Minlan Yang

Clinicopathologic significance of claudin-6, occludin, and matrix metalloproteinases −2 expression in ovarian carcinoma

BackgroundTight junctions (TJs) are mainly composed of claudins, occludin, and tight junction adhesion molecules (JAM). The invasive and metastatic phenotype of highly invasive cancer cells has been related to abnormal structure and function of TJs, and with expression of activated matrix metalloproteinases (MMPs). The relevance of these mechanisms responsible for the invasion and metastasis of ovarian carcinoma is unclear. Similarly, it is not known if the expression of claudin-6, occludin and MMP2 is related with the clinical properties of these tumors.MethodsExpression of claudin-6, occludin, and MMP2 was detected in samples of human ovarian cancer tissues by immunohistochemistry and correlated with the clinical properties of the tumors.ResultsThe positive expression rates of claudin-6 and MMP-2 were higher in ovarian papillary serous carcinomas than n ovarian serous adenomas (P < 0.05). There were no differences in the expression of occludin (P > 0.05). The expression of claudin-6 and occludin in ovarian cancer was not correlated with patient age, pathological grade, clinical stage, and metastasis (P > 0.05). MMP-2 expression was enhanced with increased clinical stage and metastasis (P < 0.05), but was unrelated to patient age or tumor grade (P > 0.05). There were no apparent correlations between expression of claudin-6, occludin and MMP-2 in ovarian cancer tissue (P > 0.05).ConclusionsOur data suggest, for the first time, that the claudin-6 and MMP-2 are up-regulated in ovarian papillary serous carcinomas, MMP-2 expression was enhanced with increased clinical stage and metastasis. Claudin-6 and MMP-2 may play a positive role in the invasion and metastasis of ovarian cancer.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1775628454106511.

The distinct expression patterns of claudin-2, -6, and 11 between human gastric neoplasms and adjacent non-neoplastic tissues

BackgroundCancers have a multifactorial etiology a part of which is genetic. Recent data indicate that expression of the tight junction claudin proteins is involved in the etiology and progression of cancer.MethodsTo explore the correlations of the tight junction proteins claudin-2,-6, and −11 in the pathogenesis and clinical behavior of gastric cancer, 40 gastric cancer tissues and 28 samples of non-neoplastic tissues adjacent to the tumors were examined for expression of claudin-2,-6, and −11 by streptavidin-perosidase immunohistochemical staining method.ResultsThe positive expression rates of claudin-2 in gastric cancer tissues and adjacent tissues were 25% and 68% respectively (P < 0.001). The positive expression rates of claudin-6 in gastric cancer tissues and adjacent tissues were 55% and 79% respectively (P = 0.045 < 0.05). In contrast, the positive expression rates of claudin-11 in gastric cancer tissues and gastric cancer adjacent tissues were 80% and 46% (P = 0.004 < 0.01). Thus in our study, the expression of claudin-2, and claudin-6 was down regulated in gastric cancer tissue while the expression of claudin-11 was up regulated. Correlations between claudin expression and clinical behavior were not observed.ConclusionOur study provides the first evidence that claudin-2,-6, and −11 protein expression varies between human gastric cancers and adjacent non-neoplastic tissues.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/5470513569630744

Transdifferentiation of Human Hair Follicle Mesenchymal Stem Cells into Red Blood Cells by OCT4

Shortage of red blood cells (RBCs, erythrocytes) can have potentially life-threatening consequences for rare or unusual blood type patients with massive blood loss resulting from various conditions. Erythrocytes have been derived from human pluripotent stem cells (PSCs), but the risk of potential tumorigenicity cannot be ignored, and a majority of these cells produced from PSCs express embryonic ε- and fetal γ-globins with little or no adult β-globin and remain nucleated. Here we report a method to generate erythrocytes from human hair follicle mesenchymal stem cells (hHFMSCs) by enforcing OCT4 gene expression and cytokine stimulation. Cells generated from hHFMSCs expressed mainly the adult β-globin chain with minimum level of the fetal γ-globin chain. Furthermore, these cells also underwent multiple maturation events and formed enucleated erythrocytes with a biconcave disc shape. Gene expression analyses showed that OCT4 regulated the expression of genes associated with both pluripotency and erythroid development during hHFMSC transdifferentiation toward erythroid cells. These findings show that mature erythrocytes can be generated from adult somatic cells, which may serve as an alternative source of RBCs for potential autologous transfusion.

The effects of shRNA-mediated gene silencing of transcription factor SNAI1 on the biological phenotypes of breast cancer cell line MCF-7

To research the effects of silencing transcription factor SNAI1 on the in vitro biological phenotypes of breast cancer cell line MCF-7, based on the gene sequence of SNAI1, we linked shRNA with the green fluorescent protein-expressing eukaryotic expression vector pGCsilencer™ U6/Neo/GFP, and transfected it into MCF-7 cells. The SNAI1 gene-silencing effect was authenticated by RT-PCR and immunofluorescence. We then examined the effect of gene silencing on the expression of epithelial and mesenchymal markers and on their biological phenotypes of the target cells. Finally, we explained that SNAI1 was bound to E-cadherin in MCF-7 cells by ChIP. Silencing SNAI1 upregulated the expression of epithelial markers claudin-4, claudin-7, and E-cadherin, while expression of the mesenchymal marker matrix metalloproteinase-2 was downregulated. The capacity for proliferation, migration, and invasion was diminished. SNAI1 binds to the E-cadherin gene promoter and inhibits its transcription. We can conclude that silencing gene SNAI1 inhibits expression of properties that are associated with the malignant phenotype of MCF-7 cells and reverses the epithelial–mesenchymal transition process by regulating relevant target gene E-cadherin.

CLDN6 enhances chemoresistance to ADM via AF-6/ERKs pathway in TNBC cell line MDAMB231

Claudin-6 (CLDN6), a critical tight junction protein acting as a tumor suppressor in breast cancer, is also considered to be a stem cell marker. Triple-negative breast cancer (TNBC) is a subtype of claudin-low and stem cell-like breast cancer which is chemoresistant to multiple anti-cancer drugs. The aim of our study was to determine whether CLDN6 plays a role in chemoresistance of TNBC. We found that overexpression of CLDN6 in TNBC cell line MDAMB231 significantly inhibited cell growth, migration, and invasion. The expression of CLDN6 increased the IC50 of adriamycin (ADM) and promoted the clonogenic survival. CLDN6 inhibited ADM-induced apoptosis and senescence in MDAMB231 cells. However, P-gp, a resistance-related protein highly associated with chemoresistance, was downregulated by CLDN6 overexpression in MDAMB231 cells. Epithelial mesenchymal transition (EMT) marker E-cadherin was increased, and vimentin was decreased by CLDN6. In addition, stem cell markers OCT4, SOX2, and Nanog were dramatically increased. CLDN6 colocalized and interacted with AF-6. Overexpression of CLDN6 increased the expression of afadin (AF-6) and hampered the activation of ERK signaling. PMA, a specific ERK activator, reversed the expression of EMT and stem cell markers, and decreased chemoresistance of MDAMB231 cells to ADM with a decreased IC50 and an increased apoptosis resulting from CLDN6. Together, we conclude that CLDN6 enhances the chemoresistance to ADM via activating the AF-6/ERK signaling pathway and up-regulating cancer stem cell characters in MDAMB231 cells.

Identification of genes and pathways associated with MDR in MCF-7/MDR breast cancer cells by RNA-seq analysis

Multidrug resistance (MDR) is a major problem in the treatment of breast cancer. In the present study, next-generation sequencing technology was employed to identify differentially expressed genes in MCF-7/MDR cells and MCF-7 cells, and aimed to investigate the underlying molecular mechanisms of MDR in breast cancer. Differentially expressed genes between MCF-7/MDR and MCF-7 cells were selected using software; a total of 2085 genes were screened as differentially expressed in MCF-7/MDR cells. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the DAVID database. Finally, a protein-protein interaction network was constructed and the hub genes in the network were analyzed using the STRING database. GO annotation demonstrated that the differentially expressed genes were enriched in various biological processes, including ‘regulation of cell differentiation’, ‘cell development’, ‘neuron development’, ‘movement of cell or subcellular component’ and ‘cell morphogenesis involved in neuron differentiation’. Cellular component analysis by GO revealed that differentially expressed genes were enriched in ‘plasma membrane region’ and ‘extracellular matrix’ terms. Furthermore, KEGG analysis demonstrated that the target genes were enriched in various pathways, including ‘cell adhesion molecules (CAMs)’, ‘calcium signaling pathway’, ‘tight junction’, ‘Wnt signaling pathway’ and ‘pathways in cancer’ terms. A protein-protein interaction network demonstrated that certain hub genes, including cyclin D1, nitric oxide synthase 3 (NOS3), NOTCH3, brain-derived neurotrophic factor (BDNF), paired box 6, neuropeptide Y, phospholipase C β (PLCB) 4, PLCB2 and actin α cardiac muscle 1, may be associated with MDR in breast cancer. Subsequently, RT-qPCR confirmed that the expression of these 9 hub genes was higher in MCF-7/MDR cells compared with MCF-7 cells, consistent with the RNA-sequencing analysis. Additionally, a Cell Counting Kit-8 assay demonstrated that specific inhibitors of NOS3 and BDNF/neurotrophic receptor tyrosine kinase, type 2 signaling reduced the IC50 of MCF-7/MDR cells in response to various anticancer drugs, including adriamycin, cisplatin and 5-fluorouracil. The results of the present study provide novel insights into the mechanism underlying MDR in MCF-7 cells and may identify novel targets for the treatment of breast cancer.

Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions

Claudin-6 (CLDN6) is an integral component of the tight junction proteins in polarized epithelial and endothelial cells and plays a crucial role in maintaining cell integrity. Deregulation of CLDN6 expression and distribution in tumor tissues have been widely documented and correlated with cancer progression and metastasis. However, a complete mechanistic understanding of CLDN6 regulation and function remains to be studied. Herein, we show new potential properties of CLDN6 regulation and functions from bioinformatics analysis. Using numerous algorithms to characterize the CLDN6 gene promoter elements and the CLDN6 protein structure, physio-chemical and localization properties, and its evolutionary relationships. CLDN6 is regulated by a diverse set of transcription factors (SP1, SPR, AML-1a, CdxA, CRE-BP and CREB) and associated with the levels of methylation of CpG islands in promoters. The structural properties of CLDN6 indicate that it promotes cancer cell behavior via the ASK1-p38/JNK MAPK secretory signaling pathway. In conclusion, this information from bioinformatics analysis will help future attempts to better understand CLDN6 regulation and functions.

SMAD2 Inactivation Inhibits CLDN6 Methylation to Suppress Migration and Invasion of Breast Cancer Cells

The downregulation of tight junction protein CLDN6 promotes breast cancer cell migration and invasion; however, the exact mechanism underlying CLDN6 downregulation remains unclear. CLDN6 silence is associated with DNA methyltransferase 1 (DNMT1) mediated DNA methylation, and DNMT1 is regulated by the transforming growth factor beta (TGFβ)/SMAD pathway. Therefore, we hypothesized that TGFβ/SMAD pathway, specifically SMAD2, may play a critical role for CLDN6 downregulation through DNA methyltransferase 1 (DNMT1) mediated DNA methylation. To test this hypothesis, we blocked the SMAD2 pathway with SB431542 in two human breast cancer cell lines (MCF-7 and SKBR-3). Our results showed that treatment with SB431542 led to a decrease of DNMT1 expression and the binding activity for CLDN6 promoter. The methylation level of CLDN6 promoter was decreased, and simultaneously CLDN6 protein expression increased. Upregulation of CLDN6 inhibited epithelial to mesenchymal transition (EMT) and reduced the migration and invasion ability of both MCF-7 and SKBR-3 cells. Furthermore, knocked down of CLDN6 abolished SB431542 effects on suppression of EMT associated gene expression and inhibition of migration and invasion. Thus, we demonstrated that the downregulation of CLDN6 is regulated through promoter methylation by DNMT1, which depends on the SMAD2 pathway, and that CLDN6 is a key regulator in the SMAD2/DNMT1/CLDN6 pathway to inhibit EMT, migration and invasion of breast cancer cells.

Genome-wide DNA methylation drives human embryonic stem cell erythropoiesis by remodeling gene expression dynamics

AIM To investigate the role of DNA methylation during erythrocyte production by human embryonic stem cells (hESCs). METHODS We employed an erythroid differentiation model from hESCs, and then tracked the genome-wide DNA methylation maps and gene expression patterns through an Infinium HumanMethylation450K BeadChip and an Ilumina Human HT-12 v4 Expression Beadchip, respectively. RESULTS A negative correlation between DNA methylation and gene expression was substantially enriched during the later differentiation stage and was present in both the promoter and the gene body. Moreover, erythropoietic genes with differentially methylated CpG sites that were primarily enriched in nonisland regions were upregulated, and demethylation of their gene bodies was associated with the presence of enhancers and DNase I hypersensitive sites. Finally, the components of JAK-STAT-NF-κB signaling were DNA hypomethylated and upregulated, which targets the key genes for erythropoiesis. CONCLUSION Erythroid lineage commitment by hESCs requires genome-wide DNA methylation modifications to remodel gene expression dynamics.