Different expression levels between miRNA targets and total mRNAs have been uncovered in the comparison between mature tissues and embryos [22
] and miRNAs have been suggested to confer precision and robustness to developmental processes. In this study, we initially reported that miRNA targets expressed less on a global scale than total mRNAs in prostate tumours, relative to benign prostate tissues. Analysis of the protein levels of miRNA targets suggests that the level of protein expression of miRNA targets may also be reduced, in agreement with a recent study which reported that the regulation of protein-coding genes by miRNAs was quite similar on the transcript and protein levels [14
]. Moreover, our data showed that the transcript abundance of the targets of androgen-repressed miRNAs was significantly lower than the abundance of the targets of all other miRNAs in androgen-independent prostate cancer. The abnormal expression pattern of miRNA targets was also seen in three other cancer types, suggesting that it may be a common feature of the human cancer transcriptome.
We also found a trend for an increased down-regulation of mRNAs with longer 3' UTRs and more target-site types, consistent with a recent study showing that proliferating cells express mRNAs with shortened 3' UTRs and fewer miRNA target sites [23
]. It has been reported that for proteins with more interacting partners, their genes tend to be regulated by more miRNA types [24
]. Genes with more interactions may require more elaborate regulation at the posttranscriptional level because unwanted output of these proteins may lead to a more severe fitness effect. Moreover, miRNAs have been proposed to primarily target downstream network components such as transcription factors [26
]. Disrupted expression of the highly regulated miRNA target genes may reflect the fact that the regulatory network in cancer cells departs from the normal regulatory routine presented in benign cells. The molecular mechanisms determining the intriguing expression patterns of miRNA targets in cancer cells presented in this study remain to be elucidated. Based on our analysis, there are three potential reasons discussed below.
First, the abnormal transcript abundance of miRNA targets may indicate a significant influence of miRNAs on the expression of their target genes in prostate tumours. This view is supported by three observations: (1) the targets of 36 up-regulated miRNAs made a strong contribution to the low expression of all miRNA targets; (2) there was a weak (but significant) negative correlation between the score of the differential expression of individual miRNAs (published by Volinia et al.) and the RR value of the miRNA's targets; and (3) the propensity for low expression increased with the number of target-site types embedded in the 3' UTRs of the miRNA targets, suggesting the possibility of synergistic regulation by multiple different miRNAs in prostate cancer. In general, lower transcript level is attributed to transcription inhibition or mRNA decay. Since miRNA target prediction relies strongly on the characteristics of 3' UTRs, translational control by 3' UTRs may play a role in the down-regulation of miRNA targets in prostate cancer. It has been demonstrated that miRNAs can promote rapid mRNA degradation by accelerating deadenylation [1
] and that miRNAs are involved in AU-rich Element (ARE)-mediated mRNA instability [27
]. Therefore, the low expression of miRNA targets may result from the action of miRNA-mediated mRNA decay in prostate cancer. Up-regulation of miRNAs in prostate tumours is common [3
] and is consistent with the known oncogenic activity of many miRNAs [28
]. It has been reported that Dicer
and other genes involved in miRNA processing are up-regulated in prostate cancer [19
], indicating that the prostate tumour is more efficient than normal prostate tissue at processing miRNA precursors into mature miRNAs. These observations support the idea that miRNAs may be up-regulated on a global scale in prostate cancer, consistent with the global down-regulation of their targets.
It should be noted that the global down-regulation of miRNA targets is an overall effect that does not negate the fact that some miRNA targets are up-regulated in prostate tumours. A recent miRNA profiling study showed a tumour gene signature that contains up-regulated and down-regulated miRNAs in prostate cancer [19
]. This study also showed that binding of miRNAs to 3' UTR sequences can lead to both degradation and accumulation of the targeted mRNA in cancer cells. In the correlation analysis between the expression level of individual miRNAs and the expression level of their putative targets, we confirmed this observation on a global scale. More specifically, both an inverse and a positive correlation could occur between a miRNA and its target mRNAs in prostate cancer cells. Since a miRNA can regulate multiple targets and a single mRNA can be targeted by several different miRNAs, the global down-regulation of miRNA targets may largely depend on the overall effect of miRNA regulation. The second potential reason is that the global down-regulation of miRNA targets is an overall effect that may depend on: (1) the reduction of mRNA expression that may be caused by the up-regulated miRNAs (such as miR-29a
); (2) the decrease of mRNA sequestration that may be caused by the down-regulated miRNAs (such as let-7i
); and (3) the moderate up-regulation of some targets of the down-regulated miRNAs (such as miR-125b
). Furthermore, this potential reason can explain the fact that global up-regulation of miRNA targets was observed for hepatocellular carcinoma, a tissue which has roughly equal numbers of up and down-regulated miRNAs [30
The third reason is related to a perplexing problem: several other miRNA profiling studies showed widespread down-regulation of miRNAs in prostate cancer [5
], and some of the miRNAs reported to be up-regulated by Volinia et al. overlapped with some of the miRNAs reported to be down-regulated by another profiling study [7
]. If most miRNAs are truly down-regulated in prostate cancer, the global down-regulation of miRNA targets may not be causatively linked to the expression levels of the miRNAs themselves. As miRNA targets have relatively long 3' UTRs (with known functions in the stability, localisation, and translation of mRNA) and more CpG islands (which may be methylated in cancers) than non-targets, a third explanation is that the target genes that are down-regulated in prostate cancer are key cellular regulators and such key regulators are themselves highly regulated at multiple levels (transcriptionally and post-transcriptionally), including regulation by miRNAs.
There are several explanations for the discrepancies in miRNA profiling studies. First, Volinia et al. [4
] and Ambs et al. [19
] used total RNA while other studies [5
] used purified small RNA samples (from 18 to 300 nt). Purification might introduce errors into miRNA expression comparisons. For example, there is presently no way to judge the different proportions of miRNAs within the pool of total RNAs [31
]. If cancer and benign tissue have different proportions of miRNA content, the validity of the analysis based on the fundamental assumption that same amount of miRNAs is extracted from the same amount of total RNAs is thrown into doubt. Second, it has been suggested that purification of the small RNA fraction could reduce nonspecific hybridisation to longer miRNA precursors. If there is a block in precursor miRNA processing in prostate cancer without a corresponding decrease in transcription, this could result in the inconsistency. A third explanation would be the differences in samples number. Volinia et al. and Ambs et al. analysed > 50 prostate cancer samples while other studies did not reach this size. Cancer is a heterogeneous disease, and the heterogeneity of tumour samples might contribute substantially to the results. It is not surprising that miRNA expression profiles published by different researchers are inconsistent, because miRNA profiling technology is still in its infancy. For example, researchers generally adopt "tried-and-true" methodologies from cDNA microarray technology for miRNA expression analysis, but the relatively small number of probes on miRNA microarrays may render these high-density approaches ineffective.
As to the basis of our investigation, the gene expression microarray and miRNA target prediction data have proved to be useful for gaining biological knowledge [13
]. Although these datasets are far from being complete and may contain noise, it is unlikely that these flaws could totally distort the results. Since consistent results were seen across various datasets generated by independent groups, the noise of microarray data and false positives in miRNA target predictions appear to have no serious effects on our study. Furthermore, the overall significances inferred from thousands of mRNAs would be strong enough to reflect real biology. The strength of our global analysis lies in the noise reduction effect, as well as the identification of general trends of miRNA target expression that would not have been discovered by individual investigation of single miRNA targets. Cancer is an extremely complex and heterogeneous disease [32
]. It should be noted that our data did not conclusively distinguish among the three possible mechanisms discussed above, and the detailed molecular mechanisms responsible for the abnormal expression of miRNA targets remain to be thoroughly elucidated. Future experiments or large microarray studies are needed to clarify the possible mechanisms.