miRs*: Innocent bystanders only?
Authors: Silvia Giordano
Journal: Hepatology (2016)

MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs regulating gene expression and protein translation. Many studies have shown that they play a crucial role during embryogenesis—driving organ and tissue differentiation—and that they finely tune fundamental biological processes, such as cell proliferation and apoptosis. The involvement of miRNAs in cancer pathogenesis is well established as they can behave as oncogenes or tumor suppressor genes, depending on the cellular function of their targets. miRNAs control gene expression through posttranscriptional gene silencing, by either blocking messenger RNA translation or inducing its degradation. The majority of miRNA genes are transcribed by RNA polymerase II, originating primary miRNAs that—upon capping, splicing, and polyadenylation—are bound by the Microprocessor complex (Fig. 1). This complex cleaves them to originate 60-70 nucleotide long, hairpin-shaped structures named precursor miRNAs, which are transferred from the nucleus to the cytoplasm in an exportin-dependent process. In a further step, precursor miRNAs are processed by the ribonuclease III enzyme DICER that originates the mature miRNA duplex. One of the two RNA strands, the so-called guide strand, is usually loaded into the miRNA-induced-silencing complex to allow interaction with the complementary target messenger RNA, which is then either degraded or translationally repressed. The other strand of the duplex, the "passenger strand" (miRNA*), is usually degraded. Very interestingly, miRNAs and their associated miRNAs* differ in their seed sequences, thus recognizing different target sequences and different target genes. miR-122/miR-122* biogenesis and activity. miR-122 gene expression is positively controlled by some transcription factors and negatively regulated by epigenetic modifications. In the nucleus, the primary miRNA is cleaved by the Microprocessor complex to originate precursor miR-122, which is then exported to the cytoplasm. Precursor miR-122 is further processed by DICER; at this stage, some factors could regulate the miRNA guide-to-passenger strand ratio. miR-122 exerts its suppressive function by targeting several genes involved in control of cell growth and differentiation and of inflammation; among the targets critical for miR-122* suppressive function is Mdm2, a negative regulator of p53. Abbreviations: ADAM10, a disintegrin and metallopeptidase 10; C/EBPa, CCAAT/enhancer binding protein alpha; HNF, hepatocyte nuclear factor; IGF1R, insulin-like growth factor 1 receptor; Pre, precursor; Pri, primary; SRF, serum response factor. Most of the work performed up to now has limited the analysis to the guide strands of the miRNAs of interest. The work of Simerzin and colleagues, in this issue of Hepatology, opens a new perspective in the miRNA field as it shows that the passenger strand of miR-122 (miR-122*) acts as a tumor suppressor through modulation of the p53–mouse double minute 2 homolog (Mdm2) circuitry.1 miR-122 is the most abundant miRNA in the adult liver and a critical player in liver biology and disease. Indeed, it regulates cholesterol, glucose and iron homeostasis, lipid metabolism, and infection with hepatitis C virus and Leishmania donovani.2, 3 Its level is decreased in patients affected by nonalcoholic steatohepatitis and in some of those presenting highly invasive hepatocellular carcinomas. The role of this miRNA as a tumor suppressor was definitively proven by the study of miR-122 null mice, which progressively develop steatohepatitis, fibrosis, and eventually hepatocellular carcinoma.4 The novelty of Simerzin and colleagues' work is that they found that in liver not only miR-122 but also the passenger strand miR-122* is expressed at a level comparable to that of the other hepatic miRNAs. Like miR-122, miR-122* is decreased in hepatocellular carcinoma compared to nontumoral liver, suggesting that the inhibition of transcription very likely takes place at the primary miR level. Why this happens has not been investigated in this context yet, but the work of Hamad and colleagues suggests that epigenetic silencing (identified in HepG2 cells) could be a possible mechanism.5 In other cases, where the ratio between the two strands is changed, different mechanisms have been proposed. For example, Winter and Diederichs showed that in the case of Let-7a the guide-to-passenger strand ratio can be actively regulated by Human Argonaute 3, which specifically enhances the passenger strand expression.6 These results strongly suggest that the passenger strand is not an "innocent bystander" but that it plays specific functions. As expected from the general structure of miRNAs, the seed sequence of miR-122 is different from that of mir-122*, which targets the messenger RNA of Mdm2, a well-known negative regulator of the tumor suppressor p53. An autoregulatory negative feedback loop exists between p53 and Mdm2 as p53 induces Mdm2 transcription, while Mdm2 promotes p53 degradation.7, 8 The decrease of miR-122* observed in hepatocellular carcinoma thus leads to an increased amount of Mdm2, which lowers p53 levels, explaining the suppressive role of this miRNA*. Thus, through different mechanisms, both strands of the precursor miR122 cooperate to suppress liver tumorigenicity. To increase the expression of endogenous miRNAs in vitro and in vivo, double-stranded miRNA mimics are widely used. These molecules bypass the natural process of miRNA biogenesis and are usually engineered to increase stability, avoid promotion of innate immune responses, and increase their entrance into cells. Upon introduction in the cells, the "mimic passenger" strand, which can be completely or partially complementary to the guide strand (but not always identical to the endogenous passenger strand), may be loaded into the miRNA-induced-silencing complex. Søkilde and colleagues have recently shown that a different chemical formulation of the miRNA mimics can alter the specificity of "guide" versus "passenger" selection, thus causing unbalance between the two strands.9 This can lead to unwanted biological effects, due to the targeting of genes whose expression is controlled by the miRNA*. Moreover, as shown by Simerzin et al., the biological activity of the two strands can be different in different tissues.1 Indeed, they found that miR122 overexpression, differently from that observed in hepatocellular carcinoma cells, had no impact on growth of cervical cancer cells, while an increase of miR122* resulted in massive cell death. Altogether, these findings suggest that our knowledge of the miRNA field is far from complete and that modulation of miRNA expression is more complex than expected. In the case of miR-122, the two strands share similar oncosuppressor activity and thus have to be lost to promote tumor growth. However, it is conceivable that the two strands of some miRNAs can display opposite biological effects, mediated by specific targets, and that the artificial unbalancing induced by exogenous modulators could lead to unwanted effects, possibly tissue-specific. Silvia Giordano, M.D., Ph.D. Department of Oncology, University of Torino Candiolo Cancer Institute, FPO-IRCCS Candiolo, Italy