The Human let-7a-3 Locus Contains an Epigenetically Regulated MicroRNA Gene with Oncogenic Function

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that repress their target mRNAs by complementary base pairing and induction of the RNA interference pathway. It has been shown that miRNA expression can be regulated by DNA methylation and it has been suggested that altered miRNA gene methylation might contribute to human tumorigenesis. In this study, we show that the human let-7a-3 gene on chromosome 22q13.31 is associated with a CpG island. Let-7a-3 belongs to the archetypal let-7 miRNA gene family and was found to be methylated by the DNA methyltransferases DNMT1 and DNMT3B. The gene was heavily methylated in normal human tissues but hypomethylated in some lung adenocarcinomas. Let-7a-3 hypomethylation facilitated epigenetic reactivation of the gene and elevated expression of let-7a-3 in a human lung cancer cell line resulted in enhanced tumor phenotypes and oncogenic changes in transcription profiles. Our results thus identify let-7a-3 as an epigenetically regulated miRNA gene with oncogenic function and suggest that aberrant miRNA gene methylation might contribute to the human cancer epigenome.

Methylation of Human MicroRNA Genes in Normal and Neoplastic Cells

“…In agreement with this notion, overexpression of let-7a-3 in lung cancer cells caused deregulation of roughly 200 genes. 25 Among those, genes involved in cell adhesion, proliferation and differentiation were significantly overrepresented, which further strengthened the connection between altered miRNA expression and tumorigenesis. 25 …”

“…25 This CpG island was hypomethylated in some human lung adenocarcinomas. Combinatorial treatment of A549 lung cancer cells with the DNA methyltransferase inhibitor DAC and the histone deacetylase inhibitor valproic acid led to a Figure 2.� Meth��lation anal��sis of miRNA genes in human cell lines.� The meth� ��lation of 9 miRNA genes was anal��zed ��� com�ined �isulfite restriction anal��sis (COBRA).� 30 The corresponding PCR reactions produced amplicons ranging from 400 to 900 �p, respecti�el�� (marked "U").� The appearance of smaller restriction fragments ("M") indicates meth��lation of the corresponding miRNA gene, resistance to digestion indicates the a�sence of meth��lation.� 5 human cell lines were tested: the human em�r��onic stem cell line HES7 (HES), primar�� human fi�ro�lasts (PHF), the HeLa cer�ical carcinoma cell line (HeLa), the HCT116 colon carcinoma cell line (HCT) and an isogenic DNA meth��ltransferase�deficient HCT116 cell line (DKO).� The CpG�islands of 3 protein�encoding genes, p15, p21 and NPM, were included as con� trols.� Genomic DNA was deaminated with sodium �isulfite using standard procedures.� 43 Primer sequences and assa�� conditions will �e pro�ided upon request.�…”

“…28 Similarly, all miRNA genes that have been linked to epigenetic regulation are closely associated with CpG islands. [23][24][25] We analyzed the genomic sequences of the miRNA genes deposited in the miRBase database, 29 release 8.0 and have found that 155 out of 332 human miRNA genes (47%) were associated with CpG islands. CpG islands were required to be located within 2000 bp upstream or downstream of the DNA sequence encoding the corresponding miRNA sequence and were defined as regions equal to or exceeding 200 basepairs of DNA with a G+C content equal to or greater than 55% and an observed CpG/ expected CpG ratio in excess of 0.65.…”

“…[32][33][34] This difference is remarkable because both gene groups seem to be methylated by similar mechanisms, as indicated by their dependency on the cooperative activities of DNMT1 and DNMT3B. 25 It is possible that the susceptibility of miRNA-associated CpG islands for DNA methylation is a consequence of their sequence composition. A role of DNA attributes in predisposing CpG islands to DNA methylation has been shown recently.…”

The impact of microRNAs and alternative splicing in pharmacogenomics

“…More recently, some reports have been showing that miRNA deregulated expression in diseases can be due to epigenetic changes such as DNA methylation and histone modifications. 48,49 microRNA variations and the impact in pharmacogenomics SNPs are frequent variations in the human genome and estimates suggest that they can have a frequency of one in every thousand base pairs. 11 SNPs have been extensively studied in order to understand the susceptibility to specific diseases in the general population.…”