LncRNA WDR11-AS1 Promotes Extracellular Matrix Synthesis in Osteoarthritis by Directly Interacting with RNA-Binding Protein PABPC1 to Stabilize SOX9 Expression.

Osteoarthritis (OA) is a degenerative disease of articular cartilage that is mainly characterized by chronic and mild inflammation of the joints. Recently, many studies have reported the crucial roles of long noncoding RNAs (lncRNAs) in OA as gene transcriptional regulatory factors, diagnostic biomarkers, or therapeutic targets. However, the exact mechanisms of lncRNAs in the regulation of OA progression remain unclear. In the present study, the lncRNA WDR11 divergent transcript (lncRNA WDR11-AS1) was shown to be downregulated in osteoarthritic cartilage tissues from patients, and to promote extracellular matrix (ECM) synthesis in osteoarthritic chondrocytes with knockdown and overexpression experiments. This function of lncRNA WDR11-AS1 was linked to its ability to interact with the polyadenylate-binding protein cytoplasmic 1 (PABPC1), which was screened by RNA pulldown and mass spectrometry analyses. PABPC1 was discovered to bind ECM-related mRNAs such as SOX9, and the inhibition of PABPC1 improved the mRNA stability of SOX9 to mitigate OA progression. Our results suggest that lncRNA WDR11-AS1 has a promising inhibitory effect on inflammation-induced ECM degradation in OA by directly binding PABPC1, thereby establishing lncRNA WDR11-AS1 and PABPC1 as potential therapeutic targets in the treatment of OA.

Interpreting the MicroRNA-15/107 family: interaction identification by combining network based and experiment supported approach.

BACKGROUND: The highly conservative miR-15/107 family (also named as miR-15/107 gene group) including ten miRNA members is currently recognized strongly implicated in multiple human disorders. Some studies focus on the entire family rather than individual miRNA for a bigger picture, while there is also certain signature dysregulation for some of the individual miRNA implicated even in the same disorder. METHODS: Faced with the exponential growth of experimental evidence, our study tries to analyze their function and target interactions using various bioinformatics tools. RESULTS: Firstly, the evolutionary conservative "AGCAGC" sequence and possible clustered transcriptional pattern were described. Secondly, both the experimentally validated and bioinformatically predicted miRNA-target gene relationship of the entire family was analyzed to understand the mechanism of underlying collective effects for target regulation from the miR-15/107 family. Moreover, pathway analysis among miR-15/107 family was performed and displayed in detail, while its impact on cell proliferation is experimentally validated. Eventually, the dysregulation of miR-15/107 in diseases was discussed. CONCLUSIONS: In summary, our study proposes that the collective functions and implication of miR-15/107 family in various human diseases are achieved relying on the massive overlapping target genes. While the minor differences within target gene interaction among family members could also explain the signature behavior for some of the individual miRNA in aspects such as its disease-specific dysregulation and various participation in pathways.

A novel long noncoding RNA Lnc-HC binds hnRNPA2B1 to regulate expressions of Cyp7a1 and Abca1 in hepatocytic cholesterol metabolism.

UNLABELLED: Cholesterol metabolism disorder in hepatocytes predicts a higher risk of metabolic syndrome (MetS). Long noncoding RNAs (lncRNAs) have emerged as critical players in cellular cholesterol metabolism, but their functions are not systematically clarified. Here, we have identified a novel lncRNA named lnc-HC negatively regulating cholesterol metabolism within hepatocytes through physical interaction with hnRNPA2B1. By further binding to the target messenger RNA of Cyp7a1 or Abca1, the lnc-HC-hnRNPA2B1 complex decreases expressions of the two genes that are implicated in cellular cholesterol excretion. lnc-HC knockdown can strongly recover the cholesterol disorder in vivo. In the upstream pathway, lnc-HC is up-regulated by high cholesterol by the transcription activator, CCAAT/enhancer-binding protein beta. CONCLUSION: These findings suggest a subtle feed-forward regulation of lnc-HC in cholesterol metabolism and define a novel line of evidence by which lncRNAs modulate the metabolic system at the post-transcriptional level. (Hepatology 2016;64:58-72).

TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species.

Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein. Se deficiency is associated with an endemic osteoarthropathy characterized by impaired cartilage formation. It is unclear whether TrxR2 have roles in cartilage function. We examined the effects of TrxR2 on chondrogenic ATDC5 cells through shRNA-mediated gene silencing of TrxR2. We demonstrated TrxR2 deficiencies could enhance chondrogenic differentiation and apoptosis of ATDC5 cells. TrxR2 deficiencies increased accumulation of cartilage glycosaminoglycans (GAGs) and mineralization. TrxR2 deficiencies also stimulated expression of extracellular (ECM) gene including Collagen II and Aggrecan. The enhanced chondrogenic properties were further confirmed by activation of Akt signaling which are required for chondrogenesis. In addition, TrxR2 deficiencies promoted chondrocyte proliferation through acceleration of cell cycle progression by increase in both S and G2/M phase cell distribution accompanied with induction of parathyroid hormone-related protein (PTHrP). Moreover, TrxR2 deficiencies induced chondrocyte death via apoptosis and increased cell sensitivity to exogenous oxidative stress. Furthermore, TrxR2 deficiencies induced emission of mitochondrial reactive oxygen species (ROS) without alteration of mitochondrial membrane potential and intracellular ATP content. Finally, treatment of TrxR2 deficiency cells with N-acetylcysteine (NAC) inhibited mitochondrial ROS production and chondrocyte apoptosis. NAC also prevented chondrogenic differentiation of TrxR2 deficiency cells by suppression of ECM gene expression, GAGs accumulation and mineralization, as well as attenuation of Akt signaling. Thus, TrxR2-mediated mitochondrial integrity is indispensable for chondrogenic differentiation of ATDC5 cells. TrxR2 deficiency-induced impaired proliferation and death of chondrocytes may be the pathological mechanism of the osteoarthropathy due to Se deficiency. Notably, this study also uncover the roles of mitochondrial ROS which could stimulate cartilage ECM synthesis that offer novel insights for development of therapeutic agent to prevent cartilage degeneration in human disease.

GPx1 knockdown suppresses chondrogenic differentiation of ATDC5 cells through induction of reductive stress.

Glutathione peroxidase 1 (GPx1) is a selenium (Se)-containing protein and is induced in cartilage formation. GPx1 eliminates reactive oxygen species (ROS), which are required for chondrogenic induction. The physiological properties of GPx1 in cartilage and the redox mechanisms involved are not known. The effects of GPx1 on chondrogenic differentiation of ATDC5 cells were examined through short hairpin RNA-mediated gene silencing. The results demonstrated that GPx1 knockdown impaired gene expression of sex determining region Y-box 9, collagen II (Col II), and aggrecan. GPx1 knockdown suppressed the accumulation of cartilage glycosaminoglycans (GAGs) and the proliferation of chondrocyte. GPx1 knockdown also induced cell apoptosis. However, cell sensitivity toward exogenous oxidative stress was not increased after GPx1 knockdown. Unexpectedly, GPx1 knockdown not only induced oxidative stress characterized by the increased production of ROS but also caused reductive stress indicated by an elevation of glutathione (GSH)/oxidized GSH (GSSG) ratio. Furthermore, GPx1 knockdown-mediated reductive and oxidative stress could be antagonized by a thiol-oxidizing agent diamide and a thiol-containing compound N-acetylcysteine (NAC), respectively. Moreover, NAC attenuated GPx1 knockdown-induced cell apoptosis, while diamide prevented GPx1 knockdown-suppressed chondrocyte proliferation. Finally, diamide but not NAC could rescue GPx1 knockdown-mediated impaired chondrogenic differentiation. In summary, GPx1 is essential for chondrogenic induction in ATDC5 cells mainly through modulation of intracellular GSH/GSSG ratio, rather than an antioxidant enzyme to detoxify ROS. In addition, GPx1 knockdown-induced impaired chondrogenesis may participate in the pathogenesis of the endemic osteoarthropathy due to Se deficiency. These observations offer novel insights for the development of therapeutic target during cartilage degeneration.

Selenoprotein O deficiencies suppress chondrogenic differentiation of ATDC5 cells.

Selenoprotein O (Sel O) is a selenium-containing protein, but its function is still unclear. In the present study, we observed that the mRNA and protein expression levels of Sel O increased during chondrogenic induction of ATDC5 cells. The effects of Sel O on chondrocyte differentiation were then examined through shRNA-mediated gene silencing technique. The expression of Sel O was significantly suppressed at both mRNA and protein levels in a stable cell line transfected with a Sel O-specific target shRNA construct. Thereafter, we demonstrated that Sel O deficiencies suppress chondrogenic differentiation of ATDC5 cells. Sel O deficiencies inhibited expression of chondrogenic gene Sox9, Col II, and aggrecan. Sel O-deficient cells also accumulated a few cartilage glycosaminoglycans (GAGs) and decreased the activity of alkaline phosphatase (ALP). In addition, Sel O deficiencies inhibited chondrocyte proliferation through delayed cell cycle progression by suppression of cyclin D1 expression. Moreover, Sel O deficiencies induced chondrocyte death through cell apoptosis. In summary, we describe the expression patterns and the essential roles of Sel O in chondrocyte viability, proliferation, and chondrogenic differentiation. Additionally, Sel O deficiency-mediated impaired chondrogenesis may illustrate the mechanisms of Se deficiency in the pathophysiological process of the endemic osteoarthropathy.

NCF4 regulates antigen presentation of cysteine peptides by intracellular oxidative response and restricts activation of autoreactive and arthritogenic T cells.

Autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematous, are regulated by polymorphisms in genes contributing to the NOX2 complex. Mutations in both Ncf1 and Ncf4 affect development of arthritis in experimental models of RA, but the different regulatory pathways mediated by NOX2-derived reactive oxygen species (ROS) have not yet been clarified. Here we address the possibility that intracellular ROS, regulated by the NCF4 protein (earlier often denoted p40phox) which interacts with endosomal membranes, could play an important role in the oxidation of cysteine peptides in mononuclear phagocytic cells, thereby regulating antigen presentation and activation of arthritogenic T cells. To study the role of NCF4 we used mice with an amino acid replacing mutation (NCF4R58A), which is known to affect interaction with endosomal membranes, leading to decreased intracellular ROS production. To study the impact of NCF4 on T cell activation, we used the glucose phosphate isomerase peptide GPI325-339, which contains two cysteine residues (325-339c-c). Macrophages from mice with the NCF458A mutation efficiently presented the peptide when the two cysteines were intact and not crosslinked, leading to a strong arthritogenic T cell response. T cell priming occurred in the draining lymph nodes (LNs) within 8 days after immunization. Clodronate treatment, which depletes antigen-presenting mononuclear phagocytes, ameliorated arthritis severity, whereas treatment with FYT720, which traps activated T cells in LNs, prohibited arthritis. We conclude that NCF4-dependent intracellular ROS maintains cysteine peptides in an oxidized crosslinked state, which prevents presentation of peptides recognized by non-tolerized T cells and thereby protects against autoimmune arthritis.

Selenium effect on selenoprotein transcriptome in chondrocytes.

Selenium is an essential micronutrient and exerts its biological functions predominantly through selenoproteins. Selenium deficiency is associated with cartilage function. This study demonstrated that all 24 selenoprotein transcripts in mouse genome were detectable in ATDC5 chondrocytes except deiodinase 1 (DIO1), DIO2, and selenoprotein V (Sel V), while all 25 selenoprotein transcripts in human genome were detectable in C28/I2 chondrocytes except glutathione peroxidase 6 (GPx6) and DIO1. In addition, gene expression of five selenoproteins (GPx1, Sel H, Sel N, Sel P, and Sel W) was up-regulated and two selenoproteins (SPS2 and Sel O) was down-regulated by sodium selenite (Se) in both ATDC5 and C28/I2 cells. Gene expression of six selenoproteins (TrxR1, Sel I, Sel M, Sel R, Sel S, Sel T) and one selenoprotein (GPx3) was up-regulated by Se in ATDC5 and C28/I2 cells, respectively. Gene expression of one selenoprotein (TrxR2) was down-regulated by Se only in ATDC5 cells. Further transcription inhibition assay showed that both transcriptional and posttranscriptional mechanisms involved in Se-regulated gene expression of GPx1, TrxR1, TrxR2, SPS2, Sel O, and Sel S. However, Se-regulated gene expression of Sel H, Sel I, Sel M, Sel N, Sel P, Sel R, Sel T, and Sel W mainly at posttranscriptional level. Moreover, new protein synthesis inhibition assay indicated that Se-mediated new protein synthesis also played roles in Se-regulated gene expression of GPx1, TrxR1, TrxR2, Sel H, Sel O, Sel P, Sel R, and Sel W. In summary, this study described the selenoprotein transcriptome, Se-regulated selenoproteins and possible mechanisms involved in chondrocytes.

Giant neurogenic tumors of mediastinum: report of two cases and literature review.

Neurogenic tumors are commonly found in the mediastinum, especially in the posterior mediastinum or in the chest wall, neurogenic tumors may reach large size before becoming symptomatic. If the neurogenic tumor occupied more than half size of the chest wall accompanied by mediastinal shift, tracheal compression, or superior vena reflux disorder, it may be called giant intrathoracic neurogenic tumors. Giant intrathoracic neurogenic tumors are relatively rare. Most of intrathoracic neurogenic tumors were benign or low-grade malignant tumors in nature. Complete surgical excision should be the rule for these patients. We report two cases of giant neurogenic tumors, and study the clinical manifestations, diagnostic methods, surgical management, and prognosis in the light of the most important published data.

Critical Roles of Protein Arginine Methylation in the Central Nervous System.

A remarkable post-transitional modification of both histones and non-histone proteins is arginine methylation. Methylation of arginine residues is crucial for a wide range of cellular process, including signal transduction, DNA repair, gene expression, mRNA splicing, and protein interaction. Arginine methylation is modulated by arginine methyltransferases and demethylases, like protein arginine methyltransferase (PRMTs) and Jumonji C (JmjC) domain containing (JMJD) proteins. Symmetric dimethylarginine and asymmetric dimethylarginine, metabolic products of the PRMTs and JMJD proteins, can be changed by abnormal expression of these proteins. Many pathologies including cancer, inflammation and immune responses have been closely linked to aberrant arginine methylation. Currently, the majority of the literature discusses the substrate specificity and function of arginine methylation in the pathogenesis and prognosis of cancers. Numerous investigations on the roles of arginine methylation in the central nervous system (CNS) have so far been conducted. In this review, we display the biochemistry of arginine methylation and provide an overview of the regulatory mechanism of arginine methyltransferases and demethylases. We also highlight physiological functions of arginine methylation in the CNS and the significance of arginine methylation in a variety of neurological diseases such as brain cancers, neurodegenerative diseases and neurodevelopmental disorders. Furthermore, we summarize PRMT inhibitors and molecular functions of arginine methylation. Finally, we pose important questions that require further research to comprehend the roles of arginine methylation in the CNS and discover more effective targets for the treatment of neurological diseases.

Selenium promotes proliferation of chondrogenic cell ATDC5 by increment of intracellular ATP content under serum deprivation.

Selenium (Se) is an essential micronutrient, and low Se intake in Se-deficient areas plays roles in an endemic osteochondropathy characterized by chondronecrosis in growth plate and articular cartilage. However, the biological activities of Se on cartilage are largely unknown. In this study, we examined the effects of Se on chondrogenic cell ATDC5 and the possible mechanisms involved. We demonstrated that Se stimulated ATDC5 cell proliferation under serum deprivation but not routine culture. Furthermore, Se promoted G1-phase cell cycle progression along with induction of cyclin D1 expression at the mRNA and protein level. Moreover, Se increased intracellular ATP content and decreased intracellular superoxide anion concentration without affecting intracellular redox status as estimated by ratio of the reduced and oxidized glutathione. In addition, suppression of intracellular ATP synthesis by glycolysis inhibitor or mitochondrial uncoupler both abrogated Se-mediated cyclin D1 induction. These findings suggest Se stimulates proliferation of chondrogenic cell ATDC5 through acceleration of cell cycle progression accompanied with cyclin D1 induction by enhancement of intracellular ATP content. This novel finding provides evidence for a role of Se in cartilage formation and degenerative processes and further supports the relationship between Se status and cartilage function that may lead to better utilization of Se for cartilage homeostasis.

DeltaEF1 promotes breast cancer cell proliferation through down-regulating p21 expression.

Although the zinc finger-homeodomain transcription factor deltaEF1 is implied as a regulatory factor at the crossroad between proliferation and differentiation in carcinogenesis, its potential effect in the regulation of cell cycle progression has not been well elucidated. In our present study, we provide novel finding that, in breast cancer, the ectopic expression of deltaEF1 in MDA-MB-231 cells significantly promoted cell proliferation by increasing the cell number in S phase of the cell cycle. In contrast, deltaEF1 knockdown by RNA interference exhibited an opposite effect, highlighting a potent role of deltaEF1 to promote G1-S transition of breast cancer cells. Moreover, we demonstrated that deltaEF1 down-regulated p21 and concurrently up-regulated the expressions of CDK2 and CDK4 during this process. Further, deltaEF1 inhibited p21 transcription by recruiting to the E(2) box element on the p21 promoter. Depletion of endogenous deltaEF1 in MDA-MB-231 cells was sufficient to allow an inherent release of p21 expression, thus resulting in the cell cycle arrest. In addition, the stimulatory effect of deltaEF1 on cell proliferation through p21 regulation was supported by an inverse correlation of deltaEF1 and p21 expressions observed in both breast cancer cell lines and clinical tumor specimens. Taken together, these observations suggest a dual effect of deltaEF1 in promoting breast cancer cell proliferation, by differentially regulating the cell cycle regulatory proteins.

miR-497 Is Implicated in the Process of Chondrogenesis and Inhibits IHH Gene Expression in Human Chondrocytes.

OBJECTIVE: The aim of this study was to examine differences in microRNA-497 (miR-497) expression during cartilage tissue formation and to test whether miR-497 directly interferes with Indian hedgehog (IHH) gene and inhibits IHH expression in human chondrocytes. DESIGN: At different cartilage development stages and different time points in bone matrix gelatin-induced endochondral ossification (BMG-ECO) rat models, the expression of miR-497 and the Ihh gene was monitored at the mRNA level. Bioinformatic analysis, gene mutation, dual luciferase reporter gene assays and gene expression assays at both the mRNA and protein levels in human chondrocytes were subsequently performed to validate the interaction between miR-497 and the IHH gene. RESULTS: The mRNA expression of miR-497 or the Ihh gene in BMG-ECO rats showed significant differences between the cartilage development stages and between different time points, and the trends in the expression of miR-497 and Ihh were reversed. Bioinformatic and dual luciferase reporter gene assays demonstrated a direct interaction between miR-497 and the IHH gene. Differential mRNA and protein expression profiles of the IHH gene in human chondrocytes after 48 hours of transfection with miR-497 mimics and a negative control indicated that miR-497 inhibited IHH expression. CONCLUSION: Our study provided new clues for further functional and molecular mechanism studies of miR-497 in chondrogenesis and demonstrated a potential target for clinical therapy for cartilage degenerative disease.

BMP6 reverses TGF-beta1-induced changes in HK-2 cells: implications for the treatment of renal fibrosis.

AIM: The aim of the study was to investigate the potential role of BMP6 in TGF-beta1-mediated changes in HK-2 cells. METHODS: BMP6 was purified via heparin affinity and reverse phase liquid chromatography. The purity, specificity, and bioactivity of BMP6 were determined by SDS-PAGE, Western blot assays, and the induction of alkaline phosphatase (ALP) activity, respectively. Cell proliferation, morphology, and expression levels of alpha-SMA and E-cadherin were assessed by cell viability, microscopy, and Western blot assays, respectively. In addition, cell adhesion abilities were determined by counting the number of attached cells. The expression of fibronectin, collagen IV, matrix metalloproteinases 2 (MMP-2), and tissue inhibitors of matrix metalloproteinases 2 (TIMP-2) were analyzed using RT-PCR. MMP-2 activity was analyzed by zymography, whereas the activation of the MAPKs and Smad signaling were analyzed using Western blot assays and a reporter gene assay, respectively. RESULTS: Our results indicated that recombinant BMP6 induced ALP activity in a dose-dependent and time-course-dependent manner. Treatment with TGF-beta1 reduced both the cell proliferation and the expression of E-cadherin, induced a morphological transformation, decreased the expression and activity of MMP-2, and increased the expression levels of alpha-SMA, fibronectin, and TIMP-2 in HK-2 cells. All of these effects were inhibited when cells were treated with TGF-beta1 in combination with rhBMP6, whereas rhBMP6 alone demonstrated no such effect. Treatment with TGF-beta1, rhBMP6, or a combination of both had no effect on the expression of collagen IV. In addition, the administration of rhBMP6 prevented the enhanced adhesion behavior triggered by TGF-beta1. Furthermore, the addition of rhBMP6 abrogated the JNK and Smad2/3 signaling that was activated by TGF-beta1. CONCLUSION: BMP6 ameliorated the TGF-beta1-induced changes in HK-2 cells. The suppression of TGF-beta1-mediated JNK and Smad2/3 signaling activation were implicated in these effects.Acta Pharmacologica Sinica (2009) 30: 994-1000; doi: 10.1038/aps.2009.56; published online 22 June 2009.

Retracted Article: CircRNA PVT1 modulates cell metastasis via the miR-181a-5p/NEK7 axis and cisplatin chemoresistance through miR-181a-5p-mediated autophagy in non-small cell lung cancer.

In the initiation and evolution of human cancers, circular RNAs (circRNAs) act as crucial regulators. The aim of this report was to ascertain the functional mechanisms of circRNA plasmacytoma variant translocation 1 (circPVT1) in the metastasis and chemoresistance of non-small cell lung cancer (NSCLC). The levels of circPVT1, microRNA-181a-5p (miR-181a-5p) and non-inherited maternal antigens-related kinase 7 (NEK7) were examined via quantitative real-time polymerase chain reaction (qRT-PCR). The levels of the associated proteins were determined through western blot. Cell counting kit-8 (CCK-8) and flow cytometry were used to assess the half inhibitory concentration (IC50) of cisplatin and cell apoptosis, respectively. Cell invasion was detected by transwell assay. A dual-luciferase reporter assay and RNA immunoprecipitation (RIP) were used to confirm the target relation. The impact of circPVT1 on cisplatin chemoresistance in vivo was investigated using xenograft experiments. CircPVT1 and NEK7 were up-regulated and miR-181a-5p was down-regulated in NSCLC. CircPVT1 knockdown refrained the cisplatin chemoresistance and metastasis of NSCLC cells. MiR-181a-5p was a target of circPVT1 and circPVT1 inhibition alleviated the effects of a miR-181a-5p inhibitor on NSCLC cells. The decrease of circPVT1 accentuated the si-NEK7-inhibited metastasis by the miR-181a-5p/NEK7 axis and relieved the 3-methyladenine (3-MA)-promoted cisplatin chemoresistance by miR-181a-5p-mediated autophagy. Down-regulation of circPVT1 facilitated the cisplatin sensitivity of NSCLC cells in vivo. Due to the modulation of cell metastasis via the miR-181a-5p/NEK7 axis and cisplatin chemoresistance by miR-181a-5p-mediated autophagy in NSCLC, circPVT1 might act as an appreciable therapeutic marker for NSCLC.

Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis involving major structural changes of peripheral joints and local or systemic inflammation and in lack of therapeutic approaches because of complexity of underlying molecular basis. Our previous work showed that HS6ST2, an enzyme involved in the transfer of sulfate, is downregulated in cartilage tissues of OA patients compared with normal donors, but little is known about its regulatory mechanism. In this study, we demonstrated that the expression of HS6ST2 was lower in OA-damaged cartilage than smooth cartilage from the same patient. In chondrocytes, HS6ST2 could be targeted by miR-23b-3p, which was higher expressed in OA-damaged cartilage. Under TNF-α stimulation, the expression of HS6ST2 was found inversely correlated with the expression of miR-23b-3p. Downregulation of HS6ST2 regulated by overexpression of miR-23b-3p and siRNAs against HS6ST2 could enhance the protein level of MMP13 and aggravate the matrix degradation in chondrocytes. Increased expression of MMP13 depended on activity of p38 MAPK rather than total p38 MAPK level and was abrogated by HS6ST2 overexpression. Together, the results indicated that downregulated HS6ST2 targeted by miR-23b-3p promotes matrix degradation by activating p38 MAPK in chondrocytes and OA cartilage.

Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells.

Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Our previous research found that BMP-6 gene expression can be activated dose-dependently by estrogen in estrogen receptor positive (ER(+)) breast cancer cell line MCF-7, but not in ER negative (ER(-)) cell line MDA-MB-231. This experiment is designed to investigate the epigenetic regulatory mechanism of the BMP-6 gene expression in breast cancer cell lines MDA-MB-231, MCF-7 and T47D with regard to the methylation status in the 5' flanking region of the human BMP-6 gene. The endogenous level of BMP-6 mRNA in ER(-) cell line MDA-MB-231 was relatively lower than that in ER(+) MCF-7 and T47D cell lines. After the treatment with 5-aza-2'-deoxycytidine (5-aza-dC, especially in the concentration of 10 microM), the BMP-6 mRNA expression in MDA-MB-231 was obviously up-regulated. However, 5-aza-dC treatment failed to regulate the expression of BMP-6 in MCF-7 and T47D cells. Using enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), methylation of human BMP-6 gene promoter was detected in MDA-MB-231; while in MCF-7 and T47D, BMP-6 gene promoter remained demethylated status. In 33 breast tumor specimens, promoter methylation of BMP-6 was detected by methylation-specific PCR, hypermethylation of BMP-6 was observed in ER negative cases (16 of 16 cases (100%)), while obviously lower methylation frequency were observed in ER positive cases (3 of 17 cases (18%)), indicating that BMP-6 promoter methylation status is correlated with ER status in breast cancer.

Activation of bone morphogenetic protein-6 gene transcription in MCF-7 cells by estrogen.

BACKGROUND: Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17beta-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER+) breast cancer cell line MCF-7. METHODS: After the treatment of MCF-7 cells with E2 at different concentrations (10(-11) mol/L, 10(-9) mol/L, 10(-7) mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0.7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10(-7) mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half-site (1/2 ERE) element and Sp1 sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (ChIP) assay was also used to confirm the binding of estrogen receptor alpha (ERalpha) on BMP-6 promoter in the presence of E2. RESULTS: E2 dose dependently increased BMP-6 mRNA expression in human ER+ breast cancer cell line MCF-7. At a dose of 10(-7) mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P < 0.05). Both the 1/2 ERE response element mutant form and the Sp1 site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter's response to E2. Through ChIP assay, the binding of ERalpha on 1/2 ERE response element in BMP-6 promoter was further validated. CONCLUSION: Estrogen induces BMP-6 expression in human ER+ breast cancer cell line MCF-7 through its receptor ERalpha binding on 1/2 ERE element in the BMP-6 promoter.

Non-Epstein-Barr virus-associated double primary lymphoepithelioma-like carcinoma of the esophagus and stomach: a case report and literature review.

A 55-year-old Chinese male was admitted to the hospital for epigastralgia and dysphagia with a two month history, and hematemesis and melena with a two-day history. Two lesions were found in the esophagus and stomach by esophago--gastroduodenoscopy and computed tomography. The patient underwent subtotal esophagectomy and gastrectomy, esophagogastric anastomosis above the aortic arch, and thoracic-abdominal two-field lymph node dissection. Pathological and immumohistochemical studies showed that both lesions had the same form of poorly differentiated carcinoma with dense lymphoid stroma, which was diagnosed as lymphoepithelioma-like carcinoma (LELC). No metastatic relationship was found between the two tumors. Therefore, the case was double primary lymphoepithelioma-like carcinoma of the esophagus and stomach. Epstein-Barr virus (EBV) in the two tumors were negative by EBV-encoded small RNA1 (EBER-1) in situ hybridization. No adjuvant therapy was performed due to his poor physical condition post-operatively, and no evidence of tumor recurrence or metastasis was found during the next 14 months of follow-up. Esophageal and gastric LELC are rare, especially the former, which has a specific geographical distribution. Literature reported cases showed upper gastrointestinal LELC were highly malignant with good prognosis, and EBV was detected less in esophageal LELC cases but more commonly in gastric LELC cases. Upper gastrointestinal LELC lesions are usually singular, and no synchronous lesions were reported in the literature. Our case is the first LELC to present as double primary lymphoepithelioma- like carcinoma of both esophagus and stomach simultaneously, which demonstrates that LELC can be multifocal in the upper gastrointestinal tract.