Xiaoying Wang

MiR‐7, Inhibited Indirectly by LincRNA HOTAIR, Directly Inhibits SETDB1 and Reverses the EMT of Breast Cancer Stem Cells by Downregulating the STAT3 Pathway

Epithelial–mesenchymal transition (EMT) contributes to tumor invasion and metastasis in many cancers and correlates highly with the acquisition of cancer stem cell (CSC) characteristics. EMT also correlates with changes in specific microRNAs (miRNAs) that have already been integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. Here, we show that miR‐7, which was downregulated in breast CSCs (BCSCs) isolated from the human MCF‐7 and MDA‐MB‐231 cell lines, inhibited cell invasion and metastasis, decreased the BCSC population and partially reversed EMT in MDA‐MB‐231 cells by directly targeting the oncogene, SETDB1. The conspicuous epigenetic transition induced by miR‐7 overexpression was found not only in MDA‐MB‐231 cells but also in BCSC xenograft tumors. MiR‐7 inhibited the metastasis of BCSCs in lungs, kidneys, and adrenal glands of NOD/SCID mice. ChIP‐polymerase chain reaction result suggested that the SETDB1 induced STAT3 expression by binding to the promoter of STAT3. MiR‐7‐mediated downregulation of SETDB1 resulted in the suppression of STAT3, which led to the downregulation of c‐myc, twist, and mir‐9. In addition, the downregulation of miR‐7 in BCSCs may be indirectly attributed to lincRNA HOTAIR by modulating the expression of HoxD10 that promotes the expression of miR‐7. These findings demonstrate that miR‐7 was a tumor suppressor and that the overexpression of miR‐7 might serve as a good strategy for treating highly invasive breast cancer. Stem Cells 2014;32:2858–2868

MicroRNA-200c overexpression inhibits tumorigenicity and metastasis of CD117 + CD44 + ovarian cancer stem cells by regulating epithelial-mesenchymal transition

BackgroundCancer stem cells (CSCs) are believed to be ‘seed cell’ in cancer recurrence and metastasis. MicroRNAs (miRNAs) can play an important role in the progression of primary tumor towards metastasis by regulating the epithelial-mesenchymal transition (EMT). The goal of this study was to investigate the effect of miRNA-200c overexpression on the EMT, tumorigenicity and metastasis of epithelial ovarian cancer (EOC) CSCs.MethodsThe EOC CD117+CD44+CSCs were isolated from the human ovarian cancer cell line SKOV3 by using a magnetic-activated cell sorting system, and the lentivirus miR-200c transduced CSCs were then selected for the study. The assays of colony forming, wound healing, cellular migration in vitro and tumor progression in vivo were performed.ResultsThe miR-200c expression was reduced in the CD117+CD44+CSCs compared with the non-CD117+CD44+CSCs. However, the stable overexpression of the miR-200c in the CD117+CD44+CSCs resulted in a significant down-regulation of ZEB-1 and the Vimentin expression, an upregulation of the E-cadherin expression as well as a decrease of colony forming, migratory and invasion in vitro. Importantly, the miR-200c overexpression significantly inhibited the CD117+CD44+CSCs xenograft growth and lung metastasis in vivo in nude mice by inhibition of the EMT. In addition, the down-regulation of ZEB-1 showed the same efficacy as the miR-200c overexpression in the CD117+CD44+CSCs.ConclusionThese findings from this study suggest that the miR-200c overexpression may be considered a critical approach for the EOC CD117+CD44+CSCs in clinical trials.

Observation of ovarian cancer stem cell behavior and investigation of potential mechanisms of drug resistance in three-dimensional cell culture.

Cancer cells behave differently in a three-dimensional (3D) cell culture compared with in the conventional two-dimensional (2D) one. Accumulated evidences indicate that the characteristics of cancer stem cells (CSCs) are different from common cancer cells due to their ability to produce tumors and resist chemoradiation. The objective of this work was to observe CSC behavior and investigate the potential mechanisms of CSC drug resistance in 3D versus 2D in vitro environment. We first demonstrated that the CD44(+)CD117(+)cells isolated from the human epithelial ovarian cancer HO8910 cell line have the properties of CSCs that revealed faster growth, larger tumorsphere and stronger survival potential in the hypoxic environment in 3D cell culture as well as more powerful tumorigenicity in a xenograft mice than the HO8910 cells. The CD44(+)CD117(+)CSCs also exhibited high chemoresistance to anticancer drugs when the cells were incubated with 5-fluorouracil, cisplatin and carboplatin, respectively in 3D versus 2D environment. This might be associated with the high expression of ABCG2, ABCB1 and the high expression of MMP-2 and MMP-9 in CD44(+)CD117(+)CSCs. Overall, these results suggest the advantages of using 3D culture model to accurately display CSC behavior in vitro. 3D model may improve the efficacy of screening anticancer drugs for treatment of ovarian CSCs.

Effect of downregulation of ZEB1 on vimentin expression, tumour migration and tumourigenicity of melanoma B16F10 cells and CSCs.

Zinc‐finger E‐box binding homeobox 1 (ZEB1) is a master regulator of epithelial‐mesenchymal transition (EMT) and has been implicated in primary epithelial cancer biological processes, such as invasion and metastasis. However, the role of ZEB1 in progression of melanoma and cancer stem cells (CSCs) remains obscure. In this study, the recombinant plasmids of t3 shRNAs targeting mouse ZEB1 were constructed and transfected into melanoma B16F10 cells. The stable transfected cells were selected and the characteristics of ZEB1 downregulated B16F10 cells was assessed. The tumourigenicity of CD44+CD133+CSCs isolated from B16F10 cells stably transfected with the ZEB1‐shRNA2 recombinant was also assessed. ZEB1‐shRNAs B16F10 showed a lower expression of ZEB1 and vimentin, weaker migration, invasiveness, colony forming, and proliferation, and a lower tumourigenicity than the control cells. The tumourigenicity of the ZEB1‐shRNA2 CD44+CD133+CSCs was also inhibited. In conclusion, ZEB1‐shRNA2‐mediated downregulation of ZEB1 expression in B16F10 cells and CSCs is involved in the inhibition of the EMT process. ZEB1 may be a potential target in melanoma targeted.

Gene therapy of ovarian cancer using IL-21-secreting human umbilical cord mesenchymal stem cells in nude mice

BackgroundThe human umbilical cord mesenchymal stem cells (hUCMSCs) have the ability to migrate into tumors and therefore have been considered as an alternative source of mesenchymal progenitors for the therapy of malignant diseases. The present study was aimed to investigate effect of hUCMSCs as vehicles for a constant source of transgenic interleukin-21 (IL-21) on ovarian cancer in vivo.MethodsThe hUCMSCs were engineered to express IL-21 via lentiviral vector- designated ‘hUCMSCs-LV-IL-21’, and then were transplanted into SKOV3 ovarian cancer xenograft-bearing nude mice. The therapeutic efficacy and mechanisms of this procedure on ovarian cancer was evaluated.ResultsThe isolated hUCMSCs were induced to differentiate efficiently into osteoblast and adipocyte lineages in vitro. The expressed IL-21 in the supernatant from hUCMSCs-LV-IL-21 obviously stimulated splenocyte’s proliferation. The hUCMSCs-LV-IL-21 significantly reduced SKOV3 ovarian cancer burden in mice indicated by tumor sizes compared with control mice. The expressed IL-21 not only regulated the levels of IFN-γ and TNF-α in the mouse serum but also increased the expression of NKG2D and MIC A molecules in the tumor tissues. The down regulation of β-catenin and cyclin-D1 in the tumor tissues may refer to the inhibition of SKOV3 ovarian cancer growth in mice. In addition, hUCMSCs did not form gross or histological teratomas up to 60 days posttransplantation in murine lung, liver, stomach and spleen.ConclusionThese results clearly indicate a safety and usability of hUCMSCs-LV- IL-21 in ovarian cancer gene therapy, suggesting the strategy may be a promising new method for clinical treatment of ovarian cancer.

Regulation gene expression of miR200c and ZEB1 positively enhances effect of tumor vaccine B16F10/GPI-IL-21 on inhibition of melanoma growth and metastasis

BackgroundGenetically modified cells have been shown to be one of the most effective tumor vaccine strategies. However, in many cases, such as in melanoma, induction of a potent immune responses against the disease still remains a major challenge. Thus, novel strategies to reinforce tumor vaccine efficacy are needed. Using microRNA (miR) and Zinc-finger E-box binding homeobox (ZEB) have received much attention for potentially regulating tumor progression. To elicit a potent antitumor efficacy against melanoma, we used tumor vaccine in combination with miR200c overexpression or ZEB1 knockdown to assess the efficacy of treatment of murine melanoma.MethodsB16F10 cell vaccine expressing interleukin 21 (IL-21) in the glycosylpho- sphatidylinositol (GPI)-anchored form (B16F10/GPI-IL-21) were developed. The vaccine was immunized into mice challenged by B16F10 cells or B16F10 cells stably transduced with lentiviral-miR200c (B16F10/miR200c) or transfected with the ZEB1-shRNA recombinant (B16F10/shZEB1) or the B16F10/GPI-IL-21 vaccine. The immune responses, tumorigenicity and lung metastasis in mice were evaluated, respectively.ResultsThe vaccination with B16F10/GPI-IL-21 markedly increased the serum cytokine levels of IFN-γ, TNF-α, IL-4 and decreased TGF-β level as well as augmented the cytotoxicity of splenocytes in immunized mice compared with control mice. In addition, the tumor vaccine B16F10/GPI-IL-21 significantly inhibited the tumor growth and reduced counts of lung metastases in mice challenged by B16F10/GPI-IL-21, B16F10/shZEB1 and B16F10/miR200c respectively compared with the control mice challenged by B16F10 cells. The efficacy mechanisms may involve in reinforcing immune responses, increasing expression of miR200c, E-cadherin and SMAD-7 and decreasing expression of TGF-β, ZEB1, Vimentin and N-cadherin in tumor tissues from the immunized mice.ConclusionsThese results indicate that the tumor vaccine B16F10/GPI-IL-21 in combination with miR200c overexpression or ZEB1 knockdown effectively inhibited melanoma growth and metastasis a murine model. Such a strategy may, therefore, be used for the clinical trials.

Decrease of ZEB1 expression inhibits the B16F10 cancer stem-like properties.

Increasing evidence supports that cancer stem cells (CSCs) are responsible for driving tumor initiation and maintenance. Zinc-finger E-box binding homeobox 1 (ZEB1) is a transcription factor for regulating tumor progression, and contributes to maintenance of CSC-like properties. The goal of the present study is to investigate the effect of decreasing ZEB1 expression on the B16F10 CSC-like properties. The recombinant shRNA targeting ZEB1 were transfected into melanoma B16F10 cells, and shZEB1-CD133(+)CD44(+) CSCs were isolated from the stable transfected cells using the magnetic-associated cell sorting method. The shZEB1-CD133(+)CD44(+) CSC-like properties were systematically analyzed. The results show the B16F10 shZEB1-CD133(+)CD44(+) CSCs significantly decreased the ability of clonogenicity, cellular proliferation, migration, and invasion. Importantly, tumorigenicity and tumor lung metastasis was significantly inhibited in B16F10 shZEB1-CD133(+)CD44(+) CSCs compared with B16F10 scramble-CD133(+)CD44(+) CSCs. The decrease of ZEB1 expression markedly resulted in down-regulation of vimentin and N-cadherin expression as well as up-regulation of E-cadherin expression in tumor tissues from the mice injected with B16F10 shZEB1-CD44(+)CD133(+) CSCs. These findings contribute to understanding the maintenance of B16F10 CD133(+)CD44(+) CSC-like properties that was closely associated with ZEB1 expression. ZEB1 may serve as a new therapeutic target for treatment of malignant melanoma.

Reinforcing B16F10/GPI-IL-21 vaccine efficacy against melanoma by injecting mice with shZEB1 plasmid or miR200c agomir.

In this study, we hypothesized that the inhibition of epithelial to mesenchymal transition (EMT) program by knockdown of Zinc-finger E-box binding homeobox 1 (ZEB1) or administration of miR200c agomir would strengthen the B16F10 cells transfected with GPI-anchored IL-21 (B16F10/GPI-IL-21) vaccine efficacy in inhibiting the melanoma metastasis. Our findings from the current study indicated that, when compared with the mice immunized with the B16F10/GPI-IL-21 vaccine alone, the mice immunized with B16F10/GPI-IL-21 vaccine combined with injection of shZEB1 plasmid or miR200c agomir not only meaningfully inhibited EMT of melanoma, reduced the EMT characteristic molecular expression in tumor tissues, but also significantly decreased the Treg cells and TGF-β1, enhanced the cytotoxicities of NK cells and cytotoxic T lymphocytes and the IFN-γ level. Furthermore, the immunotherapeutic combination resulted in inhibiting the melanoma growth and lung metastasis. Our study demonstrated that using the B16F10/GPI-IL-21 vaccine in combination with the down-regulated ZEB1 or miR200c administration effectively elicited anti-tumor immunity and reduced melanoma metastasis by inhibiting the EMT program in the B16F10 melanoma-bearing mice.

Downregulation of β-catenin decreases the tumorigenicity, but promotes epithelial-mesenchymal transition in breast cancer cells

BACKGROUND Wnt/β-catenin signaling pathway plays a key role in human breast cancer progression. In this study, we down regulated β-catenin expression in human breast cancer MDA-MB-231 cells and investigated the effect of β-catenin knockdown on the cell biological characteristics. MATERIALS AND METHODS The recombinant plasmids of pSUPER-enhancement green fluorescent protein 1 (EGFP1)-scrabble-β-catenin-short hairpin ribonucleic acid (shRNA) and pSUPER-EGFP1-β-catenin-shRNA-1 were transfected into MDA-MB-231 cells, respectively, and the stably transfected cells were isolated from G418 selected clones. The β-catenin gene silenced efficiency was measured by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) and Western blot. The biological characteristics of MDA-MB-231 cells with down regulated β-catenin were evaluated by analyzing cell proliferation, clonogenicity, cell mobility and tumorigenicity. The expression of E-cadherin and Vimentin was concurrently detected by QRT-PCR. RESULTS The β-catenin-shRNA-1 stably transfected MDA-MB-231 cells significantly decreased β-catenin expression, cell proliferation, clonogenicity, and tumorigenicity in Balb/c nude mice compared with the MDA-MB-231 cells transfected with pSUPER-EGFP1-scrabble-β-catenin-shRNA. Interestingly, knockdown of β-catenin led to the reduction of epithelial E-cadherin expression, the increase of cell mobility and mesenchymal vimentin expression in MDA-MB-231 cells, indicating an epithelial to mesenchymal transition. CONCLUSION Knockdown of β-catenin expression in human breast cancer MDA-MB-231 cells inhibits cell tumorigenicity in mice, but promotes cell epithelial-mesenchymal transition.

Hepatitis C virus core impacts expression of miR122 and miR204 involved in carcinogenic progression via regulation of TGFBRAP1 and HOTTIP expression

Background Despite the breadth of understanding the noncoding RNAs’ function in molecular biology, their functional roles in hepatocellular carcinoma (HCC) is poorly understood. In this study, we investigated the effect of hepatitis C virus (HCV) core upon the expression of noncoding RNAs. Methods The lncRNAs, mRNAs, and circRNAs were employed for identification of HCV core protein gene expression in human Huh7 hepatoma (Huh7) cell line. In data analysis, we applied a threshold that eliminated all genes that were not increased or decreased by at least a 2-fold change in a comparison between transfected and control cells. Hierarchical Clustering and the Kyoto encyclopedia of genes and genome pathway analyses were performed to show the distinguishable lncRNA, mRNAs, and circRNAs expression pattern among samples. Results The array data showed that 4,851 lncRNAs, 4,785 mRNAs, and 823 circRNAs were 2-fold up-regulated but 3,569 lncRNAs, 3,192 mRNAs, and 419 circRNAs were 2-fold down-regulated in Huh 7-core cells. The genes in the enriched set were associated with macromolecule and nucleic acid metabolic processes, DNA damage response and regulation of voltage-gated calcium channel. We identified 10 genes from the selected 14 genes that were higher or lower expression in Huh7-core cells than that of Huh7-vector cells by quantitative real-time polymerase chain reaction. Interestingly, overexpression of miR122 and miR204 partly abrogated the expression of TGFBRAP1 and HOTTIP, and increased the HPCAL1 expression in the predicted carcinogenic pathways. Conclusion Our data suggests that the pathways of miR204-HPCAL1-lncRNAHOTTIP and miR122-TGFBRAP1 were likely involved in the carcinogenic progress due to the presence of HCV core, and that overexpression of miR122 and miR204 might inhibit the HCC progress by down-regulation of TGFBRAP1 and HOTTIP expression.