Proteomics-based analysis of invasion-related proteins in malignant gliomas.

One of the insidious biological features of gliomas is their potential to extensively invade normal brain tissue, yet molecular mechanisms that dictate this locally invasive behavior remain poorly understood. To investigate the molecular basis of invasion by malignant gliomas, proteomic analysis was performed using a pair of canine glioma subclones - J3T-1 and J3T-2 - that show different invasion phenotypes in rat brains but have similar genetic backgrounds. Two-dimensional protein electrophoresis of whole-cell lysates of J3T-1 (angiogenesis-dependent invasion phenotype) and J3T-2 (angiogenesis-independent invasion phenotype) was performed. Twenty-two distinct spots were recognized when significant alteration was defined as more than 1.5-fold change in spot intensity between J3T-1 and J3T-2. Four proteins that demonstrated increased expression in J3T-1, and 14 proteins that demonstrated increased expression in J3T-2 were identified using liquid chromatography-mass spectrometry analysis. One of the proteins identified was annexin A2, which was expressed at higher levels in J3T-1 than in J3T-2. The higher expression of annexin A2 in J3T-1 was corroborated by quantitative RT-PCR of the cultured cells and immunohistochemical staining of the rat brain tumors. Moreover, immunohistochemical analysis of human glioblastoma specimens showed that annexin A2 was expressed at high levels in the tumor cells that formed clusters around dilated vessels. These results reveal differences in the proteomic profiles between these two cell lines that might correlate with their different invasion profiles. Thus, annexin A2 may be related to angiogenesis-dependent invasion.

Identification of direct targets for the miR-17-92 cluster by proteomic analysis.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally repress the expression of target genes. Many miRNAs have been implicated in a number of diseases, including cancers. The miR-17-92 miRNA cluster is known as a body of oncogenic miRNAs, and has been shown to be overexpressed in several cancers, including lung cancer. Although the overexpression of miR-17-92 is clearly implicated in the development of lung cancer, only a few direct targets for the miR-17-92 cluster have been identified thus far. In this study, we examined miR-17-92 target profiles in SBC-3 small-cell lung cancer cells using a quantitative proteomic strategy to identify direct targets of the miR-17-92 cluster. By knocking down the expression of endogenous miR-19a, miR-20a and miR-92-1, which are contained in the cluster, 112 up-regulated proteins were detected and also identified as potential targets of these miRNAs. Among these candidate targets, we validated one direct target, RAB14. In conclusion, these findings suggest that proteomic approaches are valuable for identifying direct miRNA targets, and we were able to identify a novel direct target for the miR-92-1 using our proteomic strategy.

Novel direct targets of miR-19a identified in breast cancer cells by a quantitative proteomic approach.

The miR-17-92 cluster encodes 7 miRNAs inside a single polycistronic transcript, and is known as a group of oncogenic miRNAs that contribute to tumorigenesis in several cancers. However, their direct targets remain unclear, and it has been suggested that a single miRNA is capable of reducing the production of hundreds of proteins. The majority of reports on the identification of miRNA targets are based on computational approaches or the detection of altered mRNA levels, despite the fact that most miRNAs are thought to regulate their targets primarily by translational inhibition in higher organisms. In this study, we examined the target profiles of miR-19a, miR-20a and miR-92-1 in MCF-7 breast cancer cells by a quantitative proteomic strategy to identify their direct targets. A total of 123 proteins were significantly increased after the endogenous miR-19a, miR-20a and miR-92-1 were knocked down, and were identified as potential targets by two-dimensional electrophoresis and a mass spectrometric analysis. Among the upregulated proteins, four (PPP2R2A, ARHGAP1, IMPDH1 and NPEPL1) were shown to have miR-19a or miR-20a binding sites on their mRNAs. The luciferase activity of the plasmids with each binding site was observed to decrease, and an increased luciferase activity was observed in the presence of the specific anti-miRNA-LNA. A Western blot analysis showed the expression levels of IMPDH1 and NPEPL1 to increase after treatment with anti-miR-19a, while the expression levels of PPP2R2A and ARHGAP1 did not change. The expression levels of IMPDH1 and NPEPL1 did not significantly change by anti-miR-19a-LNA at the mRNA level. These results suggest that the IMPDH1 and NPEPL1 genes are direct targets of miR-19a in breast cancer, while the exogenous expression of these genes is not associated with the growth suppression of MCF-7 cells. Furthermore, our proteomic approaches were shown to be valuable for identifying direct miRNA targets.

An outbreak of canine distemper virus in tigers (Panthera tigris): possible transmission from wild animals to zoo animals.

Canine distemper virus (CDV), a morbillivirus that causes one of the most contagious and lethal viral diseases known in canids, has an expanding host range, including wild animals. Since December 2009, several dead or dying wild raccoon dogs (Nyctereutes procyonoides) were found in and around one safari-style zoo in Japan, and CDV was isolated from four of these animals. In the subsequent months (January to February 2010), 12 tigers (Panthera tigris) in the zoo developed respiratory and gastrointestinal diseases, and CDV RNA was detected in fecal samples of the examined tigers. In March 2010, one of the tigers developed a neurological disorder and died; CDV was isolated from the lung of this animal. Sequence analysis of the complete hemagglutinin (H) gene and the signal peptide region of the fusion (F) gene showed high homology among these isolates (99.8-100%), indicating that CDV might have been transmitted from raccoon dog to tiger. In addition, these isolates belonged to genotype Asia-1 and had lower homology (<90%) to the vaccine strain (Onderstepoort). Seropositivity of lions (Panthera leo) in the zoo and wild bears (Ursus thibetanus) captured around this area supported the theory that a CDV epidemic had occurred in many mammal species in and around the zoo. These results indicate a risk of CDV transmission among many animal species, including large felids and endangered species.

Experimental infection of Japanese encephalitis virus in dogs.

A previous serosurvey of Japanese encephalitis virus (JEV) among dogs suggested that dogs are well suited for use as sentinels for assessing the risk of JEV transmission to humans. To examine the clinical symptoms and duration of anti-JEV antibodies in dogs, three dogs were experimentally challenged with JEV. All JEV-infected dogs did not show any clinical signs or abnormal blood tests, except for C-reactive protein. Virus-neutralization titers rapidly increased and were maintained until 70 days postinfection, and neither the virus nor the viral genome was detected in blood. Thus, since dogs live in close proximity to humans as companion animals, they are well suited for use as sentinels for surveying the human risk of JEV infection.

Full genome sequence and virulence analyses of the recent equine isolate of Japanese encephalitis virus.

In the past 25 years, there has been only one case of Japanese encephalitis in horses in Japan. We determined the full genome sequence of the Japanese encephalitis virus (JEV) strain JEV/eq/Tottori/2003 isolated from an afflicted horse and also analyzed its virulence in mice. The sequence analysis showed that the genome of JEV/eq/Tottori/2003 is similar to that of genotype I, a dominant genotype of JEV presently circulating in Japan. Its neurovirulence, but not neuroinvasiveness, was still as high as it was for genotype III, thus indicating the necessity for continuation of a vaccination program of horses against JEV.