Restoring expression of miR-16: a novel approach to therapy for malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is recalcitrant to treatment and new approaches to therapy are needed. Reduced expression of miR-15/16 in a range of cancer types has suggested a tumour suppressor function for these microRNAs, and re-expression has been shown to inhibit tumour cell proliferation. The miR-15/16 status in MPM is largely unknown. MATERIALS AND METHODS: MicroRNA expression was analysed by TaqMan-based RT-qPCR in MPM tumour specimens and cell lines. MicroRNA expression was restored in vitro using microRNA mimics, and effects on proliferation, drug sensitivity and target gene expression were assessed. Xenograft-bearing mice were treated with miR-16 mimic packaged in minicells targeted with epidermal growth factor receptor (EGFR)-specific antibodies. RESULTS: Expression of the miR-15 family was consistently downregulated in MPM tumour specimens and cell lines. A decrease of 4- to 22-fold was found when tumour specimens were compared with normal pleura. When MPM cell lines were compared with the normal mesothelial cell line MeT-5A, the downregulation of miR-15/16 was 2- to 10-fold. Using synthetic mimics to restore miR-15/16 expression led to growth inhibition in MPM cell lines but not in MeT-5A cells. Growth inhibition caused by miR-16 correlated with downregulation of target genes including Bcl-2 and CCND1, and miR-16 re-expression sensitised MPM cells to pemetrexed and gemcitabine. In xenograft-bearing nude mice, intravenous administration of miR-16 mimics packaged in minicells led to consistent and dose-dependent inhibition of MPM tumour growth. CONCLUSIONS: The miR-15/16 family is downregulated and has tumour suppressor function in MPM. Restoring miR-16 expression represents a novel therapeutic approach for MPM.

Effects of sequence alignment and structural domains of ribosomal DNA on phylogeny reconstruction for the protozoan family sarcocystidae.

Finding correct species relationships using phylogeny reconstruction based on molecular data is dependent on several empirical and technical factors. These include the choice of DNA sequence from which phylogeny is to be inferred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny tools provide a way of testing certain hypotheses regarding the relationship among the organisms for which phenotypic characters demonstrate conflicting evolutionary information. The protozoan family Sarcocystidae is one such group for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particularly based on small-subunit ribosomal DNA, have not resolved all of the relationships within this family. Analysis of more than one gene is necessary in order to obtain a robust species signal, and some DNA sequences may not be appropriate in terms of their phylogenetic information content. With this in mind, we tested the informativeness of our chosen molecule, the large-subunit ribosomal DNA (lsu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The segments of sequence applied correspond to areas of higher nucleotide variation in a secondary-structure alignment involving 21 taxa. We found that subdivision of the entire lsu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the lsu rDNA for certain clades, such as the one encompassing the subfamily Toxoplasmatinae. Consequently, the removal of any segment of the alignment compromises the final tree topology. We also tested the effect of using two different alignment procedures (CLUSTAL W and the structure alignment using DCSE) and three different tree-building methods on the final tree topology. This work shows that congruence between different methods in the formation of clades may be a feature of robust topology; however, a sequence alignment based on primary structure may not be comparing homologous nucleotides even though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammondia and again bring to question the relationships of Sarcocystis muris, Isospora felis, and Neospora caninum. In addition, results based on phylogenetic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monophyletic with Sarcocystis species using mammalian definitive hosts if the genus Frenkelia is synonymized with Sarcocystis.

Phylogenetic analysis based on full-length large subunit ribosomal RNA gene sequence comparison reveals that Neospora caninum is more closely related to Hammondia heydorni than to Toxoplasma gondii.

Since its first description in the late 1980s, Neospora caninum has been recognised as a prominent tissue cyst-forming parasite due to its ability to induce congenital disease and abortion in animals, especially cattle. It is found worldwide and is a cause of significant economic losses for the livestock industry. However, its place within the family Sarcocystidae, like that of several other taxa, remains unresolved. Neospora caninum shares several morphological and life cycle characters with Hammondia heydorni, although it is most commonly thought of as being a close relative of Toxoplasma gondii. This study presents information regarding the phylogenetic relationship of N. caninum to species currently classified into the genus Hammondia, as well as to two strains (RH and ME49) of T. gondii based on the full-length large subunit ribosomal RNA gene. Phylogenetic analyses using two alignment strategies and three different tree-building methods showed that the two species in the genus Hammondia are paraphyletic. Neospora caninum was shown to form a monophyletic clade with H. heydorni instead of T. gondii, which in turn was shown to be most closely related to H. hammondi. The finding that N. caninum and H. heydorni are closely related phylogenetically may aid the elucidation of currently unknown aspects of their biology and epidemiology, and suggests that H. heydorni should be considered in the differential diagnosis of N. caninum from other apicomplexan parasites.

Phylogenetic relationships of the genus Frenkelia: a review of its history and new knowledge gained from comparison of large subunit ribosomal ribonucleic acid gene sequences.

The different genera currently classified into the family Sarcocystidae include parasites which are of significant medical, veterinary and economic importance. The genus Sarcocystis is the largest within the family Sarcocystidae and consists of species which infect a broad range of animals including mammals, birds and reptiles. Frenkelia, another genus within this family, consists of parasites that use rodents as intermediate hosts and birds of prey as definitive hosts. Both genera follow an almost identical pattern of life cycle, and their life cycle stages are morphologically very similar. However, the relationship between the two genera remains unresolved because previous analyses of phenotypic characters and of small subunit ribosomal ribonucleic acid gene sequences have questioned the validity of the genus Frenkelia or the monophyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus. We therefore subjected the large subunit ribosomal ribonucleic acid gene sequences of representative taxa in these genera to phylogenetic analyses to ascertain a definitive relationship between the two genera. The full length large subunit ribosomal ribonucleic acid gene sequences obtained were aligned using Clustal W and Dedicated Comparative Sequence Editor secondary structure alignments. The Dedicated Comparative Sequence Editor alignment was then split into two data sets, one including helical regions, and one including non-helical regions, in order to determine the more informative sites. Subsequently, all four alignment data sets were subjected to different tree-building algorithms. All of the analyses produced trees supporting the paraphyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus and, thus, call for a revision of the current definition of these genera. However, an alternative, more parsimonious and more appropriate solution to the Sarcocystis/Frenkelia controversy is to synonymise the genus Frenkelia with the genus Sarcocystis.