Passenger Strands Of miRNAs Research Articles

Dual strands of the miR-223 duplex (miR-223-5p and miR-223-3p) inhibit cancer cell aggressiveness: targeted genes are involved in bladder cancer pathogenesis.

Analyses of microRNA (miRNA) expression signatures obtained by RNA sequencing revealed that some passenger miRNAs (miR-144-5p, miR-145-3p, miR-149-3p, miR-150-3p, and miR-199a-3p) acted as anti-tumor miRNAs in several types of cancer cells. The involvement of passenger strands in the pathogenesis of human cancer is a novel concept. Based on the miRNA signature of bladder cancer (BC) obtained by RNA sequencing, we focused on both strands of the miR-223-duplex (miR-223-5p and miR-223-3p) and investigated their functional significance in BC cells. Ectopic expression of these miRNAs showed that both miR-223-3p (the guide strand) and miR-223-5p (the passenger strand) inhibited cancer cell migration and invasion of BC cells. The role of miR-223-5p (the passenger strand) has not been well studied. Combining gene expression studies and in silico database analyses, we demonstrated the presence of 20 putative target genes that could be regulated by miR-223-5p in BC cells. Among these targets, high expression of five genes (ANLN, INHBA, OIP5, CCNB1, and CDCA2) was significantly associated with poor prognosis of BC patients based on The Cancer Genome Atlas (TCGA) database. Moreover, we showed that a gene (ANLN) encoding a multifunctional actin-binding protein was directly regulated by miR-223-5p in BC cells. Overexpression of ANLN was observed in BC clinical specimens and high expression of ANLN was significantly associated with poor prognosis of BC patients. We suggest that studies of regulatory cancer networks, including the passenger strands of miRNAs, may provide new insights into the pathogenic mechanisms of BC.

Passenger strand of miR-145-3p acts as a tumor-suppressor by targeting MYO1B in head and neck squamous cell carcinoma.

Analysis of the microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC) based on RNA sequencing showed that dual strands of pre-miR-145 (miR-145-5p, guide strand; and miR-145-3p, passenger strand) were significantly reduced in cancer tissues. In miRNA biogenesis, passenger strands of miRNAs are degraded and have no biological activities in cells. The aims of this study were to investigate the functional significance of the passenger strand of miR-145 and to identify miR-145-3p-regulated oncogenic genes in HNSCC cells. Expression levels of miR-145-5p and miR-145-3p were significantly downregulated in HNSCC tissues and cell lines (SAS and HSC3 cells). Ectopic expression of miR-145-3p inhibited cancer cell proliferation, migration and invasion, similar to miR-145-5p, in HNSCC cells. Myosin 1B (MYO1B) was directly regulated by miR-145-3p, and knockdown of MYO1B by siRNA inhibited cancer cell aggressiveness. Overexpression of MYO1B was confirmed in HNSCC clinical specimens by analysis of protein and mRNA levels. Interestingly, high expression of MYO1B was associated with poor prognosis in patients with HNSCC by analysis of The Cancer Genome Atlas database (p=0.00452). Our data demonstrated that the passenger strand of miR-145 acted as an antitumor miRNA through targeting MYO1B in HNSCC cells. The involvement of dual strands of pre-miR-145 (miR-145-5p and miR-145-3p) in the regulation of HNSCC pathogenesis is a novel concept in present RNA research.

Dual Strands of Pre-miR-149 Inhibit Cancer Cell Migration and Invasion through Targeting FOXM1 in Renal Cell Carcinoma.

Our recent studies revealed that dual strands of certain pre-microRNAs, e.g., pre-miR-144, pre-miR-145, and pre-miR-150, act as antitumor microRNAs (miRNAs) in several cancers. The involvement of passenger strands of miRNAs in cancer pathogenesis is a novel concept in miRNA research. The analysis of a miRNA expression signature in clear cell renal cell carcinoma (ccRCC) has revealed that the guide strand of pre-miR-149 is significantly downregulated in cancer tissues. The aims of this study were to investigate the functional significance of miR-149’s guide strand (miR-149-5p) and passenger strand (miR-149-3p), and to identify the oncogenic genes regulated by these miRNAs in ccRCC cells. The ectopic expression of these miRNAs significantly inhibited cancer cell migration and invasion in ccRCC cells. Forkhead box protein M1 (FOXM1) was directly regulated by miR-149-5p and miR-149-3p in ccRCC cells. Knockdown studies using si-FOXM1 showed that the expression of FOXM1 enhanced RCC cell aggressiveness. Interestingly, the analysis of a large number of patients in the The Cancer Genome Atlas (TCGA) database (n = 260) demonstrated that patients with high FOXM1 expression had significantly shorter survival than did those with low FOXM1 expression (p = 1.5 × 10−6). Taken together, dual strands of pre-miR-149 (miR-149-5p and miR-149-3p) acted as antitumor miRNAs through the targeting of FOXM1 in ccRCC cells.

Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy.

In response to stress, the heart undergoes extensive cardiac remodeling that results in cardiac fibrosis and pathological growth of cardiomyocytes (hypertrophy), which contribute to heart failure. Alterations in microRNA (miRNA) levels are associated with dysfunctional gene expression profiles associated with many cardiovascular disease conditions; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes. Surprisingly, evaluation of the miRNA content of cardiac fibroblast-derived exosomes revealed a relatively high abundance of many miRNA passenger strands ("star" miRNAs), which normally undergo intracellular degradation. Using confocal imaging and coculture assays, we identified fibroblast exosomal-derived miR-21_3p (miR-21*) as a potent paracrine-acting RNA molecule that induces cardiomyocyte hypertrophy. Proteome profiling identified sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) as miR-21* targets, and silencing SORBS2 or PDLIM5 in cardiomyocytes induced hypertrophy. Pharmacological inhibition of miR-21* in a mouse model of Ang II-induced cardiac hypertrophy attenuated pathology. These findings demonstrate that cardiac fibroblasts secrete star miRNA-enriched exosomes and identify fibroblast-derived miR-21* as a paracrine signaling mediator of cardiomyocyte hypertrophy that has potential as a therapeutic target.

Engineering of a conditional allele reveals multiple roles of XRN2 in Caenorhabditis elegans development and substrate specificity in microRNA turnover

Although XRN2 proteins are highly conserved eukaryotic 5′→3′ exonucleases, little is known about their function in animals. Here, we characterize Caenorhabditis elegans XRN2, which we find to be a broadly and constitutively expressed nuclear protein. An xrn-2 null mutation or loss of XRN2 catalytic activity causes a molting defect and early larval arrest. However, by generating a conditionally mutant xrn-2ts strain de novo through an approach that may be also applicable to other genes of interest, we reveal further functions in fertility, during embryogenesis and during additional larval stages. Consistent with the known role of XRN2 in controlling microRNA (miRNA) levels, we can demonstrate that loss of XRN2 activity stabilizes some rapidly decaying miRNAs. Surprisingly, however, other miRNAs continue to decay rapidly in xrn-2ts animals. Thus, XRN2 has unanticipated miRNA specificity in vivo, and its diverse developmental functions may relate to distinct substrates. Finally, our global analysis of miRNA stability during larval stage 1 reveals that miRNA passenger strands (miR*s) are substantially less stable than guide strands (miRs), supporting the notion that the former are mostly byproducts of biogenesis rather than a less abundant functional species.

Identification of microRNAs from Plutella xylostella larvae associated with parasitization by Diadegma semiclausum

MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of miRNAs in a global key pest Plutella xylostella as well as comparative analysis of miRNA expression profile of the insect in association with parasitization by Diadegma semiclausum. Combining the deep sequencing data and bioinformatics, 235 miRNAs were identified from P. xylostella. Differential expression of host cellular miRNAs in response to parasitism was examined by making small RNA libraries from parasitized and naive second instar larvae of P. xylostella. Bantam, miR-276*, miR-10, miR-31 and miR-184 were detected as five most abundant miRNAs in both libraries and 96 miRNAs were identified that were differentially expressed after parasitization. Bantam*, miR-184 and miR-281* were significantly down-regulated and two miRNAs miR-279b and miR-2944b* were highly induced in parasitized larvae. Interestingly, high copy numbers and differential expression of several miRNA passenger strands (miRNA*) suggest their potential roles in host-parasitoid interaction. In conclusion, expression profiling of miRNAs provided insights into their possible involvement in insect immune response to parasitism and offer an important resource for further studies.

Identifying Functional MicroRNAs in Macrophages with Polarized Phenotypes

Macrophages respond to external stimuli with rapid changes in expression of many genes. Different combinations of external stimuli lead to distinct polarized activation patterns, resulting in a spectrum of possible macrophage activation phenotypes. MicroRNAs (miRNAs) are small, noncoding RNAs that can repress the expression of many target genes. We hypothesized that miRNAs play a role in macrophage polarization. miRNA expression profiles were determined in monocyte-derived macrophages (MDMs) incubated in conditions causing activation toward M1, M2a, M2b, or M2c phenotypes. One miRNA guide strand and seven miRNA passenger strands were significantly altered. Changes were confirmed in MDMs from six separate donors. The amplitude of miRNA expression changes in MDMs was smaller than described studies of monocytes responding to inflammatory stimuli. Further investigation revealed this correlated with higher basal miRNA expression in MDMs compared with monocytes. The regulation of M1- and M2b-responsive miRNAs (miR-27a, miR-29b, miR-125a, miR-146a, miR-155, and miR-222) was similar in differentiated THP-1 cells and primary MDMs. Studies in this model revealed cross-talk between IFNγ- and LPS-associated pathways regulating miRNA expression. Furthermore, expression of M1-associated transcripts was increased in THP-1 cells transfected with mimics of miR-29b, miR-125a-5p, or miR-155. The apparent inflammatory property of miR-29b and miR-125a-5p can be at least partially explained by repression of TNFAIP3, a negative regulator of NF-κB signaling. Overall, these data suggest miRNAs can contribute to changes in macrophage gene expression that occur in different exogenous activating conditions.

Argonaute proteins regulate microRNA stability: Increased microRNA abundance by Argonaute proteins is due to microRNA stabilization

Argonaute proteins are key players in microRNA (miRNA) processing and function. Next to their role as RISC effector proteins mediating target silencing, they actively participate in miRNA biogenesis and increase miRNA abundance by a yet unidentified mechanism. It has been hypothesized that this increase in steady-state miRNA levels might be due to a stabilizing effect of Argonaute proteins, but this has not been analyzed so far due to a lack of test systems. Here, we provide two approaches to estimate miRNA stability and factors affecting it: in cells lacking Ago2, endogenous miRNA guide strand degradation and half-lives can be assessed using Actinomycin D or α-Amanitin. In turn, miRNA passenger strands can serve as a model in wildtype cells to ascertain the impact of miRNA stability factors.We provide evidence that Argonaute proteins stabilize mature miRNAs in a slicing-independent manner. Transcriptional inhibition reveals reduced half-lives of multiple endogenous miRNA guide strands in cells lacking Ago2. This effect is reversible upon the reconstitution of Argonaute expression. Correspondingly, overexpression of Argonaute proteins decelerates miRNA degradation and increases miRNA half-life. Taken together, this study employs two model systems to identify factors altering miRNA stability and provides evidence how Argonaute proteins post-transcriptionally elevate mature miRNA levels via increasing miRNA stability.

Target-Mediated Protection of Endogenous MicroRNAs in C. elegans

MicroRNAs (miRNAs) are tightly regulated through transcriptional and posttranscriptional mechanisms, including degradation by nucleases. Here, we report that in C. elegans, target mRNAs can protect their cognate miRNAs from degradation invivo. We show that the let-7(n2853) mutation destabilizes the mature let-7 miRNA by impairing this protection. Moreover, presence of a cognate target or depletion of the xrn-1 (XRN1) or xrn-2 (XRN2/Rat1p) exoribonucleases enforces accumulation of certain miRNA passenger (miR(∗)) strands. Thus, following biased miRNA strand loading into Argonaute, elimination of nonfunctional RNAs can further refine miRNA strand selection. Conversely, by aligning the levels of miRNAs with those of their targets, the opposing activities of mature miRNA degradation and target-mediated miRNA protection (TMMP) may enable dynamic expression of either mature strand ofa pre-miRNA, and evolution of miRNAs. Thus, it seems that mRNAs are more than inert targets and function with miRNAs in a network of mutual regulation.