Identification of Kinesin Family Member 2A (KIF2A) as a Promising Therapeutic Target for Osteosarcoma.

BACKGROUND: Osteosarcoma is known as a type of common human bone malignancy, and more therapeutic targets are still required to combat this disease. In recent years, the involvement of KIF2A in cancer progression has been widely revealed; however, its potential effect on osteosarcoma development remains unknown. This study is to assess the KIF2A expression levels in human osteosarcoma tissues and explore its potential role in osteosarcoma development. METHODS: Immunohistochemical (IHC) assays were conducted to evaluate the expression levels of KIF2A in a total of 74 samples of osteosarcoma tissues and adjacent nontumor tissues. According to the staining intensity in tumor tissues, patients were divided into highly expressed and low expression KIF2A groups. The possible links between the KIF2A expression and the clinical pathological features were explored and analyzed, and the effects of KIF2A on osteosarcoma cell proliferation, migration, and invasion were detected through colony formation assay, MTT assay, wound closure assay, and transwell assay, respectively. The effects of KIF2A on tumor growth and metastasis were detected by the use of animal models. RESULTS: KIF2A was highly expressed in human osteosarcoma tissues. Meanwhile, KIF2A was obviously correlated to the tumor size (P = 0.001∗) and clinical stage (P = 0.014∗) of osteosarcoma patients. Our results also revealed that the ablation of KIF2A dramatically blocked the proliferation, migration, and invasion capacity of osteosarcoma cells in vitro and blocked tumor growth and metastasis in mice. CONCLUSIONS: We investigated the involvement of KIF2A in the development and metastasis of osteosarcoma and therefore thought KIF2A as a promising therapeutic target for osteosarcoma treatment.

Immunologic properties and therapeutic efficacy of a multivalent epitope-based vaccine against four Helicobacter pylori adhesins (urease, Lpp20, HpaA, and CagL) in Mongolian gerbils.

BACKGROUND: Therapeutic vaccination is a desirable alternative for controlling Helicobacter pylori (H. pylori) infection. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and adhesins, which are on the surface of H. pylori, play a pivotal role in binding to human gastric mucosa. MATERIALS AND METHODS: In the present study, we constructed a multivalent epitope-based vaccine named CFAdE with seven carefully selected antigenic fragments from four H. pylori adhesins (urease, Lpp20, HpaA and CagL). The specificity, immunogenicity and ability to produce neutralizing antibodies of CFAdE were evaluated in BALB/c mice. After that, its therapeutic efficacy and protective immune mechanisms were explored in H. pylori-infected Mongolian gerbils. RESULTS: The results indicated that CFAdE could induce comparatively high levels of specific antibodies against urease, Lpp20, HpaA and CagL. Additionally, oral therapeutic immunization with CFAdE plus polysaccharide adjuvant (PA) significantly decreased H. pylori colonization compared with oral immunization with urease plus PA, and the protection was correlated with IgG and sIgA antibody and antigen-specific CD4+ T cells. CONCLUSIONS: This study indicated that the multivalent epitope-based vaccine, which targeted multiple adhesins in adherence of H. pylori to the gastric mucosa, is more effective than the univalent vaccine targeting urease only. This multivalent epitope-based vaccine may be a promising therapeutic candidate vaccine against H. pylori infection.

SPIRE, a modular pipeline for eQTL analysis of RNA-Seq data, reveals a regulatory hotspot controlling miRNA expression in C. elegans.

The interpretation of genome-wide association study is difficult, as it is hard to understand how polymorphisms can affect gene regulation, in particular for trans-regulatory elements located far from their controlling gene. Using RNA or protein expression data as phenotypes, it is possible to correlate their variations with specific genotypes. This technique is usually referred to as expression Quantitative Trait Loci (eQTLs) analysis and only few packages exist for the integration of genotype patterns and expression profiles. In particular, tools are needed for the analysis of next-generation sequencing (NGS) data on a genome-wide scale, which is essential to identify eQTLs able to control a large number of genes (hotspots). Here we present SPIRE (Software for Polymorphism Identification Regulating Expression), a generic, modular and functionally highly flexible pipeline for eQTL processing. SPIRE integrates different univariate and multivariate approaches for eQTL analysis, paying particular attention to the scalability of the procedure in order to support cis- as well as trans-mapping, thus allowing the identification of hotspots in NGS data. In particular, we demonstrated how SPIRE can handle big association study datasets, reproducing published results and improving the identification of trans-eQTLs. Furthermore, we employed the pipeline to analyse novel data concerning the genotypes of two different C. elegans strains (N2 and Hawaii) and related miRNA expression data, obtained using RNA-Seq. A miRNA regulatory hotspot was identified in chromosome 1, overlapping the transcription factor grh-1, known to be involved in the early phases of embryonic development of C. elegans. In a follow-up qPCR experiment we were able to verify most of the predicted eQTLs, as well as to show, for a novel miRNA, a significant difference in the sequences of the two analysed strains of C. elegans. SPIRE is publicly available as open source software at , together with some example data, a readme file, supplementary material and a short tutorial.

A protocol for chemical mutagenesis in Strongyloides ratti.

Genetic analysis using experimentally induced mutations has been a most valuable tool in the analysis of various organisms. However, genetic analysis of endoparasitic organisms tends to be difficult because of the limited accessibility of the sexually reproducing adults, which are normally located within the host. Nematodes of the genera Strogyloides and Parastrongyloides represent an exception to this because they can form facultative free-living sexually reproducing generations in between parasitic generations. Here we present a protocol for the chemical mutagenesis of Strongyloides ratti. Further we evaluate the feasibility of identifying the induced mutations by whole genome re-sequencing.

Conserved miRNAs are candidate post-transcriptional regulators of developmental arrest in free-living and parasitic nematodes.

Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions, Caenorhabditis elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, Pristionchus pacificus, and Strongyloides ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs, and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer versus nondauer stages and infective larvae versus free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved "dauer-infective" expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest and long-term survival in free-living and parasitic nematodes.

Global profiling of miRNAs and the hairpin precursors: insights into miRNA processing and novel miRNA discovery.

MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5' tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences.