May

25

miRBase Updates Keep Coming

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mirbase_graphAfter  couple of years with nominal growth in the number of database entries, miRBase growth has turned to exponential over the last four to five updates.  The latest deep sequencing technologies have palyed a key role in the discovery of new miRNAs.  A full 30% increase in the number of entries and a 48% increase in the number of experimentally verified functional mature sequences in the single most recent miRBase update gives little doubt that there are many more as yet undiscovered miRNAs.

May

25

Cancer Research Tech Guide

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cancer_research_thumbCancer Research Tech Guide – From Genome Technology

Download Your Copy Here

Letter from the Editor

Cancer is tricky. But equipped with a vast arsenal of tools, investigators are on the offensive against the duplicitous disease. Still, even the most robust cancer research techniques come with their own sets of challenges. From sequencing to interrogating microRNAs, this technical guide aims to give you a fresh look at what a number of cancer researchers are doing to improve their day-to-day results at the bench in an effort to supply optimal diagnostics and care at the bedside.

The following pages contain tips for genome sequence analysis, optimizing qRT-PCR procedures for the investigation of miRNAs, the merits of various methylation interrogation techniques, isolating RNA from paraffin-embedded tissues, and what to do when gene expression microarray data is inconsistent with the clinical presentation in diagnosing cancers of unknown primary site. Be sure to consult the list of resources at the end of this guide for citations to the methods and research papers our experts have referred to in their responses.

— Tracy Vence

Mar

18

Transcriptome Analysis

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by Jeffrey M. Perkel

Once the domain of microarrays – the previous decade’s hot technology — transcriptome analysis (that is, gene expression monitoring on a genome-wide scale) is now associated with the current “it” technology: Next-generation DNA sequencing.

Though such data can be collected using arrays (sequencing was previously used mostly for transcript discovery), with sequencing “You are not limited to what you probe,” he explains. “You can discover new things.” In other words, sequencing approaches, unlike microarrays, are unbiased – “hypothesis-neutral,” in Baker’s words – meaning you can find things you weren’t looking for, and reanalyze the data later as new discoveries come to light…

[Gene-expression microarrays still hold some advantages over sequencing.]

…Not everyone has access to a next-gen DNA sequencer for one thing, and even if they do, they may not be able to get instrument time when they need it.

More critically, sequencing is generally time-consuming (one to two weeks per run, typically), expensive, and bioinformatically challenging – all of which presents a problem for independent researchers outside of major sequencing centers, and especially, for diagnostics developers.  (read more)

Mar

10

MicroRNAs Regulate Kinase Activity

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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]

Feb

10

The many complex roles of microRNA in breast cancer

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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]

Jan

29

microRNA-146a

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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]

Oct

30

MicroRNAs as Potential Biomarkers in Cancer and Heart Disease

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Heart disease and cancer represent the number one and number two killer diseases in developed countries. Identifying biomarkers to detect these and other diseases at early stages remains an important research goal and researchers are scrambling for ways to identify contributing factors to the pathogenesis of diseased cells in the body.  In recent years, research has turned to the study of microRNA (miRNA) as possible biomarkers due to their extensive role in biological processes and cell functionality in normal vs. diseased cells.  Microarrays combined with quantitative real-time PCR (qRT-PCR) validation are proving to be valuable tools for miRNA expression profiling and are predicted to play a crucial role in biomarker discovery and detection.  Read more

Oct

14

Integrating microRNA and mRNA expression profiles for identification of regulatory networks

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The discovery of microRNA as an additional regulatory mechanism has been a revolution to the field of Developmental Biology. While early research has focused on the identification of miRNAs using a combination of experimental and computational techniques, subsequent studies have focused on identification of miRNA-target mRNA pairs as a means of identifying regulatory networks. It has been shown that the relationship between messenger RNA and microRNA (often an inverse relationship) plays a large role in cell functionality, especially in the early stages of cell development.  Read more

Sep

11

MicroRNAs play a critical role in immune system by regulating/fine tuning inflammatory response

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MicroRNAs (miRNAs) have proven to be extremely important part of the gene expression regulation mechanism. LC Sciences microRNA profiling services have helped many researchers to explore and examine miRNA cellular networks. Expression profiling and functional studies indicate that miRNAs participate in the regulation of almost every cellular process investigated and that changes in their expression have a profound effect on their gene targeting activities. While the role of miRNAs in cell development and tumorigenesis was recognized early on, the importance of these small RNAs on immune system development and function has only been touched on recently. Initial studies have indicated that miRNAs have a role in the regulation of immune response and inflammation. Although inflammation protects against pathogenic stimuli and promotes healing, it may actually cause more damage than the activating event if not strictly controlled by essential, internal negative regulators. Out of control immune response causes various forms of chronic inflammatory diseases. Read more