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It is known that Rho-associated kinase (ROCK) signaling plays a fundamental role in regulating cell morphology, adhesion, and motility and that aberrant expression of ROCK is related to tumor metastases and poor clinical outcome. Researchers at Tufts University proposed that ROCK may enhance the metastatic propensity of breast cancer cells by promoting the c-Myc pathway, including transcription of c-Myc–regulated miRNAs (miR-17-92 cluster)¹. They used LC Sciences microRNA microarray services to show a 2- to 6-fold increase in expression of the miR-17-92 cluster in two metastatic breast cancer cell lines compared with non metastatic cells. The miR-17-92 expression in the three cell lines was validated by endpoint and qRT-PCR. Additionally, they showed that an anti-miR can block the ROCK signaling pathway resulting in decreased breast cancer cell invasion/ migration and metastasis. Therefore, inhibition of ROCK-mediated signaling appears to be a promising and potentially specific approach to suppress breast cancer metastases.

Numerous miRNAs have been shown to act as positive and negative regulators of the phosphoinositide-3-kinase (PI3K)/Akt-signaling pathway. The miR-29 family and miR-126 negatively affect the pathway through repression of PI3K regulatory subunits and many miRNAs positively influence PI3K/Akt signaling by targeting phosphatase and tensin homolog (PTEN) for inhibition which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling. Researchers at the University of Texas Southwestern Medical Center, Dallas made use of LC Sciences microRNA microarray analysis and found that miR-486 showed a dramatic increase in expression in myocardin-related transcription factor-A (MRTF-A)–transduced cells². The induction of miR- 486 by MRTF-A was confirmed by Northern blot and real-time RT– PCR. PTEN is a strongly predicted target of miR-486 and they further demonstrated that inhibition of miR-486 expression enhances the expression of PTEN and dampens signaling through the PI3K/Akt-signaling pathway.  These findings implicate miR- 486 as another potential modulator of PI3K/Akt signaling.

1. Liu S, Goldstein RH, Scepansky EM, Rosenblatt M.  (2009) Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone.  Cancer Res 69(22), 8742-51. [abstract]
2. Small EM, O’Rourke JR, Moresi V, Sutherland LB, McAnally J, Gerard RD, Richardson JA, Olson EN. Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486. Proc Natl Acad Sci U S A 107(9), 4218-23. [abstract]

Seq-Array^SM provides an efficient pathway from an initial broad microRNA search to focused biological insights.

11. Antimicrobial Peptide Arrays for Detection of Inactivated Biothreat Agents By: Chris R. Taitt1 , Stella H. North1, Nadezhda V. Kulagina1

Arrays of immobilized antimicrobial peptides are used to detect bacterial, viral, and rickettsial pathogens, including inactivated biothreat agents. These arrays differ from the many combinatorial peptide arrays described in the literature in that the peptides used here have naturally evolved to interact with and disrupt microbial membranes with high affinity but broad specificity. The interaction of these naturally occurring peptides with membranes of pathogens has been harnessed for the purpose of detection, with immobilized antimicrobial peptides acting as “capture” molecules in detection assays. Methods are presented for immobilizing the antimicrobial peptides in planar arrays, performing direct and sandwich assays, and detecting bound targets.

Affiliation(s): (1) US Naval Research Laboratory, Washington, DC, USA

Book Title: Peptide Microarrays: Methods and Protocols Series: Methods in Molecular Biology | Volume: 570 | Pub. Date: Aug-01-2009 | Page Range: 233-255 | DOI: 10.1007/978-1-60327-394-7_11

Subject: Protein Science

Key Words: Biothreat – detection, array – antimicrobial peptide

MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells

Endothelial cells (ECs) respond to changes in mechanical forces, leading to the modulation of signaling networks and cell function; an example is the inhibition of EC proliferation by steady laminar flow. MicroRNAs (miRs) are short noncoding 20–22 nucleotide RNAs that negatively regulate the expression of target genes at the posttranscriptional level. This study demonstrates that miRs are involved in the flow regulation of gene expression in ECs. With the use of microRNA chip array, we found that laminar shear stress (12 dyn/cm2, 12 h) regulated the EC expression of manymiRs, including miR-19a.We further showed that stable transfection of miR-19a significantly decreased the expression of a reporter gene controlled by a conserved 3′-untranslated region of the cyclinD1 gene and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. Laminar flow suppressed cyclin D1 protein level, and this suppressive effect was diminished when the endogenous miR-19a was inhibited. In conclusion, we demonstrated that miR-19a plays an important role in the flow regulation of cyclin D1 expression. These results revealed a mechanism by which mechanical forces modulate endothelial gene expression.

Qin X, Wang X, Wang Y, Tang Z, Cui Q, Xi J, J Li YS, Chien S, Wang N. (2010) MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells. Proc Natl Acad Sci USA [Epub ahead of print]. [abstract]

Williams BA, Diehnelt CW, Belcher P, Greving M, Woodbury NW, Johnston SA, Chaput JC. (2009) Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds. J Am Chem Soc 131(47), 17233-41. [abstract]

A full understanding of the proteome will require ligands to all of the proteins encoded by genomes. While antibodies represent the principle affinity reagents used to bind proteins, their limitations have created a need for new ligands to large numbers of proteins. Here we propose a general concept to obtain protein affinity reagents that avoids animal immunization and iterative selection steps. Central to this process is the idea that small peptide libraries contain sequences that will bind to independent regions on a protein surface and that these ligands can be combined on synthetic scaffolds to create high affinity bivalent reagents. To demonstrate the feasibility of this approach, an array of 4000 unique 12-mer peptides was screened to identify sequences that bind to nonoverlapping sites on the yeast regulatory protein Gal80. Individual peptide ligands were screened at different distances using a novel DNA linking strategy to identify the optimal peptide pair and peptide pair separation distance required to transform two weaker ligands into a single high affinity protein capture reagent. A synthetic antibody or synbody was created with 5 nM affinity to Gal80 that functions in conventional ELISA and pull-down assays. We validated our synthetic antibody approach by creating a second synbody to human transferrin. In both cases, we observed an increase in binding affinity of  ~1000-fold (ΔΔG = ~4.1 kcal/mol) between the individual peptides and final bivalent synbody construct.

There have been several studies involving microRNAs and breast cancer to date.  The findings of these studies collectively demonstrate the wide range of function that microRNA can play in just a single disease.  Researchers at Baylor College of Medicine and the University of Houston found that dysregulation of certain microRNAs have significant effect on morphological and molecular changes such as an expansion of the progenitor cell population, decreased cell size, increased cellular proliferation, and colony-forming potential [1]. They suggest that the dysregulation of these microRNAs might be important in the causation, or origination of breast cancer.

Cancer cells that develop resistance to chemotherapeutic agents are a major clinical obstacle in the successful treatment of breast cancer.  Researchers at the University of Lethbridge, Canada found that chemoresistance may be linked to drug-induced dysregulation of microRNA function [2]. They found many dysregulated microRNAs in drug resistant MFC-7 human breast adenocarcinoma cells and that some of these microRNAs target a human multidrug resistance-associated protein. Their results suggest that dysregulated microRNA expression may underlie the abnormal functioning of critical cellular processes associated with the chemoresistance phenotype.

A major complication of breast cancer is its metastatic potential and now, some new studies have shown that microRNAs can affect breast cancer metastasis either by over or under expression.  In this first example, researchers show that over expression of a specific microRNA plays a key role in a cancer signaling pathway. It is known that Rho-associated kinase (ROCK) signaling plays a fundamental role in regulating cell morphology, adhesion, and motility and that aberrant expression of ROCK is related to tumor metastases and poor clinical outcome. Researchers at Tufts University found that addition of an anti-miR can block the ROCK signaling pathway resulting in decreased breast cancer cell invasion/ migration and metastasis [3].

In another study, it was found that microRNAs function in an opposite manner. Researchers at Memorial Sloan-Kettering Cancer Center found a specific set of microRNAs for which expression is specifically lost as human breast cancer cells develop metastatic potential [8]. Furthermore, they show that restoring the expression of these microRNAs in malignant cells reduces overall tumor growth and proliferation and suppresses metastatic cell invasion.

1. Greene SB, Gunaratne PH, Hammond SM, Rosen JM. (2010) A putative role for microRNA-205 in mammary epithelial cell progenitors. J Cell Sci [Epub ahead of print] [abstract]
2. Pogribny IP, Filkowski JN, Tryndyak VP, Golubov A, Shpyleva SI, Kovalchuk O. (2010) Alterations of microRNAs and their targets are associated with acquired resistance of MCF-7 breast cancer cells to cisplatin. Int J Cancer [Epub ahead of print] [abstract]
3. Liu S, Goldstein RH, Scepansky EM, Rosenblatt M.  (2009) Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone.  Cancer Res 69(22), 8742-751.   [abstract]
4. Dykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, Martinvalet D, Song E, Lim B, Lieberman J.  (2009) miR-200 enhances mouse breast cancer cell colonization to form distant metastases.  PLoS One 4(9), e7181.  [abstract]
5. Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y, Klinge CM. (2009) Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells. Nucleic Acids 37(8), 2584-95.  [abstract]
6. Sun Y, Wu J, Wu SH, Thakur A, Bollig A, Huang Y, Joshua Liao D. (2008) Expression profile of microRNAs in c-Myc induced mouse mammary tumors. Breast Cancer Res Treat 118(1), 185-96.  [abstract]
7. Kovalchuk O, Filkowski J, Meservy J, Ilnytskyy Y, Tryndyak VP, Chekhun VF, Pogribny IP. (2008) Involvement of microRNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin. Mol Cancer Ther 7(7), 2152-59.  [abstract]
8. Tavazoie SF, Alarcón C, Oskarsson T, Padua D, Wang Q, Bos PD, Gerald WL, Massagué J. (2008) Endogenous human microRNAs that suppress breast cancer metastasis. Nature 451(7175), 147-52.  [abstract]
9. Kovalchuk O, Tryndyak VP, Montgomery B, Boyko A, Kutanzi K, Zemp F, Warbritton AR, Latendresse JR, Kovalchuk I, Beland FA, Pogribny IP. (2007) Estrogen-induced rat breast carcinogenesis is characterized by alterations in DNA methylation, histone modifications and aberrant microRNA expression. Cell Cycle 6(16), 2010-18.  [abstract]

1. Greene SB, Gunaratne PH, Hammond SM, Rosen JM. (2010) A putative role for microRNA-205 in mammary epithelial cell progenitors. J Cell Sci [Epub ahead of print] [abstract]
2. Pogribny IP, Filkowski JN, Tryndyak VP, Golubov A, Shpyleva SI, Kovalchuk O. (2010) Alterations of microRNAs and their targets are associated with acquired resistance of MCF-7 breast cancer cells to cisplatin. Int J Cancer [Epub ahead of print] [abstract]
3. Sluijter JP, van Mil A, van Vliet P, Metz CH, Liu J, Doevendans PA, Goumans MJ. (2009) MicroRNA-1 and -499 Regulate Differentiation and Proliferation in Human-Derived Cardiomyocyte Progenitor Cells. Arterioscler Thromb Vasc Biol [Epub ahead of print] [abstract]
4. Curtale G, Citarella F, Carissimi C, Goldoni M, Carucci N, Fulci V, Franceschini D, Meloni F, Barnaba V, Macino G. (2009) An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and activation-induced cell death in T lymphocytes. Blood [Epub ahead of print] [abstract]
5. Filkowski J, Ilnytskyy Y, Tamminga J, Koturbash I, Golubov A, Bagnyukova T, Pogribny I, Kovalchuk O. (2009) Hypomethylation and genome instability in the germline of exposed parents and their progeny is associated with altered miRNA expression. Carcinogenesis [Epub ahead of print] [abstract]
6. Liu W, Gong YH, Chao TF, Peng XZ, Yuan JG, Ma ZY, Jia G, Zhao JZ. (2009) Identification of differentially expressed microRNAs by microarray: a possible role for microRNAs gene in medulloblastomas. Chin Med J (Engl) 122(20), 2405-411. [abstract]
7. Duan Z, Choy E, Nielsen GP, Rosenberg A, Iafrate J, Yang C, Schwab J, Mankin H, Xavier R, Hornicek FJ. (2009) Differential expression of microRNA (miRNA) in chordoma reveals a role for miRNA-1 in Met expression. J Orthop Res [Epub ahead of print] [abstract]
8. Lau P, Verrier JD, Nielsen JA, Johnson KR, Notterpek L, Hudson LD. (2008) Identification of dynamically regulated microRNA and mRNA networks in developing oligodendrocytes. J Neurosci 28(45), 11720-730. [abstract]

While it is clear now that microRNAs play an important regulatory role in nearly all areas of biology, what may be more interesting is the breath of function of just a single microRNA.  It is estimated that microRNAs regulate up to 60% of all genes and some say that “each microRNA can target hundreds of genes” and “a single microRNA can regulate entire networks of genes”. So here is an interesting look at a single microRNA that has a far-reaching effect in many biological systems.

Several studies have demonstrated the functional role of microRNA-146a in the immune response. MicroRNA-146a feedback inhibits production in macrophages, is upregulated by HSV-1 infection, has been associated with proinflammatory signaling in stressed brain cells and Alzheimer’s disease (AD) brain, modulates CFH gene expression to regulate an inflammatory response, and plays a functional role in T lymphocyte-mediated immune response. These and other studies demonstrate the great potential for anti-miRNAs as an effective therapeutic strategy against pathogenic inflammatory signaling.

Additionally, there have been many reports describing microRNA-146a’s role in cancer.  MicroRNA-146a suppresses prostate cancer transformation from androgen-dependent to -independent cells, suppresses a  kinase coding gene which reduces cell proliferation, invasion, and metastasis to human bone marrow endothelial cell monolayers, and is dysregulated by latent membrane protein 1 (LMP1) which contributes substantially to the oncogenic potential of Epstein-Barr virus. It is projected that microRNA-146a and other microRNAs may one day become biomarkers for clinical diagnosis of several types of cancer.

1. Curtale G, Citarella F, Carissimi C, Goldoni M, Carucci N, Fulci V, Franceschini D, Meloni F, Barnaba V, Macino G. (2010) An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and activation-induced cell death in T lymphocytes. Blood 115(2), 265-73. [abstract]
2. Hou J, Wang P, Lin L, Liu X, Ma F, An H, Wang Z, Cao X. (2009) MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2. J Immunol 183(3), 2150-58.  [abstract]
3. Hill JM, Zhao Y, Clement C, Neumann DM, Lukiw WJ.  (2009) HSV-1 infection of human brain cells induces miRNA-146a and Alzheimer-type inflammatory signaling.  Neuroreport  20(16), 1500-505.  [abstract]
4. Lukiw WJ, Zhao Y, Cui JG.  (2008) An NF-kappaB-sensitive micro RNA-146a-mediated inflammatory circuit in Alzheimer disease and in stressed human brain cells. J Biol Chem 283(46), 31315-22.  [abstract]
5. Cameron JE, Yin Q, Fewell C, Lacey M, McBride J, Wang X, Lin Z, Schaefer BC, Flemington EK. (2008) The Epstein-Barr Virus latent membrane protein 1 (LMP1) induces cellular microRNA-146a, a modulator of lymphocyte signaling pathways. J Virol 82(4), 1946-58. [abstract]
6. Lin SL, Chiang A, Chang D, Ying SY. (2008) Loss of mir-146a function in hormone-refractory prostate cancer. RNA 14(3), 417-24.  [abstract]

Identification and Sequence Composition Characterization of Chondroitin Sulfate-Binding Peptides through Peptide Array Screening

Researchers at Arizona State University made use of LC Sciences Custom Peptide Microarray Service and demonstrated an effective methodology to identify and characterize Chrondroitin sulfate (CS) binding peptides for sequence composition. 

Butterfield K, Caplan M, Panitch A. (2010) Identification and Sequence Composition Characterization of Chondroitin Sulfate-Binding Peptides through Peptide Array Screening. Biochemistry [Epub ahead of print]. [abstract]

Presented by – Dr. Christoph Eicken & Dr. Qi Zhu, LC Sciences
12:00 pm Conference Room 2

as part of – Texas Medical Center BioResearch Product Faire

Houston, TX (Marriott Medical Center) – January 16, 2010

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