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1.
Horm Mol Biol Clin Investig ; 41(2)2020 Apr 17.
Article in English | MEDLINE | ID: mdl-32304301

ABSTRACT

Background Gonadotrophins exert their functions by binding follicle-stimulating hormone receptor (FSHR) or luteinizing hormone and human chorionic gonadotropin receptor (LHCGR) present on endometrium. Within ovaries, FSH induces autophagy and apoptosis of granulosa cells leading to atresia of non-growing follicles, whereas hCG and LH have anti-apoptotic functions. Endometrial cells express functioning gonadotrophin receptors. The objective of this study was to analyze the effect of gonadotrophins on physiology and endometrial cells survival. Materials and methods Collected endometria were incubated for 48 or 72 h with 100 ng/mL of recombinant human FSH (rhFSH), recombinant human LH (rhLH) or highly purified hCG (HPhCG) alone or combined. Controls omitted gonadotrophins. The effect of gonadotrophins on cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1), hypoxia inducible factor 1α (HIF1A), and cell-death-related genes expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Immunohistochemistry for microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and apoptotic protease activating factor 1 (APAF-1) was performed. Results Gonadotrophins are able to modulate the endometrial cells survival. FSH induced autophagy and apoptosis by increasing the relative expression of MAP1LC3B and FAS receptor. In FSH-treated samples, expression of apoptosis marker APAF-1 was detected and co-localized on autophagic cells. hCG and LH does not modulate the expression of cell-death-related genes while the up-regulation of pro-proliferative epiregulin gene was observed. When combined with FSH, hCG and LH prevent autophagy and apoptosis FSH-induced. Conclusions Different gonadotrophins specifically affect endometrial cells viability differently: FSH promotes autophagy and apoptosis while LH and hCG alone or combined with rhFSH does not.


Subject(s)
Chorionic Gonadotropin/pharmacology , Endometrium/drug effects , Follicle Stimulating Hormone/pharmacology , Gene Expression Regulation/drug effects , Luteinizing Hormone/pharmacology , Adult , Apoptosis/drug effects , Apoptotic Protease-Activating Factor 1/biosynthesis , Apoptotic Protease-Activating Factor 1/genetics , Autophagy/drug effects , Cholesterol Side-Chain Cleavage Enzyme/biosynthesis , Cholesterol Side-Chain Cleavage Enzyme/genetics , Chorionic Gonadotropin/physiology , Endometrium/metabolism , Epiregulin/biosynthesis , Epiregulin/genetics , Female , Follicle Stimulating Hormone/physiology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Luteinizing Hormone/physiology , Menstrual Cycle , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Recombinant Proteins/pharmacology , fas Receptor/biosynthesis , fas Receptor/genetics
2.
Pathol Res Pract ; 215(5): 873-879, 2019 May.
Article in English | MEDLINE | ID: mdl-30738695

ABSTRACT

Epiregulin (EREG) is a novel family member of EGF-like ligands and have elevated expression in a variety of human cancers. EREG expression promotes tumor progression and metastasis and reduces patient survival. However, the expression of EREG and its prognostic value are not clear in gastric cancer (GC). We assessed EREG mRNA and protein expression in GC tissues from Chinese patients using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining of tissue microarray, and analyzed the correlation between the level of EREG expression and patient clinical characteristics and prognosis. We found that EREG expression was significantly higher in GC tissues than in matched adjacent noncancerous tissues. High EREG protein expression in GC was significantly associated with TNM stage including tumor size, lymph node metastases and distant metastases as well as poor overall survival. These finding demonstrate that EREG is an independent prognostic biomarker for GC.


Subject(s)
Biomarkers, Tumor/analysis , Carcinoma/pathology , Epiregulin/biosynthesis , Stomach Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Carcinoma/mortality , Disease-Free Survival , Epiregulin/analysis , Female , Humans , Male , Middle Aged , Prognosis , Stomach Neoplasms/mortality
3.
J Cell Biochem ; 119(3): 2911-2922, 2018 03.
Article in English | MEDLINE | ID: mdl-29091309

ABSTRACT

CFTR is a cAMP-regulated chloride channel, whose mutations produce cystic fibrosis. The impairment of CFTR activity increases the intracellular Cl- concentration, which in turn produces an increased interleukin-1ß (IL-1ß) secretion. The secreted IL-1ß then induces an autocrine positive feedback loop, further stimulating IL-1ß priming and secretion. Since IL-1ß can transactivate the epidermal growth factor receptor (EGFR), we study here the levels of expression for different EGFR ligands in Caco-2/pRS26 cells (expressing shRNA against CFTR resulting in a reduced CFTR expression and activity). The epiregulin (EREG), amphiregulin (AREG), and heparin binding EGF like growth factor (HBEGF) mRNAs, were found overexpressed in Caco-2/pRS26 cells. The EREG mRNA had the highest differential expression and was further characterized. In agreement with its mRNA levels, Western blots (WB) showed increased EREG levels in CFTR-impaired cells. In addition, EREG mRNA and protein levels were stimulated by incubation with exogenous IL-1ß and inhibited by the Interleukin 1 receptor type I (IL1R1) antagonist IL1RN, suggesting that the overexpression of EREG is a consequence of the autocrine IL-1ß loop previously described for these cells. In addition, the JNK inhibitor SP600125, and the EGFR inhibitors AG1478 and PD168393, also had an inhibitory effect on EREG expression, suggesting that EGFR, activated in Caco-2/pRS26 cells, is involved in the observed EREG upregulation. In conclusion, in Caco-2 CFTR-shRNA cells, the EGFR ligand EREG is overexpressed due to an active IL-1ß autocrine loop that indirectly activates EGFR, constituting new signaling effectors for the CFTR signaling pathway, downstream of CFTR, Cl- , and IL-1ß.


Subject(s)
Autocrine Communication , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epiregulin/biosynthesis , Epithelial Cells/metabolism , Interleukin-1beta/metabolism , Up-Regulation , Caco-2 Cells , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Epiregulin/genetics , Epithelial Cells/cytology , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Interleukin-1beta/genetics
4.
J Biol Chem ; 292(25): 10574-10585, 2017 06 23.
Article in English | MEDLINE | ID: mdl-28465351

ABSTRACT

Hepatocyte nuclear factor 4α (HNF4α) controls the expression of liver-specific protein-coding genes. However, some microRNAs are also modulated by HNF4α, and it is not known whether they are direct targets of HNF4α and whether they influence hepatic function. In this study, we found that HNF4α regulates microRNAs, indicated by marked down-regulation of miR-194 and miR-192 (miR-194/192) in liver-specific Hnf4a-null (Hnf4aΔH) mice. Transactivation of the shared miR-194/192 promoter was dependent on HNF4α expression, indicating that miR-194/192 is a target gene of HNF4α. Screening of potential mRNAs targeted by miR-194/192 revealed that expression of genes involved in glucose metabolism (glycogenin 1 (Gyg1)), cell adhesion and migration (activated leukocyte cell adhesion molecule (Alcam)), tumorigenesis and tumor progression (Rap2b and epiregulin (Ereg)), protein SUMOylation (Sumo2), epigenetic regulation (Setd5 and Cullin 4B (Cln4b)), and the epithelial-mesenchymal transition (moesin (Msn)) was up-regulated in Hnf4aΔH mice. Moreover, we also found that miR-194/192 binds the 3'-UTR of these mRNAs. siRNA knockdown of HNF4α suppressed miR-194/192 expression in human hepatocellular carcinoma (HCC) cells and resulted in up-regulation of their mRNA targets. Inhibition and overexpression experiments with miR-194/192 revealed that Gyg1, Setd5, Sumo2, Cln4b, and Rap2b are miR-194 targets, whereas Ereg, Alcam, and Msn are miR-192 targets. These findings reveal a novel HNF4α network controlled by miR-194/192 that may play a critical role in maintaining the hepatocyte-differentiated state by inhibiting expression of genes involved in dedifferentiation and tumorigenesis. These insights may contribute to the development of diagnostic markers for early HCC detection, and targeting of the miR-194/192 pathway could be useful for managing HCC.


Subject(s)
Gene Expression Regulation/physiology , Hepatocyte Nuclear Factor 4/metabolism , Hepatocytes/metabolism , MicroRNAs/metabolism , Signal Transduction/physiology , 3' Untranslated Regions/physiology , Activated-Leukocyte Cell Adhesion Molecule/biosynthesis , Activated-Leukocyte Cell Adhesion Molecule/genetics , Animals , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Epiregulin/biosynthesis , Epiregulin/genetics , Glucosyltransferases/biosynthesis , Glucosyltransferases/genetics , Glycoproteins/biosynthesis , Glycoproteins/genetics , Hepatocyte Nuclear Factor 4/genetics , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Mice , Mice, Mutant Strains , MicroRNAs/genetics , Microfilament Proteins/biosynthesis , Microfilament Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/biosynthesis , Small Ubiquitin-Related Modifier Proteins/genetics
5.
Br J Cancer ; 114(12): 1352-61, 2016 06 14.
Article in English | MEDLINE | ID: mdl-27272216

ABSTRACT

BACKGROUND: High EREG and AREG expression, and left-sided primary tumours are associated with superior efficacy of anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (CRC), but a unifying explanation of these findings is lacking. METHODS: RNA-seq, gene expression arrays, and DNA methylation profiling were completed on 179 CRC tumours. Results were validated using independent The Cancer Genome Atlas data sets. An independent cohort of 198 KRAS wild-type metastatic CRC tumours was tested for CpG island methylator phenotype (CIMP) status, and progression-free survival (PFS) with the first anti-EGFR regimen was retrospectively determined. RESULTS: EREG and AREG expression was highly inversely correlated with methylation and was inversely associated with right-sided primary tumour, BRAF mutation, and CIMP-high status. Treatment of CRC cell lines with hypomethylating agents decreased methylation and increased expression of EREG. Inferior PFS with anti-EGFR therapy was associated with CIMP-high status, BRAF mutation, NRAS mutation, and right-sided primary tumour on univariate analysis. Among known BRAF/NRAS wild-type tumours, inferior PFS remained associated with CIMP-high status (median PFS 5.6 vs 9.0 mo, P=0.023). CONCLUSIONS: EREG and AREG are strongly regulated by methylation, and their expression is associated with CIMP status and primary tumour site, which may explain the association of primary tumour site and EREG/AREG expression with anti-EGFR therapy efficacy.


Subject(s)
Amphiregulin/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , CpG Islands , DNA Methylation , Epiregulin/genetics , Amphiregulin/biosynthesis , Cohort Studies , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Epiregulin/biosynthesis , ErbB Receptors/antagonists & inhibitors , HCT116 Cells , Humans , Male , Phenotype , Promoter Regions, Genetic , Proto-Oncogene Proteins p21(ras)/biosynthesis , Proto-Oncogene Proteins p21(ras)/genetics
6.
Mol Cancer ; 14: 138, 2015 Jul 29.
Article in English | MEDLINE | ID: mdl-26215578

ABSTRACT

BACKGROUND: The epidermal growth factor (EGF) family of ligands has been implicated in promoting breast cancer initiation, growth and progression. The contributions of EGF family ligands and their receptors to breast cancer are complex, and the specific mechanisms through which different ligands regulate breast tumor initiation and growth are not well-defined. These studies focus on the EGF family member epiregulin (EREG) as a mediator of early stage breast tumorigenesis. METHODS: EREG expression levels were assessed in both cell lines and human samples of ductal carcinoma in situ (DCIS) using quantitative RT-PCR, ELISA and immunohistochemistry. Gene knock-down approaches using shRNA-based strategies were used to determine the requirement of EREG for growth of MCF10DCIS cells in vivo, and for identifying mechanisms through which EREG promotes tumor cell survival. Experiments were performed using a combination of two-dimensional culture, three-dimensional culture and tumor growth in vivo. RESULTS: In comparison with other EGF family members, EREG was induced in MCF10DCIS cells compared with MCF10A and MCF10AT cells and its expression was partially regulated by fibroblast growth factor receptor (FGFR) activity. Reduced EREG expression in MCF10DCIS cells led to decreased tumor growth in vivo, which was associated with reduced cell survival. Furthermore, treatment of MCF10A cells with exogenous EREG enhanced cell survival both in three-dimensional culture and in response to chemotherapeutic agents. Examination of EREG-induced signaling pathways demonstrated that EREG promoted survival of MCF10A cells through regulating expression of matrix metalloproteinase-1 (MMP-1). To determine the relevance of these findings in human tumors, samples of DCIS were analyzed for EREG and MMP-1 expression. EREG was induced in DCIS lesions compared to normal breast epithelium, and EREG and MMP-1 were correlated in a subset of DCIS samples. CONCLUSIONS: Together, these studies lead to identification of a novel pathway involving EREG and MMP-1 that contributes to the formation of early stage breast cancer. Understanding these complex pathways could ultimately lead to the development of novel biomarkers of neoplastic progression and/or new therapeutic strategies for patients with early stage cancer.


Subject(s)
Breast Neoplasms/genetics , Carcinogenesis/genetics , Carcinoma, Intraductal, Noninfiltrating/genetics , Epiregulin/biosynthesis , Matrix Metalloproteinase 1/biosynthesis , Breast Neoplasms/pathology , Carcinogenesis/pathology , Carcinoma, Intraductal, Noninfiltrating/pathology , Cell Line, Tumor , Epiregulin/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Matrix Metalloproteinase 1/genetics , Neoplasm Staging , Receptor, Fibroblast Growth Factor, Type 1/genetics
7.
Biol Reprod ; 93(3): 65, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26203177

ABSTRACT

Oocyte maturation and cumulus cell expansion depend on luteinizing hormone (LH)-mediated upregulation of membrane-bound epidermal growth factor (EGF)-like ligands, including amphiregulin, epiregulin, and betacellulin. These ligands then transactivate the EGF receptor (EGFR) after release by matrix metalloproteinases (MMPs). However, direct measurement of released EGF-like ligands or MMPs from granulosa cells has not been formally evaluated, nor has direct identification of responsible MMPs. Here we address these issues by analyzing LH-induced steroidogenesis, which is also MMP and EGFR dependent, in freshly isolated mouse primary granulosa cells. We demonstrate a correlation between amphiregulin and epiregulin mRNA induction and steroid production in LH-treated granulosa cells as well as in ovaries of human chorionic gonadotropin-treated mice. In contrast, LH does not alter Mmp1, Mmp2, Mmp3, Mmp8, Mmp9, or Adam17 mRNA expression. We demonstrate that, in primary mouse granulosa cells, LH triggers release of soluble amphiregulin that correlates with steroid production, both of which are blocked by MMP2/9 inhibition, confirming that MMP2/9 likely regulates LH-induced amphiregulin release and downstream processes. Notably, LH does not alter secretion of MMP2/9 from primary granulosa cells, nor does it modulate MMP activity. These findings indicate that, in the ovary, LH dictates EGFR-mediated processes not by regulating MMPs, but instead by increasing EGF-like ligand availability. In contrast, LH stimulation of primary mouse Leydig cells does not induce EGF-like ligand expression or require MMP2/9 for steroidogenesis, confirming marked differences in LH receptor-induced processes in the testes. Our results suggest that MMP inhibition may be a means of attenuating excess ovarian steroid production in diseases like polycystic ovary syndrome.


Subject(s)
ErbB Receptors/metabolism , Luteinizing Hormone/pharmacology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Ovary/metabolism , Steroids/biosynthesis , Testis/metabolism , Amphiregulin , Animals , EGF Family of Proteins/biosynthesis , Epiregulin/biosynthesis , Epiregulin/genetics , ErbB Receptors/genetics , Female , Granulosa Cells/drug effects , Granulosa Cells/enzymology , Leydig Cells/drug effects , Leydig Cells/metabolism , Ligands , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Mice , Mice, Inbred C57BL , Ovary/enzymology , Primary Cell Culture , Testis/enzymology , Up-Regulation/drug effects
8.
Eur J Med Res ; 19: 52, 2014 Oct 29.
Article in English | MEDLINE | ID: mdl-25358439

ABSTRACT

BACKGROUND: The aim of this study was to identify the candidate genes of esophageal squamous cell carcinoma (ESCC). METHODS: Gene expression profiling of 17 ESCC samples and 17 adjacent normal samples, GSE20347, was downloaded from Gene Expression Omnibus database. The raw data were preprocessed, and the differentially expressed genes (DEGs) between ESCC and normal samples were identified by using SAM software (false discovery rate <0.001). Then, the co-expression network of DEGs was constructed based on Pearson's correlation test (r-value ≥0.8). Furthermore, the topological properties of the co-expression network were analyzed through NetworkAnalyzer (default settings) of Cytoscape. The expression fold changes of DEGs and topological properties were utilized to identify the candidate genes of ESCC (Crin score >4), which were further analyzed based on DAVID functional enrichment analysis (P-value <0.05). RESULTS: A total of 1,063 DEGs were identified, including 490 up-regulated and 573 down-regulated DEGs. Then, the co-expression network of DEGs was constructed, containing 999 nodes and 46,323 edges. Based on the expression fold changes of DEGs and the topological properties of the co-expression network, DEGs were ranked, and the top 24 genes were candidate genes of ESCC, such as CRISP3, EREG, CXCR2, and CRNN. Furthermore, the 24 genes were significantly enriched in bio-functions regarding cell differentiation, glucan biosynthetic process and immune response. CONCLUSION: The present study suggested that CRISP3, EREG, CXCR2, and CRNN might be causative genes of ESCC, and play vital roles in the development of ESCC. However, further experimental studies are needed to confirm our results.


Subject(s)
Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Gene Expression Regulation, Neoplastic/genetics , Gene Regulatory Networks , Genetic Association Studies/classification , Carcinoma, Squamous Cell/pathology , Computational Biology , Databases, Genetic , Epiregulin/biosynthesis , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma , Gene Expression Profiling , Humans , Membrane Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Oligonucleotide Array Sequence Analysis , Receptors, Interleukin-8B/biosynthesis , Salivary Proteins and Peptides/biosynthesis , Seminal Plasma Proteins/biosynthesis
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