miRNAs are not directly involved in protein coding, but are believed to control the expression of more than one-third of the protein-coding genes in the human genome [18
]. Each miRNA can potentially regulate hundreds of genes at post-transcriptional levels by binding to the specific sequences in the target mRNA molecules based on ‘imperfect complementarity’. However, the precise interactions between miRNA and its target genes may be further dictated by cell types and the micro-environment. Therefore miRNAs contribute to a newly recognized level of fine-tuning regulation of gene expression. miRNA-7 has previously been characterized as a tumour-suppressor miRNA in several human cancers by targeting a number of key signalling molecules, including IRS1, IRS2, EGFR, RAF1 and PAK1 [10
]. On the basis of bioinformatics analysis, we further predicted a number of additional miR-7 targets, including IGF1R and PIK3CD. Our present experimental results confirmed that IGF1R, as well as IRS2 and PAK1, are functional targets of miR-7 in TSCC cells. Our results, together with recent reports on miR-7 and Akt and EGFR signalling pathways [10
], indicate that miR-7 contributes to tumorigenesis through multiple pathways and at multiple levels ().
Potential roles of miR-7 in tumorigenesis of TSCC
IGF1R is a receptor tyrosine kinase that consists of heterotetramers (α2β2
) held together by disulfide bridges, and it mediates IGF1-induced signalling events. Our present results clearly demonstrate that miR-7 down-regulates IGF1R by directly targeting the 3′-UTR of IGF1R
mRNA. This miR-7-mediated IGF1R down-regulation is accompanied by attenuation of Akt phosphorylation, inhibition of cell proliferation, enhanced apoptosis and cell-cycle arrest. This is consistent with the well-established IGF1R/IRS/PI3K/Akt signalling pathway that plays major roles in cell survival and proliferation. It is worth noting that IRS2, one of the previously identified miR-7 targets in glioblastoma [10
], was also down-regulated by ectopic transfection of miR-7 in our TSCC cell lines. Therefore miR-7 concurrently targets at least two major signalling molecules in the IGF1R/IRS/PI3K/Akt signalling pathway in TSCC. Furthermore, one of the PI3K members (PIK3CD) is also predicted by bioinformatics analysis to be a potential target of miR-7. One highly conserved and three poorly conserved binding sites for miR-7 were predicted in the 3′-UTR of PIK3CD
mRNA. Although our luciferase-reporter-gene assays suggested that these predicted sequences in the 3′-UTR of PIK3CD
are functional binding sites for miR-7 (results not shown), the expression level of PIK3CD was minimal in our cell lines. Furthermore, ectopic transfection or knock down of miR-7 did not change the expression level of PIK3CD to any significant extent. Therefore it appears that PIK3CD may not contribute significantly to the miR-7-mediated regulation of the Akt signalling pathway in our system. Nevertheless, it remains possible that miR-7 may functionally target PIK3CD in other cell types or different biological systems. It is worth noting that the PI3K family includes at least four members: PIK3CA (p110α
), PIK3CB (p110β
), PIK3CG (p110γ
) and PIK3CD (p110δ
). PIK3CD is selectively expressed in leucocytes [21
] and, together with PIK3CG, these two isoforms of PI3K contribute to the immune processes that underpin inflammatory responses. On a separate note, results from a study by Webster et al. [11
] suggested that PI3K was down-regulated by miR-7 overexpression in a lung adenocarcinoma cell line (A549). However, the authors did not specify which PI3K member was affected.
Although our present study has provided evidence indicating the role of miR-7 in the regulation of the IGF1R/IRS/PI3K/Akt signalling pathway, several other functional roles for miR-7 have been previously suggested. It has been reported previously that PAK1, a serine/threonine kinase that plays a pivotal role in cell migration and invasion, is targeted by miR-7 [12
]. We confirmed the miR-7 effect on PAK1 in our TSCC cell lines. miR-7 has also been suggested to be a regulator of the EGFR signalling pathway by concurrently targeting EGFR and RAF1 [10
]. However, we did not observe any apparent change in EGFR expression after the transfection of miR-7 mimic, and RAF1 was not expressed to any significant extent in our cell lines. Furthermore, miR-7 treatment did not alter the phosphorylation status of ERK1/2, one of the major downstream molecules of EGFR signalling. It is worth knowing that miR-7 precursors were used in the previous studies [10
], whereas we used mature miR-7. Nevertheless, it is possible that this apparent difference in the miR-7 effect on the EGFR signalling pathway may be due to differences in cancer types. Alternatively, this difference may be cell-line specific. The cell lines we tested in the present study (or the cell lines used in the previous study [10
]) may have specific mutation(s) that dictate the miR-7 effects on EGFR. More in-depth functional analysis will be needed to fully evaluate the effect of miR-7 on the EGFR signalling pathway.
In summary, our results indicate that miR-7 co-ordinately regulates IGF1R signalling at multiple levels. The findings shown in the present paper, in conjunction with recent reports on miR-7 and other proto-oncogenes (e.g. IRS, PAK1, RAF1 and EGFR) [10
], suggest that miR-7 plays major roles in tumorigenesis. Given the centrality of the IGF1R/IRS/PI3K/Akt signalling pathway in TSCC, delivery of miR-7 as a therapeutic agent may represent an effective approach for the treatment of this disease.