An Ultrasensitive New DNA Microarray Chip Provides Gene Expression Profiles for Preoperative Esophageal Cancer Biopsies without RNA Amplification
Division of Gastroenterology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Oncology
(Impact Factor: 2.42).
01/2007; 73(5-6):366-75. DOI: 10.1159/000136156
Gene expression profiling using pretreatment biopsies has been limited due to their small sample sizes. This study evaluated the usefulness of an ultrasensitive new DNA microarray chip, which has a unique array structure, for the clinical diagnosis of esophageal cancer using preoperative biopsies.
Paired cancer and normal esophageal epithelial tissues from 56 patients who underwent esophagectomy and from 48 patients who underwent preoperative endoscopy were studied. Among 2 feature gene sets selected by a reference DNA chip discriminating malignant status of samples, 20 feature genes were selected for the development of the new DNA chip. The new DNA chip was hybridized with 0.1 mug of total RNA per slide without RNA amplification.
Twenty feature genes, including RRM-2 and XRCC-3, for the new DNA chip could discriminate cancer from noncancer at a 95.2% rate of accuracy in 42 biopsies (sensitivity 95.7%, specificity 94.7%). A receiver operating characteristic (ROC) curve analysis showed that the area under ROC curve for the prediction was 0.966.
The gene expression profiles from the preoperative biopsies could diagnose esophageal cancer accurately, using the ultrasensitive DNA chip without RNA amplification. This new DNA chip technology might contribute further to the development of customized therapeutic strategies for various cancer patients.
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Available from: Mitsuru Sasako
- "We performed microarray analyses of BM-extracted RNA in five cases of stage IV and those of stage I using Toray's platform (Nagino et al, 2006; Ito et al, 2007; Iwahashi et al, 2007). It is possible to monitor gene expression profiles even with very small amounts of RNA (0.1–0.01 mg of total RNA) without amplification. "
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We previously reported that bone marrow (BM) was a homing site for gastric cancer (GC) cells leading to haematogenous metastases. There has been little study that microRNAs regulated pathways in malignant cells or host cells in BM, and thereby regulated the progression of GC.
Both microRNA microarray and gene expression microarray analyses of total RNA from BM were conducted, comparing five early and five advanced GC patients. We focused on miR-144-ZFX axis as a candidate BM regulator of GC progression and validated the origin of the microRNA expression in diverse cell fractions (EpCAM+CD45−, EpCAM−CD45+, and CD14+) by magnetic-activated cell sorting (MACS).
Quantitative reverse-transcriptase (RT)–PCR analysis validated diminished miR-144 expression in stage IV GC patients with respect to stage I GC patients (t-test, P=0.02), with an inverse correlation to ZFX (ANOVA, P<0.01). Luciferase reporter assays in five GC cell lines indicated their direct binding and validated by western blotting. Pre-miR144 treatment and the resultant repression of ZFX in GC cell lines moderately upregulated their susceptibility to 5-fluorouracil chemotherapy. In MACS-purified BM fractions, the level of miR-144 expression was significantly diminished in disseminated tumour cell fraction (P=0.0005). Diminished miR-144 expression in 93 cases of primary GC indicated poor prognosis.
We speculate that disseminated cancer cells could survive in BM when low expression of miR-144 permits upregulation of ZFX. The regulation of the miR-144-ZFX axis in cancer cells has a key role in the indicator of the progression of GC cases.
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ABSTRACT: Multipotent mesenchymal stem cells (MSCs) are one of the most powerful tools in regeneration medicine. Their low differentiation efficiency, however, limits further application of MSCs in clinical therapy. Here we report that a much higher multipotent differentiation efficiency of MSCs to adult cells can be achieved using a 3D spheroid culture method based on photolithography and micropatterning techniques. MSC spheroid of precise dimension and uniform quality cultured on the microdomain substrates was prepared first, and then was induced into adipocytes and osteoblasts. Both gene expression results from RT-PCR and morphology observation results revealed that the 3D spheroid culture method could greatly improve differentiation efficiency. Gene expression profiles obtained from gene microarray analysis confirmed the high differentiation efficiency and revealed that MSCs induced in 3D spheroid culture system regulated gene expression not only by increasing the expression levels of genes related to adipogenesis and osteogenesis, but also by down-regulating the gene maintaining MSCs' self-renewal phenotypes. We conclude that our 3D spheroid culture system contributes to an optimization for efficient differentiation of MSCs, offers insight into the mechanism of efficient differentiation of engineered 3D culture system, and has promise for wide applications in regeneration medicine and drug discovery fields.
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ABSTRACT: To study the differential gene expression of adriamycin-resistance of breast cancer cell line MCF-7/ADR as compared with its parent cell MCF-7 and screen the related genes.
The in vitro inhibitory rate of MCF-7/ADR and MCF-7 cell lines to ADM (adriamycin) was examined by MIT assay. Then a cDNA microarray representing 14,755 genes was used to analyze the expression profiles of these two breast cancer cell lines.
The inhibitory rate of MCF-7/ ADR to ADM was lower than that of MCF-7(P <0.05)and the drug resistance to ADR of MCF-7/ADR was stronger than that of MCF-7. There were 2374 differential expression genes in MCF-7/adr and MCF-7 cell lines, of which 1099 genes upregulated with 99 genes 10-fold upregulated and 1275 genes downregulated with 71 genes 10-fold downregulated. The important upregulated genes were Bcl-2, GSTP1, c-myc, MMP-1 and NNMT while those downregulated ones p53, p21, p27 and CYPIA1.
Drug resistance of breast cancer is a complicated process involving the expression of many upregulated or downregulated genes. The application of gene chip in screening drug-resistant genes may be a new approach for selecting the optimal chemotherapeutic regimens. [Key words]
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