Lipid Nanoparticle-Mediated Delivery of Anti-miR-17 Family Oligonucleotide Suppresses Hepatocellular Carcinoma Growth.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies with poor prognosis and urgent unmet medical need. Aberrant expression of multiple members of the miR-17 family are frequently observed in HCC, and their overexpression promotes tumorigenic properties of HCC cells. However, whether pharmacologic inhibition of the miR-17 family inhibits HCC growth remains unknown. In this study, we validated that the miR-17 family was upregulated in a subset of HCC tumors and cell lines and its inhibition by a tough decoy inhibitor suppressed the growth of Hep3B and HepG2 cells, which overexpress the miR-17 family. Furthermore, inhibition of the miR-17 family led to a global derepression of direct targets of the family in all three HCC cell lines tested. Pathway analysis of the deregulated genes indicated that the genes associated with TGFß signaling pathway were highly enriched in Hep3B and HepG2 cells. A miR-17 family target gene signature was established and used to identify RL01-17(5), a lipid nanoparticle encapsulating a potent anti-miR-17 family oligonucleotide. To address whether pharmacologic modulation of the miR-17 family can inhibit HCC growth, RL01-17(5) was systemically administrated to orthotopic Hep3B xenografts. Suppression of Hep3B tumor growth in vivo was observed and tumor growth inhibition correlated with induction of miR-17 family target genes. Together, this study provides proof-of-concept for targeting the miR-17 family in HCC therapy. Mol Cancer Ther; 16(5); 905-13. ©2017 AACR.

microRNA-17 family promotes polycystic kidney disease progression through modulation of mitochondrial metabolism.

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent genetic cause of renal failure. Here we identify miR-17 as a target for the treatment of ADPKD. We report that miR-17 is induced in kidney cysts of mouse and human ADPKD. Genetic deletion of the miR-17∼92 cluster inhibits cyst proliferation and PKD progression in four orthologous, including two long-lived, mouse models of ADPKD. Anti-miR-17 treatment attenuates cyst growth in short-term and long-term PKD mouse models. miR-17 inhibition also suppresses proliferation and cyst growth of primary ADPKD cysts cultures derived from multiple human donors. Mechanistically, c-Myc upregulates miR-17∼92 in cystic kidneys, which in turn aggravates cyst growth by inhibiting oxidative phosphorylation and stimulating proliferation through direct repression of Pparα. Thus, miR-17 family is a promising drug target for ADPKD, and miR-17-mediated inhibition of mitochondrial metabolism represents a potential new mechanism for ADPKD progression.

Relocation facilitates the acquisition of short cis-regulatory regions that drive the expression of retrogenes during spermatogenesis in Drosophila.

Retrogenes are functional processed copies of genes that originate via the retrotranscription of an mRNA intermediate and often exhibit testis-specific expression. Although this expression pattern appears to be favored by selection, the origin of such expression bias remains unexplained. Here, we study the regulation of two young testis-specific Drosophila retrogenes, Dntf-2r and Pros28.1A, using genetic transformation and the enhanced green fluorescent protein reporter gene in Drosophila melanogaster. We show that two different short (<24 bp) regions upstream of the transcription start sites (TSSs) act as testis-specific regulatory motifs in these genes. The Dntf-2r regulatory region is similar to the known ß2 tubulin 14-bp testis motif (ß2-tubulin gene upstream element 1 [ß2-UE1]). Comparative sequence analyses reveal that this motif was already present before the Dntf-2r insertion and was likely driving the transcription of a noncoding RNA. We also show that the ß2-UE1 occurs in the regulatory regions of other testis-specific retrogenes, and is functional in either orientation. In contrast, the Pros28.1A testes regulatory region in D. melanogaster appears to be novel. Only Pros28.1B, an older paralog of the Pros28.1 gene family, seems to carry a similar regulatory sequence. It is unclear how the Pros28.1A regulatory region was acquired in D. melanogaster, but it might have evolved de novo from within a region that may have been preprimed for testes expression. We conclude that relocation is critical for the evolutionary origin of male germline-specific cis-regulatory regions of retrogenes because expression depends on either the site of the retrogene insertion or the sequence changes close to the TSS thereafter. As a consequence we infer that positive selection will play a role in the evolution of these regulatory regions and can often act from the moment of the retrocopy insertion.

Cyclosporine attenuates cardiomyocyte hypertrophy induced by RAF1 mutants in Noonan and LEOPARD syndromes.

RAS activation is implicated in physiologic and pathologic cardiac hypertrophy. Cross-talk between the Ras and calcineurin pathways, the latter also having been implicated in cardiac hypertrophy, has been suspected for pathologic hypertrophy. Our recent discovery that germ-line mutations in RAF1, which encodes a downstream RAS effector, cause Noonan and LEOPARD syndromes with a high prevalence of hypertrophic cardiomyopathy provided an opportunity to elaborate the role of RAF1 in cardiomyocyte biology. Here, we characterize the role of RAF1 signaling in cardiomyocyte hypertrophy with an aim of identifying potential therapeutic targets. We modeled hypertrophic cardiomyopathy by infecting neonatal and adult rat cardiomyocytes (NRCMs and ARCMs, respectively) with adenoviruses encoding wild-type RAF1 and three Noonan/LEOPARD syndrome-associated RAF1 mutants (S257L, D486N or L613V). These RAF1 proteins, except D486N, engendered cardiomyocyte hypertrophy. Surprisingly, these effects were independent and dependent of mitogen activated protein kinases in NRCMs and ARCMs, respectively. Inhibiting Mek1/2 in RAF1 overexpressing cells blocked hypertrophy in ARCMs but not in NRCMs. Further, we found that endogenous and heterologously expressed RAF1 complexed with calcineurin, and RAF1 mutants causing hypertrophy signaled via nuclear factor of activated T cells (Nfat) in both cell types. The involvement of calcineurin was also reflected by down regulation of Serca2a and dysregulation of calcium signaling in NRCMs. Furthermore, treatment with the calcineurin inhibitor cyclosporine blocked hypertrophy in NRCMs and ARCMs overexpressing RAF1. Thus, we have identified calcineurin as a novel interaction partner for RAF1 and established a mechanistic link and possible therapeutic target for pathological cardiomyocyte hypertrophy induced by mutant RAF1. This article is part of a Special Issue entitled 'Possible Editorial'.

The limited distribution of Helitrons to vesper bats supports horizontal transfer.

Transposable elements (TEs) have the unique ability to move and replicate within the genome and therefore engender dramatic changes to genome architecture. Among different types of TEs, rolling-circle transposons (Helitrons) are well known for their ability to capture and amplify host gene fragments. Bioinformatic analysis revealed that Helitrons constitute ~3% of the Myotis lucifugus, (little brown bat) genome, while no Helitrons were found in any of the other 44+ sequenced mammalian genomes. Recently horizontal transfer has been implicated for some of the M. lucifugus Helitrons, in part explaining this disparate distribution among mammals. The purpose of this work is to determine both the distribution of Helitrons among bats and to estimate the number of independent invasions. We employed a combination of in silico, PCR and hybridization based techniques to identify Helitrons from diverse bat species belonging to ten different families. This work reveals that Helitrons invaded the vesper bat lineage, at least once. Indeed, Helitrons were not identified in the sister taxa 'Miniopterus', which suggests that the amplification of Helibat occurred (30-36 mya) only in the vesper bat lineage. The estimated age of amplification of the Helibats and the rapid radiation of vesper bats are roughly coincidental and suggest that the invasion and amplification of these elements might have influenced their evolutionary trajectory potentially contributing to phenotypic and genotypic diversity.

Turnover and lineage-specific broadening of the transcription start site in a testis-specific retrogene.

Proteasomes are large multisubunit complexes responsible for regulated protein degradation. Made of a core particle (20S) and regulatory caps (19S), proteasomal proteins are encoded by at least 33 genes, of which 12 have been shown to have testis-specific isoforms in Drosophila melanogaster. Pros28.1A (also known as Prosalpha4T1), a young retroduplicate copy of Pros28.1 (also known as Prosalpha4), is one of these isoforms. It is present in the D. melanogaster subgroup and was previously shown to be testis-specific in D. melanogaster. Here, we show its testis-specific transcription in all D. melanogaster subgroup species. Due to this conserved pattern of expression in the species harboring this insertion, we initially expected that a regulatory region common to these species evolved prior to the speciation event. We determined that the region driving testis expression in D. melanogaster is not far from the coding region (within 272 bp upstream of the ATG). However, different Transcription Start Sites (TSSs) are used in D. melanogaster and D. simulans, and a "broad" transcription start site is used in D. yakuba. These results suggest one of the following scenarios: (1) there is a conserved motif in the 5' region of the gene that can be used as an upstream or downstream element or at different distance depending on the species; (2) different species evolved diverse regulatory sequences for the same pattern of expression (i.e., "TSS turnover"); or (3) the transcription start site can be broad or narrow depending on the species. This work reveals the difficulties of studying gene regulation in one species and extrapolating those findings to close relatives.