Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although ... more Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although both offer considerable theranostic potential. In this study, we identified frequent upregulation of miR-196a and lincRNA HOTAIR in high-risk gastrointestinal stromal tumors (GIST). Overexpression of miR-196a was associated with high-risk grade, metastasis and poor survival among GIST specimens. miR-196a genes are located within the HOX gene clusters and microarray expression analysis revealed that the HOXC and HOTAIR gene were also coordinately upregulated in GISTs which overexpress miR-196a. In like manner, overexpression of HOTAIR was also strongly associated with high-risk grade and metastasis among GIST specimens. RNA interference–mediated knockdown of HOTAIR altered the expression of reported HOTAIR target genes and suppressed GIST cell invasiveness. These findings reveal concurrent overexpression of HOX genes with noncoding RNAs in human cancer in this setting, revealing miR-196a ...
Colorectal sessile serrated adenoma/polyps (SSA/Ps) are well-known precursors of colorectal cance... more Colorectal sessile serrated adenoma/polyps (SSA/Ps) are well-known precursors of colorectal cancer (CRC) characterized bymutation and microsatellite instability. By contrast, the molecular characteristics of traditional serrated adenoma (TSAs) are not fully understood. We analyzed genome-wide DNA methylation in TSAs having both protruding and flat components. We identified 11 genes, including, methylation of which progressively increased during the development of TSAs.was prevalently methylated in TSAs, but was rarely methylated in SSA/Ps (< 0.001). RT-PCR and immunohistochemistry revealed that SMOC1 was expressed in normal colon and SSA/Ps, but its expression was decreased in TSAs. Ectopic expression of SMOC1 suppressed proliferation, colony formation andtumor formation by CRC cells. Analysis of colorectal lesions (= 847) revealed thatis frequently methylated in TSAs, high-grade adenomas and CRCs. Among these,methylation was strongly associated withmutation and CpG island methyl...
Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, alth... more Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced aden...
Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mech... more Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mechanism is not fully understood. 5-hydroxymethylcytosine levels and TET1 expression are both reduced in CRC, while epigenetic silencing of TET1 is reportedly associated with the CpG island methylator phenotype. In the present study, we aimed to clarify the relationship between loss of TET1 and aberrant DNA methylation in CRC. Stable TET1 knockdown clones were established using Colo320DM cells, which express high levels of TET1, and HCT116 cells, which express TET1 at a level similar to that in normal colonic tissue. Infinium HumanMethylation450 BeadChip assays revealed increased levels of 5-methylcytosine at more than 10,000 CpG sites in TET1-depleted Colo320DM cells. Changes in DNA methylation were observed at various positions within the genome, including promoters, gene bodies and intergenic regions, and the altered methylation affected expression of a subset of genes. By contrast, TET1...
Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association, Jan 28, 2016
Although genetic alterations in patients with advanced gastric cancer have been extensively studi... more Although genetic alterations in patients with advanced gastric cancer have been extensively studied, those in patients with intramucosal neoplasia (IMN) are still poorly understood. We evaluated genetic differences in 158 IMNs, including 51 low-grade dysplasias, 58 high-grade dysplasias (HGDs), 30 intramucosal cancers (IMCs), and 19 mixed tumors (composed of IMC and HGD within the same tumor), using PCR-based microsatellite analysis [allelic imbalance (AI) and microsatellite instability (MSI)]. We classified the DNA methylation status as a hypermethylated epigenome, a moderately methylated epigenome, or a hypomethylated epigenome. In addition, p53 overexpression, β-catenin nuclear localization, and mucin expression were also examined. From cluster analysis, the IMNs examined were categorized into four subgroups as follows. Tumors in subgroup 1 were characterized by MSI-high status, a hypermethylated epigenome, and loss or reduction of expression of MLH-1. Tumors in subgroup 2 showed...
Our aim was to identify DNA methylation changes associated with the growth pattern and invasivene... more Our aim was to identify DNA methylation changes associated with the growth pattern and invasiveness of colorectal cancers (CRCs). Comparison of the methylation statuses of large (≥20 mm in diameter along the colonic surface) noninvasive tumors (NTs) and small (<20 mm in diameter along the colonic surface) invasive tumors (ITs) using CpG island microarray analysis showed neurotensin receptor 1 (NTSR1) to be hypermethylated in large NTs. Quantitative bisulfite pyrosequencing revealed that NTSR1 is frequently methylated in colorectal tumors, with large NTs exhibiting the highest methylation levels. The higher NTSR1 methylation levels were associated with better prognoses. By contrast, NTSR1 copy number gains were most frequent among small ITs. Methylation of NTSR1 was associated with the gene's silencing in CRC cell lines, whereas ectopic expression of NTSR1 promoted proliferation and invasion by CRC cells. Analysis of primary tumors composed of adenomatous and malignant portion...
Cancer prevention research (Philadelphia, Pa.), Jan 10, 2015
Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associa... more Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer (CRC). The relationship between TET inactivation and CIMP in CRCs is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) CRCs. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in CRCs, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of CRCs as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P<0.0001) CRCs. This trend was also observed in colon polyps (CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P=0.002), suggesting th...
Cancer prevention research (Philadelphia, Pa.), 2014
Aberrant DNA methylation could potentially serve as a biomarker for colorectal neoplasms. In this... more Aberrant DNA methylation could potentially serve as a biomarker for colorectal neoplasms. In this study, we assessed the feasibility of using DNA methylation detected in bowel lavage fluid (BLF) for colorectal cancer screening. A total of 508 BLF specimens were collected from patients with colorectal cancer (n = 56), advanced adenoma (n = 53), minor polyp (n = 209), and healthy individuals (n = 190) undergoing colonoscopy. Methylation of 15 genes (miR-1-1, miR-9-1, miR-9-3, miR-34b/c, miR-124-1, miR-124-2, miR-124-3, miR-137, SFRP1, SFRP2, APC, DKK2, WIF1, LOC386758, and ZNF582) was then analyzed in MethyLight assays, after which receiver operating characteristic (ROC) curves were analyzed to assess the diagnostic performance of BLF methylation. Through analyzing BLF specimens in a training set (n = 345), we selected the three genes showing the greatest sensitivity for colorectal cancer detection (miR-124-3, 71.8%; LOC386758, 79.5%; and SFRP1, 74.4%). A scoring system based on the m...
MicroRNAs (miRNAs) play pivotal roles in numerous biological processes, and their dysregulation i... more MicroRNAs (miRNAs) play pivotal roles in numerous biological processes, and their dysregulation is a common feature of human cancer. Thanks to recent advances in the analysis of the cancer epigenome, we now know that epigenetic alterations, including aberrant DNA methylation and histone modifications, are major causes of miRNA dysregulation in cancer. Moreover, the list of miRNA genes silenced in association with CpG island hypermethylation is rapidly growing, and various oncogenic miRNAs are now known to be upregulated via DNA hypomethylation. Histone modifications also play important roles in the dysregulation of miRNAs, and histone deacetylation and gain of repressive histone marks are strongly associated with miRNA gene silencing. Conversely, miRNA dysregulation is causally related to epigenetic alterations in cancer. Thus aberrant methylation of miRNA genes is a potentially useful biomarker for detecting cancer and predicting its outcome. Given that many of the silenced miRNAs ...
World journal of gastroenterology : WJG, Jan 7, 2014
To investigate insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypome... more To investigate insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypomethylation in relation to clinicopathological and molecular features in colorectal serrated lesions. To accurately analyze the association between the histological types and molecular features of each type of serrated lesion, we consecutively collected 1386 formalin-fixed paraffin-embedded tissue specimens that comprised all histological types [hyperplastic polyps (HPs, n = 121), sessile serrated adenomas (SSAs, n = 132), traditional serrated adenomas (TSAs, n = 111), non-serrated adenomas (n = 195), and colorectal cancers (CRCs, n = 827)]. We evaluated the methylation levels of IGF2 DMR0 and long interspersed nucleotide element-1 (LINE-1) in HPs (n = 115), SSAs (n = 120), SSAs with cytological dysplasia (n = 10), TSAs (n = 91), TSAs with high-grade dysplasia (HGD) (n = 15), non-serrated adenomas (n = 80), non-serrated adenomas with HGD (n = 105), and CRCs (n = 794). For the accurate quan...
Adenylate cyclase 3 (ADCY3) is a widely expressed membrane-associated protein in human tissues, w... more Adenylate cyclase 3 (ADCY3) is a widely expressed membrane-associated protein in human tissues, which catalyzes the formation of cyclic adenosine-3',5'-monophosphate (cAMP). However, our transcriptome analysis of gastric cancer tissue samples (NCBI GEO GSE30727) revealed that ADCY3 expression was specifically altered in cancer samples. Here we investigated the tumor-promoting effects of ADCY3 overexpression and confirmed a significant correlation between the upregulation of ADCY3 and Lauren's intestinal-type gastric cancers. ADCY3 overexpression increased cell migration, invasion, proliferation, and clonogenicity in HEK293 cells; conversely, silencing ADCY3 expression in SNU-216 cells reduced these phenotypes. Interestingly, ADCY3 overexpression increased both the mRNA level and activity of matrix metalloproteinase 2 (MMP2) and MMP9 by increasing the levels of cAMP and phosphorylated cAMP-responsive element-binding protein (CREB). Consistent with these findings, treatmen...
Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although ... more Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although both offer considerable theranostic potential. In this study, we identified frequent upregulation of miR-196a and lincRNA HOTAIR in high-risk gastrointestinal stromal tumors (GIST). Overexpression of miR-196a was associated with high-risk grade, metastasis and poor survival among GIST specimens. miR-196a genes are located within the HOX gene clusters and microarray expression analysis revealed that the HOXC and HOTAIR gene were also coordinately upregulated in GISTs which overexpress miR-196a. In like manner, overexpression of HOTAIR was also strongly associated with high-risk grade and metastasis among GIST specimens. RNA interference–mediated knockdown of HOTAIR altered the expression of reported HOTAIR target genes and suppressed GIST cell invasiveness. These findings reveal concurrent overexpression of HOX genes with noncoding RNAs in human cancer in this setting, revealing miR-196a ...
Colorectal sessile serrated adenoma/polyps (SSA/Ps) are well-known precursors of colorectal cance... more Colorectal sessile serrated adenoma/polyps (SSA/Ps) are well-known precursors of colorectal cancer (CRC) characterized bymutation and microsatellite instability. By contrast, the molecular characteristics of traditional serrated adenoma (TSAs) are not fully understood. We analyzed genome-wide DNA methylation in TSAs having both protruding and flat components. We identified 11 genes, including, methylation of which progressively increased during the development of TSAs.was prevalently methylated in TSAs, but was rarely methylated in SSA/Ps (< 0.001). RT-PCR and immunohistochemistry revealed that SMOC1 was expressed in normal colon and SSA/Ps, but its expression was decreased in TSAs. Ectopic expression of SMOC1 suppressed proliferation, colony formation andtumor formation by CRC cells. Analysis of colorectal lesions (= 847) revealed thatis frequently methylated in TSAs, high-grade adenomas and CRCs. Among these,methylation was strongly associated withmutation and CpG island methyl...
Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, alth... more Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced aden...
Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mech... more Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mechanism is not fully understood. 5-hydroxymethylcytosine levels and TET1 expression are both reduced in CRC, while epigenetic silencing of TET1 is reportedly associated with the CpG island methylator phenotype. In the present study, we aimed to clarify the relationship between loss of TET1 and aberrant DNA methylation in CRC. Stable TET1 knockdown clones were established using Colo320DM cells, which express high levels of TET1, and HCT116 cells, which express TET1 at a level similar to that in normal colonic tissue. Infinium HumanMethylation450 BeadChip assays revealed increased levels of 5-methylcytosine at more than 10,000 CpG sites in TET1-depleted Colo320DM cells. Changes in DNA methylation were observed at various positions within the genome, including promoters, gene bodies and intergenic regions, and the altered methylation affected expression of a subset of genes. By contrast, TET1...
Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association, Jan 28, 2016
Although genetic alterations in patients with advanced gastric cancer have been extensively studi... more Although genetic alterations in patients with advanced gastric cancer have been extensively studied, those in patients with intramucosal neoplasia (IMN) are still poorly understood. We evaluated genetic differences in 158 IMNs, including 51 low-grade dysplasias, 58 high-grade dysplasias (HGDs), 30 intramucosal cancers (IMCs), and 19 mixed tumors (composed of IMC and HGD within the same tumor), using PCR-based microsatellite analysis [allelic imbalance (AI) and microsatellite instability (MSI)]. We classified the DNA methylation status as a hypermethylated epigenome, a moderately methylated epigenome, or a hypomethylated epigenome. In addition, p53 overexpression, β-catenin nuclear localization, and mucin expression were also examined. From cluster analysis, the IMNs examined were categorized into four subgroups as follows. Tumors in subgroup 1 were characterized by MSI-high status, a hypermethylated epigenome, and loss or reduction of expression of MLH-1. Tumors in subgroup 2 showed...
Our aim was to identify DNA methylation changes associated with the growth pattern and invasivene... more Our aim was to identify DNA methylation changes associated with the growth pattern and invasiveness of colorectal cancers (CRCs). Comparison of the methylation statuses of large (≥20 mm in diameter along the colonic surface) noninvasive tumors (NTs) and small (<20 mm in diameter along the colonic surface) invasive tumors (ITs) using CpG island microarray analysis showed neurotensin receptor 1 (NTSR1) to be hypermethylated in large NTs. Quantitative bisulfite pyrosequencing revealed that NTSR1 is frequently methylated in colorectal tumors, with large NTs exhibiting the highest methylation levels. The higher NTSR1 methylation levels were associated with better prognoses. By contrast, NTSR1 copy number gains were most frequent among small ITs. Methylation of NTSR1 was associated with the gene's silencing in CRC cell lines, whereas ectopic expression of NTSR1 promoted proliferation and invasion by CRC cells. Analysis of primary tumors composed of adenomatous and malignant portion...
Cancer prevention research (Philadelphia, Pa.), Jan 10, 2015
Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associa... more Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer (CRC). The relationship between TET inactivation and CIMP in CRCs is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) CRCs. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in CRCs, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of CRCs as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P<0.0001) CRCs. This trend was also observed in colon polyps (CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P=0.002), suggesting th...
Cancer prevention research (Philadelphia, Pa.), 2014
Aberrant DNA methylation could potentially serve as a biomarker for colorectal neoplasms. In this... more Aberrant DNA methylation could potentially serve as a biomarker for colorectal neoplasms. In this study, we assessed the feasibility of using DNA methylation detected in bowel lavage fluid (BLF) for colorectal cancer screening. A total of 508 BLF specimens were collected from patients with colorectal cancer (n = 56), advanced adenoma (n = 53), minor polyp (n = 209), and healthy individuals (n = 190) undergoing colonoscopy. Methylation of 15 genes (miR-1-1, miR-9-1, miR-9-3, miR-34b/c, miR-124-1, miR-124-2, miR-124-3, miR-137, SFRP1, SFRP2, APC, DKK2, WIF1, LOC386758, and ZNF582) was then analyzed in MethyLight assays, after which receiver operating characteristic (ROC) curves were analyzed to assess the diagnostic performance of BLF methylation. Through analyzing BLF specimens in a training set (n = 345), we selected the three genes showing the greatest sensitivity for colorectal cancer detection (miR-124-3, 71.8%; LOC386758, 79.5%; and SFRP1, 74.4%). A scoring system based on the m...
MicroRNAs (miRNAs) play pivotal roles in numerous biological processes, and their dysregulation i... more MicroRNAs (miRNAs) play pivotal roles in numerous biological processes, and their dysregulation is a common feature of human cancer. Thanks to recent advances in the analysis of the cancer epigenome, we now know that epigenetic alterations, including aberrant DNA methylation and histone modifications, are major causes of miRNA dysregulation in cancer. Moreover, the list of miRNA genes silenced in association with CpG island hypermethylation is rapidly growing, and various oncogenic miRNAs are now known to be upregulated via DNA hypomethylation. Histone modifications also play important roles in the dysregulation of miRNAs, and histone deacetylation and gain of repressive histone marks are strongly associated with miRNA gene silencing. Conversely, miRNA dysregulation is causally related to epigenetic alterations in cancer. Thus aberrant methylation of miRNA genes is a potentially useful biomarker for detecting cancer and predicting its outcome. Given that many of the silenced miRNAs ...
World journal of gastroenterology : WJG, Jan 7, 2014
To investigate insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypome... more To investigate insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypomethylation in relation to clinicopathological and molecular features in colorectal serrated lesions. To accurately analyze the association between the histological types and molecular features of each type of serrated lesion, we consecutively collected 1386 formalin-fixed paraffin-embedded tissue specimens that comprised all histological types [hyperplastic polyps (HPs, n = 121), sessile serrated adenomas (SSAs, n = 132), traditional serrated adenomas (TSAs, n = 111), non-serrated adenomas (n = 195), and colorectal cancers (CRCs, n = 827)]. We evaluated the methylation levels of IGF2 DMR0 and long interspersed nucleotide element-1 (LINE-1) in HPs (n = 115), SSAs (n = 120), SSAs with cytological dysplasia (n = 10), TSAs (n = 91), TSAs with high-grade dysplasia (HGD) (n = 15), non-serrated adenomas (n = 80), non-serrated adenomas with HGD (n = 105), and CRCs (n = 794). For the accurate quan...
Adenylate cyclase 3 (ADCY3) is a widely expressed membrane-associated protein in human tissues, w... more Adenylate cyclase 3 (ADCY3) is a widely expressed membrane-associated protein in human tissues, which catalyzes the formation of cyclic adenosine-3',5'-monophosphate (cAMP). However, our transcriptome analysis of gastric cancer tissue samples (NCBI GEO GSE30727) revealed that ADCY3 expression was specifically altered in cancer samples. Here we investigated the tumor-promoting effects of ADCY3 overexpression and confirmed a significant correlation between the upregulation of ADCY3 and Lauren's intestinal-type gastric cancers. ADCY3 overexpression increased cell migration, invasion, proliferation, and clonogenicity in HEK293 cells; conversely, silencing ADCY3 expression in SNU-216 cells reduced these phenotypes. Interestingly, ADCY3 overexpression increased both the mRNA level and activity of matrix metalloproteinase 2 (MMP2) and MMP9 by increasing the levels of cAMP and phosphorylated cAMP-responsive element-binding protein (CREB). Consistent with these findings, treatmen...
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