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Ji J.,Xi'an Jiaotong University | Zheng P.-S.,Xi'an Jiaotong University | Zheng P.-S.,Section of Cancer Stem Cell Research
Human Pathology | Year: 2010

Sox2 is a key transcription factor for embryonic development and plays a critical role in determining the fate of stem cells. Recently, Sox2 has been detected in several human tumors, indicating a potential function in tumorigenesis. We initially reported remarkably increased nuclear Sox2 staining in cervical carcinomas compared with normal cervix (P < .05). Furthermore, Sox2 staining was detected in most tumorsphere cells isolated from fresh cervical cancer tissues but not among the differentiated tumorsphere cells. When Sox2 was stably expressed in cervical cancer cells (SiHa and HeLa), Sox2-overexpressing cells had increased proliferation, clonogenicity, and tumorigenicity in vitro and in vivo than control cells. These results suggest that Sox2 may participate in carcinogenesis of cervical carcinomas and may be a potential therapeutic target molecule for cervical cancers. © 2010 Elsevier Inc. All rights reserved.


Yang W.-T.,Xi'an Jiaotong University | Zheng P.-S.,Xi'an Jiaotong University | Zheng P.-S.,Section of Cancer Stem Cell Research
PLoS ONE | Year: 2014

Objective: The KLF4 gene has been shown to be inactivated in cervical carcinogenesis as a tumor suppressor. However, the mechanism of KLF4 silencing in cervical carcinomas has not yet been identified. DNA methylation plays a key role in stable suppression of gene expression. Methods: The methylation status of the KLF4 promoter CpG islands was analyzed by bisulfite sequencing (BSQ) in tissues of normal cervix and cervical cancer. KLF4 gene expression was detected by RT-PCR, immunohistochemistry and western blot. KLF4 promoter methylation in cervical cancer cell line was determined by BSQ and methylation-specific polymerase chain reaction (MS-PCR). Cell proliferation ability was detected by cell growth curve and MTT assay. Results: The methylated allele was found in 41.90% of 24 cervical cancer tissues but only in 11.11% of 11 normal cervix tissues (P<0.005). KLF4 mRNA levels were significantly reduced in cervical cancer tissues compared with normal cervix tissues (P<0.01) and KLF4 mRNA expression showed a significant negative correlation with the promoter hypermethylation (r = 20.486, P = 0.003). Cervical cancer cell lines also showed a significant negative correlation between KLF4 expression and hypermethylation. After treatment with the demethylating agent 5-Azacytidine (5-Aza), the expression of KLF4 in the cervical cancer cell lines at both mRNA and protein levels was drastically increased, the cell proliferation ability was inhibited and the chemosensitivity for cisplatin was significantly increased. Conclusion: KLF4 gene is inactivated by methylation-induced silencing mechanisms in a large subset of cervical carcinomas and KLF4 promoter hypermethylation inactivates the gene's function as a tumor suppressor in cervical carcinogenesis. © 2014 Yang, Zheng.


Liu X.,Xi'an Jiaotong University | Yang W.-T.,Xi'an Jiaotong University | Zheng P.-S.,Xi'an Jiaotong University | Zheng P.-S.,Section of Cancer Stem Cell Research
Oncotarget | Year: 2014

Musashi RNA-binding protein1 (Msi1), a member of the RNA-binding protein family, has been reported to be a diagnostic marker and potential therapeutic target in some cancers, its function in cervical cancer remains unknown. In this study, we found Msi1 was highly expressed in cervical cancer tissues, and over-expressing Msi1 in cervical cancer cells enhanced tumor formation and cell proliferation and accelerated cells into the S phase. Whereas, down-regulating Msi1 by shRNA in cervical cancer cells inhibited tumor formation and cell proliferation and slowed cell into the S phase, suggesting that Msi1 might act as cell cycle regulator. Immunohistochemistry assay showed the negative correlation between Msi1 and p21, p27 and p53, suggesting that Msi1 might regulate these cycle regulators in cervical cancer. Moreover, the expression of the p21, p27 and p53 proteins were down-regulated in Msi1 overexpressing cervical cancer cells and up-regulated in shMsi1 cervical cancer cells. Luciferase assays and RNA-protein binding assays confirmed that Msi1 could bind to the mRNA 3'UTRs of p21, p27 and p53 and suppress the translation of these proteins. Our findings provide new evidence that Msi1 might promote cell proliferation by accelerating the cell cycle by directly targeting p21, p27 and p53.


Li B.,Xi'an Jiaotong University | Xiao L.,XiAn Honghui Hospital | Wang Z.Y.,Xi'an Jiaotong University | Zheng P.S.,Xi'an Jiaotong University | Zheng P.S.,Section of Cancer Stem Cell Research
Free Radical Research | Year: 2014

Stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca2+ signaling systems and are considered to be important players in regulating neuronal Ca2+ homeostasis under normal ageing and pathological conditions. Here, we investigated the potential role of STIM1 in 6-hydroxydopamine (6-OHDA)-induced toxicity in undifferentiated PC12 cell lines. Cells exposed to 6-OHDA demonstrated alterations in the generation of reactive oxygen species (ROS) in a Ca2+-dependent manner. Downregulation of STIM1 expression by specific small interfering RNA (siRNA) attenuated apoptotic cell death, reduced intracellular ROS production, and partially prevented the impaired endogenous antioxidant enzyme activities after 6-OHDA treatment. Furthermore, STIM1 knockdown significantly attenuated 6-OHDA-induced intracellular Ca2+ overload by inhibiting endogenous store-operated calcium entry (SOCE). The effect of STIM1 siNRA on SOCE was related to orai1 and L-type Ca2+ channels, but not to transient receptor potential canonical type 1 (TRPC1) channel. In addition, silencing of STIM1 increased the Ca2+ buffering capacity of the endoplasmic reticulum (ER) in 6-OHDA-injured cells. ER vacuoles formed from the destruction of ER structural integrity and activation of ER-related apoptotic factors (CHOP and Caspase-12) were partially prevented by STIM1 knockdown. Moreover, STIM1 knockdown attenuated 6-OHDA-induced mitochondrial Ca2+ uptake and mitochondrial dysfunction, including the collapse of mitochondrial membrane potential (MMP) and the decrease of ATP generation. Taken together, our data provide the first evidence that inhibition of STIM1-meditated intracellular Ca2+ dyshomeostasis protects undifferentiated PC12 cells against 6-OHDA toxicity and indicate that STIM1 may be responsible for neuronal oxidative stress induced by ER stress and mitochondrial dysfunction in PD. © 2014 Informa UK, Ltd.


Liu S.-Y.,Xi'an Jiaotong University | Zheng P.-S.,Xi'an Jiaotong University | Zheng P.-S.,Section of Cancer Stem Cell Research
Oncotarget | Year: 2013

High aldehyde dehydrogenase (ALDH) activity characterizes a subpopulation of cells with cancer stem cell (CSC) properties in several malignancies. To clarify whether ALDH can be used as a marker of cervical cancer stem cells (CCSCs), ALDHhigh and ALDHlow cells were sorted from 4 cervical cancer cell lines and 5 primary tumor xenografts and examined for CSC characteristics. Here, we demonstrate that cervical cancer cells with high ALDH activity fulfill the functional criteria for CSCs: (1) ALDHhigh cells, unlike ALDHlow cells, are highly tumorigenic in vivo; (2) ALDHhigh cells can give rise to both ALDHhigh and ALDHlow cells in vitro and in vivo, thereby establishing a cellular hierarchy; and (3) ALDHhigh cells have enhanced self-renewal and differentiation potentials. Additionally, ALDHhigh cervical cancer cells are more resistant to cisplatin treatment than ALDHlow cells. Finally, expression of the stem cell self-renewal-associated transcription factors OCT4, NANOG, KLF4 and BMI1 is elevated in ALDHhigh cervical cancer cells. Taken together, our data indicated that high ALDH activity may represent both a functional marker for CCSCs and a target for novel cervical cancer therapies.


Zhang Y.,Xi'an Jiaotong University | Li B.,Xi'an Jiaotong University | Ji Z.-Z.,Xi'an Jiaotong University | Zheng P.-S.,Xi'an Jiaotong University | Zheng P.-S.,Section of Cancer Stem Cell Research
Cancer | Year: 2010

BACKGROUND: The aberrant activation of the Notch signaling has been associated with the development of colon cancers. However, the role of Notch1 in the pathogenesis of colon cancers is poorly understood. METHODS: The expression of Notch1 in colon cancer tissues and nontumor tissues and in colon cancer cell lines was examined by Western blot analysis and immunohistochemistry. The impact of small interfering RNA (siRNA)-mediated Notch1 knockdown or Notch1 intracellular domain (NICD)-based transgene-induced Notch1 overexpression on the proliferation, cell cycling, apoptosis, colony formation, and tumorsphere formation in vitro and the development and growth of implanted tumors in vivo was characterized. RESULTS: Notch1 was overexpressed in colon cancer tissues, and the levels of Notch1 expression in different types of colon cancers were associated with the pathologic grade, progression, and metastasis of colon cancers. Furthermore, knockdown of Notch1 significantly inhibited the proliferation, colony formation, and tumorsphere formation of SW480 and HT-29 cells, induced apoptosis and cell cycle arrest at G0/G1 phase, and mitigated the development and growth of implanted colon cancers in vivo. In contrast, Notch1 overexpression promoted the proliferation, colony formation, cell cycling, and tumorsphere formation of colon cancer cells in vitro and the development and growth of implanted colon cancers in vivo, but it inhibited spontaneous apoptosis. CONCLUSIONS: The current results indicated that Notch1 signaling positively regulates the growth of colon cancers. Conceivably, the modulation of Notch1-related signaling may be a promising therapy for human colon cancers. © 2010 American Cancer Society.


Cui N.,Xi'an Jiaotong University | Yang W.-T.,Xi'an Jiaotong University | Yang W.-T.,Section of Cancer Stem Cell Research | Zheng P.-S.,Xi'an Jiaotong University
Oncotarget | Year: 2016

Slug (Snai2) has been demonstrated to act as an oncogene or tumor suppressor in different human cancers, but the function of Slug in cervical cancer remains poorly understood. In this study, we demonstrated that Slug could suppress the proliferation of cervical cancer cells in vitro and tumor formation in vivo. Further experiments found that Slug could trans-suppress the expression of Akt1/p-Akt1 by binding to E-box motifs in the promoter of the Akt1 gene and then inhibit the cell proliferation and tumor formation of cervical cancer cells by up-regulating p21/p27 and/or down-regulating the activity of the Wnt/β-catenin signaling pathway. Therefore, Slug acts as a tumor suppressor during cervical carcinogenesis.


Chen Q.,Xi'an Jiaotong University | Zheng P.-S.,Xi'an Jiaotong University | Zheng P.-S.,Section of Cancer Stem Cell Research | Yang W.-T.,Xi'an Jiaotong University
Oncotarget | Year: 2016

Enhancer of zeste homolog 2 (EZH2), a catalytic core component of the Polycomb repressive complex 2 (PRC2), stimulates the silencing of target genes through histone H3 lysine 27 trimethylation (H3K27me3). Recent findings have indicated EZH2 is involved in the development and progression of various human cancers. However, the exact mechanism of EZH2 in the promotion of cervical cancer is largely unknown. Here, we show that EZH2 expression gradually increases during the progression of cervical cancer. We identified a significant positive correlation between EZH2 expression and cell proliferation in vitro and tumor formation in vivo by the up-regulation or downregulation of EZH2 using CRISPR-Cas9-mediated gene editing technology and shRNA in HeLa and SiHa cells. Further investigation indicated that EZH2 protein significantly accelerated the cell cycle transition from the G0/G1 to S phase. TOP/FOP-Flash reporter assay revealed that EZH2 significantly activated Wnt/β-catenin signaling and the target genes of Wnt/β-catenin pathway were up-regulated, including β-catenin, cyclin D1, and c-myc. Moreover, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays confirmed that EZH2 inhibited the expression of glycogen synthase kinase-3β (GSK-3β) and TP53 through physically interacting with motifs in the promoters of the GSK-3β and TP53 genes. Additionally, blockage of the Wnt/β-catenin pathway resulted in significant inhibition of cell proliferation, and activation of the Wnt/β-catenin pathway resulted in significant enhancement of cell proliferation, as induced by EZH2. Taken together, our data demonstrate that EZH2 promotes cell proliferation and tumor formation in cervical cancer through activating the Wnt/β-catenin pathway by epigenetic silencing via GSK-3β and TP53.


Wang Y.-D.,Xi'an Jiaotong University | Cai N.,Xi'an Jiaotong University | Wu X.-L.,Xi'an Jiaotong University | Cao H.-Z.,Xi'an Jiaotong University | And 3 more authors.
Cell Death and Disease | Year: 2013

Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene that maintains the pluripotency and self-renewal properties of embryonic stem cells. Although there is emerging evidence that it can function as oncogene in several cancers, the role in mediating cervical cancer remains unexplored. Here we found that OCT4 protein expression showed a pattern of gradual increase from normal cervix to cervical carcinoma in situ and then to invasive cervical cancer. Overexpression of OCT4 in two types of cervical cancer cells promotes the carcinogenesis, and inhibits cancer cell apoptosis. OCT4 induces upregulation of miR-125b through directly binding to the promoter of miR-125b-1 confirmed by chromatin immunoprecipitation analysis. MiRNA-125b overexpression suppressed apoptosis and expression of BAK1 protein. In contrast, miR-125b sponge impaired the anti-apoptotic effect of OCT4, along with the upregulated expression of BAK1. Significantly, Luciferase assay showed that the activity of the wild-type BAK1 30-untranslated region reporter was suppressed and this suppression was diminished when the miR-125b response element was mutated or deleted. In addition, we observed negative correlation between levels of BAK1 and OCT4, and positive between OCT4 and miR-125b in primary cervical cancers. These findings suggest an undescribed regulatory pathway in cervical cancer, by which OCT4 directly induces expression of miR-125b, which inhibits its direct target BAK1, leading to suppression of cervical cancer cell apoptosis. © 2013 Macmillan Publishers Limited All rights reserved.


Gu T.-T.,Xi'an Jiaotong University | Gu T.-T.,Section of Cancer Stem Cell Research | Liu S.-Y.,Xi'an Jiaotong University | Liu S.-Y.,Section of Cancer Stem Cell Research | And 2 more authors.
American Journal of Pathology | Year: 2012

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell gene NANOG, a divergent homeodomain transcription factor that is independent of leukemia inhibitory factor, has been reported to be expressed in germ cells and in several tumor types. However, the short-term expression and role of NANOG in cervical cancer remain unclear. In the present study, we demonstrate that NANOG exhibits cellular shuttling behavior and increasing stromal distribution during the progression of cervical cancer. Our molecular data using RT-PCR and restriction enzyme digestion show that NANOG is mainly transcribed from the NANOG gene in cervical cancer. In addition, IHC using confocal microscopy suggests that mesenchymal stem cells (MSCs) are one type of cytoplasmic NANOG-positive cells in cervical cancer stroma. Co-culture of cervical cancer-derived MSCs with SiHa cells showed increased proliferation characteristics in vitro and enhanced tumor growth in vivo. Our results show, for the first time to our knowledge, that MSCs are a source of cytoplasmic NANOG expression in the cervical cancer stroma and that they participate in the progression of cervical cancer both in vitro and in vivo. Our study provides evidence that NANOG is a cervical cancer progression marker and also serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and the multifunctionality of the stromal microenvironment. © 2012 American Society for Investigative Pathology.

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