivo to inhibit tumors.

In the miRNA database (15), the miR-9 sequences between humans and mice showed no difference. Therefore, the efficacy and toxicity of CSSDs in the mouse models were markedly close to those in human experiments. In vivo experiments demonstrated that CSSD-9 inhibited tumor growth and pulmonary metastasis. The results of the PDX model showed that CSSD-9 was highly selective for tumors with high miR-9 levels. In addition, CSSD-9s were transfected into cells or infected into tumors by special nanoparticles. The nanoparticles were spherical and uniform in size, and the average size was 80 to 100 nm. Displaying large specific surface areas, they were characterized by surface and small-size effects. They can bind to nucleic acids stably and protect the nucleic acids from degradation by nucleases. Given the special structures of the nanoparticles, we transfected CSSD-9 into tumor cells by the nucleic acid–nanoparticle complex efficiently. In addition, cytotoxicity was considerably low. In vivo, there was no obvious effect on complete blood count, liver function, or kidney function after CSSD-9 treatment. Moreover, we designed other CSSDs that targeted carcinogenic miRNAs and had a substantial inhibitory effect on tumor cells.

In summary, our study indicated that CSSD-9 transfected into cells on nanoparticles effectively inhibited the proliferation, migration, and invasion and promoted the apoptosis of multiple tumors. CSSD-9 acted as a sponge and directly inhibited miR-9 activity, thereby restoring the functions of miR-9 target tumor suppressor genes KLF17, CDH1, and LASS2 in tumor cells. The schematic diagram of the proposed mechanisms underlying antitumor of CSSD is shown in fig. S13. We found that CSSD-9 targeted not only tumor cells but also MDSCs. Further research is needed to determine how CSSD-9 may affect tumor immunity. Although CSSD-9 can stably inhibit the expression of miR-9 for more than 72 hours, further improvement in its stability should be able to enhance its tumor inhibitory effect. The introduction of chemically modified or highly stable nucleic acid structures may possibly help achieve this. In addition, we can improve the CSSD adsorption efficiency by designing different sequences or considering the seed sequence of multiple miRNAs. The efficiency of CSSD administration via nanoparticles should also be further optimized, and the carrier could be further modified to aid CSSD visualization in the tumor. These findings revealed a miRNA inhibitor, namely, our artificial CSSD, which effectively decreased tumor-promoting miRNA activities by transfecting nanoparticles in vitro and in vivo. This study provides new insights into the miRNA-based resurrection of “co-silenced” tumor suppressor genes and reports a precise multitarget drug for potential tumor therapy.

MATERIALS AND METHODS

Study design

This study aimed to demonstrate that co-silenced tumor suppressor genes can be reactivated by nanoparticle-loaded CSSDs, which absorb miRNAs to suppress tumor malignancy. We first analyzed the effect of 51 tumor suppressor genes on the median survival time of breast, lung, and ovarian cancers and screened three common tumor suppressor genes, namely, KLF17, CDH1, and LASS2, whose associations with the median survival time in cancers were relatively greater than others. Hepatocellular carcinoma pathological analysis also demonstrated that co-silencing of KLF17, CDH1, and LASS2 is correlated with escalated malignancy.

Because miR-9 contributed to KLF17, CDH1, and LASS2 co-silencing, we designed CSSD-9 containing miR-9 binding sites to up-regulate the expression of co-silenced tumor suppressor genes by absorbing miR-9. Nanoparticle-loaded CSSD-9 served as a miR-9 sponge that inhibited multiple tumor malignancy by increasing tumor suppressor gene expression in vitro and in vivo in a dose-dependent manner. Whole-genome gene expression chip analysis revealed key biological processes and molecular functions influenced by CSSD-9. Primary tumor fragments (hepatocellular carcinoma, lung cancer, cervical cancer, ovarian cancer, and breast cancer) were collected from 61 patients to construct 43 PDX models to test whether CSSD-9 was more effective in inhibiting tumors that have high miR-9 expression. Details on sampling and experimental replicates are provided in each figure legend. All treatments were randomly allocated, and all analyses were done by investigators who were blinded to the treatment allocation.

CSSD design

Two single-stranded DNAs (ssDNAs) contained two miRNA binding sites linked by oligo(dT) (table S1). Each miRNA binding site included a bulged site. The ends of the two ssDNAs were complementary sequences. They were combined in annealing buffer (Beyotime Biotechnology), heated for 2 min at 95°C, and allowed to cool at room temperature and form circular DNA. After exonuclease VII (New England Biolabs) treatment, the ssDNAs without cyclization were removed, and the CSSDs were purified.

Cell culture and transfections

Cells were obtained from the American Type Culture Collection and KeyGEN. HeLa, SiHa, HepG2, HEK293, and HaCaT were cultured in Dulbecco’s modified Eagle’s medium. A549, H1299, SMMC-7721, PLC-PRF-5, Bel-7402, MCF-7, MX-1, and LO2 were cultured in RPMI 1640 medium. SKOV-3 was cultured in McCoy’s 5A medium (modified), and HUVECs were cultured in endothelial cell medium. The medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C in 5% CO2. All CSSDs, miRNA mimics, inhibitors, scramble controls, and pcDNA3.1-KLF17, pcDNA3.1-CDH1, and pcDNA3.1-LASS2 were transfected into cells by nanoparticles (Nantong Micropoly Bio Technologies Ltd.), a transfection reagent based on nanogene transfer technology. The nanoparticles adsorb nucleic acids on their surfaces by electrostatic adsorption. Even small-volume particles can have a large effective surface area, which can be combined with nucleic acid. The nanoparticles help protect the nucleic acids, circumventing their degradation and destruction by nucleases. Primers used are shown in table S2.

Online prediction of target genes

The following bioinformatics software was used: miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/) (15), TargetScan (http://targetscan.org) (16), miRanda (https://omictools.com/miranda-tool) (17), and CircNet (http://circnet.mbc.nctu.edu.tw/) (35). MiRTarBase is a comprehensive, experimentally validated miRNA-target interactions database. TargetScan and miRanda predict biological targets of miRNAs. miRNAs targeting KLF17, CDH1, and LASS2 included both experimentally validated and predicted data.

Kaplan-Meier plotter

The Kaplan-Meier plotter database (http://kmplot.com) (14) was used to analyze the median survival time and survival curve of tumor suppressor genes in breast, lung, and ovarian cancers. The patient samples selected were divided into two cohorts according to high and low expression of KLF17, CDH1, and LASS2 by autoselecting the best cutoff, whereby the lower and upper quartiles are computed, and the best performing threshold was used as a cutoff. Breast cancer data from all versions of the database were used, and in lung and ovarian cancers, the current version of the database was used for analysis. For array quality control, we excluded biased or outlier arrays.

Gelatin zymography

MMP activity was measured by gelatin zymography using precast zymogram gelatin gels (10% polyacrylamide containing 0.1% gelatin; Novex/Invitrogen Corp.) following the manufacturer’s instructions. The enzyme sample (15 μg of protein) was denatured in SDS buffer (without reducing conditions) using tris-glycine-SDS sample buffer (Novex/Invitrogen Corp.). After electrophoresis, the gel was rinsed overnight and incubated at 37°C for 20 hours to allow gelatinolytic activity. The gel was stained with Coomassie Brilliant Blue. MMP activity was detected as unstained bands against the background of blue-stained gelatin (36).

Scanning electron microscope

The CSSD-9 and nanoparticles (Nantong Micropoly Bio Technologies Ltd.) were mixed for 15 min at room temperature according to the manufacturer’s instructions and then added dropwise into HeLa cells. After 48 hours, cells were fixed in 2.5% glutaraldehyde at 4°C for 4 hours and 1% osmic acid at 4°C for 1 hour, then dehydrated in acetone/isoamyl acetate (1:1) for 10 min and isoamyl acetate for 30 min, and dried with acetonitrile for 15 to 20 min for each concentration. After vacuum freeze-drying and coating with gold, cells were photographed using a scanning electron microscope (JEOL6000).

Cell proliferation assay

At 12, 24, 36, 48, 60, and 72 hours after transfection, cell culture medium was removed and 100 μl of methylthiazolyldiphenyl-tetrazolium bromide (MTT) (1 mg/ml) was added to each well of a 96-well plate. Afterward, the cells were incubated for 5 hours at 37°C in 5% CO2. The MTT solution was discarded, and 100 μl of dimethyl sulfoxide was added to each well. Absorbance was measured at a wavelength of 490 nm using an ELISA microplate reader (Bio-Rad). Assays were repeated at least three times.

Cell migration assays

For wound-healing assays, cells were plated into 24-well culture plates until a monolayer was formed. A straight scratch was created in the center of each well using a micropipette tip. Cell migration was assessed by measuring the movement of the cells into the scratch in the well. The distance of wound closure at 24 and 48 hours was measured and normalized by wound length at 0 hours. Each experiment was performed in triplicate.

Cell invasion assays

Cells in serum-free medium were seeded onto a chamber coated with Matrigel (BD Biosciences) and inserted in a well of a 24-well plate. Medium with 10% FBS was added to the well below the chamber as a chemoattractant. After 24 hours, invasive cells located on the lower surface of the chamber were stained with 0.1% crystal violet and counted.

Cell apoptosis

Cells were transfected with CSSDs. After 48 hours, they were harvested with 0.25% trypsin without EDTA. They were also washed twice with ice-cold phosphate-buffered saline (PBS), resuspended in 250 μl of binding buffer, adjusted to 1 × 106/ml, and fixed in 1% paraformaldehyde. Subsequently, they were stained with the Annexin V/PI Apoptosis Detection Kit (KeyGen Biotech). They were analyzed with a flow cytometer (Millipore) after incubation in the dark for 30 min.

Colony formation assay

Cells were transfected with KLF17, CDH1, LASS2, or CSSDs for 48 hours. Subsequently, they were seeded into six-well plates (800 cells per well) and incubated for 14 days. The plates were washed with PBS and stained with 0.1% crystal violet for 15 min at room temperature. The number of colonies with more than 50 cells was manually counted under a microscope.

Whole-genome gene expression chip analysis

HeLa cells were treated with CSSD-9 for 48 hours. The cells were lysed to isolate RNA, and gene expression was detected with whole-genome gene expression chip (Genergy). Data analysis was performed to reveal critical pathways. GO analysis (DAVID; https://david.ncifcrf.gov/) (37) was used to detect changes in molecular functions, biological processes, and cellular components induced by CSSD-9. The STRING database (https://string-db.org) (38) was used to analyze protein-protein interactions.

Xenograft tumor model

About 1 × 106 SiHa, A549, and HepG2 cells were injected subcutaneously in the mid-dorsal region of each BALB/c nude mouse. Each group (control and CSSD-9) consisted of eight mice. Tumor-bearing mice began to receive treatment when the average tumor size reached 100 to 200 mm3. The CSSD-9 group was treated every 3 days with I.V. or I.T. injection of CSSD-9 (20 μg) in nanoparticles (20 μl). Control groups were treated with control DNA (20 μg) in nanoparticles (20 μl) every 3 days. Tumor diameters were serially measured with a digital caliper every 3 days. Tumor volumes were calculated by length × width2/2. On day 27, the mice were sacrificed. Tumor tissues and lung tissues were collected, fixed with 10% formalin, and embedded in paraffin, followed by H&E or IHC staining. The remaining tissues were kept in a deep freezer at −80°C for isolating protein and RNA. The blood routine index of mice was detected by automatic blood analyzer (SYSMEX XT-2000i), and the full automatic biochemical analyzer (Hitachi 7020) was used for the analysis of liver and kidney function (LEADMAN).

Experimental metastasis assay

A549-GFP cells (2 × 105), which were stably labeled by GFP, were injected I.V. through the tail vein of BALB/c nude mice (4 to 6 weeks old). The CSSD-9 group was treated every 3 days with I.V. CSSD-9 (20 μg) in nanoparticles (20 μl) after cell injection. After 6 weeks, peripheral blood (500 to 700 μl) was collected from the inferior vena cava of each animal under anesthesia (2% pentobarbital, 40 to 50 mg/kg, intraperitoneal injection). Whole blood was processed within 1 hour of collection to lyse red blood cells with Red Blood Cell Lysis Buffer (Beyotime Biotechnology), and GFP-positive mononuclear cells in peripheral blood samples were detected by flow cytometry (39).

PDX models

Forty-three fresh surgical tumor tissues (F0, hepatocellular carcinoma, lung cancer, cervical cancer, ovarian cancer, and breast cancer) were collected immediately after surgery without any other treatment from 61 patients in S. G. Hospital (Shandong, China), Tianjin Medical University General Hospital, and the Hospital of Shunyi District, Beijing. A written informed consent was obtained from each patient. According to the Declaration of Helsinki, studies were performed after approval of the ethics committee of Nankai University, including the use of animal experiments and tumor specimens. The tumors were divided into three parts. One part was fixed for detection of KLF17, CDH1, and LASS2 expression in IHC. Another part was used for the detection of miR-9 expression in quantitative real-time PCR, and the remaining fresh tumor tissues were cut into 1- to 2-mm3 pieces in antibiotic–RPMI 1640 medium containing penicillin (100 U/ml) and streptomycin (0.1 mg/ml). Tumor fragments were implanted subcutaneously into the right axilla of 4- to 6-week-old BALB/c nude mice. By palpation of the skin at the tumor site, we selected mice that bore tumor nodules and began to measure the tumor volumes. When the tumor size reached 100 to 200 mm3, the samples (F1) were divided into pieces for in vivo passaging to construct F2 and then F3 tumors as described above. These transplanted tumors were analyzed by pathology, and the results were consistent with the clinical pathological analysis (40). When the F3 tumor size reached 100 to 200 mm3, the mice bearing different tumor types were randomly divided into control and CSSD groups consisting of four mice per group. Twenty micrograms of CSSD-9 with 20 μl of nanoparticles was I.T. administered in mice in the CSSD-9 group every 3 days for 18 days. The tumor inhibitory rate of CSSD-9 was calculated using the following formula: Inhibition rate = (tumor volumecontrol − tumor volumeCSSD)/tumor volumecontrol.

Quantitative real-time PCR

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Afterward, complementary DNAs were synthesized from total RNA using the PrimeScript RT Reagent Kit (Tiangen). U6 was used as an internal control. All primers were synthesized by Invitrogen. The primers used in qPCR are shown in table S3. All results are expressed as the means ± SD of three independent experiments.

Western blot analysis

Collected tumor tissues were lysed in a buffer containing Hepes (25 mM; pH 7.5), NaCl (150 mM), 1% Triton X-100, 10% glycerol, EDTA (5 mM), and a protease inhibitor cocktail. Protein concentration was determined using bicinchoninic acid assay. Cells were washed with PBS and lysed in ice-cold lysis buffer with protease inhibitor cocktail (Sigma) for 30 min. Lysates were separated by electrophoresis and transferred on polyvinylidene difluoride membranes (Millipore). The membranes were blocked and incubated with primary antibody of KLF17 (1:500 dilution; Santa Cruz Biotechnology), CDH1 (1:500 dilution; Abcam), and LASS2 (1:500 dilution; Santa Cruz Biotechnology). Subsequently, they were incubated with a goat anti-rabbit immunoglobulin G–horseradish peroxidase (HRP) secondary antibody (Thermo Fisher Scientific). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as loading control. Protein expression was assessed by enhanced chemiluminescence substrate (Millipore) and exposed to the chemiluminescence film.

IHC assay and analysis

The hepatocellular carcinoma tissue microarrays containing 171 cases were purchased from US Biomax for IHC, followed by analysis of correlation between survival time, clinical stage, pathological grade, metastasis, AFP content, and KLF17, CDH1, and LASS2 expression. The tissues were incubated with xylene for deparaffinization and decreasing concentrations of ethanol for rehydration. Next, 3% hydrogen peroxide was applied to block endogenous peroxidase activity. Antigen retrieval was performed by boiling the slides in citrate buffer (10 mM; pH 6.0) in microwave (Midea) and keeping the temperature between 92° and −98°C for 20 min (41). After blocking, the samples were incubated with primary antibodies overnight at 4°C: Mouse polyclonal anti-KLF17 (1:200 dilution; Santa Cruz Biotechnology), mouse polyclonal anti-LASS2 (1:200 dilution; Santa Cruz Biotechnology), rabbit polyclonal anti-CDH1 (1:100 dilution; Abcam), and PBS were used as negative control. The secondary antibody was added using the HRP-Polymer Anti-Mouse/Rabbit IHC Kit (Maixin Biotech) for 1 hour at room temperature. Subsequently, samples were developed with diaminobenzidine reagent, counterstained with hematoxylin, and mounted with Permount. IHC score was calculated by multiplying the intensity (0, negative; 1, canary yellow colors; 2, claybank; 3, brown) and percentage of cells with positive scores (1, less than 25%; 2, 25 to 50%; 3, 51 to 75%; 4, more than 75%).

Fluorescence in situ hybridization

FAM-labeled locked nucleic acid–modified oligonucleotide probes complementary to mature miR-9 were purchased from GenePharma. The probe sequences were as follows: 5′-TCATACAGCTAGATAACCAAAGA-3′. After deparaffinization and deproteinization, the slides were prehybridized with 1× hybridization buffer without probes. The hybridization was carried out overnight in a 1× hybridization buffer (30 to 70 μl) with predenatured miRNA probes. After washing, the slides were stained with 4′,6-diamidino-2-phenylindole (DAPI) and analyzed by confocal laser scanning microscopy.

Statistical analysis

The significance of the overlap between the tumor suppressor genes associated with median survival time represented in Venn diagrams was calculated using a hypergeometric distribution test. When normality assumptions were met, two-tailed unpaired Student’s t test was used for comparing two groups of data. For paired observations and repeated measures over time, we used Wilcoxon matched-pairs signed-rank test and two-way ANOVA with Bonferroni’s multiple comparison post-test or one-way ANOVA analysis. When normality assumptions were not met (Kolmogorov-Smirnov test, P < 0.05), nonparametric statistical tests were performed. Mann-Whitney or Kruskal-Wallis test with Dunn’s multiple comparison post hoc test was performed in the comparison of two or more groups. For survival studies, Kaplan-Meier was used to analyze the survival time comparing two and three groups. Pearson’s correlation was used to measure the linear correlation for two random variables. Values are expressed as means ± SEM unless otherwise indicated, and P < 0.05 was deemed significant.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/10/442/eaao6321/DC1

Fig. S1. Kaplan-Meier survival analysis of KLF17, CDH1, and LASS2 expression in breast, lung, and ovarian cancer.

Fig. S2. Correlations between KLF17, CDH1, and LASS2 expression in breast cancer.

Fig. S3. The inhibitory effects of carcinogenic miRNAs on KLF17, CDH1, and LASS2 expression and cell proliferation in HeLa cells.

Fig. S4. miR-9 expression in different tumors and normal tissues.

Fig. S5. miR-9–targeted KLF17, CDH1, and LASS2.

Fig. S6. The relative expression of miR-9 in tumor cell lines.

Fig. S7. mRNA expression of KLF17, CDH1, and LASS2 in different cancer cell lines.

Fig. S8. The absorbance of HEK293, LO2, HUVEC, and HaCaT cells after CSSD-9 treatment.

Fig. S9. The inhibitory effect of CSSD-190, CSSD-21, CSSD-17, and CSSD-10 on HeLa cells.

Fig. S10. mRNA expression relative to control of chemokines/receptors and growth factors in tumor tissues treated with CSSD-9.

Fig. S11. White blood cell counts, liver function, and kidney function of mice under CSSD-9 treatment.

Fig. S12. The inhibitory effect of CSSD-9 on lung metastasis in an experimental model.

Fig. S13. Schematic diagram of the mechanisms underlying the antitumor activity of CSSD.

Table S1. The sequences of CSSD-9 and circR-9.

Table S2. Primers used in cloning (vector: pcDNA3.1).

Table S3. Primers for real time RT-PCR.

REFERENCES AND NOTES

Acknowledgments: We are grateful to Nantong Micropoly Biotechnologies Ltd. for providing the nanoparticles. Funding: This study was supported by the National Natural Science Foundation of China (grant nos. 81572838 to T.S., 81402973 to Y.-r.L., and 81703581 to H.-j.L.), Tianjin Natural Science and Technology Fund (grant no. 15JCYBJC26400 to T.S.), Tianjin Science and Technology Project (grant no. 15PTGCCX00140 to C.Y.), and the National Science and Technology Major Project (grant no. 2017ZX09306007 to T.S.). Author contributions: T.S., C.Y., J.M., and S.C. are responsible for this study. T.S. conceived and designed the study. T.S., S.C., and J.M. proposed the concept of artificial CSSD and targeting multiple tumor suppressor genes for the treatment of tumor. C.Y. revised the paper and provided financial support. X.-r.W., H.-z.W., and Y.-y.L. analyzed the Kaplan-Meier plotter database. W.-l.Z. and W.-f.G. carried out the bioinformatics analysis. Q.T. and H.-j.L. performed the animal experiment. Y.-r.L. performed the pathological analysis. H.-g.Z. constructed the cloning vector. J.M. and S.C. performed all other experiments. J.M. and J.-x.H. analyzed data. K.-l.Q., C.Z., and Y.Q. performed the miRNA analysis. J.M., T.S., S.C., and C.Y. wrote the manuscript. Competing interests: The authors declare that they have no competing interests.
View Abstract

Navigate This Article