Th i P i l f i RNA i T i iTherapeutic Potential of microRNAs in ToxicogenomicsTherapeutic Potential of microRNAs in ToxicogenomicsTherapeutic Potential of microRNAs in Toxicogenomicsp gId ifi i f i RNA h l i l l i di d l h i iIdentification of microRNAs that play essential roles in disease and may act as novel therapeutics or preventive targetsIdentification of microRNAs that play essential roles in disease and may act as novel therapeutics or preventive targetsp y y p p g

LC Sciences LLC - 2575 W Bellfort Suite 270 Xiaolian Gao PhD Chris HebelLC Sciences, LLC - 2575 W. Bellfort, Suite 270, Xiaolian Gao, PhD Chris Hebel Houston TX 77054 Tel 713 664-7087 www lcsciences com Xiaochuan Zhou PhD Christoph Eicken PhDHouston, TX 77054, Tel. 713 664 7087, www.lcsciences.com Xiaochuan Zhou, PhD Christoph Eicken, PhD

Introduction miRNA Detection and Expression Profiling Application ExamplesIntroduction miRNA Detection and Expression Profiling Application ExamplesIntroduction miRNA Detection and Expression Profiling Application ExamplesMicroRNAs (miRNAs) are effectors of environmental influences on gene expression. Thus Which method to choose? Garcinol sensitizes human pancreatic adenocarcinoma cellsMicroRNAs (miRNAs) are effectors of environmental influences on gene expression. Thus iRNA l i l i h ll l i j i d

Which method to choose? Garcinol sensitizes human pancreatic adenocarcinoma cells miRNAs play an important role in the cellular response to injury, exposure to toxicants, and  pp y p p j y, p ,disease Insight into the mechanisms through which these small molecules function may lead qRT‐PCR – Fast, quantitative and very specific, but low throughput – Choose when the specific

to gemcitabine in association with microRNA signaturesdisease. Insight into the mechanisms through which these small molecules function may lead  qRT PCR Fast, quantitative and very specific, but low throughput  Choose when the specific iRNA f i k to gemcitabine in association with microRNA signatures

to development of therapeutics that target miRNA pathways Identification and quantitation of miRNAs of interest are known. g gto development of therapeutics that target miRNA pathways. Identification and quantitation of miRNA expression is an important first step in understanding the potential mechanisms RNA‐Seq – No prior sequence knowledge required, but expensive and low throughput –

Alterations in miRNA/miR genes are ofmiRNA expression is an important first step in understanding the potential mechanisms  RNA Seq No prior sequence knowledge required, but expensive and low throughput 

Ch f di li ti ith l i l h diff i Alterations in miRNA/miR genes are of bi l i l i t i th th h i linvolved in these cellular and molecular responses. Here, we present an advanced microfluidic Choose for discovery applications, with unusual species samples or when difference in biological importance in the pathophysiology involved in these cellular and molecular responses. Here, we present an advanced microfluidic 

bi hi h l h d l d bl h i iRNA i filiy pp , p p

expression between isomirs is required of cancers including pancreatic cancer (PaCa)biochip technology that was developed to enable comprehensive miRNA expression profiling.  expression between isomirs is required. of cancers, including pancreatic cancer (PaCa). p gy p p p p gDetection of miRNA using an array offers the opportunity to examine all known and/or Mi F t hi h th h t i i l b l i fil b t lt ti It has been demonstrated that garcinol Detection of miRNA using an array offers the opportunity to examine all known and/or  Microarray – Fast, high‐throughput, inexpensive, global expression profile, but alternatives are  g

induces PaCa cell growth arrest and apoptosispredicted miRNA transcripts in a single experiment providing a comprehensive view of ally g g p p g p p

better suited for discovery or absolute quantitation Choose for global comparisons of miRNA induces PaCa cell growth arrest and apoptosis predicted miRNA transcripts in a single experiment providing a comprehensive view of all  better suited for discovery or absolute quantitation – Choose for global comparisons of miRNA in vitro.miRNAs that may be involved in the system being investigated The detection probes are in expression profiles in model species in vitro. miRNAs that may be involved in the system being investigated. The detection probes are in  expression profiles in model species. 

situ synthesized using PGR (Photo‐Generated Reagent) chemistry to afford the high synthesisResearchers evaluated the miRNA expression

situ synthesized using PGR (Photo Generated Reagent) chemistry to afford the high synthesis i ld d l fl ibili d difi d l id i d h Researchers evaluated the miRNA expression yield and complete sequence flexibility, and modified nucleotides are incorporated to enhance 

profile of gemcitabine‐resistant Panc‐1 cells y p q y, pthe binding to short miRNAs without sacrificing specificity Application examples will be p g

treated with garcinol and/or gemcitabinethe binding to short miRNAs without sacrificing specificity. Application examples will be 

treated with garcinol and/or gemcitabine.  provided to demonstrate how the technology is enabling breakthrough discoveries They found that garcinol synergizes withprovided to demonstrate how the technology is enabling breakthrough discoveries. They found that garcinol synergizes with 

it bi t i hibit ll lif ti dgemcitabine to inhibit cell proliferation and induce apoptosis in PaCa cells with significantWh t iRNA ’ f ti i t i i ? induce apoptosis in PaCa cells with significant What are miRNAs’ functions in toxicogenomics? modulation of key cancer regulators including What are miRNAs functions in toxicogenomics?  y g gPARP VEGF MMPs ILs caspases and NF κBi ff f i l i fl PARP, VEGF, MMPs, ILs, caspases, and NF‐κB.   1 miRNAs are effectors of environmental influences on geneThey identified garcinol‐specific miRNA

1. miRNAs are effectors of environmental influences on gene They identified garcinol specific miRNA bi k th t iti P C ll texpression Thus miRNAs play an important role in the cellular biomarkers that sensitize PaCa cells to expression. Thus miRNAs play an important role in the cellular gemcitabine treatment thus attenuating the

p p y pt t i t d di gemcitabine treatment, thus attenuating the response to toxicants and disease.

drug‐resistance phenotype. response to toxicants and disease. 

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2 The expression of miRNAs like many of the genes important in2. The expression of miRNAs, like many of the genes important in  Colin C. Pritchard, Heather H. Cheng & Muneesh These results prompt further interest in

p , y g pi l b l d b bi i d DNA h l i

Tewari. (2012) MicroRNA profiling: approaches and These results prompt further interest in i l d it bi bi titoxicology can be regulated by xenobiotics and DNA methylation

Tewari. (2012) MicroRNA profiling: approaches and considerations Nature Reviews Genetics 13 358‐369 garcinol and gemcitabine combination toxicology, can be regulated by xenobiotics and DNA methylation. considerations Nature Reviews Genetics 13, 358 369.

strategy as a drug modality to improve Fi 1 S i ti t t i it I P C ll B PC 3 ll3 X bi ti di t d iRNA i h b di tl li k d strategy as a drug modality to improve  Fig 1 ‐ Synergistic cytotoxicity. In PaCa cells, BxPC‐3 cells 3. Xenobiotic‐mediated miRNA expression has been directly linked treatment outcome in patients diagnosed with  (A and C) and Panc‐1 cells (B and D). (A and B) Apoptotic 

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( ) ( ) ( ) p pmorphological changes were observed due to single agentwith downstream protein expression and cell proliferation µParaflo™ Microfluidics Chip Platform PaCa. morphological changes were observed due to single agent 

i l d/ it bi (G ) t t t i DAPIwith downstream protein expression and cell proliferation. µParaflo Microfluidics Chip Platform 

garcinol and/or gemcitabine (Gz) treatment using DAPI µ p

Parasramka MA, Ali S, Banerjee S, Deryavoush T, Sarkar FH, Gupta S. (2013) G i l iti h ti d i ll t it bi i

stain. (CI value is suggestive of the potential of garcinol in µParaflo™ miRNA microarrays are in situ synthesized directly in a microfluidics chip, not Garcinol sensitizes human pancreatic adenocarcinoma cells to gemcitabine in association with microRNA signatures Mol Nutr Food Res 57(2) 235 48

( gg p gsensitizing PaCa cells to effects of gemcitabineBi k f E A t iRNA fili i

µParaflo  miRNA microarrays are in situ synthesized directly in a microfluidics chip, not d l lid hi d d i i li l id h i h l association with microRNA signatures. Mol Nutr Food Res 57(2), 235‐48.  sensitizing PaCa cells to effects of gemcitabine.Biomarkers for Exposure Assessment – miRNA profiling in spotted on a glass slide.  This advanced in situ oligonucleotide synthesis technology Biomarkers for Exposure Assessment  miRNA profiling in  p g g y gyti ht t l hi hl if t d hi h d ibilit l tresponse to toxic compounds can provide toxicant specific profiles ensures tight process control, highly uniform spots and high reproducibility across lots 

Restoring miR 342 a novel therapeutic approach to sensitizingresponse to toxic compounds can provide toxicant‐specific profiles g p g y p g p yof arrays yet permits total customization of contents on each individual array Restoring miR‐342 ‐ a novel therapeutic approach to sensitizing p p p p p of arrays, yet permits total customization of contents on each individual array. g p pp g

d h h f f b1 iRNA i fil di ti i h th i i ff t f h i l t i i Th h f h P fl ™ h l and suppressing the growth of tamoxifen resistant breast tumors1. miRNA expression profiles distinguish the carcinogenic effects of chemical toxins in  Three components are at the core of the μParaflo™ technology: and suppressing the growth of tamoxifen resistant breast tumorsp p g gspecific organs

p μ gyspecific organs.

Programmable DLP Photolithography Di it l Li ht P j ti d i li ht di t d h i l ti2 miRNAs expression can be used as biomarkers of chemical exposure in risk assessment of

Programmable DLP Photolithography ‐ Digital Light Projection drives light directed chemical reactions Tumor resistance to the selective estrogen2. miRNAs expression can be used as biomarkers of chemical exposure in risk assessment of  at specific sites in an array format and eliminates the need for expensive inconvenient microfabricated Tumor resistance to the selective estrogen 

t d l t t if ichemical carcinogenesisat specific sites in an array format and eliminates the need for expensive, inconvenient microfabricated h k h d l k d l h ( ) h l h receptor modulator tamoxifen remains a chemical carcinogenesis photomasks. A computer generates the digital mask and a Digital Light Projector (DLP) projects the light 

serious clinical problem especially inp p g g g g j ( ) p j gbeam very accurately into the micro reaction chambers where a photogenerated reagent is produced serious clinical problem especially in beam very accurately into the micro reaction chambers where a photogenerated reagent is produced.

patients with tumors that also l l i f ibili & i

poverexpress HER2 However a unifyingMolecular Basis for Susceptibility & Resistance – miRNAs play a overexpress HER2. However, a unifying Molecular Basis for Susceptibility & Resistance  miRNAs play a molecular mechanism of tamoxifenfundamental role in toxicological phenomenon such as cellular molecular mechanism of tamoxifen 

i t h i d l ifundamental role in toxicological phenomenon such as cellular 

resistance has remained elusive.g p

b b l dresponses to xenobiotic stress susceptibility and resistanceresponses to xenobiotic stress, susceptibility and resistance.Researchers analyzed multiple cell models y pof tamoxifen resistance derived from1 miRNAs can suppress resistance to anticancer cytotoxic therapy Digital mask of tamoxifen resistance derived from 1. miRNAs can suppress resistance to anticancer cytotoxic therapy

Sequence input &Digital mask projection Programmable light

MCF‐7 cells to examine the influence of2 Differences in the susceptibility to carcinogenesis may be determined by the variations inSequence input & mask generation

projection Programmable light driven reactions MCF 7 cells to examine the influence of 

iRNA t if i t Th2. Differences in the susceptibility to carcinogenesis may be determined by the variations in  mask generation driven reactions

miRNAs on tamoxifen resistance. They miRNA expression response to toxins h d d ( ) h observed significant and dramaticmiRNA expression response to toxins. Photo Generated Acid (PGA) Deprotection Chemistry ‐ A observed significant and dramatic 

3 miRNA expression profiling can be used to identify genetic susceptibility to pollutantsPhoto Generated  Acid (PGA) Deprotection Chemistry  A 

l l ti h t h i l h l i li ht di t d downregulation of miR‐342 in tamoxifen 3. miRNA expression profiling can be used to identify genetic susceptibility to pollutants. novel solution photochemical approach employing light directed  gresistant MCF 7 variant cell linesparallel synthesis and deprotection with a photogenerated resistant MCF‐7 variant cell lines. parallel synthesis and deprotection with a photogenerated 

bl hi h i ld ll l h i i d d Restoring miR‐342 expression in the cellreagent enables high‐yield parallel synthesis using standard DMT  Restoring miR 342 expression in the cell li iti d th ll t t ifTherapeutic Potential of miRNA Identification of miRNAs that

g g y p y gprotected phosphoramidites R ti lines sensitized these cells to tamoxifen‐Therapeutic Potential of miRNA – Identification of miRNAs that  protected phosphoramidites.

Fl idReaction

Fig 1 ‐ Restored miR‐342 expression sensitizes resistant cells toinduced apoptosis with a dramaticp

l l l d d bl hCover GlassFluid Chamber Fig 1 ‐ Restored miR‐342 expression sensitizes resistant cells to 

t if T if i t t ll li t f t d ithinduced apoptosis with a dramatic play essential roles in disease to act as drugs or possible therapeutic Microfluidic Reaction Devices

Cover GlassChannel

tamoxifen. Tamoxifen resistant cell lines were transfected with reduction in cell growth. play essential roles in disease to act as drugs or possible therapeutic  Microfluidic Reaction Devices ‐transfection reagent alone, 20 nM of scrambled precursor negative 

gtargets Microfluidic chip (4K‐30K reaction  Distribution

g , p gcontrol (miR‐NC) or 20 nM of miR‐342‐3p precursor (miR‐342) At 24 hrtargets.  p (

chambers) provides an enclosed systemDistribution Ch l control (miR‐NC), or 20 nM of miR‐342‐3p precursor (miR‐342). At 24 hr 

t t f ti ll t t d f 96 h ith 100 M f E2 lThese findings suggest that miR‐342g

chambers) provides an enclosed system  Channelpost‐transfection cells were treated for 96 hr with 100 pM of E2 alone or These findings suggest that miR 342 

l t t if i b t1 D bi i i i ll i iRNA h that facilitates the use of the solutionin combination with 1.0 μM TAM. (A) MTT assay was used to measure regulates tamoxifen response in breast 1. Drug combinations can sensitize cancer cells via miRNA pathways. that facilitates the use of the solution 

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proliferation as a function of metabolism (B) apoptosis was assayed bytumor cell lines and our clinical datag p y

photochemistry, promotes spot uniformity  Light Beam proliferation as a function of metabolism, (B) apoptosis was assayed by ll d th ELISA (C) ll t i d ith DAPI d idi i did

tumor cell lines and our clinical data 2. Restoring miRNA expression is a novel therapeutic approach to sensitizing andp y p p yand reproducibility and enhances kinetics

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Ph t t d cell death ELISA, or (C) cells were stained with DAPI and propidium iodide indicates a trend towards reduced miR‐2. Restoring miRNA expression is a novel therapeutic approach to sensitizing and 

i th th f i t t tand reproducibility and enhances kinetics  Photogenerated

(PI). 342 expression and tamoxifen resistancesuppressing the growth of resistant tumors. for chemical synthesis as well as various  Reagent ( )Cittelly DM, Das PM, Spoelstra NS, Edgerton SM, Richer JK, Thor AD, Jones 

342 expression and tamoxifen resistance. pp g g ybinding/capture assays U if Fl

y , , p , g , , ,FE. (2010) Downregulation of miR‐342 is Associated with Tamoxifen binding/capture assays. Uniform Flow Resistant Breast Tumors. Mol Cancer 9(1), 317.Si Substrate Distribution

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