Aim: To identify genetic markers associated with late treatment-related skeletal morbidity in survivors of childhood acute lymphoblastic leukemia (ALL). Patients & methods: To this end, we measured the association between reduction in bone mineral density or vertebral fractures prevalence and variants from 1039 genes derived through whole exome sequencing in 242 childhood ALL survivors. Top-ranking variants were confirmed through genotyping, and further explored with stratified analyses and multivariable models. Results: The minor allele of rs1944294 in CDH2 gene was associated with bone geometrical parameter, trabecular cross-sectional area (p = 0.001). The association was modulated by radiation therapy (p = 0.001) and post-treatment time (p = 0.0002). Conclusion: The variant in CDH2 gene is a potential novel risk factor of bone morbidity in survivors of childhood ALL.

Drugs that inhibit the erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2) are the standard treatment of patients with different types of cancer, including HER2-overexpressing gastroesophageal tumors. Unfortunately, cancer cells become resistant to these drugs, so overall these drugs provide little benefit to patients with these tumors. We investigated mechanisms that mediate resistance of esophageal adenocarcinoma (EAC) cells and patient-derived xenograft tumors to ERBB inhibitors.

We cultured primary tumor cells, isolated from EAC patient samples, and OE19 and OE33 EAC cell lines with trastuzumab (an inhibitor of HER2), with or without pertuzumab (which inhibits dimerization of HER2 with HER3) or a specific antibody against HER3 (anti-HER3). HER2 was knocked down by expression of small hairpin RNAs. In addition, cells were incubated with NRG1-β, a mediator of HER2-HER3 signaling, or A83-01, an inhibitor of transforming growth factor beta (TGFβ) signaling. Cells were analyzed for markers of the epithelial to mesenchymal transition (EMT) using flow cytometry, immunofluorescence, and quantitative reverse transcription polymerase chain reaction. We performed limiting dilution, transwell, and cell viability assays to study the functional effects of HER2 and HER3 inhibition and reactivation. We analyzed publicly available EAC gene expression datasets to correlate expression of ERBB genes with genes encoding epithelial and mesenchymal proteins. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were given subcutaneous injections of AMC-EAC-007B cells and also given injections of single or combined inhibitors; growth of these patient-derived xenograft tumors was quantified.

EAC cells incubated with trastuzumab decreased expression of epithelial markers (CD24, CD29, and CDH1) and increased expression of mesenchymal markers (CXCR4, VIM, ZEB1, SNAI2, and CDH2), compared with cells not exposed to trastuzumab, indicating induction of EMT. Addition of NRG1-β to these cells restored their epithelial phenotype. Incubation of EAC cells with trastuzumab and pertuzumab accelerated the expression of EMT markers, compared with cells incubated with trastuzumab alone. EAC cells cultured for 2 months with a combination of trastuzumab and pertuzumab became resistant to chemotherapeutic agents (5-fluoruracil, carboplatin, cisplatin, eribulin, and paclitaxel), based on their continued viability, which was accompanied with an enhanced migratory capacity in transwell assays and clonogenicity in limiting dilution analyses. In comparisons of EAC gene expression patterns, we associated high expression of ERBB3 with an epithelial gene expression signature; expression of TGFβ correlated with expression of EMT-related genes, and we found an inverse correlation between expression of TGFB1 and ERBB3. EAC cells incubated with ERBB inhibitors began to secrete ligands for the TGFβ receptor and underwent EMT. Incubation of EAC cells with trastuzumab, followed by 10 days of incubation with the TGFβ receptor inhibitor in the presence of trastuzumab, caused cells to regain an epithelial phenotype. EAC patient-derived xenograft tumors grew more slowly in mice given the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor than in mice given single agents or a combination of trastuzumab and pertuzumab. Tumors exposed to trastuzumab and pertuzumab expressed EMT markers and were poorly differentiated, whereas tumors exposed to the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor expressed epithelial markers and were more differentiated.

EAC cells become resistant to trastuzumab and pertuzumab by activating TGFβ signaling, which induces EMT. Agents that block TGFβ signaling can increase the anti-tumor efficacies of trastuzumab and pertuzumab.

OBJECTIVE

In our previous study, we displayed that knockdown of Opa interacting protein 5 (OIP5) inhibited cell growth, disturbed cell cycle and increased cell apoptosis in bladder cancer (BC) cell line. Our present study aimed to explore the underlying pathways and interaction network involved in the roles of OIP5 in BC.

METHODS

Microarray analysis was conducted to obtain mRNA expression profiling of OIP5 knockdown (shOIP5) and control (shCtrl) BC cell lines. Bioinformatics analyses were performed including differentially expressed mRNAs (DEGs) identification, protein-protein interaction network construction, biological functions of prediction and ingenuity pathways analysis (IPA). Western Blotting (WB) was subjected to validate the protein expression levels of candidate DEGs in shOIP5 BC cell line.

RESULTS

Respective 255 up- and 184 down-regulated DEGs were identified in shOIP5 group compared with shCtrl group. In the PPI network, CAND1 and MYC had the highest connectivity with DEGs. 439 DEGs were significantly enriched in inflammatory response, regulation of cell proliferation, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and bladder cancer. In the disease and function enrichment, DEGs were obviously involved in cellular movement, cellular growth and proliferation, cancer, inflammatory response, cell death and survival. In the OIP5 regulatory network, CDH2, IRS1, IRAK3, ID1, TNF, IL6, ITGA6, MYC and SOD2 interacted with OIP5. The WB validation results were compatible with our bioinformatics analyses.

CONCLUSIONS

OIP5 interaction network might function as an oncogene in BC progression based on aberrant inflammatory responses. Our study might provide valuable information for investigation of tumorigenesis mechanism in BC.

Proinflammatory cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β) secreted by infiltrating lymphocytes or macrophages may play a role in triggering RPE dysfunction associated with age-related macular degeneration (AMD). Binding of these proinflammatory cytokines to their specific receptors residing on the RPE cell surface can activate signaling pathways that, in turn, may dysregulate cellular gene expression. The purpose of the present study was to investigate whether IFN-γ, TNF-α, and IL-1β have an adverse effect on the expression of genes essential for RPE function, employing the RPE cell line ARPE-19 as a model system.

ARPE-19 cells were cultured for 3-4 months until they exhibited epithelial morphology and expressed mRNAs for visual cycle genes. The differentiated cells were treated with IFN-γ, TNF-α, and/or IL-1β, and gene expression was analyzed with real-time PCR analysis. Western immunoblotting was employed for the detection of proteins.

Proinflammatory cytokines (IFN-γ + TNF-α + IL-1β) greatly increased the expression of chemokines and cytokines in cultured ARPE-19 cells that exhibited RPE characteristics. However, this response was accompanied by markedly decreased expression of genes important for RPE function, such as CDH1, RPE65, RDH5, RDH10, TYR, and MERTK. This was associated with decreased expression of the genes MITF, TRPM1, and TRPM3, as well as microRNAs miR-204 and miR-211, which are known to regulate RPE-specific gene expression. The decreased expression of the epithelial marker gene CDH1 was associated with increased expression of mesenchymal marker genes (CDH2, VIM, and CCND1) and epithelial-mesenchymal transition (EMT) promoting transcription factor genes (ZEB1 and SNAI1).

RPE cells exposed to proinflammatory cytokines IFN-γ, TNF-α, and IL-1β showed decreased expression of key genes involved in the visual cycle, epithelial morphology, and phagocytosis. This adverse effect of proinflammatory cytokines, which could be secreted by infiltrating lymphocytes or macrophages, on the expression of genes indispensable for RPE function may contribute to the RPE dysfunction implicated in AMD pathology.

Background: Thyroid carcinoma (THCA) is a common endocrine malignant tumor. Papillary carcinoma with low degree of malignancy and good prognosis is the most common. It can occur at any age, but it is more common in young adults. Although the mortality rate is decreased due to early diagnosis, the survival rate varies depending on the type of tumor. Therefore, the purpose of this study is to identify hub biomarkers and novel therapeutic targets for THCA.

Methods: The GSE3467, GSE3678, GSE33630 and GSE53157 were obtained from the GEO database, including 100 thyroid tumors and 64 normal tissues to obtain the intersection of differentially expressed genes, and a protein-protein interaction network was constructed to obtain the HUB gene. The corresponding overall survival information from The Cancer Genome Atlas Project-THCA was then included in this research. The signature mechanism was studied by analyzing the gene ontology and the Kyoto Encyclopedia of Genes and Genome database.

Results: In this research, we identified eight candidate genes (FN1, CCND1, CDH2, CXCL12, MET, IRS1, DCN and FMOD) from the network. Also, expression verification and survival analysis of these candidate genes based on the TCGA database indicate the robustness of the above results. Finally, our hospital samples validated the expression levels of these genes.

Conclusion: The research identified eight mRNA (four up-regulated and four down-regulated) which serve as signatures and could be a potential prognostic marker of THCA.

Keywords: Biomarker; GEO; Prognosis; Signature; TCGA; Thyroid carcinoma.

UNASSIGNED

Prostate cancer (PCa) is one of the most common malignancies in men. However, a lack of understanding of the mechanism underlying PCa metastasis has strongly limited the effectiveness of therapy for this disease. Thus, investigating the mechanism of PCa may help improve the prognosis of PCa patients. The goal of this study was to investigate the role of microRNA-194 (miR-194) in PCa.

UNASSIGNED

The expression of miR-194 and cadherin 2 (CDH2) at the transcriptional level was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The MTT assay cell apoptosis assay and Western blotting were used to determine the role of miR-194 and CDH2 in the PC3 human PCa cell line. The dual luciferase reporter assay system was performed to clarify the relationship between miR-194 and CDH2. qRT-PCR results showed that miR-194 was downregulated and CDH2 was upregulated in PC3 cells.

UNASSIGNED

Transfection with miR-194 mimics decreased cell viability and increased the rate of apoptosis compared with the control group of PC3 cells. Bioinformatics and the luciferase reporter assay indicated that CDH2 was a target of miR-194, and Western blot analysis suggested that CDH2 was negatively regulated by miR-194. Further studies revealed that the downregulation of CDH2 suppressed cell viability and promoted the apoptosis of PC3 cells and that miR-194 directly targeted CDH2 in PC3 cells. Finally, the in vivo experiments showed that miR-194 mimics suppressed tumor growth and induced apoptosis in a greater proportion of cells by decreasing the expression of CDH2 compared with the control group.

UNASSIGNED

The results of this study showed that miR-194 targeted CDH2 to regulate PCa cell survival in vitro and suppress tumor growth in vivo. These findings suggest that miR-194 may be a useful therapeutic target in PCa.

Background: Peritoneal dissemination (PD) frequently occurs in gastric cancer (GC) and is incurable. In this study, we aimed to identify novel PD-associated genes and clarify their clinical and biological significance in GC.

Materials and methods: We identified LOXL1 as a PD-associated candidate gene by in silico analysis of GC datasets (highly disseminated peritoneal GC cell line and two freely available GC datasets, GSE15459 and TCGA). Next, we evaluated the clinical significance of LOXL1 expression using RT-qPCR and immunohistochemistry staining (IHC) in a validation cohort (Kyushu cohort). Moreover, we performed gene expression analysis, including gene set enrichment analysis (GSEA) with GSE15459 and TCGA datasets. Finally, we performed a series of in vitro experiments using GC cells.

Results: In silico analysis showed that LOXL1 was overexpressed in tumor tissues of GC patients with PD and in highly disseminated peritoneal GC cells, relative to that in the control GC patients and cells, respectively. High expression of LOXL1 was a poor prognostic factor in the TCGA dataset. Next, IHC showed that LOXL1 was highly expressed in GC cells. High LOXL1 mRNA expression was associated with poorly differentiated histological type, lymph node metastasis, and was an independent poor prognostic factor in the Kyushu validation cohort. Moreover, LOXL1 expression was positively correlated with the EMT (epithelial-mesenchymal transition) gene set in GSEA. Finally, LOXL1-overexpressing GC cells changed their morphology to a spindle-like form. LOXL1 overexpression reduced CDH1 expression; increased the expression of VIM, CDH2, SNAI2, and PLS3; and promoted the migration capacity of GC cells.

Conclusions: LOXL1 is associated with PD in GC, possibly through the induction of EMT.

The endothelial-to-mesenchymal transition (EndoMT) is a cellular process featuring decreased expression of endothelial marker genes but increased expression of mesenchymal marker genes. The EndoMT is involved in endothelial dysfunction and the pathogenesis of atherosclerosis. To investigate the dynamic expression of EndoMT genes in vascular endothelial cells under atheroprotective pulasatile shear stress (PS) and atheroprone oscillatory shear stress (OS), we analyzed RNA sequencing data from multi-timepoint shear stress experiments. This unbiased analysis involving next-generation sequencing confirmed that PS and OS had an opposite effect in regulating EndoMT genes. Further experimental validations with H2O2 and gain- and loss-of-function approaches indicated that reactive oxygen species are involved in the OS-induced EndoMT, whereas AMP-activated protein kinase and sirtuin-1 could inhibit the OS-induced EndoMT. Furthermore, as compared with PS, OS increased the DNA methylation of the promoter regions of vWF, CD31, and CDH5 genes but decreased that of CDH2, FSP1, and vimentin. The translational implication of the current study builds on the ability of the anti-diabetic drug metformin and cholesterol-lowering drug atorvastatin to suppress EndoMT in cultured endothelial cells and in mouse aortae.

Cadherins constitute a family of transmembrane proteins that mediate calcium-dependent cell-cell adhesion. The extracellular domain of cadherins consists of extracellular cadherin (EC) domains, separated by calcium binding sites. The EC interacts with other cadherin molecules in cis and in trans to mechanically hold apposing cell surfaces together. CDH2 encodes N-cadherin, whose essential roles in neural development include neuronal migration and axon pathfinding. However, CDH2 has not yet been linked to a Mendelian neurodevelopmental disorder. Here, we report de novo heterozygous pathogenic variants (seven missense, two frameshift) in CDH2 in nine individuals with a syndromic neurodevelopmental disorder characterized by global developmental delay and/or intellectual disability, variable axon pathfinding defects (corpus callosum agenesis or hypoplasia, mirror movements, Duane anomaly), and ocular, cardiac, and genital anomalies. All seven missense variants (c.1057G>A [p.Asp353Asn]; c.1789G>A [p.Asp597Asn]; c.1789G>T [p.Asp597Tyr]; c.1802A>C [p.Asn601Thr]; c.1839C>G [p.Cys613Trp]; c.1880A>G [p.Asp627Gly]; c.2027A>G [p.Tyr676Cys]) result in substitution of highly conserved residues, and six of seven cluster within EC domains 4 and 5. Four of the substitutions affect the calcium-binding site in the EC4-EC5 interdomain. We show that cells expressing these variants in the EC4-EC5 domains have a defect in cell-cell adhesion; this defect includes impaired binding in trans with N-cadherin-WT expressed on apposing cells. The two frameshift variants (c.2563_2564delCT [p.Leu855Valfs^∗∗CDH2 impair the adhesive activity of N-cadherin, resulting in a multisystemic developmental disorder, that could be named ACOG syndrome (agenesis of corpus callosum, axon pathfinding, cardiac, ocular, and genital defects).

Sinapic acid (SA) is a derivative of hydroxycinnamic acid and found in various vegetables and fruit species. Aim was to evaluate the anticancer effects of SA in PC-3 and LNCaP human prostate cancer cells. The effect of SA on cell viability was determined using XTT assay. Expressions of 8 genes for apoptosis and 6 genes for metastasis were evaluated by qPCR. Caspase-3 activity was determined using caspase-3 colorimetric assay kit. Effect of SA on cell invasion was evaluated with cell invasion assay. The IC50 dose of SA in PC-3 and LNCaP cells was found to be 1000 μM for 72 h. SA treatment increased the expression of BAX, CASP3, CASP8, CYCS, FAS, TIMP-1 and CDH1 however significant decreased the expression of MMP-9 in PC-3 cells. In LNCaP cells, the expressions of BAX, CASP3, CASP7 and CYCS were significantly elevated; however, a decrease was seen in the expressions of CDH2, MMP-2 and MMP-9 in the SA treatment. Moreover, SA significantly increased caspase-3 activity and suppressed the cell invasion. In conclusion, it is thought that SA has anticancer effect on prostate cancer cells. However, more detailed studies should be conduct to illuminate molecular mechanism of apoptotic and antimetastatic activity of SA.

Epithelial-to-mesenchymal transition (EMT) plays an important role in invasion and metastasis of various cancers including gliomas. EMT has also been linked to cancer stem cells and resistance to chemotherapy. An initial in-silico data mining in a published ependymoma (EPN) patient series (GSE21687) revealed upregulation of EMT-Transcription factors (EMT-TFs) in tumor samples. Further, quantitative real-time polymerase chain reaction (q-RT-PCR) based gene expression analysis of EMT-TFs in 96 EPNs showed significant up-regulation of SNAI1, SNAI2, ZEB1, and TWIST1 as compared to normal brain, associated with upregulation of CDH2/N-Cadherin and downregulation of CDH1/E-Cadherin. Although this was observed in varying degrees in all clinico-pathological-molecular subgroups of EPNs, it was most evident in supratentorial EPNs harboring fusions of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) gene and in posterior fossa EPNs. Immunohistochemistry performed in 60 of the above cases corroborated with gene expression patterns and immunopositivity for Snail, Slug, Zeb1, and Twist1 was observed in 80%, 80%, 81%, and 63% of all EPNs. Immunopositivity for N-Cadherin and E-Cadherin was observed in 76.6% and 2% cases respectively. Univariate Cox regression analysis showed that low expression of CDH1/E-Cadherin (P=.002) and high expression levels of CDH2/N-Cadherin (P<.001), SNAI1/Snail (P=.023), SNAI2/Slug (P<.001) and ZEB1 (P<.001) to be associated with shorter progression free survival. Here, we report for the first time the existence of EMT- like phenotype in EPNs. These factors could represent new prognostic and therapeutic targets in EPN.

OBJECTIVE

To investigate how Placental Mesenchymal Stem Cells (P-MSCs) would adapt themselves and survive under hypoxic conditions which are prevalent in most injury sites.

METHODS

P-MSCs were isolated from term placenta and characterised under normoxia and hypoxia (2-2.5% O2). Cells were examined for morphology and surface marker variations by flow cytometry analysis. Glucose stimulated insulin secretion was assayed by Insulin ELISA Kit. Gene expression levels were estimated using Real Time PCR for hypoxia inducible factor1 alpha, Insulin (INS), Glucose transporters (GLUT-1, GLUT-2 and GLUT-3), Adhesion Proteins- Integrins, Fibronectin1 (FN1), E-Cadherin (CDH1), and N-Cadherin (CDH2) and angiogenesis marker VEGFA. Immunofluorescence assay was done to confirm the presence of C-Peptide, GLUT 2, E-Cadherin and ITGB3. Adhesion was confirmed assessed on fibronectin binding.

RESULTS

We show that insulin secretion is not hampered under hypoxia. We found an upregulation of glucose transporters under hypoxia indicating enhanced glucose uptake needed to cater to metabolic demands of proliferating cells. Up regulation of adhesion molecules was seen under hypoxia indicative of a favoured environment for retention of cells at the injury site. We also found increased level of angiogenesis of P-MSCs under hypoxia.

CONCLUSIONS

Our present study thus demonstrates for the first time that P-MSCs modulate themselves under hypoxic conditions by secreting insulin, up regulating glucose transporters and adhesion molecules and eventually exhibiting an increased angiogenic potential. We thus infer that priming P-MSCs under hypoxia, could make them more suitable for wound healing applications.

The high invasive phenotype of glioblastoma is one of the main causes of therapy inefficacy and tumor relapse. Cell adhesion molecules of the cadherin family are involved in cell migration and are known as master regulators of epithelial tumor invasiveness, but their role in glioblastoma is less understood. In particular, we recently demonstrated, in the syngeneic murine model, the occurrence of a previously undescribed cadherin switch between Cdh2 and Cdh4 during gliomagenesis, which is necessary for the acquisition of the highly infiltrative and tumorigenic phenotype of these cells. In the present study, we tested the role of Cdh4 in human gliomas. Our results on patient-derived glioma cells demonstrate a positive correlation between Cdh4 expression levels and the loss of cell-cell contact inhibition of proliferation controls that allows cells to proliferate over confluence. Moreover, the silencing of Cdh4 by artificial microRNAs induced a decrease in the infiltrative ability of human glioma cells both in vitro and in vivo. More strikingly, Cdh4 silencing induced an impairment of the tumorigenic potential of these cells after orthotopic transplantation in immunodeficient mice. Overall, we conclude that in human glioblastoma, Cdh4 can also actively contribute in regulating cell invasiveness and malignancy.

Epithelial to mesenchymal transition (EMT) is a key trans-differentiation process, which plays a critical role in physiology and pathology. Although gene expression changes in EMT have been scrutinized, study of epigenome is in its infancy. To understand epigenetic changes during TWIST-driven EMT, we used the AcceSssIble assay to study DNA methylation and chromatin accessibility in human mammary epithelial cells (HMECs). The DNA methylation changes were found to have functional significance in EMT - i.e. methylated genes were enriched for E-box motifs that can be recognized by TWIST, at the promoters suggesting a potential targeting phenomenon, whereas the demethylated regions were enriched for pro-metastatic genes, supporting the role of EMT in metastasis. TWIST-induced EMT triggers alterations in chromatin accessibility both independent of and dependent on DNA methylation changes, primarily resulting in closed chromatin conformation. By overlapping the genes, whose chromatin structure is changed during early EMT and a known "core EMT signature", we identified 18 driver candidate genes during EMT, 14 upregulated and 4 downregulated genes with corresponding chromatin structure changes. Among 18 genes, we focused on TRIM29 as a novel marker of EMT. Although loss of TRIM29 is insufficient to suppress CDH, it is enough to induce CDH2 and VIM. Gene functional annotation analysis shows the involvement of TRIM29 in epidermal development, cell differentiation and cell migration. Taken together, our results provide a robust snapshot of chromatin state during human EMT and identify TRIM29 as a core mediator of EMT.

N-cadherin is a well-studied classic cadherin involved in multiple developmental processes and is also known to have a signaling function. Using the zebrafish (Danio rerio) as a model, we tested the hypothesis that tooth morphogenesis is accompanied by dynamic changes in N-cadherin distribution and that absence of N-cadherin disturbs tooth development. N-cadherin, encoded by the gene cdh2, is absent during the initiation and morphogenesis stages of both primary (first-generation) and replacement teeth, as demonstrated by immunohistochemistry. However, N-cadherin is up-regulated at the onset of differentiation of cells of the inner dental epithelium and the dental papilla, i.e., the ameloblasts and odontoblasts, respectively. In the inner dental epithelium, N-cadherin is co-expressed with E-cadherin, excluding the occurrence of cadherin switching such as observed during human tooth development. While early lethality of N-cadherin knockout mice prevents any functional study of N-cadherin in mouse odontogenesis, zebrafish parachute (pac) mutants, deficient for N-cadherin, survive beyond the age when primary teeth normally start to form. In these mutants, the first tooth forms, but its development stops at the early cytodifferentiation stage. N-cadherin deficiency also completely inhibits the development of the other first-generation teeth, possibly due to the absence of N-cadherin signaling once the first tooth has differentiated.

Myocardial infarction (MI) induced by acute coronary arterial occlusion is usually secondary to atherosclerotic plaque rupture. Dysregulated response of vascular smooth muscle cells (VSMCs) in atherosclerotic plaques may promote plaque rupture. Cadherins (CDHs) form adherens junctions and are known stabilizers of atherosclerotic plaques. To date, the expression patterns of cadherin have not been well investigated in MI aortic VSMCs. We aimed to investigate the expression of cadherin genes in the aortic wall of patients with and without MI. Laser capture microdissected VSMCs were obtained from aortic tissue samples of patients undergoing coronary artery bypass graft surgery. Integrative bioinformatic analysis of the microarray profiles of the VSMCs revealed that MI is discriminated at the whole transcriptome level by hundreds of differentially expressed genes, including genes involved in cell adhesion, of which the cadherin superfamily genes were among the top structural category. Eleven significantly deregulated candidates of the cadherin superfamily were chosen and formed a new classifier that collectively discriminated MI vs. non-MI with ~95% accuracy. Significance validation was performed with an independent cohort by quantitative RT-quantitative PCR, confirming overexpression of CDH2, CDH12, PCDH17, and PCDH18 in MI VSMCs. The dysregulation of these cadherin superfamily genes might be related to an MI-induced remote effect on aortic wall VSMCs and to imbalances in signaling pathways and myocardial repair mechanisms. Although pathophysiological significance of our findings requires functional studies, mRNA upregulation of the identified cadherin superfamily members in VSMCs might be associated with the progression of atherosclerosis and angiogenesis activation in MI.

N-cadherin (CDH2) is a member of the cadherin family of Ca2(+)-dependent cell-cell adhesion molecules. To investigate mechanisms controlling CDH2 transcription, we isolated and analyzed a genomic DNA sequence containing 2.8 kb of 5' flanking region and the first two exons of chicken CDH2. Sequence analysis of the promoter region of CDH2 revealed no CCATT or TATA boxes, but showed a high overall GC content, high CpG dinucleotide content, and several consensus Sp1 and Ap2 binding sequences. When fused to the cat reporter gene in transient transfection experiments, the sequence from positions -3231 to -118 (relative to the translation start site) directed high-level expression in CDH2-expressing chicken primary retinal cells and mouse N2A cells, but was much less active in chicken embryonic fibroblast cells and mouse 3T3 cells which do not express CDH2. Similarly, this promoter fragment directed variable, but neuronal-specific, expression of reporter genes in adult transgenic mice, but failed to produce the correct pattern of expression in other tissues, implying that additional sequences further upstream and/or within introns of CDH2 may play important roles in the transcriptional control.

Differences in matrix compositions in human nucleus pulposus (NP) clinical samples demand different cell isolation protocols for optimal results but there is no clear guide about this to date. Sub-optimal protocols may result in low cell yield, limited reliability of results or even failure of experiments. Cell yield, viability and attachment of cells isolated from bovine NP tissue with different protocols were estimated by cell counting, Trypan blue staining and cell culturing respectively. RNA was extracted from isolated cells and quantified by Nanodrop spectrometry and RT-qPCR. Higher collagenase concentration, longer digestion duration and pronase pre-treatment increased the cell yield. Cell viability remained high (<5% dead cells) even after 0.2% collagenase treatment for overnight. NP cells remained to have high ACAN, COL2A1, CDH2, KRT18, and KRT19 expression compared to muscle cells for different cell isolation conditions tested. Digestion by collagenase alone without the use of pronase could isolate cells from human degenerated NP tissue but clusters of cells were observed. We suggest the use of the disappearance of tissue as an indirect measure of cells released. This study provides a guide for researchers to decide the parameters involved in NP cell isolation for optimal outcome.

Lipotransfer has been applied in breast augmentation surgery for several years and the resident adipose-derived stem cells (ASCs) play an important role in enhancing fat graft survival. However, the interaction between ASCs and mammary epithelium is not fully understood. Many studies have shown that ASCs have a tumor-supportive effect in breast cancer. To the best of our knowledge, this is the first study on the effect of mammary epithelial cells on the human ASCs in 3D culture. ASCs were cultivated on matrigel in the conditioned medium (CM) prepared from a human breast epithelial cell line (HBL-100). The ASCs formed KRT18-positive acini-like structures after stimulation with breast epithelial cells. The expression of epithelial genes (CDH1 and KRT18) was up-regulated while the expression of mesenchymal specific genes (CDH2 and VIM) was down-regulated as determined by qRT-PCR. The stemness marker (CD29) and angiogenic factors (CD31 and VEGF) were also down-regulated as examined by immunofluorescence. In addition, the CM obtained from HBL-100 enhanced the migration and inhibited the adipogenic differentiation of ASCs. These results demonstrate that ASCs have the ability to transform into epithelial-like cells when cultured with mammary epithelial cells. Given these observations, we infer that ASCs have a positive effect on lipotransfer, not only due to their ability to secrete growth factors, but also due to their direct participation in the formation of new breast tissue.

OBJECTIVE

3,5,3'-Triiodothyronine (T(3))-predominant Graves' disease is characterized by the increasing volume of thyroid goiter resulting in poor prognosis. Although type 1 and type 2 iodothyronine deiodinases (DIO1 and DIO2 respectively) are known to be overexpressed in the thyroid tissues of T(3)-predominant Graves' disease, the pathogenesis of this disease is still unclear. The aim of our study is to identify genes that characterize T(3)-predominant Graves' disease tissue in order to clarify the molecular mechanism of this disease.

METHODS

mRNAs from two thyroid tissues of both typical T(3)-predominant and common-type Graves' disease were analyzed with DNA microarrays with probes for 28 869 genes. Genes identified to be differentially expressed between the two groups were further analyzed in the second and third screenings using 70 Graves' thyroid tissues by real-time quantitative RT-PCR.

RESULTS

Twenty-three candidate genes were selected as being differentially expressed in the first screening with microarrays. Among these, seven genes, leucine-rich repeat neuronal 1 (LRRN1), bone morphogenetic protein 8a (BMP8A), N-cadherin (CDH2), phosphodiesterase 1A (PDE1A), creatine kinase mitochondrial 2 (CKMT2), integrin beta-3 (ITGB3), and protein tyrosine phosphatase non-receptor type 4 (PTPN4), were confirmed to be differentially expressed in DIO1 or DIO2 over- and underexpressing Graves' tissues.

CONCLUSIONS

These genes are related to the characteristics of T(3)-predominant Graves' disease, such as high titer level of serum anti-TSH receptor antibody, high free T(3) to free thyroxine ratio, and a large goiter size. They might play a role in the pathogenesis of T(3)-predominant Graves' disease.

Adhesion tissue is formed following injury to the uterine basal layer. Currently, there is no effective treatment for severe intrauterine adhesion (IUA), which causes loss of reproductive function. Enhanced understanding of the molecular mechanisms driving severe IUA would be beneficial for the treatment.

Differentially expressed microRNAs (miRNAs) and messenger RNAs (mRNAs) in severe IUA (n = 3) and normal (n = 3) endometrium were analyzed by high-throughput microarray analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and found to overlap with the differentially expressed mRNAs. Gene Ontology and pathway analyses were performed for the intersecting genes. Three of the significantly dysregulated miRNAs and 4 of their target mRNAs were further assessed using quantitative real-time polymerase chain reaction (PCR) in 10 severe IUA and 10 normal endometrium samples.

Microarray analysis indicated that 26 miRNAs and 1180 mRNAs were significantly different between the 2 groups. Of these, 16 miRNAs and 54 mRNAs overlapped with putative miRNA target genes and prediction of target gene. Real-time PCR revealed upregulation of hsa-miR-513a-5p and has-miR-135a-3p and downregulation of hsa-miR-543 and their corresponding target genes, plus downregulation of ADAM9 (a disintegrin-containing and metalloproteinases) and lysyl oxidase and upregulation of CDH2 (N-cadherin) and COL16A1 (collagen 16A1). Both CDH2 and COL16A1 were bioinformatically predicted and confirmed in vitro as target genes of miR-543.

This study provides an integrated data set of the miRNA and mRNA profiles in severe IUA, showing involvement of many miRNAs and their target genes. Further analysis of these genes will help in understanding of the molecular mechanism of IUA formation.

Nasopharyngeal cancer or nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. The factors that induce nasopharyngeal cancer are still not clear. Additional information about the chemicals or genes related to nasopharyngeal cancer will promote a better understanding of the pathogenesis of this cancer and the factors that induce it. Thus, a computational method NPC-RGCP was proposed in this study to identify the possible relevant chemicals and genes based on the presently known chemicals and genes related to nasopharyngeal cancer. To extensively utilize the functional associations between proteins and chemicals, a heterogeneous network was constructed based on interactions of proteins and chemicals. The NPC-RGCP included two stages: the searching stage and the screening stage. The former stage is for finding new possible genes and chemicals in the heterogeneous network, while the latter stage is for screening and removing false discoveries and selecting the core genes and chemicals. As a result, five putative genes, CXCR3, IRF1, CDK1, GSTP1, and CDH2, and seven putative chemicals, iron, propionic acid, dimethyl sulfoxide, isopropanol, erythrose 4-phosphate, β-D-Fructose 6-phosphate, and flavin adenine dinucleotide, were identified by NPC-RGCP. Extensive analyses provided confirmation that the putative genes and chemicals have significant associations with nasopharyngeal cancer.