The aberrant expression of miR-196a is a frequent event in various cancers, suggesting that miR-196a may play an important role in tumorigenesis and tumor progression. Indeed, several key oncogenic functions have been attributed to miR-196a in the context of tumorigenesis. In esophageal cancer, miR-196a over-expression promotes cell proliferation, anchorage-independent growth and suppresses apoptosis by directly regulating ANXA1 . In colorectal cancer, high levels of miR-196a were observed to activate the Akt signaling pathway, promote cancer cell detachment, migration, invasion and chemosensitivity, and increase the development of lung metastases in mice [10, 17]. In previous studies from our group, we reported that aberrant over-expression of miR-196a and the resultant down-regulation of its target p27kip1, contributes to gastric carcinogenesis . We also analyzed miRNA expression profiles in NSCLC patient tissues by miRNA microarray, and identified miR-196a as the most highly up-regulated miRNA compared with corresponding normal tissues . Inspired by the above observation, we investigated the biological role of miR-196a and explored the molecular mechanisms by which miR-196a modulates the behavior of NSCLC cells.
In this study, we first examined the expression of miR-196a in 34 paired normal/tumor tissues from NSCLC patients, and then investigated the clinical implications. Consistent with our previous microarray analysis, miR-196a was dramatically upregulated in NSCLC tissues. Specifically, miR-196a expression was significantly higher at later stages of NSCLC development or in specimens displaying more extensive metastasis compared with their normal counterparts. This suggests that high expression of miR-196a may be involved in NSCLC carcinogenesis. In addition, we demonstrate that miR-196a expression is elevated in NSCLC cell lines compared with the 16HBE normal human bronchial epithelial cell line, with the exception of A549 cells. We also show that DNA demethylation may underlie the aberrant expression of miR-196a in NSCLC, and these findings are currently under further investigation in our laboratory.
To assess the role of miR-196a in NSCLC, we investigated the gain-or-loss of function effects of miR-196a on various aspects of NSCLC biology. First, we demonstrated that targeted knock-down of miR-196a expression in SPC-A1 cells led to significant inhibition of cell proliferation, migration and invasion. Conversely, introducing miR-196a into A549 cells, which express relatively low levels of endogenous miR-196a, induced corresponding malignant tumor cell behaviors. However, unlike previous descriptions in breast cancer, alteration of miR-196a expression did not impact apoptosis in NSCLC cells. Therefore, the functional role of miR-196a may be tissue- and cell type-specific, and the detailed mechanisms of miR-196a and its targets are worthy of further study.
Identification of putative miRNA targets is important for a complete understanding of the specific functions of miRNAs. In this study, we identify HOXA5 as a direct target of miR-196a, and demonstrate that upregulation of miR-196a significantly reduces HOXA5 expression at both transcriptional and protein level in NSCLC cells.
Mammals have four HOX gene clusters (HOX A-D), which act as master regulators that specify body patterning during embryonic development. As transcription factors, HOX proteins control a battery of target genes encoding cellular functions required for cell identity, cell growth and differentiation, as well as cell–cell and cell–matrix interactions [19–22]. Deregulation of HOX genes in cancer is now well established, although in general rather less is known about their function [23–28]. The homeobox protein, HOXA5, has been shown to participate in the developmental regulation of the lung. Mandeville et al. observed impaired postnatal lung development in HOXA5
mice, indicating that HOXA5 has a critical role in lung ontogeny, and implying its involvement in lung maturation and function . Similarly, Packer et al. reported that HOXA5 is likely to be involved in development and patterning of the mouse lung . Moreover, dysregulation of HOXA5 expression has been associated with lung tumorigenesis and other diseases in humans [26, 31]. In breast cancer, down-regulation of HOXA5 may impact p53 gene expression, contributing to the oncogenic process [32, 33]. Additionally, induction of HOXA5 is important for retinoic acid (RA)-mediated apoptosis and cellular growth inhibition acting directly downstream of RARβ, and plays an important role in RA-mediated anti-cancer activity [34, 35].
Currently, the role of HOXA5 in NSCLC has not been well established. Our results reveal that inhibition of HOXA5 expression in A549 cells significantly promotes cell proliferation, migration and invasion, consistent with the results of ectopic miR-196a expression in the same cells. Furthermore, our observation of a correlation between elevated miR-196a levels and decreased HOXA5 levels in NSCLC tissues, indicates that down-regulation of HOXA5 may be a mechanism by which miR-196a exerts its oncogenic functions.
While our study provides critical insight into NSCLC pathogenesis, the existence of several limitations should be noted. First, as the number of tissue samples in this study was limited, further investigation of a larger patient cohort is essential to confirm the clinical significance of miR-196a. Second, to further understand the biological role of miR-196a in regulating NSCLC development and progression, a series of in vivo studies using xenograft models are required. Third, many studies have clearly indicated that one miRNA is capable of controlling multiple genes. Therefore, the observed miR-196a-mediated inhibition of cell growth and invasion is likely due to simultaneous targeting of multiple targets in NSCLC. Further studies of miR-196a will undoubtedly enhance our knowledge of how miR-196a functions in regulating NSCLC cell growth and metastasis.