OBJECTIVES: Increasing detection of low-risk papillary thyroid carcinoma (PTC) is associated with overdiagnosis and overtreatment. N6-methyladenosine (m⁶A)-mediated microRNA (miRNA) dysregulation plays a critical role in tumor metastasis and progression. However, the functional role of m⁶A-miRNAs in PTC remains unclear. This study aims to elucidate the regulatory mechanism of m⁶A-miR-139-5p expression in PTC, determine its association with PTC metastasis, and evaluate its potential as a diagnostic biomarker for PTC metastasis, thereby providing experimental evidence for precision diagnosis and therapy. METHODS: Expression profiles of m⁶A-miRNAs were compared between the The Cancer Genome Atlas (TCGA) and GSE130512 cohorts to identify metastasis-associated candidates. Clinical specimens from 13 metastasis and 18 non-metastasis PTC patients were analyzed to assess m⁶A-miR-139-5p expression and its correlation with metastasis. Functional experiments were conducted to investigate the effect of fat mass and obesity-associated protein (FTO) on pri-miR-139 methylation and processing, clarifying its regulatory role in miR-139-5p expression. In TPC-1 cells, MTT assays were performed to evaluate whether miR-139-5p overexpression could counteract FTO-mediated cell proliferation. Transwell invasion assays were used to determine the impact of miR-139-5p on PTC cell invasion, exploring whether it functions through the ZEB1/E-cadherin axis. RESULTS: By comparing TCGA and GSE130512 cohorts, it was found that circulating m⁶A-miR-139-5p could serve as a biological indicator for detecting PTC metastasis. Detection of 13 metastatic and 18 non-metastatic clinical specimens showed that FTO inhibited the processing of pri-miR-139 by reducing its methylation level, leading to the dysregulation of miR-139-5p in PTC (P<0.05). In TPC-1 cells, MTT assay showed that overexpression of miR-139-5p could partially reverse FTO overexpression-mediated cell proliferation (P<0.05). In addition, miR-139-5p inhibited the invasive ability of PTC cells by targeting the ZEB1/E-cadherin axis, while FTO overexpression could partially weaken this inhibitory effect. CONCLUSIONS Circulating miR-139-5p can be a potential marker for evaluating PTC metastasis. FTO affects the expression and function of miR-139-5p by regulating m⁶A modification of pri-miR-139, but its clinical value needs further verification.
Humans ; MicroRNAs/metabolism* ; Thyroid Cancer, Papillary/metabolism* ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism* ; Thyroid Neoplasms/metabolism* ; Cell Line, Tumor ; Neoplasm Metastasis ; Adenosine/genetics* ; Gene Expression Regulation, Neoplastic ; Female ; Male ; Cadherins/metabolism* ; Cell Proliferation ; Zinc Finger E-box-Binding Homeobox 1/genetics*

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

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*

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

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 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