Noriko Emoto

Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer†

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non‐small‐cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza‐CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5‐aza‐CdR treatment. After a detailed DNA methylation analysis, we found that mir‐34b and mir‐126 were silenced by DNA methylation. Mir‐34b was silenced by the DNA methylation of its own promoter, whereas mir‐126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir‐34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir‐34b and mir‐126 decreased the expression of c‐Met and Crk, respectively. The 5‐aza‐CdR treatment of lung cancer cell line resulted in increased mir‐34b expression and decreased c‐Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir‐34b and mir‐126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir‐34b was not associated with c‐Met expression determined by immunohistochemistry, but both mir‐34b methylation (p = 0.007) and c‐Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir‐34b can be used as a biomarker for an invasive phenotype of lung cancer.

Identification of G0S2 as a gene frequently methylated in squamous lung cancer by combination of in silico and experimental approaches

Epigenetic changes can lead to abnormal expression of genes in cancer, and several genes have been reported to have aberrant promoter DNA methylation in non‐small‐cell lung cancer (NSCLC). We identified aberrantly methylated genes in NSCLC by combination of in silico and experimental approaches. We first applied bioinformatics, and from microarray datasets, we selected genes with low expression and having functions related to cancer. Next, combined bisulfite restriction analysis was carried out in 10 pooled resected lung cancer tissues to screen for genes that were aberrantly methylated, and the methylation ratio (the fraction of methylated DNA in extracted DNA from a cancer tissue sample) was quantified using quantitative analysis of methylated alleles. We identified 8 methylated genes (ARPC1B, DNAH9, FLRT2, G0S2, IRS2, PKP1, SPOCK1 and UCHL1) previously unreported in NSCLC. Analyses of methylation profiles of 101 resected lung cancer tissue samples revealed quantitatively low methylation in whole, methylation ratios were almost less than 30% even in the methylated samples, and no significant correlation to prognosis after 2 years of follow‐up using hierarchical clustering. DNA methylation of G0S2 gene was significantly more frequent in squamous lung cancer (n = 18, mean of methylation ratios: 15%) compared with nonsquamous lung cancer (n = 83, mean of methylation ratios: 2.6%) (Mann‐Whitney U test, p < 0.001). DNA methylation of G0S2 can be an important biomarker for squamous lung cancer.

CpG island methylation of microRNAs is associated with tumor size and recurrence of non-small-cell lung cancer

We investigated whether the CpG island methylation of certain microRNAs was associated with the clinicopathological features and the prognosis of non‐small‐cell lung cancer. The methylation of mir‐152, ‐9‐3, ‐124‐1, ‐124‐2, and ‐124‐3 was analyzed in 96 non‐small‐cell lung cancer specimens using a combined bisulfite restriction analysis. The median observation period was 49.5 months. The methylation of mir‐9‐3, ‐124‐2, and ‐124‐3 was individually associated with an advanced T factor independent of age, sex, and smoking habit. Moreover, the methylation of multiple microRNA loci was associated with a poorer progression‐free survival in a univariate analysis. Our result enlightens the accumulation of aberrant DNA methylation which occurs in concordance with the tumor progression. (Cancer Sci 2011; 102: 2126–2131)

Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis.

DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 degrees C or 65 degrees C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE).

Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5′- and 3′-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.

Abstract B39: Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer

microRNA expression is frequently altered in human cancers. Epigenetic silencing, especially DNA methylation, is one of many mechanisms of microRNA suppression in cancer. To search for epigenetically silenced microRNAs in non-small-cell lung cancer (NSCLC), we mapped human microRNAs on autosomal chromosomes and selected in silico 55 microRNAs that met one of the following criteria: (i) microRNAs within CpG islands, (ii) microRNAs within 1 kbp downstream of CpG islands, and (iii) microRNAs within gene introns whose promoters have CpG islands. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 microRNAs. Fourteen microRNAs (mir-375, mir-196b, mir-126, mir-34b, mir-127, mir-203, mir-148a, mir-181c, mir-30e, mir-449a, mir-340, mir-486, mir-483, mir-139) were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. Among the 14 microRNAs, seven (mir-126, mir-34b, mir-203, mir-30e, mir-449a, mir-486, mir-139) were frequently suppressed in primary NSCLCs. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, while mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. A significant enrichment of H3K27me3 was observed in lung cancer cell lines with decreased mir-126 expression, suggesting the involvement of a polycomb complex in the regulation of EGFL7 and mir-126. The overexpression of mir-34b and mir-126 by plasmid vectors decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these microRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41% and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.

Abstract A24: CpG island methylation of microRNAs is associated with tumor size and the recurrence of non-small cell lung cancer

We investigated whether the CpG island methylation of certain microRNAs was associated with the clinicopathological features and the prognosis of non-small cell lung cancer. The methylation of mir-152, -9-3, -124-1, -124-2, and -124-3 was analyzed in 96 non-small cell lung cancer specimens using a combined bisulfite restriction analysis. The median observation period was 49.5 months. The methylation of mir-9-3, -124-2, and -124-3 was individually associated with an advanced T factor independently of age, sex, and smoking habit. Moreover, the methylation of multiple microRNA loci was associated with a poorer progression-free survival in a univariate analysis. Our result enlightens the accumulation of aberrant DNA methylation which occurs in concordance with the tumor progression.