MiR-155-targeted IcosL controls tumor rejection.

Elevated levels of miR-155 in solid and liquid malignancies correlate with aggressiveness of the disease. In this manuscript, we show that miR-155 targets transcripts encoding IcosL, the ligand for Inducible T-cell costimulator (Icos), thus impairing the ability of T cells to recognize and eliminate malignant cells. We specifically found that overexpression of miR-155 in B cells of Eµ-miR-155 mice causes loss of IcosL expression as they progress toward malignancy. Similarly, in mice where miR-155 expression is controlled by a Cre-Tet-OFF system, miR-155 induction led to malignant infiltrates lacking IcosL expression. Conversely, turning miR-155 OFF led to tumor regression and emergence of infiltrates composed of IcosL-positive B cells and Icos-positive T cells forming immunological synapses. Therefore, we next engineered malignant cells to express IcosL, in order to determine whether IcosL expression would increase tumor infiltration by cytotoxic T cells and reduce tumor progression. Indeed, overexpressing an IcosL-encoding cDNA in MC38 murine colon cancer cells before injection into syngeneic C57BL6 mice reduced tumor size and increased intratumor CD8+ T cell infiltration, that formed synapses with IcosL-expressing MC38 cells. Our results underscore the fact that by targeting IcosL transcripts, miR-155 impairs the infiltration of tumors by cytotoxic T cells, as well as the importance of IcosL on enhancing the immune response against malignant cells. These findings should lead to the development of more effective anticancer treatments based on maintaining, increasing, or restoring IcosL expression by malignant cells, along with impairing miR-155 activity.

Effect of ZEB1 Associated with microRNAs on Tumor Stem Cells in Head and Neck Cancer.

Cancer biologists have focused on studying cancer stem cells (CSCs) because of their ability to self-renew and recapitulate tumor heterogeneity, which increases their resistance to chemotherapy and is associated with cancer relapse. Here, we used two approaches to isolate CSCs: the first involved the metabolic enzyme aldehyde dehydrogenase ALDH, and the second involved the three cell surface markers CD44, CD117, and CD133. ALDH cells showed a higher zinc finger E-box binding homeobox 1 (ZEB1) microRNA (miRNA) expression than CD44/CD117/133 triple-positive cells, which overexpressed miRNA 200c-3p: a well-known microRNA ZEB1 inhibitor. We found that ZEB1 inhibition was driven by miR-101-3p, miR-139-5p, miR-144-3p, miR-199b-5p, and miR-200c-3p and that the FaDu Cell Line inhibition occurred at the mRNA level, whereas HN13 did not affect mRNA expression but decreased protein levels. Furthermore, we demonstrated the ability of the ZEB1 inhibitor miRNAs to modulate CSC-related genes, such as TrkB, ALDH, NANOG, and HIF1A, using transfection technology. We showed that ALDH was upregulated upon ZEB1-suppressed miRNA transfection (Mann-Whitney ** p101 = 0.009, t-test ** p139 = 0.009, t-test ** p144 = 0.002, and t-test *** p199 = 0.0006). Overall, our study enabled an improved understanding of the role of ZEB1-suppressed miRNAs in CSC biology.

Binding sites of miRNA on the overexpressed genes of oral cancer using 7mer-seed match.

The microRNAs having a length of ~ 19-22 nucleotides are the small, non-coding RNAs. The evolution of microRNAs in many disorders may hold the key to tackle complex challenges. Oral cancer belongs to the group of head and neck cancer. It occurs in the mouth region that appears as an ulcer. In this study, we collected information on the overexpressed genes of oral cancer. The coding sequences of the genes were derived from NCBI and the entire set of human microRNAs present in miRBASE 21 was retrieved. The human microRNAs that can target the overexpressed genes of oral cancer were determined with the aid of our in-house software. The interaction between microRNAs and the overexpressed genes was evaluated with 7mer-m8 model of microRNA targeting. The genes DKK1 and APLN paired with only one miRNA i.e., miR-447 and miR-6087, respectively. But the genes INHBA and MMP1 were found to be targeted by 2 miRNAs, while the genes FN1, FAP, TGFPI, COL4A1, COL4A2, and LOXL2 were found to be targeted by 16, 5, 9, 18, 29, and 11 miRNAs. Subsequently, several measures such as free energy, translation efficiency, and cosine similarity metric were used to estimate the binding process. It was found that the target region's stability was higher than the upstream and downstream zones. The overexpressed genes' GC contents were calculated, revealing that the codons in target miRNA region were overall GC rich as well as GC3 rich. Lastly, gene ontology was performed to better understand each gene's involvement in biological processes, molecular function, and cellular component. Our study showed the role of microRNAs in gene repression, which could possibly aid in the prognosis and diagnosis of oral cancer.

Determinants of Functional MicroRNA Targeting.

MicroRNAs (miRNAs) play cardinal roles in regulating biological pathways and processes, resulting in significant physiological effects. To understand the complex regulatory network of miRNAs, previous studies have utilized massivescale datasets of miRNA targeting and attempted to computationally predict the functional targets of miRNAs. Many miRNA target prediction tools have been developed and are widely used by scientists from various fields of biology and medicine. Most of these tools consider seed pairing between miRNAs and their mRNA targets and additionally consider other determinants to improve prediction accuracy. However, these tools exhibit limited prediction accuracy and high false positive rates. The utilization of additional determinants, such as RNA modifications and RNA-binding protein binding sites, may further improve miRNA target prediction. In this review, we discuss the determinants of functional miRNA targeting that are currently used in miRNA target prediction and the potentially predictive but unappreciated determinants that may improve prediction accuracy.

MicroRNAs miR-584-5p and miR-425-3p Are Up-Regulated in Plasma of Colorectal Cancer (CRC) Patients: Targeting with Inhibitor Peptide Nucleic Acids Is Associated with Induction of Apoptosis in Colon Cancer Cell Lines.

Liquid biopsy has dramatically changed cancer management in the last decade; however, despite the huge number of miRNA signatures available for diagnostic or prognostic purposes, it is still unclear if dysregulated miRNAs in the bloodstream could be used to develop miRNA-based therapeutic approaches. In one author's previous work, nine miRNAs were found to be dysregulated in early-stage colon cancer (CRC) patients by NGS analysis followed by RT-dd-PCR validation. In the present study, the biological effects of the targeting of the most relevant dysregulated miRNAs with anti-miRNA peptide nucleic acids (PNAs) were verified, and their anticancer activity in terms of apoptosis induction was evaluated. Our data demonstrate that targeting bloodstream up-regulated miRNAs using anti-miRNA PNAs leads to the down-regulation of target miRNAs associated with inhibition of the activation of the pro-apoptotic pathway in CRC cellular models. Moreover, very high percentages of apoptotic cells were found when the anti-miRNA PNAs were associated with other pro-apoptotic agents, such as sulforaphane (SFN). The presented data sustain the idea that the targeting of miRNAs up-regulated in the bloodstream with a known role in tumor pathology might be a tool for the design of protocols for anti-tumor therapy based on miRNA-targeting molecules.

miRNA-mediated control of exogenous OCT4 during mesenchymal-epithelial transition increases measles vector reprogramming efficiency.

OCT4 is a key mediator of induced pluripotent stem cell (iPSC) reprogramming, but the mechanistic insights into the role of exogenous OCT4 and timelines that initiate pluripotency remain to be resolved. Here, using measles reprogramming vectors, we present microRNA (miRNA) targeting of exogenous OCT4 to shut down its expression during the mesenchymal to the epithelial transition phase of reprogramming. We showed that exogenous OCT4 is required only for the initiation of reprogramming and is dispensable for the maturation stage. However, the continuous expression of SOX2, KLF4, and c-MYC is necessary for the maturation stage of the iPSC. Additionally, we demonstrate a novel application of miRNA targeting in a viral vector to contextually control the vector/transgene, ultimately leading to an improved reprogramming efficiency. This novel approach could be applied to other systems for improving the efficiency of vector-induced processes.

Detecting and Characterizing A-To-I microRNA Editing in Cancer.

Adenosine to inosine (A-to-I) editing consists of an RNA modification where single adenosines along the RNA sequence are converted into inosines. Such a biochemical transformation is catalyzed by enzymes belonging to the family of adenosine deaminases acting on RNA (ADARs) and occurs either co- or post-transcriptionally. The employment of powerful, high-throughput detection methods has recently revealed that A-to-I editing widely occurs in non-coding RNAs, including microRNAs (miRNAs). MiRNAs are a class of small regulatory non-coding RNAs (ncRNAs) acting as translation inhibitors, known to exert relevant roles in controlling cell cycle, proliferation, and cancer development. Indeed, a growing number of recent researches have evidenced the importance of miRNA editing in cancer biology by exploiting various detection and validation methods. Herein, we briefly overview early and currently available A-to-I miRNA editing detection and validation methods and discuss the significance of A-to-I miRNA editing in human cancer.

Gene Reporter Assays to Study miRNA Function.

This chapter describes one of the most reliable quantitative assays to test the silencing of a possible target gene by a specific miRNA using a luciferase reporter gene. The experimental procedure first consists in cloning both the wild-type and mutated forms of the 3'UTR of the miRNA-predicted mRNA target downstream of a firefly luciferase reporter. Next, each construct is co-transfected together with the miRNA into HeLa cells, and the reporter expression is monitored. Changes in luciferase levels will indicate whether or not an miRNA can bind to the UTR and regulate its expression.

ADAR Mediated RNA Editing Modulates MicroRNA Targeting in Human Breast Cancer.

RNA editing by RNA specific adenosine deaminase acting on RNA (ADAR) is increasingly being found to alter microRNA (miRNA) regulation. Editing of miRNA transcripts can affect their processing, as well as which messenger RNAs (mRNAs) they target. Further, editing of target mRNAs can also affect their complementarity to miRNAs. Notably, ADAR editing is often increased in malignancy with the effect of these RNA changes being largely unclear. In addition, numerous reports have now identified an array of miRNAs that directly contribute to various malignancies although the majority of their targets remain largely undefined. Here we propose that modulating the targets of miRNAs via mRNA editing is a frequent occurrence in cancer and an underappreciated participant in pathology. In order to more accurately characterize the relationship between these two regulatory processes, this study examined RNA editing events within mRNA sequences of two breast cancer cell lines (MCF-7 and MDA-MB-231) and determined whether or not these edits could modulate miRNA associations. Computational analyses of RNA-Seq data from these two cell lines identified over 50,000 recurrent editing sites within human mRNAs, and many of these were located in 3' untranslated regions (UTRs). When these locations were screened against the list of currently-annotated miRNAs we discovered that editing caused a subset (~9%) to have significant alterations to mRNA complementarity. One miRNA in particular, miR-140-3p, is known to be misexpressed in many breast cancers, and we found that mRNA editing allowed this miRNA to directly target the apoptosis inducing gene DFFA in MCF-7, but not in MDA-MB-231 cells. As these two cell lines are known to have distinct characteristics in terms of morphology, invasiveness and physiological responses, we hypothesized that the differential RNA editing of DFFA in these two cell lines could contribute to their phenotypic differences. Indeed, we confirmed through western blotting that inhibiting miR-140-3p increases expression of the DFFA protein product in MCF-7, but not MDA-MB-231, and further that inhibition of miR-140-3p also increases cellular growth in MCF-7, but not MDA-MB-231. Broadly, these results suggest that the creation of miRNA targets may be an underappreciated function of ADAR and may help further elucidate the role of RNA editing in tumor pathogenicity.

Hypoxia-Induced MicroRNA-210 Targets Neurodegenerative Pathways.

Hypoxia-regulated microRNA-210 (miR-210) is a highly conserved microRNA, known to regulate various processes under hypoxic conditions. Previously we found that miR-210 is also involved in honeybee learning and memory, raising the questions of how neural activity may induce hypoxia-regulated genes and how miR-210 may regulate plasticity in more complex mammalian systems. Using a pull-down approach, we identified 620 unique target genes of miR-210 in humans, among which there was a significant enrichment of age-related neurodegenerative pathways, including Huntington's, Alzheimer's, and Parkinson's diseases. We have also validated that miR-210 directly regulates various identified target genes of interest involved with neuronal plasticity, neurodegenerative diseases, and miR-210-associated cancers. This data suggests a potentially novel mechanism for how metabolic changes may couple plasticity to neuronal activity through hypoxia-regulated genes such as miR-210.

Inhibition of v-rel-Induced Oncogenesis through microRNA Targeting.

Several studies have shown that microRNA-targeting is an effective strategy for the selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. However, the exploitation of microRNA-targeting for the inhibition of transformation by oncogenic viruses has not been studied. The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transforming primary chicken spleen and bone marrow cells. Here, by engineering the target sequence of endogenous microRNA miR-142 downstream of the v-rel gene in a Replication-Competent ALV (avian leukosis virus) long terminal repeat (LTR) with a splice acceptor (RCAS) vector and using a v-rel-induced transformation model of chicken embryonic splenocyte cultures, we show that hematopoietic-specific miR-142 can inhibit the v-rel-induced transformation, and that this inhibition effect is due to the silencing of v-rel expression. The data supports the idea that microRNA-targeting can be used to inhibit viral oncogene-induced oncogenesis.