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1.  Detection of Genetic Variations in Coagulopathy-Related Genes Using Ramified Rolling Circle Amplification 
BioMed Research International  2014;2014:641090.
We evaluated single nucleotide polymorphism (SNP) detection via a target-capture, C-probe ligation, and RAM assay in a single-blind comparison to clinical samples that had been tested with FDA-cleared tests for up to 4 different vascular disease-related SNPs. In the RAM assay circulizable linear probes (C- or padlock probes) were annealed directly to genomic DNA, processed on a largely automated platform, and ligated C-probes were amplified by real-time RAM. After allele determinations were made with the experimental system, the sample genotypes were unblinded and the experimentally determined genotypes were found to be completely consistent with the FDA-cleared test results. The methods and results presented here show that a combination of C-probes, automated sample processing, and isothermal RAM provides a robust, and specific, nucleic acid detection platform that is compatible with automated DNA sample preparation and the throughput requirements of the clinical laboratory.
PMCID: PMC3955693  PMID: 24719880
2.  microRNA 126 Inhibits the Transition of Endothelial Progenitor Cells to Mesenchymal Cells via the PIK3R2-PI3K/Akt Signalling Pathway 
PLoS ONE  2013;8(12):e83294.
Endothelial progenitor cells (EPCs) are capable of proliferating and differentiating into mature endothelial cells, and they have been considered as potential candidates for coronary heart disease therapy. However, the transition of EPCs to mesenchymal cells is not fully understood. This study aimed to explore the role of microRNA 126 (miR-126) in the endothelial-to-mesenchymal transition (EndMT) induced by transforming growth factor beta 1 (TGFβ1).
Methods and Results
EndMT of rat bone marrow-derived EPCs was induced by TGFβ1 (5 ng/mL) for 7 days. miR-126 expression was depressed in the process of EPC EndMT. The luciferase reporter assay showed that the PI3K regulatory subunit p85 beta (PIK3R2) was a direct target of miR-126 in EPCs. Overexpression of miR-126 by a lentiviral vector (lenti-miR-126) was found to downregulate the mRNA expression of mesenchymal cell markers (α-SMA, sm22-a, and myocardin) and to maintain the mRNA expression of progenitor cell markers (CD34, CD133). In the cellular process of EndMT, there was an increase in the protein expression of PIK3R2 and the nuclear transcription factors FoxO3 and Smad4; PI3K and phosphor-Akt expression decreased, a change that was reversed markedly by overexpression of miR-126. Furthermore, knockdown of PIK3R2 gene expression level showed reversed morphological changes of the EPCs treated with TGFβ1, thereby giving the evidence that PIK3R2 is the target gene of miR-126 during EndMT process.
These results show that miR-126 targets PIK3R2 to inhibit EPC EndMT and that this process involves regulation of the PI3K/Akt signalling pathway. miR-126 has the potential to be used as a biomarker for the early diagnosis of intimal hyperplasia in cardiovascular disease and can even be a therapeutic tool for treating cardiovascular diseases mediated by the EndMT process.
PMCID: PMC3862723  PMID: 24349482
3.  Long Chain Fatty Acyl-CoA Synthetase 4 Is a Biomarker for and Mediator of Hormone Resistance in Human Breast Cancer 
PLoS ONE  2013;8(10):e77060.
The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.
PMCID: PMC3796543  PMID: 24155918
4.  Regulation of a Novel Androgen Receptor Target Gene, the Cyclin B1 Gene, through Androgen-Dependent E2F Family Member Switching 
Molecular and Cellular Biology  2012;32(13):2454-2466.
The malignant transformation of human prostatic epithelium is associated with the loss of androgen receptor (AR) in the surrounding stroma. However, the function and mechanisms of AR signaling in prostate cancer (PCa) stroma remain elusive. Here we report, by using proteomics pathway array analysis (PPAA), that androgen and its receptor inhibit the proliferation of prostate stromal cells through transcriptional suppression of cyclin B1, and we confirmed our findings at mRNA and protein levels using AR-negative or -positive primary prostate stromal cells. Furthermore, AR showed a negative correlation with cyclin B1 expression in stroma of human PCa samples in vivo. Mechanistically, we identify cyclin B1 as a bona fide AR target gene in prostate stromal cells. The negative regulation of cyclin B1 by AR is mediated through switching between E2F1 and E2F4 on the promoter of cyclin B1. E2F1 binds to the cyclin B1 promoter and maintains its expression and subsequent cell cycle progression in AR-negative stromal cells or AR-positive stromal cells when androgens are depleted. Upon stimulation with androgen in AR-positive stromal cells, E2F1 is displaced from the binding site by AR and replaced with E2F4, leading to the recruitment of the silencing mediator for retinoid and thyroid hormone receptor (SMRT)/histone deacetylase 3 (HDAC3) corepressor complex and repression of cyclin B1 at the chromatin level. The switch between E2F1 and E2F4 at the E2F binding site of the cyclin B1 promoter coincides with an androgen-dependent interaction between AR and E2F1 as well as the cytoplasmic-to-nuclear translocation of E2F4. Thus, we identified a novel mechanism for E2F factors in the regulation of cell cycle gene expression and cell cycle progression under the control of AR signaling.
PMCID: PMC3434485  PMID: 22508987
5.  Global Profiling of Signaling Networks: Study of Breast Cancer Stem Cells and Potential Regulation 
The Oncologist  2011;16(7):966-979.
Microarray analysis was used to examine gene expression profiles in breast cancer stem cells in an attempt to identify signature patterns and mechanisms of signaling networks in these cells.
There is overwhelming evidence that breast cancer may be driven by a small subset of breast cancer stem cells (BCSCs) that display stem/progenitor cell properties. In the present study, we identified the rare population of BCSCs, the so-called side population (SP) cells, using flow cytometry. Then, we used microarray analysis to study the differential gene expression profiles between SP and non-SP cells. Sixty-three probe sets showed a more than fourfold difference. Next, we compared the levels of proteins with Pathway Array using 154 antibodies, focusing on the proteins and phosphorylation sites that differed among SP cells, malignant mammary cells, and breast cancer tissues. Our results revealed that 40 proteins and phosphorylation sites were more than 1.5-fold different in SP cells than in non-SP cells. By comparing SP cells, MCF7 cells, and nontumorigenic MCF10A cells, we found 12 proteins that were significantly upregulated in SP cells; these proteins—cAMP-response element binding protein (CREB), cyclic AMP-dependent transcription factor 1, mesothelin, thyroid transcription factor 1, phosphorylated (p)-focal adhesion kinase, p38, Bad, p-CREB, p-protein kinase C (PKC)δ, Wee1, cell division cycle 42, and Twist—were more likely to play important roles in the signaling regulation of BCSCs. Further, 16 proteins and phosphoproteins showed differential expression in SP cells and tumor tissues. β-catenin, p-PKCα, and p-CREB were upregulated in both SP cells and breast tumors. Finally, we filtered the differential expression proteins, summarized the pathway interactions of these proteins, and rebuilt Path-Net in order to determine molecular mechanisms and core regulators. This process will allow us to identify signature patterns and mechanisms of signaling networks in BCSCs.
PMCID: PMC3228139  PMID: 21665913
Breast cancer; Stem cell; Signaling network; Proteome profile
6.  Loss of the Heparan Sulfate Sulfotransferase, Ndst1, in Mammary Epithelial Cells Selectively Blocks Lobuloalveolar Development in Mice 
PLoS ONE  2010;5(5):e10691.
Considerable evidence indicates that heparan sulfate is essential for the development of tissues consisting of branching ducts and tubules. However, there are few examples where specific sulfate residues regulate a specific stage in the formation of such tissues.
Methodology/Principal Findings
We examined the role of heparan sulfation in mammary gland branching morphogenesis, lactation and lobuloalveolar development by inactivation of heparan sulfate GlcNAc N-deacetylase/N-sulfotransferase genes (Ndst) in mammary epithelial cells using the Cre-loxP system. Ndst1 deficiency resulted in an overall reduction in glucosamine N-sulfation and decreased binding of FGF to mammary epithelial cells in vitro and in vivo. Mammary epithelia lacking Ndst1 underwent branching morphogenesis, filling the gland with ductal tissue by sexual maturity to the same extent as wildtype epithelia. However, lobuloalveolar expansion did not occur in Ndst1-deficient animals, resulting in insufficient milk production to nurture newly born pups. Lactational differentiation of isolated mammary epithelial cells occurred appropriately via stat5 activation, further supporting the notion that the lack of milk production was due to lack of expansion of the lobuloalveoli.
These findings demonstrate a selective, highly penetrant, cell autonomous effect of Ndst1-mediated sulfation on lobuloalveolar development.
PMCID: PMC2872662  PMID: 20502530
7.  Proteomics, pathway array and signaling network-based medicine in cancer 
Cell Division  2009;4:20.
Cancer is a multifaceted disease that results from dysregulated normal cellular signaling networks caused by genetic, genomic and epigenetic alterations at cell or tissue levels. Uncovering the underlying protein signaling network changes, including cell cycle gene networks in cancer, aids in understanding the molecular mechanism of carcinogenesis and identifies the characteristic signaling network signatures unique for different cancers and specific cancer subtypes. The identified signatures can be used for cancer diagnosis, prognosis, and personalized treatment. During the past several decades, the available technology to study signaling networks has significantly evolved to include such platforms as genomic microarray (expression array, SNP array, CGH array, etc.) and proteomic analysis, which globally assesses genetic, epigenetic, and proteomic alterations in cancer. In this review, we compared Pathway Array analysis with other proteomic approaches in analyzing protein network involved in cancer and its utility serving as cancer biomarkers in diagnosis, prognosis and therapeutic target identification. With the advent of bioinformatics, constructing high complexity signaling networks is possible. As the use of signaling network-based cancer diagnosis, prognosis and treatment is anticipated in the near future, medical and scientific communities should be prepared to apply these techniques to further enhance personalized medicine.
PMCID: PMC2780394  PMID: 19863813
9.  Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification 
Nucleic Acids Research  2006;34(11):e81.
Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.
PMCID: PMC1488881  PMID: 16822854
10.  Use of Ramification Amplification Assay for Detection of Escherichia coli O157:H7 and Other E. coli Shiga Toxin-Producing Strains 
Journal of Clinical Microbiology  2005;43(12):6086-6090.
Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx2). Our results showed that as few as 10 copies of stx2 could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx2 gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.
PMCID: PMC1317159  PMID: 16333102
11.  Detection of Chlamydia trachomatis by Isothermal Ramification Amplification Method: a Feasibility Study 
Journal of Clinical Microbiology  2002;40(1):128-132.
Chlamydia trachomatis is the leading cause of sexually transmitted disease in the United States. Effective screening for this agent can facilitate prompt treatment and prevent its sequelae. The recent introduction of liquid-based cytology has made possible the simultaneous screening of cervical intraepithelial lesions and detection of C. trachomatis in a single collection vial. In this study we determined whether cytological fluid could support DNA-based amplification for the detection of C. trachomatis. Three methods were compared, including ramification amplification (RAM), real-time PCR with molecular beacon, and Abbott’s ligase chain reaction (LCx). RAM is a novel, recently introduced, isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. Our results show that RAM can detect as few as 10 C. trachomatis elementary bodies in less than 2 h, comparable to results with real-time PCR. Thirty clinical specimens collected in PreservCyt solution were tested by LCx, real-time PCR, and RAM. Among 30 specimens, 15 were positive by PCR and LCx and 14 were positive by RAM. One specimen missed by RAM had an inadequate amount of residual cellular material. Our results show that nucleic acid amplification methods can serve to detect C. trachomatis and presumably other sexually transmitted agents in cytological fluid and that the RAM assay can be an alternative to PCR and LCx because of its simplicity and isothermal amplification.
PMCID: PMC120135  PMID: 11773105
12.  TT Virus Infection Is Widespread in the General Populations from Different Geographic Regions 
Journal of Clinical Microbiology  1999;37(8):2703-2705.
By PCR screening, we found an extremely high prevalence of TT virus (TTV) in the general populations from different geographic regions. This suggests that TTV may be a common DNA virus with no clear disease association in humans. TTV genotyping by phylogenetic analysis was also performed.
PMCID: PMC85320  PMID: 10405426

Results 1-12 (12)