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CircRNA is a kind of covalent head-to-tail looped RNA and plays an important role in tumor development. However, the identification of new potential targetable circRNAs to inhibit cancer development is still a huge challenge. In this study, we found that circEHMT1 inhibited migration and invasion of breast cancer cells. Mechanistically, we identified miR-1233-3p as a target of circEHMT1, and the circEHMT1/miR-1233-3p axis regulated matrix metalloprotease 2 (MMP2) by modulating the transcription factor Krϋppel-like factor 4 (KLF4). In summary, we showed that circEHMT1 has potential as a prognostic factor in breast cancer and played a tumor suppressor role via the circEHMT1/miR-1233-3p/KLF4/MMP2 axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aim: To study the epigenetic alternations and biological functions of ZFP82 in esophageal squamous cell carcinoma., Materials & Methods: Analysis of ZFP82 expression was carried out by quantitative real-time PCR. Cell function was tested by MTS cell proliferation assay, transwell assay and flow cytometry. Gene mechanisms were studied by reverse transcription-PCR (RT-PCR), quantitative real-time PCR, luciferase reporter assay and Western blot., Results: ZFP82 promoter methylation was downregulated in esophageal squamous cell carcinoma. ZFP82 ectopic expression suppressed cell function and regulated NF-κB phosphorylation and genes involved in cell cycle arrest and apoptosis. Moreover, ZFP82 methylation was correlated with age, tumor stage and outcome., Conclusion: ZFP82 is a tumor suppressor and is disrupted by promoter CpG methylation during esophageal carcinogenesis.

The abnormal expression of microRNAs is a key hallmark of breast cancer. Nevertheless, the biological roles of miR-1247-5p in breast cancer remain unknown. In this study, we revealed that miR-1247-5p expression was markedly decreased in breast cancer. It was a valuable diagnostic biomarker for breast cancer with the area under the curve of more than 0.80. Reduced miR-1247-5p expression was significantly correlated with patient age, tumor size, and poor prognosis in The Cancer Genome Atlas cohort including 839 breast cancer patients. Multivariate Cox regression analysis demonstrated that miR-1247-5p was an independent prognostic indicator for overall survival (hazard radio [HR] = 1.683, 95% confidence interval [CI] = 1.087-2.606, p = 0.020) and recurrence-free survival (HR = 2.496, 95% CI = 1.576-3.951, p < 0.001). Moreover, functional studies showed that overexpression of miR-1247-5p inhibited proliferation and induced apoptosis in breast cancer cells. Bioinformatics analysis and mechanistic investigations revealed that Dishevelled 1 (DVL1) was a direct target of miR-1247-5p. Inhibition of DVL1 by miR-1247-5p resulted in the suppression of Wnt/β-catenin signaling, whereas overexpression of DVL1 abrogated the miR-1247-5p-mediated effect. These data reveal that miR-1247-5p, as an oncosuppressor in breast cancer, may be a promising prognostic biomarker and therapeutic target., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Zinc finger proteins (ZFPs) are the largest transcription factor family in mammals. About one-third of ZFPs are Krüppel-associated box domain (KRAB)-ZFPs and involved in the regulation of cell differentiation/proliferation/apoptosis and neoplastic transformation. We recently identified ZNF382 as a novel KRAB-ZFP epigenetically inactivated in multiple cancers due to frequent promoter CpG methylation. However, its epigenetic alterations, biological functions/mechanism and clinical significance in oesophageal squamous cell carcinoma (ESCC) are still unknown. Here, we demonstrate that ZNF382 expression was suppressed in ESCC due to aberrant promoter methylation, but highly expressed in normal oesophagus tissues. ZNF382 promoter methylation is correlated with ESCC differentiation levels. Restoration of ZNF382 expression in silenced ESCC cells suppressed tumour cell proliferation and metastasis through inducing cell apoptosis. Importantly, ZNF382 suppressed Wnt/β-catenin signalling and downstream target gene expression, likely through binding directly to FZD1 and DVL2 promoters. In summary, our findings demonstrate that ZNF382 functions as a bona fide tumour suppressor inhibiting ESCC pathogenesis through inhibiting the Wnt/β-catenin signalling pathway.

Deregulation of msh homeobox 1 (MSX1) has been identified to be associated with multiple human malignant neoplasms. However, the association of the expression and biological function of MSX1 with breast tumorigenesis, and the underlying mechanism remain largely unknown. Therefore, the present study examined the expression and promoter methylation of MSX1 in breast tumor cell lines, primary breast tumors and normal breast tissues using semi-quantitative, quantitative and methylation-specific reverse transcription‑polymerase chain reaction. Colony formation assays, flow cytometric analysis, and wound healing and Transwell assays were used to assess various functions of MSX1. Western blot analyses were also conducted to explore the mechanism of MSX1. The results revealed that MSX1 was broadly expressed in normal human tissues, including breast tissues, but was frequently downregulated or silenced in breast cancer cell lines and primary tumors by promoter methylation. Methylation of the MSX1 promoter was observed in 7/9 (77.8%) breast cancer cell lines and 47/99 (47.5%) primary tumors, but not in normal breast tissues or surgical margin tissues, suggesting that tumor-specific methylation of MSX1 occurs in breast cancer. Pharmacological demethylation reduced MSX1 promoter methylation levels and restored the expression of MSX1. The ectopic expression of MSX1, induced by transfection with a lentiviral vector, significantly inhibited the clonogenicity, proliferation, migration and invasion of breast tumor cells by inducing G1/S cell cycle arrest and apoptosis. Ectopic MSX1 expression also inhibited the expression of active β-catenin and its downstream targets c-Myc and cyclin D1, and also increased the cleavage of caspase-3 and poly (ADP-ribose) polymerase. In conclusion, MSX1 exerts tumor-suppressive functions by inducing G1/S cell cycle arrest and apoptosis in breast tumorigenesis. Its methylation may be used as an epigenetic biomarker for the early detection and diagnosis of breast cancer.

Recent studies suggest that nucleotide-binding domain leucine-rich repeat protein 1 (NLRP1) is a pivotal factor in the inflammatory process. However, the role of NLRP1 in breast cancer pathogenesis remains unclear. The aim of this study was to examine the expression and function of NLRP1 in breast cancer. We found that NLRP1 was widely expressed in 83% (60/72) of primary breast cancer tissue. NLRP1 expression level was higher in primary breast cancer tissue than in adjacent noncancerous tissue ( p < 0.001) and NLRP1 expression was associated with lymph node metastasis ( p = 0.003), TNM stage ( p = 0.003), and Ki-67 levels ( p < 0.001). Overexpression of NLRP1 in the breast cancer cell line MCF-7 promotes proliferation, migration, invasion, and tumorigenicity in nude mice. Restoration of NLRP1 expression resulted in the EMT occurrence that downregulation of epithelial marker E-cadherin and upregulation mesenchymal marker vimentin, C-myc, MMP-9, and snail. In summary, NLRP1 promotes cell line MCF-7 the proliferation, migration, and invasion through inducing EMT.

Background/aims: Aberrant activation of the Wnt/β-catenin signaling pathway plays a key role in the pathogenesis of multiple tumors including digestive cancers. Recent studies have reported that Dickkopf-related protein 2 (DKK2) is epigenetically inactivated in numerous types of cancers and that its gene products exhibit tumor-suppressive properties. However, the biological functions and underlying molecular mechanisms of DKK2 in colon carcinoma remains obscure., Methods: We examined the expression of DKK2 in colon tumor cell lines by RT-PCR and its promoter methylation status in colon tumor cell lines and primary tumors by methylation-specific PCR (MSP). Ectopic expression of DKK2 was measured by RT-PCR prior to the other experiments. To investigate the function of DKK2, we assayed colony formation and cell proliferation, utilized flow cytometric analyses of the cell cycle and acridine orange/ethidium bromide (AO/EB) fluorescence staining for apoptosis, and examined wound healing, transwell migration and tumor growth in vivo. Western blots were used to explore the mechanisms of DKK2 in epithelial- mesenchymal transition and canonical Wnt/β-catenin signaling., Results: We show here that downregulation or silencing of DKK2 was closely associated with the hypermethylation status of its promoter and that DKK2 expression could be restored by demethylation treatment. Methylation of the DKK2 promoter was detected in nearly all tumors and tumor-adjacent tissues, but not in normal colon tissues. Ectopic expression of DKK2 in colon cell lines HCT116 and HT-29 inhibited colony formation and cell viability by inducing cell cycle G0/G1 arrest and apoptosis, and growth of stable DKK2-infected HCT116 cells in nude mice was decreased compared to controls. Furthermore, DKK2 restrained cell migration through partial reversal of epithelial-to- mesenchymal transition and also by downregulating several stem cell markers. Our data further showed that restoration of DKK2 expression resulted in downregulation of active β-catenin and its downstream target genes., Conclusion: DKK2 appears to be a functional tumor suppressor regulating tumorigenesis of colorectal cancer by antagonizing Wnt/β-catenin signaling., (© 2017 The Author(s)Published by S. Karger AG, Basel.)

Wnt signaling plays an important role in breast carcinogenesis. DAPPER2 (DACT2) functions as an inhibitor of canonical Wnt signaling and plays distinct roles in different cell contexts, with its role in breast tumorigenesis unclear. We investigated DACT2 expression in breast cancer cell lines and primary tumors, as well as its functions and molecular mechanisms. Results showed that DACT2 expression was silenced in 9/9 of cell lines. Promoter CpG methylation of DACT2 was detected in 89% (8/9) of cell lines, as well as in 73% (107/147) of primary tumors, but only in 20% (1/5) of surgical margin tissues and in none of normal breast tissues. Demethylation of BT549 and T47D cell lines with 5-aza-2'-deoxycytidine restored DACT2 expression along with promoter demethylation, suggesting that its downregulation in breast cancer is dependent on promoter methylation. Furthermore, ectopic expression of DACT2 induced breast cell apoptosis in vitro, and further inhibited breast tumor cell proliferation, migration and EMT, through antagonizing Wnt/β-catenin and Akt/GSK-3 signaling. Thus, these results demonstrate that DACT2 functions as a tumor suppressor for breast cancer but was frequently disrupted epigenetically in this cancer.

Recent studies suggest that paired box 5 (PAX5) is down-regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non-small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down-regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non-cancerous tissues (P = 0.0201). PAX5 down-regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down-regulation of β-catenin and up-regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down-regulating the β-catenin pathway and up-regulating GADD45G expression., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The incidence of young cases of breast cancer is higher in China compared to the western world. We aimed to explore differences in risk factors, clinicopathological features and treatment modes of young female breast cancer compared to older patients in West China. We collected clinical information from 12,209 female breast cancer patients in West China, including risk factors, clinicopathological features and treatment modes, from January 2010 to December 2012. Chi-square tests and the multivariate logistic regression analysis were applied for statistical analysis. There were 2,682 young (≤40 years) cases and 9,527 older cases at the time of breast cancer diagnosis. Young patients had a greater tumor diameter at diagnosis, and a higher probability of axillary lymph node and distant metastasis (P < 0.05). The progesterone receptor positive expression rate, estrogen receptor/progesterone receptor double positive expression rate, and human epidermal growth factor receptor 2 (HER2) negative expression rate was higher in young patients compared to older patients (P < 0.05). For young patients, the age at menarche was earlier, they had lower marriage rates, fewer pregnancies and births, and a lower breastfeeding rate (P < 0.05). A higher proportion of young patients underwent advanced operations, neoadjuvant and adjuvant chemotherapy, radiotherapy, and endocrine therapy compared to older patients (P < 0.05). We found significant differences in the clinicopathological features, risk factors and treatment modes between young (≤40 years) and older (>40 years) female breast cancer patients in West China. As some of these results differ from those found in the western female population, it is likely that the mechanism of tumorigenesis of young female breast cancer patients in West China may differ from that in western developed countries. Further investigation into the regional differences in breast cancer tumorigenesis is warranted.

The Krüppel-like factor 5 (KLF5) has been suggested to promote breast cell proliferation, survival and tumorigenesis. KLF5 protein degradation is increased by several E3 ubiquitin ligases, including WWP1 and SCFFbw7, through the ubiquitin-proteasome pathway. However, the deubiquitinase (DUB) of KLF5 has not been demonstrated. In this study, we identified ATXN3L as a KLF5 DUB by genome-wide siRNA screening. ATXN3L directly binds to KLF5, decreasing its ubiquitination and thus degradation. Functionally, knockdown of ATXN3L inhibits breast cancer cell proliferation partially through KLF5. These findings reveal a previously unrecognized role of ATXN3L in the regulation of KLF5 stability in breast cancer. ATXN3L might be a therapeutic target for breast cancer.

Protocadherin8 (PCDH8), an integral membrane protein, was reported to be a tumor suppressor involved in tumorigenesis in cancers. We aimed to investigate the epigenetic inactivation of PCDH8 and its tumor-suppressor function in nasopharyngeal carcinoma (NPC). Frequent downregulation/silencing of PCDH8 was found in NPC cell lines using semiquantitative PCR. Promoter methylation of PCDH8 was observed in 100% (5/5) of cell lines and 85.3% (35/41) of primary tumors by methylation-specific PCR, but not in normal nasopharyngeal tissues. Treatment of NPC cells with 5-aza-2'-deoxycytidine and/or trichostatin A could restore PCDH8 expression. Ectopic expression of PCDH8 in silenced NPC cells significantly inhibited cell colony formation and cell migration. For the first time, our study demonstrates the epigenetic inactivation of PCDH8 by promoter methylation and its tumor-suppressive function in NPC. Thus, PCDH8 could be identified as a tumor suppressor in NPC.

The estrogen receptors (ERs) play important roles in hepatocarcinogenesis, which is always fostered by persistent hepatitis B virus (HBV) infection. Recent studies have linked microRNAs (miRNAs) to viral pathogenesis or oncogenesis. ERα could lead to cell cycle progression or inhibition of apoptosis, while ERβ had opposite effects. Here we proposed that HBV affected ERs expression in viral oncogenesis, which might be triggered by miRNAs. The protein expression of ERα in HepG2.2.15 cells was much stronger than that in HepG2 cells, which was not consistent with its mRNA expression in these cells. MicroRNA-130a (miR-130a) was predicted to be a regulator of ERα by targeting its 3' untranslated region (3'-UTR). The enhanced green fluorescence protein (EGFP) reporter experiment confirmed the direct interaction of miR-130a and ERα. Moreover, ERα protein level was inversely correlated with the miR-130a level. Taken together, our studies supported that HBV infection might attenuate miR-130a expression and ERα was a direct target of miR-130a. Difference in miR-130a levels between HepG2 and HepG2.2.15 cells resulted in the difference in ERα expression, implying host-virus crosstalk in viral pathogenesis mediated by miRNAs.

Chromosome 3p harbors multiple tumor-suppressor genes. PLCD1, located at 3p22, encodes an enzyme that mediates regulatory signaling of energy metabolism, calcium homeostasis and intracellular movement. We investigated the epigenetic alterations of PLCD1 and its tumor suppressor function in breast cancer. Frequent downregulation/silencing of PLCD1 was shown in most breast cancer cell lines by using semi-quantitative PCR. Promoter methylation of PLCD1 was detected in 78% (7/9) of cell lines and 52% (13/25) of primary tumors by Methylation-specific PCR (MSP), but not in any tumor adjacent breast tissues and normal breast tissues, which was further confirmed by bisulfite genomic sequencing (BGS). The silencing of PLCD1 could be reversed by pharmacological demethylation, indicating a methylation-mediated mechanism. Ectopic expression of PLCD1 in silenced breast cancer cells significantly inhibited their colony formation. In addition, PLCD1 inhibited tumor cell migration and induced cell cycle G(2)/M arrest. Thus, this study for the first time demonstrates the frequent inactivation of PLCD1 by promoter methylation and its tumor inhibitory function in breast cancer. Tumor-specific methylation of PLCD1 might serve as a biomarker for possible early detection and prognosis prediction of breast cancer.