scholarly journals Identification of novel DNA hypermethylation of the adenylate kinase 5 promoter in colorectal adenocarcinoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bokyung Ahn ◽  
Yang Seok Chae ◽  
Soo Kyung Lee ◽  
Moa Kim ◽  
Hyeon Soo Kim ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Lines ◽  
Adenylate Kinase ◽  
Dna Methyltransferase ◽  
Colorectal Adenocarcinoma ◽  
Adenine Nucleotides ◽  
Inverse Correlation ◽  
Dna Hypermethylation ◽  
Normal Tissues ◽  
The Difference

AbstractAdenylate kinase 5 (AK5) belongs to the adenylate kinase family that catalyses reversible phosphate transfer between adenine nucleotides, and it is related to various energetic signalling mechanisms. However, the role of AK5 in colorectal cancer (CRC) has not been reported. In this study, AK5 was significantly hypermethylated in CRC compared to adjacent normal tissues (P < 0.0001) and normal tissues (P = 0.0015). Although the difference in mRNA expression was not statistically significant in all of them, the selected 49 cases of CRC tissues with AK5 hypermethylation with the cut off value of 40% showed a significant inverse correlation with mRNA expression (P = 0.0003). DNA methylation of AK5 promoter significantly decreased and AK5 expression recovered by 5-aza-2′-deoxycytidine, DNA methyltransferase inhibitor in CRC cell lines. In addition, AK5 promoter activity significantly decreased due to DNA methyltransferase, and it increased due to 5-aza. Moreover, AK5 regulated the phosphorylated AMPK and mTOR phosphorylation and inhibited the cell migration and cell invasion in CRC cell lines. Furthermore, low AK5 expression is associated with poor differentiation (P = 0.014). These results demonstrate that the AK5 promoter is frequently hypermethylated and induced methylation-mediated gene down-regulation. AK5 expression regulates AMPK/mTOR signalling and may be closely related to metastasis in colorectal adenocarcinoma.

Frequent Epigenetic Inactivation of MSX2 In Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4645-4645 ◽  
Author(s):  
Chen Zhao ◽  
Xin Han ◽  
Yu H. Zhang ◽  
Xiaoyan Huang ◽  
Aili Dai ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Functional Role ◽  
Methylation Status ◽  
Dna Hypermethylation ◽  
Expression Levels ◽  
Mrna Expression Levels ◽  
Acute Myeloid

Abstract Abstract 4645 DNA hypermethylation has been implicated in the tumorigenesis and prognosis in acute myeloid leukemia (AML). To identify and validate relevant methylated genes in AML, we have compared expression levels and methylation status of 26 candidate genes. One of the interesting candidates identified in our study is MSX2. MSX2 is a member of muscle segment homeobox gene family. MSX2 plays a role in promoting cell growth under certain conditions and may be an important target for RAS signaling pathways. However, the mechanism of transcriptional regulation and functional role of MSX2 in hematological malignancies, especially AML, are poorly understood. In our study, we determined the methylation status, and analyzed the expression levels of MSX2 in AML cell lines and primary AML cells using RT-PCR and/or Taqman real-time PCR. MSX2 mRNA expression was robust in the normal granulocytes and blasts of human bone-marrow, but was either absent or significantly diminished in 6 of 9 (66.7%) AML cell lines. The expression levels of MSX2 in those 6 AML cell lines were restored after treatment of 5-aza 2′-deoxycytidine. In addition, COBRA (Combined Bisulfite Restriction) analysis demonstrated hypermethylation of MSX2 in those AML cell lines (6 of 9, 66.7%), and partial methylation in 3 of 9 AML cell lines. The methylation status was inversely correlated with the mRNA expression levels of MSX2 in those cell lines. Furthermore, the expression levels and methylation status of MSX2 in human primary AML cells were evaluated. COBRA analysis demonstrated frequent hypermethylation of MSX2 in primary AML patient samples (19 of 32, 59.3%). Importantly, the mRNA expression levels of MSX2 as shown by Taqman real-time PCR in those 19 primary AML patient samples were inversely correlated with the methylation status of MSX2. These findings confirmed the role of frequent DNA hypermethylation in silencing MSX2 in AML. We are in the process of determining the functional role of MSX2 in the pathogenesis of AML. In addition, diagnostic and prognostic values of MSX2 in AML are being pursued. Disclosures: No relevant conflicts of interest to declare.

Overexpression of melanoma-associated antigen D4 as an independent prognostic factor in squamous cell carcinoma of the esophagus.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 71-71
Author(s):  
Mitsuro Kanda ◽  
Hisaharu Oya ◽  
Soki Hibino ◽  
Hideki Takami ◽  
Dai Shimizu ◽  
...  
Keyword(s):  
Prognostic Factor ◽  
Mrna Expression ◽  
Cell Lines ◽  
Squamous Cell ◽  
Univariate Analysis ◽  
Independent Prognostic Factor ◽  
Protein Distribution ◽  
Rt Pcr ◽  
Carcinoma Of The Esophagus ◽  
Normal Tissues

71 Background: To pursue an urgently needed treatment target for esophageal cancer (EC), we investigated the function of the recently discovered melanoma-associated antigen (MAGE)-D4 in squamous cell EC. Methods: MAGE-D4 mRNA expression was analyzed in nine EC cell lines using quantitative reverse transcription-polymerase chain reaction (RT-PCR). In 65 surgical specimens of squamous cell EC with no prior neoadjuvant therapy, MAGE-D4 mRNA expression in EC tissues and corresponding normal tissues was analyzed and compared, and evaluated in terms of clinicopathological factors. In representative cases, MAGE-D4 protein distribution was analyzed immunohistochemically. Results: The heterogeneity of MAGE-D4 mRNA expression was confirmed in EC cell lines by quantitative RT-PCR. In surgical specimens, MAGE-D4 mRNA expression was significantly higher in EC tissues than in corresponding normal tissues (P < 0.001). Patients with the highest MAGE-D4 mRNA expression in EC tissues (top quartile, n=17) had significantly shorter overall survival than patients with low expression (2-year survival: 44% and 73%, respectively, P = 0.006). Univariate analysis identified age (≥ 65 years), lymphatic involvement and high MAGE-D4 mRNA expression as significant prognostic factors; high MAGE-D4 mRNA expression was also an independent prognostic factor in multivariable analysis (hazard ratio: 2.194; P = 0.039), and was significantly associated with Brinkman index (P = 0.008) and preoperative carcinoembryonic antigen level (P = 0.002). Immunohistochemical MAGE-D4b expression was consistent with MAGE-D4mRNA profiling. Conclusions: Our results suggest that MAGE-D4 overexpression influences tumor progression and MADE-D4 can be a prognostic marker and a potential molecular target in squamous cell EC.

scholarly journals Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylation of FLT1 gene in choriocarcinoma cells

2020 ◽  
Author(s):  
Tadashi Sasagawa ◽  
Atsushi Jinno-Oue ◽  
Takeshi Nagamatsu ◽  
Kazuki Morita ◽  
Tetsushi Tsuruga ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Mrna Expression ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Bisulfite Sequencing ◽  
Methylation Status ◽  
Angiogenic Factor ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Choriocarcinoma Cells

Abstract Background: Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells. Methods: qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulfite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model. Results: Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells. Conclusions: Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo . We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.

Impact of Overexpression of Immune Suppressor Siglec-15 on The Prognosis of Clear Renal Cell Carcinoma and Fibrosis Level

2021 ◽  
Author(s):  
Wenbo Yang ◽  
Caipeng Qin ◽  
Yiqing Du ◽  
Songchen Han ◽  
Wenjun Bai ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Carcinoma ◽  
Mrna Expression ◽  
Second Harmonic ◽  
Inverse Correlation ◽  
The Cancer Genome Atlas ◽  
Fuhrman Grade ◽  
Normal Tissues ◽  
Significant Difference ◽  
Immune Suppressor

Abstract Background The expression of Siglec-15, as a critical immune suppressor, in renal clear cell carcinoma (ccRCC) was few evaluated and remains unclear, especially in protein level. As previous studies reported, tumor fibrosis plays an essential role in assessing the prognosis of ccRCC, but the exact mechanism is not precise. This study evaluated the expression of Siglec-15, its role in prognosis, and the association with tumor fibrosis in ccRCC.Methods: Immunohistochemistry was used to analyze the Siglec-15 expression in one tissue microarray (cohort A, tumor: n=134, adjacent normal tissues: n=29). Subsequently, the mRNA expression of Siglec-15 and its clinical significance in ccRCC were analyzed using The Cancer Genome Atlas database (TCGA, cohort B, n = 534) and samples. Spearman correlation coefficients were calculated for correlation analysis of correlated expression genes of Siglec-15, and then functional annotation analysis was obtained with correlated expression genes. We detected the tumor fibrosis grade in cohort C (n=32) via second harmonic generation/two-photon excitation fluorescence. Results: Siglec-15 was overexpressed in tumor tissues compared with adjacent normal tissues in both cohort A (n=29, p<0.001) and cohort C (n=25, p<0.001). However, there was no significant difference in mRNA expression of Siglec-15 between tumor and adjacent normal tissues in cohort B (p>0.05). Moreover, over-expression of Siglec-15 is associated with higher Fuhrman grade in cohort A＆C (n=166, p=0.001, OR=3.132, 1.563-6.275), cohort B (n=534, p=0.008, OR=1.606, 1.138-2.267). Univariate Kaplan-Meir survival analysis showed that patients with high Siglec-15 mRNA expression had shorter survival periods without significance in cohort B (p=0.073). Multivariate analysis employing the Siglec-15 regression model revealed that AJCC and Fuhrman grade was the only significant independent prognostic indicators. Besides, an inverse correlation was found between Siglec-15 protein expression and the tumor's fibrosis level (p = 0.02).Conclusions: Siglec-15 expression increases in ccRCC compared with adjacent normal tissues. Siglec-15 was frequently expressed and positively associated with pathology grade in ccRCC. This study indicated a significant role of Siglec-15 in the prognosis and immunotherapy target of ccRCC. This study also found an inverse correlation between Siglec-15 protein expression and the fibrosis level of the tumor.

A novel preclinical model of cholangiocarcinoma based on human aberrant FBXW7 expression.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e15624-e15624
Author(s):  
Jingxiao Wang ◽  
Haichuan Wang ◽  
Michele Peters ◽  
Ning Ding ◽  
Silvia Ribback ◽  
...  
Keyword(s):  
Mouse Model ◽  
Mrna Expression ◽  
Cell Lines ◽  
Inverse Correlation ◽  
Tumor Burden ◽  
Dominant Negative ◽  
Preclinical Model ◽  
Dominant Negative Form ◽  
Clinical Models

e15624 Background: Pre-clinical models that mimic human genetic events occurring in intrahepatic cholangiocarcinoma (iCCA) are limited. The ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7) is recognized as a tumor suppressor in many cancer types. Methods: Firstly, we determined the FBXW7 mutation frequency (n = 120) and mRNA expression (n = 87) in a collection of human iCCA. Based on the preliminary findings in human data, we generated a mouse model by hydrodynamic tail vein injection of activated/myristylated (myr-)AKT with Fbxw7ΔF, a dominant negative form of Fbxw7. Subsequently, we investigated the role of established targets of Fbxw7, namely Notch2, Yap, and c-Myc in this novel mouse model and in human CCA cell lines. Results: FBXW7 mRNA expression is almost ubiquitously downregulated (71/82; 86.6%) in human iCCA specimens, while only 0.8% of samples showed FBXW7 somatic mutations. In vivo, co-expression of AKT and Fbxw7ΔF triggered the development of iCCA lesions and mice were euthanized by 15 weeks post-injection due to high tumor burden. At the molecular level, a strong induction of FBXW7 canonical targets, including Yap, Notch2, and c-Myc oncoproteins, was detected. However, only c-Myc was consistently confirmed as a FBXW7 target in human CCA cell lines. Interestingly, selected ablation of c-Myc completely impaired iCCA formation in AKT/Fbxw7ΔF mice, whereas deletion of either Yap or Notch2 delayed cholangiocarcinogenesis in the same model. Furthermore, in human iCCA specimens, a strong, inverse correlation between the expression levels of FBXW7 and c-Myc was observed. Conclusions: Downregulation of FBXW7 is almost ubiquitous in human iCCA and cooperates with AKT to induce cholangiocarcinogenesis in mice. This pre-clinical mouse model could be used to test novel therapeutics targeting c-Myc, Notch2, and/or Yap.

scholarly journals Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylayion of FLT1 gene in choriocarcinoma cells

2019 ◽  
Author(s):  
Tadashi Sasagawa ◽  
Atsushi Jinno-Oue ◽  
Takeshi Nagamatsu ◽  
Kazuki Morita ◽  
Tetsushi Tsuruga ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Mrna Expression ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Angiogenic Factor ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Choriocarcinoma Cells

Abstract Background: Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells. Methods: qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulphite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model. Results: Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells. Conclusions: Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo. We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.

scholarly journals The epithelial splicing regulator ESRP2 is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor

2020 ◽  
Author(s):  
Danny Legge ◽  
Ling Li ◽  
Whei Moriarty ◽  
David Lee ◽  
Marianna Szemes ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Cellular Proliferation ◽  
Kidney Development ◽  
Regulatory Protein ◽  
Wilms Tumour ◽  
Fetal Kidney ◽  
Dna Hypermethylation

ABSTRACTWilms tumour (WT), a childhood kidney cancer with embryonal origins, has been extensively characterised for genetic and epigenetic alterations, but a proportion of WTs still lack identifiable abnormalities. To uncover DNA methylation changes critical for WT pathogenesis, we compared the epigenome of fetal kidney with two WT cell lines, using methyl-CpG immunoprecipitation. We filtered our results to remove common cancer-associated epigenetic changes, and to enrich for genes involved in early kidney development. This identified four candidate genes that were hypermethylated in WT cell lines compared to fetal kidney, of which ESRP2 (epithelial splicing regulatory protein 2), was the most promising gene for further study. ESRP2 was commonly repressed by DNA methylation in WT, and this was shown to occur early in WT development (in nephrogenic rests). ESRP2 expression could be reactivated by DNA methyltransferase inhibition in WT cell lines. When ESRP2 was overexpressed in WT cell lines, it acted as an inhibitor of cellular proliferation in vitro, and in vivo it suppressed tumour growth of orthotopic xenografts in nude mice. RNA-seq of the ESRP2-expressing WT cell lines identified several novel splicing targets, in addition to well-characterised targets of ESRP2. We propose a model in which the mesenchymal to epithelial transition that is essential for early kidney development, can be disrupted in to generate WT, either by genetic abnormalities such as WT1 mutations, or by epigenetic defects, such as ESRP2 methylation.

scholarly journals mRNA Expression of MMP-28 (Epilysin) in Gingival Tissues of Chronic and Aggressive Periodontitis Patients: A Reverse Transcriptase PCR Study

2013 ◽  
Vol 35 ◽  
pp. 113-118 ◽  
Author(s):  
P. Padmavati ◽  
S. Savita ◽  
B. M. Shivaprasad ◽  
Krishna Kripal ◽  
K. Rithesh
Keyword(s):  
Reverse Transcriptase ◽  
Mrna Expression ◽  
Chronic Periodontitis ◽  
Aggressive Periodontitis ◽  
Gingival Tissue ◽  
Tissue Samples ◽  
Normal Tissues ◽  
Periodontal Tissues ◽  
Probing Depth ◽  
The Difference

Background and Objectives. Matrix metalloproteinases degrade extracellular membrane and also release bioactive fragments and growth factors, thus influencing fundamental biological and pathological processes. Epilysin (MMP-28) differs from most other MMPs as it is expressed in a number of normal tissues, suggestive of functions in tissue homeostasis. The aim of the present study was to quantitatively evaluate and compare the mRNA expression of epilysin (MMP-28) in gingival tissues of healthy patients and of patients affected by chronic or aggressive periodontitis.Methods. A total of 60 subjects, 20 periodontally healthy subjects, 20 with chronic periodontitis, and 20 with aggressive periodontitis, were included in this study. Periodontal status was evaluated by measuring gingival index, probing depth and clinical attachment level. mRNA expression of MMP-28 was determined by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) in gingival tissue samples collected.Results. Relative quantification of mRNA expression of MMP-28 was highest in healthy tissues () when compared to subjects with chronic periodontitis () and aggressive periodontitis (), but the difference was not statistically significant.Conclusion. mRNA expression of MMP-28 was highest in healthy tissues when compared to diseased periodontal tissues suggesting that MMP-28 could act as a biomarker for periodontal health.

scholarly journals Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylation of FLT1 gene in choriocarcinoma cells

2020 ◽  
Author(s):  
Tadashi Sasagawa ◽  
Atsushi Jinno-Oue ◽  
Takeshi Nagamatsu ◽  
Kazuki Morita ◽  
Tetsushi Tsuruga ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Mrna Expression ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Bisulfite Sequencing ◽  
Methylation Status ◽  
Angiogenic Factor ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Choriocarcinoma Cells

Abstract Background: Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells. Methods: qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulfite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model. Results: Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells. Conclusions: Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo . We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.

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