Publications by authors named "Aaron Buechlein"

21 Publications

  • Page 1 of 1

Constitutive activation of MEK5 promotes a mesenchymal and migratory cell phenotype in triple negative breast cancer.

Oncoscience 2021 18;8:64-71. Epub 2021 May 18.

Department of Medicine, Division of Hematology and Oncology, Tulane University, New Orleans, LA 70118, USA.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited targeted therapeutic options. A defining feature of TNBC is the propensity to metastasize and acquire resistance to cytotoxic agents. Mitogen activated protein kinase (MAPK) and extracellular regulated kinase (ERK) signaling pathways have integral roles in cancer development and progression. While MEK5/ERK5 signaling drives mesenchymal and migratory cell phenotypes in breast cancer, the specific mechanisms underlying these actions remain under-characterized. To elucidate the mechanisms through which MEK5 regulates the mesenchymal and migratory phenotype, we generated stably transfected constitutively active MEK5 (MEK5-ca) TNBC cells. Downstream signaling pathways and candidate targets of MEK5-ca cells were based on RNA sequencing and confirmed using qPCR and Western blot analyses. MEK5 activation drove a mesenchymal cell phenotype independent of cell proliferation effects. Transwell migration assays demonstrated MEK5 activation significantly increased breast cancer cell migration. In this study, we provide supporting evidence that MEK5 functions through FRA-1 to regulate the mesenchymal and migratory phenotype in TNBC.
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http://dx.doi.org/10.18632/oncoscience.535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131078PMC
May 2021

Dual inhibition of MEK1/2 and MEK5 suppresses the EMT/migration axis in triple-negative breast cancer through FRA-1 regulation.

J Cell Biochem 2021 Apr 20. Epub 2021 Apr 20.

Department of Medicine, Section of Hematology and Oncology, Tulane University School of Medicine, New Orleans, Louisiana, USA.

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). Although MAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration (FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.
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http://dx.doi.org/10.1002/jcb.29916DOI Listing
April 2021

Experimental competition induces immediate and lasting effects on the neurogenome in free-living female birds.

Proc Natl Acad Sci U S A 2021 Mar;118(13)

Department of Biology, Indiana University, Bloomington, IN 47405.

Periods of social instability can elicit adaptive phenotypic plasticity to promote success in future competition. However, the underlying molecular mechanisms have primarily been studied in captive and laboratory-reared animals, leaving uncertainty as to how natural competition among free-living animals affects gene activity. Here, we experimentally generated social competition among wild, cavity-nesting female birds (tree swallows, ). After territorial settlement, we reduced the availability of key breeding resources (i.e., nest boxes), generating heightened competition; within 24 h we reversed the manipulation, causing aggressive interactions to subside. We sampled females during the peak of competition and 48 h after it ended, along with date-matched controls. We measured transcriptomic and epigenomic responses to competition in two socially relevant brain regions (hypothalamus and ventromedial telencephalon). Gene network analyses suggest that processes related to energy mobilization and aggression (e.g., dopamine synthesis) were up-regulated during competition, the latter of which persisted 2 d after competition had ended. Cellular maintenance processes were also down-regulated after competition. Competition additionally altered methylation patterns, particularly in pathways related to hormonal signaling, suggesting those genes were transcriptionally poised to respond to future competition. Thus, experimental competition among free-living animals shifts gene expression in ways that may facilitate the demands of competition at the expense of self-maintenance. Further, some of these effects persisted after competition ended, demonstrating the potential for epigenetic biological embedding of the social environment in ways that may prime individuals for success in future social instability.
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http://dx.doi.org/10.1073/pnas.2016154118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020798PMC
March 2021

Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

Nat Commun 2020 09 21;11(1):4755. Epub 2020 Sep 21.

Rogel Cancer Center, University of Michigan, Ann Arbor, MI, 48109, USA.

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance.
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http://dx.doi.org/10.1038/s41467-020-18411-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505984PMC
September 2020

Pten and Dicer1 loss in the mouse uterus causes poorly differentiated endometrial adenocarcinoma.

Oncogene 2020 10 25;39(40):6286-6299. Epub 2020 Aug 25.

Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, IN, USA.

Endometrial cancer remains the most common gynecological malignancy in the United States. While the loss of the tumor suppressor, PTEN (phosphatase and tensin homolog), is well studied in endometrial cancer, recent studies suggest that DICER1, the endoribonuclease responsible for miRNA genesis, also plays a significant role in endometrial adenocarcinoma. Conditional uterine deletion of Dicer1 and Pten in mice resulted in poorly differentiated endometrial adenocarcinomas, which expressed Napsin A and HNF1B (hepatocyte nuclear factor 1 homeobox B), markers of clear-cell adenocarcinoma. Adenocarcinomas were hormone-independent. Treatment with progesterone did not mitigate poorly differentiated adenocarcinoma, nor did it affect adnexal metastasis. Transcriptomic analyses of DICER1 deleted uteri or Ishikawa cells revealed unique transcriptomic profiles and global miRNA downregulation. Computational integration of miRNA with mRNA targets revealed deregulated let-7 and miR-16 target genes, similar to published human DICER1-mutant endometrial cancers from TCGA (The Cancer Genome Atlas). Similar to human endometrial cancers, tumors exhibited dysregulation of ephrin-receptor signaling and transforming growth factor-beta signaling pathways. LIM kinase 2 (LIMK2), an essential molecule in p21 signal transduction, was significantly upregulated and represents a novel mechanism for hormone-independent pathogenesis of endometrial adenocarcinoma. This preclinical mouse model represents the first genetically engineered mouse model of poorly differentiated endometrial adenocarcinoma.
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http://dx.doi.org/10.1038/s41388-020-01434-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541676PMC
October 2020

A novel screening approach comparing kinase activity of small molecule inhibitors with similar molecular structures and distinct biologic effects in triple-negative breast cancer to identify targetable signaling pathways.

Anticancer Drugs 2020 09;31(8):759-775

Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana.

Breast cancer affects women globally; the majority of breast cancer-related mortalities are due to metastasis. Acquisition of a mesenchymal phenotype has been implicated in the progression of breast cancer cells to an invasive, metastatic state. Triple-negative breast cancer (TNBC) subtypes have high rates of metastases, recurrence, and have poorer prognoses compared to other breast cancer types, partially due to lack of commonly targeted receptors. Kinases have diverse and pivotal functions in metastasis in TNBC, and discovery of new kinase targets for TNBC is warranted. We previously used a screening approach to identify intermediate-synthesis nonpotent, nonselective small-molecule inhibitors from the Published Kinase Inhibitor Set that reversed the mesenchymal phenotype in TNBC cells. Two of these inhibitors (GSK346294A and GSK448459A) are structurally similar, but have unique kinase activity profiles and exhibited differential biologic effects on TNBC cells, specifically on epithelial-to-mesenchymal transition (EMT). Here, we further interrogate these effects and compare activity of these inhibitors on transwell migration, gene (qRT-PCR) and protein (western blot) expressions, and cancer stem cell-like behavior. We incorporated translational patient-derived xenograft models in these studies, and we focused on the lead inhibitor hit, GSK346294A, to demonstrate the utility of our comparative analysis as a screening modality to identify novel kinase targets and signaling pathways to pursue in TNBC. This study introduces a new method for discovering novel kinase targets that reverse the EMT phenotype; this screening approach can be applied to all cancer types and is not limited to breast cancer.
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http://dx.doi.org/10.1097/CAD.0000000000000962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092417PMC
September 2020

The neurogenomic transition from territory establishment to parenting in a territorial female songbird.

BMC Genomics 2019 Nov 7;20(1):819. Epub 2019 Nov 7.

Department of Biology, Indiana University, Bloomington, IN, 47405, USA.

Background: The brain plays a critical role in upstream regulation of processes central to mating effort, parental effort, and self-maintenance. For seasonally breeding animals, the brain is likely mediating trade-offs among these processes within a short breeding season, yet research thus far has only explored neurogenomic changes from non-breeding to breeding states or select pathways (e.g., steroids) in male and/or lab-reared animals. Here, we use RNA-seq to explore neural plasticity in three behaviorally relevant neural tissues (ventromedial telencephalon [VmT], hypothalamus [HYPO], and hindbrain [HB]), comparing free-living female tree swallows (Tachycineta bicolor) as they shift from territory establishment to incubation. We additionally highlight changes in aggression-related genes to explore the potential for a neurogenomic shift in the mechanisms regulating aggression, a critical behavior both in establishing and maintaining a territory and in defense of offspring.

Results: HB had few differentially expressed genes, but VmT and HYPO had hundreds. In particular, VmT had higher expression of genes related to neuroplasticity and processes beneficial for competition during territory establishment, but down-regulated immune processes. HYPO showed signs of high neuroplasticity during incubation, and a decreased potential for glucocorticoid signaling. Expression of aggression-related genes also shifted from steroidal to non-steroidal pathways across the breeding season.

Conclusions: These patterns suggest trade-offs between enhanced activity and immunity in the VmT and between stress responsiveness and parental care in the HYPO, along with a potential shift in the mechanisms regulating aggression. Collectively, these data highlight important gene regulatory pathways that may underlie behavioral plasticity in females.
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http://dx.doi.org/10.1186/s12864-019-6202-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836416PMC
November 2019

Reciprocal control of motility and biofilm formation by the PdhS2 two-component sensor kinase of Agrobacterium tumefaciens.

Microbiology (Reading) 2019 02 8;165(2):146-162. Epub 2019 Jan 8.

1​Department of Biology, Indiana University, Bloomington, IN 47405, USA.

A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1 and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study, the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue implicated in phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 may function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA, which is inhibited by DivK. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, most likely functioning through CtrA to separately control motility and regulate the levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting the production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through the addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment.
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http://dx.doi.org/10.1099/mic.0.000758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003649PMC
February 2019

Changes in mRNA/protein expression and signaling pathways in in vivo passaged mouse ovarian cancer cells.

PLoS One 2018 21;13(6):e0197404. Epub 2018 Jun 21.

Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

The cure rate for late stage epithelial ovarian cancer (EOC) has not significantly improved over several decades. New and more effective targets and treatment modalities are urgently needed. RNA-seq analyses of a syngeneic EOC cell pair, representing more and less aggressive tumor cells in vivo were conducted. Bioinformatics analyses of the RNA-seq data and biological signaling and function studies have identified new targets, such as ZIP4 in EOC. Many up-regulated tumor promoting signaling pathways have been identified which are mainly grouped into three cellular activities: 1) cell proliferation and apoptosis resistance; 2) cell skeleton and adhesion changes; and 3) carbohydrate metabolic reprograming. Unexpectedly, lipid metabolism has been the major down-regulated signaling pathway in the more aggressive EOC cells. In addition, we found that hypoxic responsive genes were at the center stage of regulation and detected functional changes were related to cancer stem cell-like activities. Moreover, our genetic, cellular, biochemical, and lipidomic analyses indicated that cells grown in 2D vs. 3D, or attached vs. suspended had dramatic changes. The important clinical implications of peritoneal cavity floating tumor cells are supported by the data proved in this work. Overall, the RNA-seq data provide a landscape of gene expression alterations during tumor progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197404PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013233PMC
November 2018

Epigenetic Targeting of Adipocytes Inhibits High-Grade Serous Ovarian Cancer Cell Migration and Invasion.

Mol Cancer Res 2018 08 14;16(8):1226-1240. Epub 2018 May 14.

Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana.

Ovarian cancer (OC) cells frequently metastasize to the omentum, and adipocytes play a significant role in ovarian tumor progression. Therapeutic interventions targeting aberrant DNA methylation in ovarian tumors have shown promise in the clinic, but the effects of epigenetic therapy on the tumor microenvironment are understudied. Here, we examined the effect of adipocytes on OC cell behavior in culture and impact of targeting DNA methylation in adipocytes on OC metastasis. The presence of adipocytes increased OC cell migration and invasion, and proximal and direct coculture of adipocytes increased OC proliferation alone or after treatment with carboplatin. Treatment of adipocytes with hypomethylating agent guadecitabine decreased migration and invasion of OC cells toward adipocytes. Subcellular protein fractionation of adipocytes treated with guadecitabine revealed decreased DNA methyltransferase 1 (DNMT1) levels even in the presence of DNA synthesis inhibitor, aphidicolin. Methyl-Capture- and RNA-sequencing analysis of guadecitabine-treated adipocytes revealed derepression of tumor-suppressor genes and epithelial-mesenchymal transition inhibitors. , a secreted tumor suppressor downregulated by promoter CpG island methylation in adipocytes, was upregulated after guadecitabine treatment, and recombinant SUSD2 decreased OC cell migration and invasion. Integrated analysis of the methylomic and transcriptomic data identified pathways associated with inhibition of matrix metalloproteases and fatty acid α-oxidation, suggesting a possible mechanism of how epigenetic therapy of adipocytes decreases metastasis. In conclusion, the effect of DNMT inhibitor on fully differentiated adipocytes suggests that hypomethylating agents may affect the tumor microenvironment to decrease cancer cell metastasis. Epigenetic targeting of tumor microenvironment can affect metastatic behavior of ovarian cancer cells. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072573PMC
August 2018

MicroRNA-335-5p and -3p synergize to inhibit estrogen receptor alpha expression and promote tamoxifen resistance.

FEBS Lett 2017 01 12;591(2):382-392. Epub 2017 Jan 12.

Department of Medicine-Section of Hematology and Medical Oncology, Tulane University, New Orleans, LA, USA.

microRNAs (miRNAs) are small noncoding RNA molecules involved in the regulation of gene expression and play critical roles in human malignancies. Next-generation sequencing analysis of the MCF-7 breast cancer cell line overexpressing miR-335-5p and miR-335-3p demonstrated that the miRNA duplex repressed genes involved in the ERα signaling pathway, and enhanced resistance of MCF-7 cells to the growth inhibitory effects of tamoxifen. These data suggest that despite its conventional role in tumor suppression, the miR-335 transcript can also play an oncogenic role in promoting agonistic estrogen signaling in a cancerous setting.
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http://dx.doi.org/10.1002/1873-3468.12538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853108PMC
January 2017

Complete Transcriptome RNA-Seq.

Methods Mol Biol 2017 ;1513:141-162

Medical Sciences Program, Indiana University School of Medicine, 1001 East 3rd Street, Bloomington, IN, 47405, USA.

RNA-Seq is the leading technology for analyzing gene expression on a global scale across a broad spectrum of sample types. However, due to chemical modifications by fixation or degradation due to collection methods, samples often contain an abundance of RNA that is no longer intact, and the capability of current RNA-Seq protocols to accurately quantify such samples is often limited. We have developed an RNA-Seq protocol to address these key issues as well as quantify gene expression from the whole transcriptome. Furthermore, for compatibility with improved sequencing platforms, we use restructured adapter sequences to generate libraries for Illumina HiSeq, MiSeq, and NextSeq platforms. Our protocol utilizes duplex-specific nuclease (DSN) to remove abundant ribosomal RNA sequences while retaining other types of RNA for superior transcriptome profiling from low quantity input. We employ the Illumina sequencing platform, but this method is described in sufficient detail to adapt to other platforms.
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http://dx.doi.org/10.1007/978-1-4939-6539-7_10DOI Listing
January 2018

Discrete Responses to Limitation for Iron and Manganese in Agrobacterium tumefaciens: Influence on Attachment and Biofilm Formation.

J Bacteriol 2015 Dec 28;198(5):816-29. Epub 2015 Dec 28.

Department of Biology, Indiana University, Bloomington, Indiana, USA

Unlabelled: Transition metals such as iron and manganese are crucial trace nutrients for the growth of most bacteria, functioning as catalytic cofactors for many essential enzymes. Dedicated uptake and regulatory systems have evolved to ensure their acquisition for growth, while preventing toxicity. Transcriptomic analysis of the iron- and manganese-responsive regulons of Agrobacterium tumefaciens revealed that there are discrete regulatory networks that respond to changes in iron and manganese levels. Complementing earlier studies, the iron-responsive gene network is quite large and includes many aspects of iron-dependent metabolism and the iron-sparing response. In contrast, the manganese-responsive network is restricted to a limited number of genes, many of which can be linked to transport and utilization of the transition metal. Several of the target genes predicted to drive manganese uptake are required for growth under manganese-limited conditions, and an A. tumefaciens mutant with a manganese transport deficiency is attenuated for plant virulence. Iron and manganese limitation independently inhibit biofilm formation by A. tumefaciens, and several candidate genes that could impact biofilm formation were identified in each regulon. The biofilm-inhibitory effects of iron and manganese do not rely on recognized metal-responsive transcriptional regulators, suggesting alternate mechanisms influencing biofilm formation. However, under low-manganese conditions the dcpA operon is upregulated, encoding a system that controls levels of the cyclic di-GMP second messenger. Mutation of this regulatory pathway dampens the effect of manganese limitation.

Importance: Responses to changes in transition metal levels, such as those of manganese and iron, are important for normal metabolism and growth in bacteria. Our study used global gene expression profiling to understand the response of the plant pathogen Agrobacterium tumefaciens to changes of transition metal availability. Among the properties that are affected by both iron and manganese levels are those required for normal surface attachment and biofilm formation, but the requirement for each of these transition metals is mechanistically independent from the other.
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http://dx.doi.org/10.1128/JB.00668-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810603PMC
December 2015

Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer.

Oncotarget 2015 Jun;6(18):16638-52

Department of Medicine, Section of Hematology and Medical Oncology, Tulane University, New Orleans, LA, USA.

Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599295PMC
http://dx.doi.org/10.18632/oncotarget.3184DOI Listing
June 2015

Stranded Whole Transcriptome RNA-Seq for All RNA Types.

Curr Protoc Hum Genet 2015 Jan 20;84:11.14.1-11.14.23. Epub 2015 Jan 20.

Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana.

Stranded whole transcriptome RNA-Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to, miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non-coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA), and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications.
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http://dx.doi.org/10.1002/0471142905.hg1114s84DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337225PMC
January 2015

Effects of the endocrine-disrupting chemical DDT on self-renewal and differentiation of human mesenchymal stem cells.

Environ Health Perspect 2015 Jan 11;123(1):42-8. Epub 2014 Jul 11.

Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA.

Background: Although the global use of the endocrine-disrupting chemical DDT has decreased, its persistence in the environment has resulted in continued human exposure. Accumulating evidence suggests that DDT exposure has long-term adverse effects on development, yet the impact on growth and differentiation of adult stem cells remains unclear.

Objectives: Human mesenchymal stem cells (MSCs) exposed to DDT were used to evaluate the impact on stem cell biology.

Methods: We assessed DDT-treated MSCs for self-renewal, proliferation, and differentiation potential. Whole genome RNA sequencing was performed to assess gene expression in DDT-treated MSCs.

Results: MSCs exposed to DDT formed fewer colonies, suggesting a reduction in self-renewal potential. DDT enhanced both adipogenic and osteogenic differentiation, which was confirmed by increased mRNA expression of glucose transporter type 4 (GLUT4), lipoprotein lipase (LpL), peroxisome proliferator-activated receptor gamma (PPARγ), leptin, osteonectin, core binding factor 1 (CBFA1), and FBJ murine osteosarcoma viral oncogene homolog (c-Fos). Expression of factors in DDT-treated cells was similar to that in estrogen-treated MSCs, suggesting that DDT may function via the estrogen receptor (ER)-mediated pathway. The coadministration of ICI 182,780 blocked the effects of DDT. RNA sequencing revealed 121 genes and noncoding RNAs to be differentially expressed in DDT-treated MSCs compared with controls cells.

Conclusion: Human MSCs provide a powerful biological system to investigate and identify the molecular mechanisms underlying the effects of environmental agents on stem cells and human health. MSCs exposed to DDT demonstrated profound alterations in self-renewal, proliferation, differentiation, and gene expression, which may partially explain the homeostatic imbalance and increased cancer incidence among those exposed to long-term EDCs.
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http://dx.doi.org/10.1289/ehp.1408188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286277PMC
January 2015

A new method for stranded whole transcriptome RNA-seq.

Methods 2013 Sep 1;63(2):126-34. Epub 2013 Apr 1.

Medical Sciences, Indiana University School of Medicine, 1001 East 3rd St., Bloomington, IN 47405, United States. Electronic address:

This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).
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http://dx.doi.org/10.1016/j.ymeth.2013.03.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739992PMC
September 2013

De novo transcriptome sequencing in a songbird, the dark-eyed junco (Junco hyemalis): genomic tools for an ecological model system.

BMC Genomics 2012 Jul 9;13:305. Epub 2012 Jul 9.

Dept. of Biology, Center for Integrated Study of Animal Behavior, Indiana University, Bloomington, IN, USA.

Background: Though genomic-level data are becoming widely available, many of the metazoan species sequenced are laboratory systems whose natural history is not well documented. In contrast, the wide array of species with very well-characterized natural history have, until recently, lacked genomics tools. It is now possible to address significant evolutionary genomics questions by applying high-throughput sequencing to discover the majority of genes for ecologically tractable species, and by subsequently developing microarray platforms from which to investigate gene regulatory networks that function in natural systems. We used GS-FLX Titanium Sequencing (Roche/454-Sequencing) of two normalized libraries of pooled RNA samples to characterize a transcriptome of the dark-eyed junco (Junco hyemalis), a North American sparrow that is a classically studied species in the fields of photoperiodism, speciation, and hormone-mediated behavior.

Results: From a broad pool of RNA sampled from tissues throughout the body of a male and a female junco, we sequenced a total of 434 million nucleotides from 1.17 million reads that were assembled de novo into 31,379 putative transcripts representing 22,765 gene sets covering 35.8 million nucleotides with 12-fold average depth of coverage. Annotation of roughly half of the putative genes was accomplished using sequence similarity, and expression was confirmed for the majority with a preliminary microarray analysis. Of 716 core bilaterian genes, 646 (90 %) were recovered within our characterized gene set. Gene Ontology, orthoDB orthology groups, and KEGG Pathway annotation provide further functional information about the sequences, and 25,781 potential SNPs were identified.

Conclusions: The extensive sequence information returned by this effort adds to the growing store of genomic data on diverse species. The extent of coverage and annotation achieved and confirmation of expression, show that transcriptome sequencing provides useful information for ecological model systems that have historically lacked genomic tools. The junco-specific microarray developed here is allowing investigations of gene expression responses to environmental and hormonal manipulations - extending the historic work on natural history and hormone-mediated phenotypes in this system.
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http://dx.doi.org/10.1186/1471-2164-13-305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476391PMC
July 2012

Genome sequences of eight morphologically diverse Alphaproteobacteria.

J Bacteriol 2011 Sep 24;193(17):4567-8. Epub 2011 Jun 24.

Department of Biology, Indiana University, Bloomington, IN 47405, USA.

The Alphaproteobacteria comprise morphologically diverse bacteria, including many species of stalked bacteria. Here we announce the genome sequences of eight alphaproteobacteria, including the first genome sequences of species belonging to the genera Asticcacaulis, Hirschia, Hyphomicrobium, and Rhodomicrobium.
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http://dx.doi.org/10.1128/JB.05453-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165538PMC
September 2011

An Ergatis-based prokaryotic genome annotation web server.

Bioinformatics 2010 Apr 1;26(8):1122-4. Epub 2010 Mar 1.

Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA.

Summary: Ergatis is a flexible workflow management system for designing and executing complex bioinformatics pipelines. However, its complexity restricts its usage to only highly skilled bioinformaticians. We have developed a web-based prokaryotic genome annotation server, Integrative Services for Genomics Analysis (ISGA), which builds upon the Ergatis workflow system, integrates other dynamic analysis tools and provides intuitive web interfaces for biologists to customize and execute their own annotation pipelines. ISGA is designed to be installed at genomics core facilities and be used directly by biologists.

Availability: ISGA is accessible at http://isga.cgb.indiana.edu/ and the system is also freely available for local installation.
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http://dx.doi.org/10.1093/bioinformatics/btq090DOI Listing
April 2010