Identification of epigenetic silencing of GCNT2 expression by comprehensive real-time PCR screening in colorectal cancer.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 506-506
Author(s):  
Kazunorii Nakamura ◽  
Horomichi Sawaki ◽  
Keishi Yamashita ◽  
Masahiko Watanabe ◽  
Hisashi Narimatsu
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Critical Role ◽  
Normal Mucosa ◽  
Primary Cancer ◽  
Normal Tissues ◽  
Cancer Tissues

506 Background: Glycoprotein expression profile has been proved to be dramatically altered in human cancers, however specific glycogenes which are aberrant in expression in cancer cells has not been fully identified. Recent accumulated evidence supported notion that the reduced expression of tumor suppressor genes is explained by DNA promoter methylation in human cancer. Methods: We used Comprehensive Real time PCR system (CRPS) for glycogenes (189 genes) to identify genes aberrantly expressed in colorectal cancer tissues (CRC) as compared to the corresponding normal mucosa tissues. GCNT2 was of particular interest among the identified genes in CRC. Results: (1) GCNT2 harbors 3 isoforms which have different promoter regions. (2) All of the 3 isoforms of GCNT2 genes were remarkably decreased in CRC as compared to the corresponding normal mucosa, and each isoform expression was strongly associated with other 2 isoforms in primary cancer tissues by TaqMan real time PCR (R = 0.99-995, p < 0.0001). (3) Among the 5 CRC cell lines (DLD1, HCT116, CACO2, LOVO), those which were silenced in expression were reactivated by demethylating agents such as 5-aza-2’ deoxycytidine and trichostatin A. (4) Promoter region of the variant 2 of GCNT2 was consistent with its silenced expression in CRC cell lines by cloned sequence, so we examined DNA methylation status of the promoter of the GCNT2 variant 2 in 50 primary cancer tissues and the corresponding normal tissues. Quantitative MSP revealed that almost half of normal tissues have methylation as high as tumor tissues, while, in the primary CRC with less methylation in the corresponding normal tissues, DNA methylation was higher in primary CRC tissues than in the corresponding normal tissues. Finally, GCNT2 variant 2 stable transfection induced expression of other 2 isoform variants. Conclusions: We identified novel methylation gene GCNT2 among the glycoenes. Glycoenes that were altered in genomic or epigenetic manner have been few, so GCNT2 may play a critical role in cancer progression through glycan change.

scholarly journals MiR-382 functions as tumor suppressor and chemosensitizer in colorectal cancer

2019 ◽  
Vol 39 (8) ◽  
Author(s):  
Hui Yao ◽  
Dong Xia ◽  
Zong-lin Li ◽  
Lei Ren ◽  
Ming-ming Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Cancer Cell ◽  
Critical Role ◽  
Colorectal Cancer Cell ◽  
Cancer Cell Proliferation ◽  
Interacting Protein ◽  
Colorectal Cancer Cell Lines ◽  
Cancer Tissues

Abstract Increasing evidence suggests that microRNAs (miRNAs) play a critical role in tumorigenesis. Decreased expression of miR-382 has been observed in various types of cancers. However, the biological function of miR-382 in colorectal cancer (CRC) is still largely unknown. Here, we found that miR-382 was down-regulated in human colorectal cancer tissues and cell lines associated with it. MiR-382 inhibited colorectal cancer cell proliferation, migration, invasion, and enhance chemosensitivity. Furthermore, we identified Krüppel-like factor 12 (KLF12) and homeodomain-interacting protein kinase 3 (HIPK3) as the target of miR-382, and miR-382 rescued the promotion effect of KFL12 on migration and enhanced chemosensitivity in colorectal cancer cell lines. Collectively, these findings revealed that miR-382 inhibits migration and enhances chemosensitivity by targeting KLF12 and HIPK3 in colorectal cancer. These findings might serve as a tumor suppressor in CRC.

67 CELL TYPE AND CULTURE OVER TIME EFFECT DNA METHYLTRANSFERASE 1 EXPRESSION IN BOVINE DONOR FIBROBLAST CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 151 ◽  
Author(s):  
K. Moore ◽  
E. Wroclawska ◽  
J. M. Kramer ◽  
S. L. Goicoa
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Culture Conditions ◽  
Fibroblast Cells ◽  
Cell Type ◽  
Donor Cells ◽  
Dnmt1 Expression ◽  
Over Time

Aberrant chromatin remodeling has been implicated in the low success rates achieved from cloned embryos. Following fertilization, DNA methylation within a normal embryo is rapidly reduced to a very low level and remains low until the 8–16 cell stage when DNA methylation once again increases. In contrast, the majority of cloned embryos fail to exhibit a similar methylation pattern. This may be due to somatic cell-associated DNMT1s keeping methylation high. However, attempts to chemically modify methylation patterns of donor cells prior to cloning have proven problematic. The objective of this study was to determine if a more natural approach, such as culture conditions, time in culture, and/or cell type, could alter DNMT1 expression in donor fibroblast cells. Two experiments were designed to meet these objectives. Donor fibroblast cell lines were produced from biopsies taken from male and female skin, ovaries, and testes, and were grown in Dulbecco&apos;s modified Eagle&apos;s medium (DMEM) supplemented with 15&percnt; fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 0.1 mM β-mercaptoethanol, in a humidified environment of 5&percnt; CO2 in air, at 39&deg;C. In Experiment 1, cell lines were maintained at 70&percnt; confluence to passage 4, 8, and 12, and analyzed by reverse transcription real-time PCR. In Experiment 2, cell lines were evaluated under 3 culture conditions: proliferating (70&percnt; confluence), serum-starved (0.5&percnt; FBS), and confluent (100&percnt;), and analyzed by reverse transcription real-time PCR. RNA was isolated from cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA), reverse transcribed, and analyzed for DNMT1 expression using Taqman real-time PCR, with β-actin as the reference standard. All samples and no template controls were run in triplicate. Final quantitation was done using the comparative CT method, and relative DNMT1 expression was analyzed using one-way ANOVA followed by LS means multiple comparisons. Cell type and passage number had a significant effect on DNMT1 expression. Ovarian fibroblasts had an overall increase in expression in DNMT1 over time (P &lt; 0.05), whereas male skin fibroblasts demonstrated an opposite trend (P &equals; 0.05). Female skin fibroblasts and testes fibroblasts also had a decrease in DNMT1 expression over time, but only approached significance (P &lt; 0.10). For Experiment 2, culture conditions tested did not affect DNMT1 expression for any except one skin cell line. In that case, proliferating cells had significantly higher DNMT1 than quiescent cells (P &lt; 0.005). This research emphasizes the importance of donor cell type and culture effects over time on gene expression. These important aspects should be considered when selecting and growing donor cells to be utilized in somatic cell nuclear transfer. This project was supported by National Research Initiative Competitive Grant no. 2006-35203-16620 from the USDA Cooperative State Research, Education, and Extension Service and the Florida Agricultural Experiment Station.

Production of Monoclonal Antibodies Against ADAMTS13 Determining Its Expression in Different Human Tissues.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3948-3948
Author(s):  
Weiqiang Gao ◽  
Xiaojun Chow ◽  
Xia Bai ◽  
Jiang Su ◽  
Changgeng Ruan
Keyword(s):  
Dna Methylation ◽  
Hepatitis B ◽  
Cell Lines ◽  
Hepatic Function ◽  
Expression Levels ◽  
Normal Tissues ◽  
Hepatocarcinoma Cell ◽  
Cancer Tissues ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The deficiency of ADAMTS13/von Willebrand factor-cleaving protease in cancer patients, which can lead to the accumulation of ultra-large von Willebrand factor (UL-vWF) in plasma, has been confirmed by several previous reports. Nevertheless, the results of them did not avoid the heterogeneity between different tumors and the reasons accounted for its deficiency still remain to be elucidated. Since ADAMTS13 is mainly synthesized and released from liver and tumors derived from liver often accompanies with metastasis, we have studied the expression levels of ADAMTS13 in patients with primary hepatocarcinoma and in different hepatic cancer cell lines. Firstly, we detected the ADAMTS13 mRNA and protein levels in paired primary cancer tissues and adjacent normal tissues using semi-quantitative reverse transcription PCR (RT-PCR) and immunohistochemistry. It showed that most of the cancer tissues expressed lower ADAMTS13 levels compared with their paired adjacent normal tissues. Significant low expression levels of ADAMTS13 had a close relationship with distal metastasis of hepatocarcinoma, but the abnormal of hepatic function did not have an association with the deficiency of ADAMTS13 expression. Interestingly, most of the patients suffering with hepatitis B and cirrhosis did not show significantly decreased ADAMTS13 levels, and most of the cancer patients with distal metastasis while without hepatitis B and cirrhosis accompanied markedly deficiency of the protease. The hepatocarcinoma cell lines, HepG2 and BEL7702, also expressed lower ADAMTS13 mRNA levels compared with SMMC7721 and a normal hepatic cell line L02. However, when the hepatocarcinoma cell lines were treated with a demethylation drug 5-aza-2′-deoxycytidine, its ADAMTS13 mRNA expression levels elevated with the increase of treatment dosage, especially in HepG 2 (P<0.05). It seems that the deficiency of ADAMTS13 in hepatocarcinoma might be associated with DNA methylation. The data above revealed that the ADAMTS13 expression levels were lower in hepatocarcinoma tissues. Furthermore, the activity of ADAMTS13 was significantly decreased in aged people older than 65 years, and the extent of methylation of genes are closely related with age. Therefore, based on our results, the deficiency of ADAMTS13 in hepatocarcinoma patients might not be caused by the abnormal of hepatic function, but be related to the DNA methylation of the protease.

scholarly journals Identification of Aberrantly Expressed miRNAs in Gastric Cancer

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Dan Liu ◽  
Xiaowei Hu ◽  
Hongfeng Zhou ◽  
Guangyue Shi ◽  
Jin Wu
Keyword(s):  
Gastric Cancer ◽  
Gene Ontology ◽  
Real Time ◽  
Real Time Pcr ◽  
Malignant Disease ◽  
Gene Ontology Analysis ◽  
Normal Tissues ◽  
Therapy Targets ◽  
New Perspective ◽  
Cancer Tissues

The noncoding components of the genome, including miRNA, can contribute to pathogenesis of gastric cancer. Their expression has been profiled in many human cancers, but there are a few published studies in gastric cancer. It is necessary to identify novel aberrantly expressed miRNAs in gastric cancer. In this study, the expression profile of 1891 miRNAs was analyzed using a miRCURY array LNA miRNA chip from three gastric cancer tissues and three normal tissues. The expression levels of 4 miRNAs were compared by real-time PCR between cancerous and normal tissues. We found that 31 miRNAs are upregulated in gastric cancer(P<0.05)and 10 miRNAs have never been reported by other studies; 30 miRNA are downregulated(P<0.05)in gastric cancer tissues. Gene ontology analysis revealed that those dysregulated miRNAs mainly take part in regulating cell proliferation. The levels of has-miR-105, -213*, -514b, and -548n were tested by real-time PCR and have high levels in cancerous tissues. Here, we report a miRNA profile of gastric cancer and provide new perspective to understand this malignant disease. This novel information suggests the potential roles of these miRNAs in the diagnosis, prognosis biomarkers, or therapy targets of gastric cancer.

RPS24c Isoform Facilitates Tumor Angiogenesis Via Promoting the Stability of MVIH in Colorectal Cancer

2020 ◽  
Vol 20 (5) ◽  
pp. 388-395 ◽  
Author(s):  
Yue Wang ◽  
Youjun Wu ◽  
Kun Xiao ◽  
Yingjie Zhao ◽  
Gang Lv ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Ribosomal Protein ◽  
Real Time ◽  
Tumor Angiogenesis ◽  
Real Time Pcr ◽  
Molecular Mechanisms ◽  
Migration And Invasion ◽  
Binding Interaction ◽  
Tubule Formation ◽  
The Stability

Background: Colorectal cancer (CRC) is the second leading cause of death worldwide, and distant metastasis is responsible for the poor prognosis in patients with advanced-stage CRC. RPS24 (ribosomal protein S24) as a ribosomal protein, multiple transcript variant encoding different isoforms have been found for this gene. Our previous studies have demonstrated that RPS24 is overexpressed in CRC. However, the mechanisms underlying the role of RPS24 in tumor development have not been fully defined. Methods: Expression of RPS24 isoforms and lncRNA MVIH in CRC tissues and cell lines were quantified by real-time PCR or western blotting assay. Endothelial tube formation assay was performed to determine the effect of RPS24 on tumor angiogenesis. The cell viability of HUVEC was determined by MTT assay, and the migration and invasion ability of HUVEC were detected by transwell assay. PGK1 secretion was tested with a specific ELISA kit. Results: Here, we found that RPS24c isoform was a major contributor to tumor angiogenesis, a vital process in tumor growth and metastasis. Real-time PCR revealed that RPS24c isoform was highly expressed in CRC tissues, while other isoforms are present in both normal and CRC tissues with no statistical difference. Moreover the change of RPS24 protein level is mainly due to the fluctuation of RPS24c. Furthermore, we observed that silencing RPS24c could decrease angiogenesis by inhibiting tubule formation, HUVEC cell proliferation and migration. Additionally, we investigated the molecular mechanisms and demonstrated that RPS24c mRNA interacted with lncRNA MVIH, the binding-interaction enhanced the stability of each other, thereby activated angiogenesis by inhibiting the secretion of PGK1. Conclusion: RPS24c facilitates tumor angiogenesis via the RPS24c/MVIH/PGK1 pathway in CRC. RPS24c inhibition may be a novel option for anti-vascular treatment in CRC.

scholarly journals Genome-wide analysis identifies critical DNA methylations within NTRKs genes in colorectal cancer

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Zijian Chen ◽  
Zenghong Huang ◽  
Yanxin Luo ◽  
Qi Zou ◽  
Liangliang Bai ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Promoter Methylation ◽  
Expression Profiles ◽  
Methylation Status ◽  
Gene Promoter ◽  
Normal Mucosa ◽  
Suppressor Gene ◽  
Survival Outcome ◽  
Prognostic Models

Abstract Background Neurotrophic tropomyosin receptor kinases (NTRKs) are a gene family function as oncogene or tumor suppressor gene in distinct cancers. We aimed to investigate the methylation and expression profiles and prognostic value of NTRKs gene in colorectal cancer (CRC). Methods An analysis of DNA methylation and expression profiles in CRC patients was performed to explore the critical methylations within NTRKs genes. The methylation marker was validated in a retrospectively collected cohort of 229 CRC patients and tested in other tumor types from TCGA. DNA methylation status was determined by quantitative methylation-specific PCR (QMSP). Results The profiles in six CRC cohorts showed that NTRKs gene promoter was more frequently methylated in CRC compared to normal mucosa, which was associated with suppressed gene expression. We identified a specific methylated region within NTRK3 promoter targeted by cg27034819 and cg11525479 that best predicted survival outcome in CRC. NTRK3 promoter methylation showed independently predictive value for survival outcome in the validation cohort (P = 0.004, HR 2.688, 95% CI [1.355, 5.333]). Based on this, a nomogram predicting survival outcome was developed with a C-index of 0.705. Furthermore, the addition of NTRK3 promoter methylation improved the performance of currently-used prognostic model (AIC: 516.49 vs 513.91; LR: 39.06 vs 43.64, P = 0.032). Finally, NTRK3 promoter methylation also predicted survival in other tumors, including pancreatic cancer, glioblastoma and stomach adenocarcinoma. Conclusions This study highlights the essential value of NTRK3 methylation in prognostic evaluation and the potential to improve current prognostic models in CRC and other tumors.

scholarly journals The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2341
Author(s):  
Normann Steiner ◽  
Karin Jöhrer ◽  
Selina Plewan ◽  
Andrea Brunner-Véber ◽  
Georg Göbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

scholarly journals Increased Expression of the Pro-Protein Convertase Furin Predicts Decreased Survival in Ovarian Cancer

2007 ◽  
Vol 29 (4) ◽  
pp. 289-299
Author(s):  
Robert E. Page ◽  
Andrés J. P. Klein-Szanto ◽  
Samuel Litwin ◽  
Emmanuelle Nicolas ◽  
Raid Al-Jumaily ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Progression ◽  
Real Time ◽  
Cell Lines ◽  
Cancer Cell ◽  
Real Time Pcr ◽  
Ovarian Tumor ◽  
Cancer Cell Lines ◽  
Rt Pcr ◽  
Primary Tumors

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

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