miRNAs are small noncoding regulatory RNAs that have been studied in various types of cancers. Many miRNAs that regulate epithelial to mesenchymal transition (EMT)  and pro-metastatic [5, 6] or anti-metastatic functions  have been identified. Previously miR-135 has been reported to regulate genes in many other types of cancer [11, 14, 16, 33, 34], but its roles in breast cancer was unknown. Our current study provides the first evidence to demonstrate that miR-135a plays a role in promoting migration and invasion of breast cancer cells.
The ability of miR-135a to promote cell migration and invasion was assessed by both over-expression and down-regulation experiments. Remarkably, we observed apparent high levels of miR-135a expression in „triple negative" malignant invasive breast tumors (Figure 1A), and the highly invasive phenotype of the BT549 cell line (Figure 1B), suggesting that miR-135a might play an important role in maintaining metastatic functions. This hypothesis is supported by our experiments showing that inhibition of miR-135a activity impaired the invasion and migration of BT549 cells in vitro (Figure 2A). To further verify, this relationship up-regulation experiments were performed in cell lines with different invasive phenotypes. In addition to the enhanced migration and invasive ability of SKBr3 cells induced by increased miR-135a expression (Figure 2B), we also observed enhanced invasive ability of miR-135a-transfected MDA-MB-231 cells (Figure 2C). Although endogenous miR-135a was not detectable in MDA-MB-231 cells they are highly invasive, and we speculate that migration and invasion may not be miR-135a-dependent processes in those cells due to genetic differences between different breast cancer cell lines. Furthermore, miR-135a could not affect the low invasive property of MCF-7 cells even though it was forced overexpressed and led to decreased endogenous HOXA10 protein expression in those cells (Figure 4D). We speculate this is also due to genetic cell specificity. For example, E-cadherin is highly expressed in MCF-7, so we believe that the effect of miR-135a over-expression is not strong enough to induce a change of the migration status of the MCF-7 cells. This suggests that the native non-metastatic character of MCF-7 may have other miR-135a-independent mechanisms that are responsible to maintain the non-aggressiveness of this particular cell type. It is also possible that HOXA10 3'UTR is mutated and cannot be targeted by miR-135a in MCF-7 cells. Future studies are required to define these two possibilities. Indeed it has been reported that HOXA10 is not regulated by miR-135a in MCF-7 cells  which is in contrast to our results for other cancer cell lines tested. Unlike the migration and invasion phenotypes, up- or down-regulation of miR-135a did not affect cell proliferation (Figure 2D).
Several HOX genes are regulated by miRNAs [6, 19–21]. This work is the first to implicate miR-135a down-regulation of HOXA10 expression in breast cancer cell invasiveness. The mechanism by miR-135a targets HOXA10 for repression was verified by in vitro 3'-UTR luciferase assays. HOXA10 over-expression in miR-135a expressing cells was dependent on the absence of the HOXA10 3'-UTR (Figure 3C-D) supporting our conclusion that miR-135a inhibits HOXA10 via targeting its 3'-UTR. Our results showed that endogenous HOXA10 in MDA-MB-231 was not detectable (Figure 3B) and this could be attributable to methylation of the HOXA10 promoter as previously reported . Combined with the fact that individual miRNAs have the potential to modulate the expression of many mRNAs, our result showing miR-135a expression in MDA-MB-231 cells that increased migration and invasion (Figure 2C) suggest there may be miR-135a targets other than HOXA10 that can promote migration/invasion events. Indeed, miR135a was reported to be up-regulated in portal vein tumor thrombus and these cells showed increased migration and invasion in vitro. However, the metastasis suppressor 1 gene and not HOXA10 was found to be the direct, functional target of miR-135a in this tissue . We propose that this gene may function in some breast cancers to suppress migration and invasion rather than HOXA10, and we intend to test this in the near future. Our results also suggested that miR-135a post-transcriptional down-regulation of the HOXA10 target gene was not restricted to translation repression (Figure 4A-B, 4D-E) but also occurred by inducing mRNA degradation (Figure 4C), which agrees with previous reports on the action of miRNAs and highly homologous targets .
To further investigate the effect of HOXA10 on breast cancer cell invasiveness, we overexpressed HOXA10 in already highly invasive breast cancer cell lines. Overexpression of HOXA10 without 3' UTR targeting by miR-135a led to decreased invasion of MDA-MB-231 cells (Figure 5A), and significantly inhibited the miR-135a-regulated invasion of BT549 cells (Figure 5D). Knockdown of HOXA10 increased invasiveness of BT549 cells and could partially rescue the decreased invasive property caused by 135a inhibitor (Figure 5B). Other members in the same gene family have also been shown to play roles in breast cancer. It has previously been reported that HOXA9, a paralog of HOXA10, is a tumor suppressor in breast cancer , and expression of HOXD10 in MDA-MB-231 significantly impaired migration . Interestingly, in BT549 cells the expression of a full-length HOXA10 cDNA was repressed. It is highly likely that the high endogenous miR-135a level inhibits HOXA10 expression through targeting its 3'-UTR, since deletion of 3'-UTR led to a higher expression of HOXA10 protein than the full-length cDNA HOXA10 (Figure 5C), and the effect of HOXA10 overexpression on cell invasion varied depending on the absence or presence of the HOXA10 3'-UTR (Figure 5D). These results illustrate that miR-135a promoted cell migration and invasion, at least partially, through repression of HOXA10 via its 3'-UTR and also verified the in vitro luciferase assay results (Figure 3C-D). However, there is no evidence that miR-135a regulation of HOXA10 is exclusive. HOXA10 may also be targeted by other miRNAs and it appears that promoter methylation is also an important regulatory mechanism for HOXA10 in some tissues [35, 39]. Our result is in an agreement with a former study that reported HOXA10 expression inhibited matrigel invasion by breast cancer cells . The study also reported that HOXA10 expression induced p53 production. Therefore, understanding the molecular mechanism of the regulation of HOXA10 by miR-135a may provide a method to explore upstream regulation of HOXA10 and connect it with p53 tumor suppressor signalling pathways in breast cancer. Also, the "triple negative" invasive breast cancer type has a very poor prognosis and as yet no antibody target has been reported for the treatment of this type of breast cancer. So we believe future investigation on in vivo studies as well as on clinical specimens will confirm the importance of miR-135 and identify additional markers for diagnosis and treatment.