MicroRNAs have emerged as an important factor in posttranscriptional regulation of gene expression with ties to cellular processes such as proliferation and differentiation. It has been demonstrated that microRNAs can contribute to the deregulation of such processes . Consequently, the use of microRNAs may help a virus to attain e.g. persistence and, indeed, some viruses bring their own microRNAs [30, 31]. Lacking any self-encoded microRNAs, HTLV could still interfere with host cell microRNAs in order to drive it towards accelerated proliferation and longevity.
This study approached HTLV-1's impact on cellular microRNA expression, focussing on a defined subset of oncogenesis-related specimens rather than relying on undirected microarray screening techniques. The selection procedure took into account (a) Treg-specific expression patterns of microRNAs and (b) available functional characterization, i.e., links to oncogenicity. While the patterns were based on data gathered in mice, comparability was maintained by phenotypically characterizing the cell lines used in this study (data not shown). This ensured continuity with both the mouse data and published descriptions of HTLV-transformed T cells' phenotype. Out of a panel of seven microRNAs, four were highly and significantly upregulated in HTLV-/Tax-positive samples as compared to controls, namely miR-21, miR-24, miR-146a and miR-155, whereas miR-223 was downregulated. The remaining two microRNAs, miR-191 and miR-214, were present in small amounts yet not regulated. Overall, the emerging pattern of microRNA expression in HTLV-transformed cells even within the investigated group of seven miRs illustrates the validity of our selective approach. Moreover, the pattern might contribute to a more detailed phenotypic characterization of HTLV-transformed cells, complementing well established protein markers. BIC and its mature product, miR-155, can be regarded as a prototypical oncomiR since it was first described as an integration site for avian leukosis virus before being identified as capapble of cooperation with myc in cancerogenesis . Moreover, miR-155 appears to be involved in regulating a variety of lymphoid processes, particularly differentiation . The overexpression of miR-155 in HTLV-transformed cell lines fits into that picture, hinting at a possible involvement in the pathogenesis of HTLV-associate disease at some point. Because the expression level of the BIC primary transcripts mirrored – insomuch as they were elevated – those of the mature miR-155, this indicated that miR-155 expression is probably controlled on a transcriptional level. Direct viral interference – in particular one of HTLV-1 Tax – with BIC/miR-155 expression could not be detected. The differences seen in Tesi cells, with and without Tax, could not be reproduced in an independent system. Clarifying this issue probably would have to take into account aspects of hematpoietic differentiation. It is worth mentioning that, though the association of hematologic malignancies with high miR-155 expression has been repeatedly described, no mechanism for its upregulation has been presented. Involvement of transcription factors AP-1 and NF- κ B during normal immune function of B cells has been described, however [59, 60].
While expression levels of miR-191 and miR-214 did not significantly differ between HTLV-positive and -negative samples, miR-223 was downregulated in an HTLV context. The latter is particularly interesting since reduced miR-223 abundance has been described recently in hepatocellular carcinoma . When overexpressed, miR-223 led to a decrease in cell viability by targeting stathmin. According to this link between miR-223 and stathmin, the observed downregulation of miR-223 in our study could potentially be involved in HTLV-1 cell-to cell spread in vivo [62, 63].
Ectopically expressing HTLV-1 Tax in Jurkat T cells, which express only low levels of miR-146a, entailed a marked increase in miR-146a expression, thus, demonstrating that Tax is able to boost the cellular signals underlying that expression. Subsequent promoter analysis refined the initial observation, showing a circa 15-fold activation by Tax and explained the observed high levels of miR-146a in HTLV-transformed cell lines. Using mutated forms of Tax and the coexpression of a dominant active NF- κ B inhibitor, we were able to pinpoint the transactivation as being mediated via NF- κ B. These findings are in line with data by other groups who described NF- κ B regulation of miR-146a expression . The previously published upregulation of miR-146a expression by EBV LMP-1 is of interest because it adds to the relevance of our findings [64, 65]. Conceivably, this constitutes an important facet in both viruses' efforts to establish perstistence or their potential to bring about malignant transformation. In contrast to activation by LMP-1, Tax uses only one of the two NF- κ B sites present in the promoter sequence proximal to the transcriptional start site. Other transcription factors do not seem to participate in the Tax effect since the deletion of that aforementioned singular NF- κ B site completely abrogated promoter activity. Recent data by Bhaumik et al. describe a suppressive effect of miR-146a on NF- κ B signaling . This could constitute a negative feedback loop of miR-146a on its own expression, which is rendered inoperative in an HTLV context. Taken together, our studies indicate that miR-146a stimulation in HTLV-transformed cells can be traced back to promoter transactivation by HTLV-1 Tax via NF- κ B.
Defining target genes for microRNAs bioinformatically is difficult owing to inherent inaccuracies in predictive algorithms, because, unlike siRNAs, microRNAs do not depend on perfect sequence complementarity to targets. Nevertheless, some targets have been described, like IRAK6 and TRAF1 3'-UTRs for miR-146a . Another study  found endogenous IRAK6 and TRAF1 mRNA levels to be not or barely (respectively) miR-146a-regulated which could hint at the involvement of further regulatory elements, like additional microRNAs. Looking at cooperative effects of several microRNAs on a given mRNA might help avoiding such conflicting data. Interestingly, two targets ascribed to miR-155, SMAD5  and TP53INP1 , came up in our analysis as having predicted binding sites for all four upregulated microRANs, miR-21, miR-24, miR-146a and miR-155. In the latter case, this was bolstered by congruent predictions from different databases. An upcoming paper describes that TP53INP1 is targeted by miRs 93 and 130b which were found to be overexpressed in both samples from patients suffering from acute ATLL and ATLL-derived cell lines . The same paper also mentions the upregulation of miR-155 in ATLL patient samples. This strengthens our point that microRNAs do play a role in the pathogenesis of HTLV-associated disease. With regard to our findings, the data from Yeung at al. open up the possibility that a combined 'attack' of miR-155, miR-93, miR130b and maybe one the microRNAs investigated in this study on TP53INP1 might even increase documented microRNA effects on its mRNA. In summary, a combinatorial approach to the search for microRNA targets might help finding new targets or refining the regulation of known ones.