Since miRNAs are known to down-regulate proteins, we selected a series of miRNA-protein pairs to evaluate this property on populations of patient specimens on TMAs. Our breast cancer cohort TMA has been extensively used by our lab and has been tested with 116 proteins previously. Thus we were able to assess this premise by comparing the AQUA scores of the protein targets to the miRNA AQUA scores. The following pairs were tested: miR-92a and ERbeta1, miR-34a and c-Met, miR-221 and Estrogen Receptor α and finally miR-21 and PTEN (Figure 4) (19, 22, 27-29). In no case was an inverse relationship observed. We also attempted to discover an inverse relationship between miR-221 and any of the 116 proteins in our database (Supplementary Table 1). While inverse relationships could be found in non-randomly selected subsets of cases, a compelling (statistically significant) inverse relationship was not discovered between miR-221 and any of the proteins in our database. To further assess this principal, we also evaluated miR-205 in non-small cell lung cancer where we had previously tested several validated targets of miR-205 including Zeb1, ERbB3, PTEN, BCL2, ERbB4, and E2F4 (Supplementary Figure 2) (30-33). Again, in 145 cases, we did not establish an inverse relationship between miR-205 and these targets, even when subclassifying tumors by histotype. Thus, in breast and lung cancer, the inverse relationships between a miRNA and its predicted targets are not readily seen in large patient cohorts.
Numerous in vitro and cell line based models suggest a tight inverse relationship between a miRNA and its protein targets. Many papers are identifying new “targets” of miRNAs based on cell line luciferase assays and forced overexpression conditions. These studies convincingly describe potential targets of miRNAs in forced situations, but often lack strong evidence for biological relevance for an interaction. We sought to validate an inverse relationship between the miRNAs investigated and their targets, but were unable to find an inverse relationship in real human tumors. This lack of inverse relationships could be due to tissue heterogeneity since the protein and miRNAs assays were not done in serial sections, but often several sections away or on different cores. It is also possible that the inverse relationship is seen only by examination on an individual cell basis, which we could not discern since our method scores on the basis of a field of view. Alternatively, the regulation of protein expression may be complicated by numerous additional regulators at pre- and post-translational stages such that a single miRNA cannot be used to differentiate high versus low expression of a single protein target, except in the contrived situation of forced expression in model systems.
Given the vast regulatory network where an mRNA can be targeted by many miRNAs and miRNAs can target hundreds of mRNAs, we believe the absence of an inverse relationship in human tumors is not surprising. In each case here, we evaluated a single miRNA and compared it to the expression of one or more proteins. It is possible that in order to see decreased protein expression, multiple miRNAs are simultaneously required. A key recent report by Mukherji et al. (34) suggests a nonlinear relationship between target protein level and mRNA level where miRNAs can highly repress mRNA only when it is below a certain threshold level leading to concomitant repression of target protein level. But, if the mRNA is above the threshold level then the miRNA has almost no measurable repressive effect on the total level of mRNA, and hence no effect on the total level of protein target. They also show that this interaction has substantial cell-to-cell variability in strength of target protein repression. Thus, visualization of an inverse relationship may require simultaneous measurement of miRNA, mRNA and protein within individual cells. Even then, the Mukherji et al. work predicts an inverse relationship might only be seen in a subset of cells where the relative amounts of mRNA and microRNA are specifically coordinated. In addition, the recent evidence for “competitive endogenous RNAs” (ceRNAs) or RNAs which can act as miRNA decoys further complicates the level of regulation between a miRNA and target mRNA (35). Overall, these studies support the absence of an inverse relationship observed here.