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

Abstract

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in nonsmall-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 0 -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 5 0.007) and c-Met expression (p 5 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.MicroRNAs (miRNAs) are broadly conserved small noncoding RNA that regulate gene expression by binding to the 3 0 UTR of target mRNAs in a complementary manner. 1Through the posttranscriptional regulation of many target genes, miRNAs are involved in many biological processes, such as development and human carcinogenesis. MicroRNA expression is altered in human cancers, and some miRNAs have oncogenic or tumor suppressive functions in human malignancies, including lung cancer. 2-5Chromosomal deletions or amplifications are important mechanisms of miRNA expression change in cancers. For example, mir-15 and mir-16 are frequently deleted and downregulated in chronic lymphocytic leukemia.2 The mir-17-92 miRNA cluster is amplified and overexpressed in B-cell lymphoma 6 and lung cancer. 4 However, the precise mechanisms responsible for changes in miRNA expression in cancer remain largely unknown.DNA methylation plays an important role in inactivating tumor suppressor genes in many types of human cancers. 7,8 Recently, DNA methylation in cancerous tissue has been shown to cause the silencing of miRNAs located in the vicinity of CpG islands. 9,10 As the epigenetic silencing of tumor suppressor genes is a common event in lung carcinogenesis 11-14 and miRNA expression is altered in lung cancer, 5 we decided to search for epigenetically silenced miRNAs in lung cancer.In our study, we selected 55 candidate miRNAs in silico based on the genome structure and tre...

Serum Kynurenic Acid: Possible Association with Invasiveness of Non-small Cell Lung Cancer

“…It has been previously shown that bronchial adenocarcinoma is characterized by aggressive development and poor prognosis compared to other histologic types of NSCLC (Cooke et al, 2010;Maeda et al, 2011;Tomaszek et al, 2011). This behavior may be attributed to the properties of the tumor itself, as well as to impaired anti-tumor immune response (Dai et al, 2010;Watanabe et al, 2011). Tryptofan degradation along the kynurenine pathway has been shown to be a major factor contributing to escape of tumor cells from immune surveillance (Munn et al, 1999).…”

“…Overall invasiveness of malignant tumors is dependent on the properties of the tumor at the molecular level (Watanabe et al, 2011), as well as on the proper response of the immune system (Dai et al, 2010 tumors, including lung cancer, may be attributed to metabolites of tryptophan degradation via kynurenine pathway (Munn and Mellor, 2007;Suzuki et al, 2010). It is the main pathway for tryptophan metabolism catalyzed by indoleamine 2,3-dioxygenase (IDO) and kynurenine aminotransferases (KATs), resulting in synthesis of kynurenic acid (KYNA) (Figure 1) (Fulop et al, 2007).…”

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

“…Systematic assessments of miRNA expression and epigenetic modifications among cell lines and primary tumor specimens have revealed the existence of the epigenetic regulation of miRNAs in multiple tumor types. (6)(7)(8)(9) The goals of this study were to explore possible relationships between the methylation profiles of miRNAs and the clinicopathological characteristics of NSCLC patients and to identify new specific methylation markers capable of detecting advanced pathological features.In the present study, the methylation status of five miRNA loci within five separate CpG islands was determined in 96 NSCLC tissue specimens. The choice of the miRNAs was based on our previous study (9) as well as other published reports.…”

“…(6)(7)(8)(9) The goals of this study were to explore possible relationships between the methylation profiles of miRNAs and the clinicopathological characteristics of NSCLC patients and to identify new specific methylation markers capable of detecting advanced pathological features.…”

“…The choice of the miRNAs was based on our previous study (9) as well as other published reports. (10,11) Our data show that the CpG island methylation of miRNAs is common in NSCLC, and that the methylation of multiple miR-NA loci is associated with an advanced T status as well as the progression-free survival (PFS) of patients with NSCLC.…”