The aim of the present study was to investigate the regulatory effects of histone methylation modifications on the expression of miR-200c, as well as invasion and migration of gastric carcinoma cells. Gastric carcinoma cell line, MGC-803, were treated by 2.5 μmol/L histone methyltransferase inhibitor, DZNep. The expression of miR-200c was detected by real-time quantitative PCR (qRT-PCR). The epithelial-mesenchymal transition (EMT) indicators (ZEB1/2 and E/N-cadherin), EZH2, EED, SUZ12 and H3K27me3 expressions were detected by Western blot. Cell migration and invasion abilities were detected by Transwell and scratch tests. The result showed that, compared with DMSO (control) group, DZNep significantly increased the expression of miR-200c to about 2.1 times, inhibited ZEB1, ZEB2, and N-cadherin expressions, and activated E-cadherin expression; Also, DZNep decreased the protein expressions of EZH2, EED, SUZ12 and H3K27me3; Moreover, DZNep could inhibit MGC-803 cell invasive and migrative abilities, as well as MMP9 expression. These results suggest DZNep raises miR-200c expression to delay the invasion and migration of gastric carcinoma cells, and the underlying mechanisms involve the regulations of EMT-related proteins and polycomb repressive complex 2.
Adenosine ; analogs & derivatives ; pharmacology ; Cadherins ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Epithelial-Mesenchymal Transition ; Gene Expression Regulation, Neoplastic ; Homeodomain Proteins ; metabolism ; Humans ; MicroRNAs ; metabolism ; Protein Methyltransferases ; antagonists & inhibitors ; Repressor Proteins ; metabolism ; Transcription Factors ; metabolism ; Zinc Finger E-box Binding Homeobox 2 ; Zinc Finger E-box-Binding Homeobox 1

miR-200c has been shown to regulate the epithelial-mesenchymal transition (EMT) by inhibiting ZEB1 and ZEB2 expression in breast cancer cells. This study further examined the role of miR-200c in the invasion and metastasis of breast cancer that goes beyond the regulation on ZEB1 and ZEB2 expression. In this study, the bioinformatics software (miRanda) was used to predict the target gene of miR-200c and Renilla luciferase assay to verify the result. The metastatic breast cancer cells MDA-MB-231 were cultured and transfected with the miR-200c mimic or inhibitor. The expressions of miR-200c and HMGB1 were detected by RT-PCR and Western blotting, respectively. Transwell assay and wound healing assay were employed to examine the invasive and migrating ability of transfected cells. Target prediction and Renilla luciferase analysis revealed that HMGB1 was a putative target gene of miR-200c. After transfection of MDA-MB-231 cells with the miR-200c mimic or inhibitor, the expression of miR-200c was significantly increased or decreased when compared with cells transfected with the miR-200c mimic NC or inhibitor NC. Moreover, the expression of HMGB1 was reversely correlated with that of miR-200c in transfected cells. Tranwell assay showed that the number of invasive cells was significantly reduced in miR-200c mimic group when compared with miR-200c inhibitor group. It was also found that the migrating ability of cells transfected with miR-200c mimics was much lower than that of cells transfected with miR-200c inhibitors. It was suggested that miR-200c can suppress the invasion and migration of breast cancer cells by regulating the expression of HMGB1. miR-200c and HMGB1 may become useful biomarkers for progression of breast cancer and targets of gene therapy.
Biomarkers, Tumor ; Breast Neoplasms ; genetics ; metabolism ; Cell Movement ; genetics ; Epithelial-Mesenchymal Transition ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; HMGB1 Protein ; genetics ; Homeodomain Proteins ; biosynthesis ; Humans ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; genetics ; Neoplasm Metastasis ; genetics ; pathology ; Repressor Proteins ; biosynthesis ; Transcription Factors ; biosynthesis ; Zinc Finger E-box Binding Homeobox 2 ; Zinc Finger E-box-Binding Homeobox 1

OBJECTIVETo investigate the expression of zinc finger E-box binding homebox 1 (ZEB1) in the prepuce of hypospadias children and its relationship to the incidence of hypospadias.

METHODSPrepuce tissues were collected from 37 children aged 6-15 months undergoing hypospadias repair and 11 age-matched controls receiving circumcision. Based on the position of the urethral meatus, the hypospadias cases were classified as severe (n = 13) and mild-moderate (n = 24). The mRNA and protein expressions of ZEB1 were determined by immunohistochemistry and RT-PCR.

RESULTSThe expression of the ZEB1 protein was remarkably higher in the severe (100% [13/13]) and mild-moderate hypospadias patients (75.0% [18/24]) than in the controls (9.1% [1/11]), with statistically significant differences between any two groups (P < 0.05). RT-PCR showed the integrated density value (IDV) of the ZEB1 mRNA expression to be (0.67 ± 0.21), (0.81 ± 0.24), and (1.55 ± 0.29) in the control, mild-moderate, and severe hypospadias patients, respectively, significantly higher in the severe hypospadias than in the control and mild-moderate hypospadias groups (P < 0.05), but with no significant difference between the latter two (P = 0.64).

CONCLUSIONThe expression of ZEB1 is significantly increased in hypospadias patients, and its upregulation is positively correlated with the severity of hypospadias, which suggests that the overexpression of ZEB1 may contribute to the development of hypospadias.

Biomarkers ; metabolism ; Case-Control Studies ; Circumcision, Male ; Foreskin ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Hypospadias ; classification ; etiology ; metabolism ; Immunohistochemistry ; Infant ; Male ; Penis ; RNA, Messenger ; metabolism ; Transcription Factors ; genetics ; metabolism ; Up-Regulation ; Urethra ; Zinc Finger E-box-Binding Homeobox 1

It has been reported that one of the factors that promotes tumoral progression is the abnormal activation of the epithelial-mesenchymal transition program. This process is associated with tumoral cells acquiring invasive and malignant properties and has the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1) as one of its main activators. However, the role of ZEB1 in promoting malignancy in prostate cancer (PCa) is still unclear. Here, we report that ZEB1 expression correlates with Gleason score in PCa samples and that expression of ZEB1 regulates epithelial-mesenchymal transition and malignant characteristics in PCa cell lines. The results showed that ZEB1 expression is higher in samples of higher malignancy and that overexpression of ZEB1 was able to induce epithelial-mesenchymal transition by upregulating the mesenchymal marker Vimentin and downregulating the epithelial marker E-Cadherin. On the contrary, ZEB1 silencing repressed Vimentin expression and upregulated E-Cadherin. ZEB1 expression conferred enhanced motility and invasiveness and a higher colony formation capacity to 22Rv1 cells whereas DU145 cells with ZEB1 silencing showed a decrease in those same properties. The results showed that ZEB1 could be a key promoter of tumoral progression toward advanced stages of PCa.
Cadherins/metabolism* ; Cell Line, Tumor ; Cell Movement/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Male ; Neoplasm Grading ; Neoplasm Invasiveness/genetics* ; Prostatic Neoplasms/pathology* ; Vimentin/metabolism* ; Zinc Finger E-box-Binding Homeobox 1/metabolism*

One of the factors promoting tumoral progress is the abnormal activation of the epithelial-mesenchymal transition (EMT) program which has been associated with chemoresistance in tumoral cells. The transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), a key EMT activator, has recently been related to docetaxel resistance, the main chemotherapeutic used in advanced prostate cancer treatment. The mechanisms involved in this protective effect are still unclear. In a previous work, we demonstrated that ZEB1 expression induced an EMT-like phenotype in prostate cancer cell lines. In this work, we used prostate cancer cell lines 22Rv1 and DU145 to study the effect of ZEB1 modulation on docetaxel resistance and its possible mechanisms. The results showed that ZEB1 overexpression conferred to 22Rv1 cell resistance to docetaxel while its silencing made DU145 cells more sensitive to it. Analysis of resistance markers showed no presence of ATP-binding cassette subfamily B member 1 (MDR1) and no changes in breast cancer resistance protein (BCRP) or ATP-binding cassette subfamily C member 10 (MRP7). However, a correlation between ZEB1, multidrug resistance-associated protein 1 (MRP1), and ATP-binding cassette subfamily C member 4 (MRP4) expression was observed. MRP4 inhibition, using MK571, resensitized cells with ZEB1 overexpression to docetaxel treatment. In addition, modulation of ZEB1 and subsequent change in MRP4 expression correlated with a lower apoptotic response to docetaxel, characterized by lower B-cell lymphoma 2 (Bcl2), high BCL2-associated X protein (Bax), and high active caspase 3 expression. The response to docetaxel in our model seems to be mediated mainly by activation of the apoptotic death program. Our results showed that modulation of MRP4 could be a mediator of ZEB1-related resistance to docetaxel in prostate cancer, making it a possible marker for chemotherapy response in patients who do not express MDR1.
Antineoplastic Agents/therapeutic use* ; Blotting, Western ; Cell Line, Tumor ; Docetaxel/therapeutic use* ; Drug Resistance, Neoplasm ; Epithelial-Mesenchymal Transition/drug effects* ; Gene Silencing ; Humans ; Male ; Prostatic Neoplasms/metabolism* ; Zinc Finger E-box-Binding Homeobox 1/metabolism*

OBJECTIVE: To explore the role of SIRT1 in the occurrence of epithelial-mesenchymal transition (EMT) in EC-9706 and Eca-109 cells and the possible mechanism. METHODS: Three chemically synthesized siRNA targeting SIRT1 were transfected into EC-9706 and Eca-109 cells with the non-transfected cells and cells transfected with the negative siRNAs as controls. Real-time PCR and Western blotting were used to detect the expressions of SIRT1, E-cadherin, vimentin, Snail, Twist1 and ZEB in the cells. Transwell invasion assay and wounding healing assay were used to examine the changes in the invasion and metastasis abilities of the cells after transfection. RESULTS: EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3 showed significantly decreased mRNA and protein expressions of SIRT1 ( < 0.05). Transwell invasion assay and wounding healing assay showed that transfection with SIRT1 siRNA1 and SIRT1 siRNA3 caused significantly lowered invasion and metastasis abilities in EC-9706 and Eca-109 cells ( < 0.05). In EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3, the expression level of E-cadherin was significantly increased while the expressions of vimentin, Snail and Twist were significantly lowered ( < 0.05). CONCLUSIONS SIRT1 participates in the invasion and metastasis of EC-9706 and Eca- 109 cells probably by inducing EMT via regulating the expression of Snail.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Epithelial-Mesenchymal Transition ; physiology ; Humans ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; metabolism ; Sirtuin 1 ; genetics ; metabolism ; Snail Family Transcription Factors ; metabolism ; Transfection ; Twist-Related Protein 1 ; metabolism ; Vimentin ; metabolism ; Zinc Finger E-box-Binding Homeobox 1 ; metabolism

OBJECTIVETo investigate the effect and mechanism of B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine on pulmonary metastasis in mouse model of melanoma.

METHODSTwelve 8-week old female C57BL/6 mice were used in this study. The mice were injected with wild-type B16F10 cells through tail vein after immunization with B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine, and the pulmonary metastasis was observed. The CD4(+) and CD8(+) T cells were isolated by magnetic activated cell sorting, and then used for the detection of CFSE/7-AAD cytotoxicity by flow cytometry. Serum from the mice immunized with tumor-cell vaccine was used to detect IFN-γ expression by ELISA. The expression of TGF-β2, ZEB1, E-cadherin, and N-cadherin of tumor tissues was detected by RT-PCR and immunofluorescence, respectively.

RESULTSThe mice vaccinated with B16F10-ESAT-6-gpi/IL-21 had significantly fewer nodules in the lung and lower lung weight [(285.8 ± 19.01) mg vs. (406.3 ± 27.12) mg], with lower levels of TGF-β2, ZEB1 and N-cadherin proteins but higher level of E-cadherin protein within the tumor tissue, as compared with the control mice. Meanwhile, the immunized mice had significantly increased CD8(+) T cell killing activity [(42.62 ± 3.465)% vs. (22.29 ± 1.804)%] and IFN-γ expression level [(55.200 ± 7.173) pg/ml vs. (6.435 ± 1.339) pg/ml] over the control mice.

CONCLUSIONSThe B16F10-ESAT-6-gpi/IL-21 vaccine can inhibit the metastasis of melanoma in the lung in vaccinated melanoma-bearing mice. This inhibitory effect is associated with CD8(+) T cell immune response and a higher level of IFN-γ, which may influence on the mesenchymal-epithelial transition of tumor cells.

Animals ; CD8-Positive T-Lymphocytes ; immunology ; Cadherins ; metabolism ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition ; Female ; Homeodomain Proteins ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukins ; immunology ; Lung ; pathology ; Lung Neoplasms ; metabolism ; secondary ; Melanoma ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Organ Size ; Transcription Factors ; metabolism ; Transforming Growth Factor beta2 ; metabolism ; Zinc Finger E-box-Binding Homeobox 1

Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; physiology ; Cadherins ; metabolism ; Cell Transformation, Neoplastic ; metabolism ; pathology ; Epithelial Cells ; pathology ; Homeodomain Proteins ; metabolism ; physiology ; Humans ; Mesenchymal Stromal Cells ; pathology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasms ; metabolism ; pathology ; Snail Family Transcription Factors ; Transcription Factors ; metabolism ; physiology ; Zinc Finger E-box-Binding Homeobox 1

OBJECTIVETo identify potential markers for predicting invasion, metastasis, and prognosis of gastric adenocarcinoma (GAC).

METHODSThe expressions of Slug, ZEB1 and KISS-1 were detected immunohistochemically in 261 GAC tissues and 80 normal gastric tissues.

RESULTSThe positivity rates of Slug, ZEB1, and KISS-1 in gastric tissues were 2.5%, 1.3%, and 87.5%, respectively, significantly different from the rates of 62.1%, 28.4%, and 41.1% in GAC tissues (P<0.05). The expression level of Slug was significantly correlated with the depth of invasion, lymph node metastasis, and pTNM stages; the positivity rates of both ZEB1 and KISS-1 were significantly correlated with the tumor grade, depth of invasion, lymph node metastasis and pTNM stages. Slug expression was positively correlated with ZEB1 expression, and KISS-1 expression was inversely correlated with Slug and ZEB1 expressions. Kaplan-Meier analysis showed that the overall survival time of patients with positive expressions of Slug and ZEB1 was significantly shorter than that of the negative patients, and the survival time of patients positive for KISS-1 was significantly longer than the negative patients. COX multivariate analysis showed that positive Slug, ZEB1 and KISS-1 protein expressions and pTNM stages were independent prognostic factors of GAC (P<0.05).

CONCLUSIONThe abnormal expressions of Slug, ZEB1 and KISS-1 may contribute to the tumorigenesis of GAC and are related with lymph node metastasis, pTNM stages, and prognosis of GAC. The combined detection of Slug, ZEB1, and KISS-1 expression has an important value in predicting the progression and prognosis of GAC.

Adenocarcinoma ; metabolism ; pathology ; Disease Progression ; Homeodomain Proteins ; metabolism ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Kisspeptins ; metabolism ; Lymphatic Metastasis ; Neoplasm Grading ; Prognosis ; Proportional Hazards Models ; Snail Family Transcription Factors ; Stomach Neoplasms ; metabolism ; pathology ; Transcription Factors ; metabolism ; Zinc Finger E-box-Binding Homeobox 1

OBJECTIVETo investigate the antiproliferative and anti-metastasis effect of Xihuang Pill (, XP) on human colorectal cancer cell and to explore the molecular mechanism by which it produces the effects.

METHODSHighly metastatic human colorectal cancer cell line LoVo was treated with low-, medium-, and highdose XP-containing serum (XP-L, XP-M, XP-H) groups for 48 h, cells intervened with no drug rat serum and PD98059 [extracellular signal-regulated kinase (ERK) inhibitor] as negative and positive controls (NC and PC) groups. Cell proliferation assay was made using cell counting kit-8 (CCK8). The 8 μm pore-size transwell chamber and 4', 6-diamidino-2-phenylindole (DAPI) staining were applied to examine the ability of invasion and migration of the cells. The protein expression of ERK1/2, zinc fifi nger E-box-binding homeobox 1 (ZEB1), Scrib and lethal giant larvae homolog 2 (Lgl2) was detected by Western blotting while the relative mRNA quantity of E-cadherin, N-cadherin, Occludin and junctional adhesion molecule-1 (JAM1) was measured by realtime fluorescent quantitative polymerase chain reaction (RT-qPCR).

RESULTSXP induced a dose-dependent suppression on the proliferation of LoVo cells (P <0.05 or P<0.01), with the inhibition rates varied from 27.30% to 31.08%. Transwell assay showed that when preprocessed with PD98059 and XP-containing serum, the number of cells that passed the filter decreased significantly compared with that of NC group (P <0.05 or P<0.01). Moreover, XP inhibited the protein expression of ERK1/2 and ZEB1 (P <0.05); and up-regulated the protein expression of Scrib and Lgl2 (P <0.05). The mRNA levels of E-cadherin, Occludin and JAM1 of the XP intervened groups and PC group markedly ascended (P <0.05) while that of N-cadherin showed a descending tendency (P>0.05).

CONCLUSIONXP intervention suppressed the ability of proliferation, invasion and migration of the LoVo cells. Regulating ZEB1-SCRIB Loop so as to recover epithelial phenotype and apical junctional complex might be one of the mechanisms by which XP produces the anti-metastasis effect.

Animals ; Cadherins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Polarity ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; genetics ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Epithelial-Mesenchymal Transition ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Homeodomain Proteins ; metabolism ; Humans ; Intercellular Junctions ; drug effects ; metabolism ; Membrane Proteins ; metabolism ; Neoplasm Invasiveness ; Phenotype ; RNA, Messenger ; genetics ; metabolism ; Rats, Wistar ; Transcription Factors ; metabolism ; Tumor Suppressor Proteins ; metabolism ; Zinc Finger E-box-Binding Homeobox 1