MicroRNA-218 is deleted and downregulated in lung squamous cell carcinoma

Abstract

MicroRNAs (miRNAs) are a family of small, non-coding RNA species functioning as negative regulators of multiple target genes including tumour suppressor genes and oncogenes. Many miRNA gene loci are located within cancer-associated genomic regions. To identify potential new amplified oncogenic and/or deleted tumour suppressing miRNAs in lung cancer, we inferred miRNA gene dosage from high dimensional arrayCGH data. From miRBase v9.0 (http://microrna.sanger.ac.uk), 474 human miRNA genes were physically mapped to regions of chromosomal loss or gain identified from a high-resolution genome-wide arrayCGH study of 132 primary non-small cell lung cancers (NSCLCs) (a training set of 60 squamous cell carcinomas and 72 adenocarcinomas). MiRNAs were selected as candidates if their immediately flanking probes or host gene were deleted or amplified in at least 25% of primary tumours using both Analysis of Copy Errors algorithm and fold change (≥ ± 1.2) analyses. Using these criteria, 97 miRNAs mapped to regions of aberrant copy number. Analysis of three independent published lung cancer arrayCGH datasets confirmed that 22 of these miRNA loci showed directionally concordant copy number variation. MiR-218, encoded on 4p15.31 and 5q35.1 within two host genes (SLIT2 and SLIT3), in a region of copy number loss, was selected as a priority candidate for follow-up as it is reported as underexpressed in lung cancer. We confirmed decreased expression of mature miR-218 and its host genes by qRT-PCR in 39 NSCLCs relative to normal lung tissue. This downregulation of miR-218 was found to be associated with a history of cigarette smoking, but not human papilloma virus. Thus, we show for the first time that putative lung cancer-associated miRNAs can be identified from genome-wide arrayCGH datasets using a bioinformatics mapping approach, and report that miR-218 is a strong candidate tumour suppressing miRNA potentially involved in lung cancer.

Figure 1. Identification of miR-218 from concordant copy number variation and gene expression from independent data sets.

Expression levels of 38/89 candidate miRNAs and 19/51 published arrayCGH miRNAs have not been studied due to their recent inclusion in miRBase. The genomic location of miR-218 within its two host genes, SLIT2 and SLIT3, is also illustrated.

Figure 2. Expression of miR-218 and its host genes in primary NSCLCs and their paired normal lung.

*p<0.05; **p<0.01. (a) Down-regulation of miR-218 expression was observed in both ACs (FC -2; p = 0.001; Z score  = −3.258) and SCCs (FC −4.365; p<0.0001; Z score  = −4.406). (b) A reduction in host gene expression was observed in both ACs (SLIT2: FC −12.77, p<0.0001, Z score  = −4.277; SLIT3: FC −13.73, p<0.0001, Z score  = −3.839) and SCCs (SLIT2: FC −10.85, p<0.0001, Z score  = −5.182; SLIT3: FC −5.46, p<0.0001, Z score  = −4.540). Abbreviations: FC, Fold change.

Figure 3. Relationship of miR-218 expression to smoking (a), HPV (b), asbestos (c), recurrence (d) and survival (e).

*p = 0.05; **p = 0.01. Abbreviations: FC, Fold change. Statistically significant downregulation of miR-218 expression was observed in current and former smokers of both NSCLC subtypes. No significant associations were observed between miR-218 expression and HPV, asbestos, disease recurrence or survival.