The Dugesia ryukyuensis Database as a Molecular Resource for Studying Switching of the Reproductive System

The planarian Dugesia ryukyuensis reproduces both asexually and sexually, and can switch from one mode of reproduction to the other. We recently developed a method for experimentally switching reproduction of the planarian from the asexual to the sexual mode. We constructed a cDNA library from sexualized D. ryukyuensis and sequenced and analyzed 8,988 expressed sequence tags (ESTs). The ESTs were analyzed and grouped into 3,077 non-redundant sequences, leaving 1,929 singletons that formed the basis of unigene sets. Fifty-six percent of the cDNAs analyzed shared similarity (E-value<1E -20) with sequences deposited in NCBI. Highly redundant sequences encoded granulin and actin, which are expressed in the whole body, and other redundant sequences encoded a Vasa-like protein, which is known to be a component of germ-line cells and is expressed in the ovary, and Y-protein, which is expressed in the testis. The sexualized planarian expressed sequence tag database (http://planaria.bio.keio.ac.jp/planaria/) is an open-access, online resource providing access to sequence, classification, clustering, and annotation data. This database should constitute a powerful tool for analyzing sexualization in planarians

Use of Non-Amplified RNA Samples for Microarray Analysis of Gene Expression

Demand for high quality gene expression data has driven the development of revolutionary microarray technologies. The quality of the data is affected by the performance of the microarray platform as well as how the nucleic acid targets are prepared. The most common method for target nucleic acid preparation includes in vitro transcription amplification of the sample RNA. Although this method requires a small amount of starting material and is reported to have high reproducibility, there are also technical disadvantages such as amplification bias and the long, laborious protocol. Using RNA derived from human brain, breast and colon, we demonstrate that a non-amplification method, which was previously shown to be inferior, could be transformed to a highly quantitative method with a dynamic range of five orders of magnitude. Furthermore, the correlation coefficient calculated by comparing microarray assays using non-amplified samples with qRT-PCR assays was approximately 0.9, a value much higher than when samples were prepared using amplification methods. Our results were also compared with data from various microarray platforms studied in the MicroArray Quality Control (MAQC) project. In combination with micro-columnar 3D-Gene™ microarray, this non-amplification method is applicable to a variety of genetic analyses, including biomarker screening and diagnostic tests for cancer

MicroRNA-8073: Tumor suppressor and potential therapeutic treatment.

The comprehensive screening of intracellular and extracellular microRNAs was performed to identify novel tumor suppressors. We found that miR-8073 was present in exosome and predominantly exported from colorectal cancer cells. Treatment with a synthetic miR-8073 mimic resulted in a dramatic decrease in the proliferation of various types of cancer cells, which was not observed in similarly treated normal cells. As little is known about the biological functions of miR-8073, its target mRNAs were analyzed by both mRNA expression and in silico sequence analyses, leading to five probable target candidates (FOXM1, MBD3, CCND1, KLK10, and CASP2) that enhance survival during the regulation of the cell cycle, cell proliferation, and apoptosis. We experimentally confirmed that miR-8073 binds the 3'-UTR of each of these mRNA target candidates and that the introduction of a synthetic miR-8073 mimic into cancer cells reduced levels of protein expression. Finally, the antiproliferative effects of miR-8073 were validated in vivo: the subcutaneous injection of a synthetic miR-8073 mimic suppressed colorectal tumor volume to 43% in tumor-bearing xenografted mice. These results suggest that because miR-8073 binds, and thus reduces the levels of, these oncogenic targets, cancer cells must actively downregulate miR-8073 as a survival mechanism. The introduction of miR-8073 into tumors could thus inhibit tumor growth, indicating its great potential for cancer therapeutics

Spearman's rank correlation between the microarray assay and qRT-PCR assay.

<p>UHRR, HBRR, breast, and colon RNA derived from human tissue or cells were processed by the non-amplification (black bar), the 1-round amplification (grey bar), or the 2-round amplification (white bar) method, analyzed by microarray. The obtained PolR2A-normalized signal intensity (log₂) of the 42 genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031397#pone.0031397.s001" target="_blank">Table S1</a>) was compared with ΔCt values of the same samples analyzed by qRT-PCR assays.</p

Qualitative concordance of detected genes by three sample preparation methods.

<p>Among 24,267 gene spots on the microarray used, genes that were detected in at least 60% of the sample replicates were used to create the Venn diagram.</p

MicroRNA Markers for the Diagnosis of Pancreatic and Biliary-Tract Cancers

<div><p>It is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology. Utilizing microRNA (miRNA) markers that are stably present in peripheral blood, we aimed to identify pancreatic and biliary-tract cancers in patients. With “3D-Gene”, a highly sensitive microarray, we examined comprehensive miRNA expression profiles in 571 serum samples obtained from healthy patients, patients with pancreatic, biliary-tract, or other digestive cancers, and patients with non-malignant abnormalities in the pancreas or biliary tract. The samples were randomly divided into training and test cohorts, and candidate miRNA markers were independently evaluated. We found 81 miRNAs for pancreatic cancer and 66 miRNAs for biliary-tract cancer that showed statistically different expression compared with healthy controls. Among those markers, 55 miRNAs were common in both the pancreatic and biliary-tract cancer samples. The previously reported miR-125a-3p was one of the common markers; however, it was also expressed in other types of digestive-tract cancers, suggesting that it is not specific to cancer types. In order to discriminate the pancreato-biliary cancers from all other clinical conditions including the healthy controls, non-malignant abnormalities, and other types of cancers, we developed a diagnostic index using expression profiles of the 10 most significant miRNAs. A combination of eight miRNAs (miR-6075, miR-4294, miR-6880-5p, miR-6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR-4476) achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively. In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort. Our results suggest that the assessment of these miRNA markers is clinically valuable to identify patients with pancreato-biliary cancers who could benefit from surgical intervention.</p></div

Brain metastasis-related microRNAs in patients with advanced breast cancer.

Brain metastasis is a major distant metastasis occurring in patients with advanced breast cancer, and is associated with poor prognosis. MicroRNAs (miRNAs) have a strong influence on various oncological functions and have been reported as potential biomarkers for detecting distant metastasis. Specific biomarkers and unique miRNAs for brain metastasis have yet to be reported. The aim of this study was to identify novel miRNAs in serum, to assist in the diagnosis of brain metastasis in patients with advanced breast cancer. We retrospectively analyzed the medical records of patients with breast cancer and collected clinical data. In addition, we evaluated serum miRNA profiles in patients with breast cancer, with and without brain metastasis, using high-sensitivity microarrays. All patients underwent computed tomography or magnetic resonance imaging brain imaging tests. A total of 51 serum samples from patients with breast cancer and brain metastasis, stored in the National Cancer Center Biobank, were used, and 28 serum samples were obtained from controls without brain metastasis. Two miRNAs, miR-4428 and miR-4480, could significantly distinguish patients with brain metastasis, with area under the receiver operating characteristic curve (AUC) values of 0.779 and 0.781, respectively, while a combination of miR-4428 and progesterone receptor had an AUC value of 0.884. No significant correlations were identified between the expression levels of these two miRNAs in serum and clinical data. We conclude that serum miR-4428 and miR-4480 may be useful as biomarkers for predicting brain metastasis in patients with breast cancer

Plots of miR-6075 (A) and miR-125a-3p (B) expression signals on a log2 scale in patients with pancreato-biliary cancers, healthy control individuals, and patients with colon, stomach, esophageal, or liver cancer, or with non-malignant abnormalities in either the pancreas or the biliary tract.

<p>Plots of miR-6075 (A) and miR-125a-3p (B) expression signals on a log2 scale in patients with pancreato-biliary cancers, healthy control individuals, and patients with colon, stomach, esophageal, or liver cancer, or with non-malignant abnormalities in either the pancreas or the biliary tract.</p