Upload File

specific knockdown of egfr and plk1 gene expression in u87

  • Home
  • Documents
  • Specific knockdown of EGFR and PLK1 gene expression in U87
1
Specific knockdown of EGFR and PLK1 gene expression in U87 GBM cells with radio frequency energy Ilya V Ulasov 1 , Haidn Foster 1 , Mike Butters 2 , Jae-Geun Yoon 1 , Xavier Figueroa 2 , Parvinder Hothi 1 , John Butters 2 , & Charles Cobbs 1 1 Ivy Center for Advanced Brain Tumor Treatment, Swedish Neuroscience Institute. 550 17 th Ave, Seattle, WA, 98104, USA; 2 Nativis Inc, 219 Terry Ave North, Seattle, WA, 98109, USA Mock ABSTRACT Every molecule contains a unique electromagnetic radio frequency energy (RFE) potential. Using superconducting quantum interference device (SQUID) technology, we have developed a technique to specifically record the RFE of a given molecule, and then expose living tissues to the specific and precise oscillating magnetic RFE field of that molecule. We discovered that exposure of a molecule’s unique RFE through a transmitting electromagnetic coil device can specifically impact biological systems as if the authentic molecule were present. We have recently acquired the RFE signals of siRNA oligonucleotide molecules. Here, we demonstrate for the first time that disruption of signaling pathway using ultra-low radio frequency energy (ulRFE™) can significantly inhibit target mRNA and protein expression. EGFR and PLK1 blockade by ulRFE caused a 30-70% reduction of mRNA expression and ~40% protein expression vs. control treated cells. Moreover, using RNA microarray and protein array we validated specificity of cellular EGFR knockdown. Finally, we demonstrated that RFE-mediated knockdown of EGFR and PLK1 was associated with decreased U87 GBM cell proliferation. These are the first results to demonstrate that specific ulRFE signals can be used to knock down specific gene products, impacting key pathways associated with cancer signaling. Materials & Methods Cell lines: U87 Human glioblastoma (ATCC) PCR: RT-PCR was performed using SYBR Green Mastermix (Life Technologies-Thermo Fisher, USA) or TaqMan Universal Master Mix II (Applied Biosystems, CA, USA). Gene Array:Total RNA from U87 cells treated with Mock, NCsiRNA, siEGFR and RFE-siEGFR for 4 or 12 hrs was isolated using RNeasy Mini Kit (Qiagen, CA, USA). The expression of 84 cancer-related genes was assessed by real-time PCR using Human Cancer Drug Targets RT2 ProfilerTM PCR Array (Qiagen, PAHS-507z). Western Blot: mouse monoclonal anti-EGFR antibodies (#sc-03, Santa Cruz Biotechnology, TX, USA) mouse monoclonal anti-PLK1 (#sc-17783, Santa Cruz Biotechnology, TX, USA) and polyclonal rabbit anti-GAPDH antibody (sc-25778, Santa Cruz Biotechnology, TX, USA). As secondary antibodies: goat anti-rabbit HRP-conjugated (#1706515, Bio-Rad, CA, USA), goat anti-mouse HRP- conjugated (#1721011, Bio-Rad, CA, USA) antibodies used for the Western blotting. Figure 1 – Plastic incubator (A) and ulRFE transmission system (B) used in the experiments with U87 cells. The blue ring is the transmission antenna and the white box with the LCD display is the Voyager transmitter. (C) Diagram of exposure set up for U87 Cells in (A). A B C Figure 2 – Reduction in mRNA and protein levels using ulRFE that targets EGFR expression (EGFR ulRFE). Transfection without siRNA (Mock), transfection with non- coding siRNA (NC siRNA) and EGFR targeting siRNA (EGFR siRNA). The EGFR ulRFE reduces both EGFR mRNA (A) and protein (B) levels in U87 cells. Values between blots (B) are GAPDH- adjusted EGFR expression. Both EGFR siRNA and EGFR ulRFE significantly reduced expression of EGFR in U87 cells (*P< 0.05). A B Figure 3 – The EGFR ulRFE specifically targets EGFR expression in a similar fashion as physical EGFR siRNA. Gene chip array (A) demonstrates a near identical profile in gene expression between the two treatments. Fold regulation (B) reveals subtle differences in gene expression between siEGFR and ulRFE EGFR. (C) Reduction in protein levels associated with downstream EGFR targets are 2-4 fold for ulRFE vs. Mock. A B C A B Figure 4 – Reduction in mRNA and protein levels using ulRFE that targets PLK1 expression (PLK1 ulRFE). Transfection without siRNA (Mock), transfection with non- coding siRNA (NC siRNA) and PLK1 targeting siRNA (PLK1 siRNA). The PLK1 ulRFE reduces both PLK1 mRNA (A) and protein (B) levels in U87 cells. Values between blots (B) are GAPDH- adjusted PLK1 expression. Both PLK1 siRNA and PLK1 ulRFE significantly reduced mRNA expression (*P< 0.05). * * * * * * Figure 5 – Effects of EGFR and PLK1 inhibition in cultured human GBM. Left, primary cultures from 2 patients with resected GBM tumors (GBM2 and GBM3). Expression levels of EGFR vary from each sample, but exposure to the EGFR ulRFE reduces the expression of EGFR and the production of neurospheres. Right, effects of PLK1 siRNA and PLK1 ulRFE exposure in U87 cultures. Both assays are at 72 hours exposure. PLK1

Upload: others

Post on 14-Mar-2022

2 views

Category:

Documents


0 download

Report

TRANSCRIPT

Page 1: Specific knockdown of EGFR and PLK1 gene expression in U87

SpecificknockdownofEGFRandPLK1geneexpressioninU87GBMcellswithradiofrequencyenergy

IlyaVUlasov1,Haidn Foster1,MikeButters2,Jae-Geun Yoon1,XavierFigueroa2,Parvinder Hothi1,JohnButters2,&CharlesCobbs11IvyCenterforAdvancedBrainTumorTreatment,SwedishNeuroscienceInstitute.55017th Ave,Seattle,WA,98104,USA;2NativisInc,219TerryAveNorth,Seattle,WA,98109,USA

Mock

ABSTRACTEverymoleculecontainsauniqueelectromagneticradiofrequencyenergy(RFE)potential.Usingsuperconductingquantuminterferencedevice(SQUID)technology,wehavedevelopedatechniquetospecificallyrecordtheRFEofagivenmolecule,andthenexposelivingtissuestothespecificandpreciseoscillatingmagneticRFEfieldofthatmolecule.Wediscoveredthatexposureofamolecule’suniqueRFEthroughatransmittingelectromagneticcoildevicecanspecificallyimpactbiologicalsystemsasiftheauthenticmoleculewerepresent.WehaverecentlyacquiredtheRFEsignalsofsiRNAoligonucleotidemolecules.Here,wedemonstrateforthefirsttimethatdisruptionofsignalingpathwayusingultra-lowradiofrequencyenergy(ulRFE™)cansignificantlyinhibittargetmRNAandproteinexpression.EGFRandPLK1blockadebyulRFE causeda30-70%reductionofmRNAexpressionand~40%proteinexpressionvs.controltreatedcells.Moreover,usingRNAmicroarrayandproteinarraywevalidatedspecificityofcellularEGFRknockdown.Finally,wedemonstratedthatRFE-mediatedknockdownofEGFRandPLK1wasassociatedwithdecreasedU87GBMcellproliferation.ThesearethefirstresultstodemonstratethatspecificulRFE signalscanbeusedtoknockdownspecificgeneproducts,impactingkeypathwaysassociatedwithcancersignaling.

Materials&MethodsCelllines:U87Humanglioblastoma(ATCC)PCR:RT-PCRwasperformedusingSYBRGreenMastermix (LifeTechnologies-Thermo Fisher,USA)orTaqMan UniversalMasterMixII(AppliedBiosystems,CA,USA).GeneArray:Total RNAfromU87cellstreatedwithMock,NCsiRNA,siEGFR andRFE-siEGFR for4or12hrs wasisolatedusingRNeasyMiniKit(Qiagen,CA,USA).Theexpressionof84cancer-relatedgeneswasassessedbyreal-timePCRusingHumanCancerDrugTargetsRT2ProfilerTM PCRArray(Qiagen,PAHS-507z).WesternBlot:mousemonoclonalanti-EGFRantibodies(#sc-03,SantaCruzBiotechnology,TX,USA)mousemonoclonalanti-PLK1(#sc-17783,SantaCruzBiotechnology,TX,USA)andpolyclonalrabbitanti-GAPDHantibody(sc-25778,SantaCruzBiotechnology,TX,USA).Assecondaryantibodies:goatanti-rabbitHRP-conjugated(#1706515,Bio-Rad,CA,USA),goatanti-mouseHRP-conjugated(#1721011,Bio-Rad,CA,USA)antibodiesusedfortheWesternblotting.

Figure1– Plasticincubator(A)andulRFE transmissionsystem(B)usedintheexperimentswithU87cells.TheblueringisthetransmissionantennaandthewhiteboxwiththeLCDdisplayistheVoyagertransmitter.(C)DiagramofexposuresetupforU87Cellsin(A).

A B C

Figure2 – ReductioninmRNAandproteinlevelsusingulRFE thattargetsEGFRexpression(EGFRulRFE).TransfectionwithoutsiRNA(Mock),transfectionwithnon-codingsiRNA(NCsiRNA)andEGFRtargetingsiRNA(EGFRsiRNA).TheEGFRulRFEreducesbothEGFRmRNA(A)andprotein(B)levelsinU87cells.Valuesbetweenblots(B)areGAPDH- adjustedEGFRexpression.BothEGFRsiRNAandEGFRulRFE significantlyreducedexpressionofEGFRinU87cells(*P<0.05).

A B

Figure3 – TheEGFRulRFE specificallytargetsEGFRexpressioninasimilarfashionasphysicalEGFRsiRNA.Genechiparray(A)demonstratesanearidenticalprofileingeneexpressionbetweenthetwotreatments.Foldregulation(B)revealssubtledifferencesingeneexpressionbetweensiEGFR andulRFE EGFR.(C)ReductioninproteinlevelsassociatedwithdownstreamEGFRtargetsare2-4foldforulRFE vs.Mock.

A B

C

A B

Figure4– ReductioninmRNAandproteinlevelsusingulRFE thattargetsPLK1expression(PLK1ulRFE).TransfectionwithoutsiRNA(Mock),transfectionwithnon-codingsiRNA(NCsiRNA)andPLK1targetingsiRNA(PLK1siRNA).ThePLK1ulRFE reducesbothPLK1mRNA(A)andprotein(B)levelsinU87cells.Valuesbetweenblots(B)areGAPDH- adjustedPLK1expression.BothPLK1siRNAandPLK1ulRFE significantlyreducedmRNAexpression(*P<0.05).

* * * * **

Figure5– EffectsofEGFRandPLK1inhibitioninculturedhumanGBM.Left,primaryculturesfrom2patientswithresectedGBMtumors(GBM2andGBM3).ExpressionlevelsofEGFRvaryfromeachsample,butexposuretotheEGFRulRFE reducestheexpressionofEGFRandtheproductionofneurospheres.Right,effectsofPLK1siRNAandPLK1ulRFE exposureinU87cultures.Bothassaysareat72hoursexposure.

PLK1

Dentate gyrus-specific knockdown of adult …whoville.ucsd.edu/PDFs/446_Jessberger_etal_LearnMem_2009.pdfResearch Dentate gyrus-specific knockdown of adult neurogenesis impairs spatial

PLK1 Is Transcriptionally Activated by NF-kB during Cell ... · in anoikis resistance of esophageal squamous cell carcinoma (ESCC) cells. Experimental Design: The role of PLK1 in

The Pyrethroid Knockdown Resistance - InTech - Opencdn.intechopen.com/.../InTech-The_pyrethroid_knockdown_resistance.pdf · The Pyrethroid Knockdown Resistance ... Laboratório de

U87 non-stem

Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole

KNOCKDOWN BOOKCASE - Woodworker's Journal

Phosphorylation- and Polo-Box–dependent Binding of Plk1 to ...Phosphorylation- and Polo-Box–dependent Binding of Plk1 to Bub1 Is Required for the Kinetochore Localization ... (Casenghi

Social media knockdown

Targeted Treatment of Metastatic Breast Cancer by PLK1 ... 763-full.pdfModels and Technologies Targeted Treatment of Metastatic Breast Cancer by PLK1 siRNA Delivered by an Antioxidant

A U87 non-stem U87 stem-like U251 non-stem U251 stem-like 0 200 400 600 800 MFI/FOV:TLR9 ** 0 200 400 1,000 1,400 MFI/FOV:pSTAT3 600 800 1,200 *** 0 200

RNAterference -mediated knockdown 3,4

Small interfering RNA induced knockdown of green

September 2018 Safety Recall U87 / NHTSA 18V-524 ... · Safety Recall U87 – Powertrain Control Module Page 2 NOTE: This recall applies only to the above vehicles equipped with a

Gene Knockdown

Coles Flying Insect Killer Fast Knockdown 300g

KNOCKDOWN - National Foam

Knockdown of Minichromosome Maintenance Proteins Inhibits

Knockdown furniture process work

Gemcitabine Sensitivity on Podocalyxin Knockdown

Knockdown resistance mutations predict DDT resistance …archive.lstmed.ac.uk/7070/1/Plos_NTD_11_4_e0005504.pdf · RESEARCH ARTICLE Knockdown resistance mutations predict DDT resistance

Feed-forward mechanism of converting biochemical ... · kinase 1 (Plk1) rapidly accumulates at centrosomes and kineto-chores as cells enter mitosis. Plk1 function is spatially regulated

KNOCKDOWN AND CAPSIZING SAIL TRAINING YACHT …

Revision 2 October 2018 Safety Recall U87 / NHTSA 18V-524

Knockdown of osteonectin expression upregulates collagen2003

Ariane 6, Buckling, cylinder, imperfection, knockdown

Knockdown Shooting Bench - GunTactics...Knockdown Shooting Bench Construction Plans Page 1 A portable, knockdown shooting bench that is a perfect accessory for shooting at a primitive

Modeling bacterial phenotypes using conditional knockdown mutants

Knockdown Installation Manual 230 v

Manual u87 Neumann

PLK1 and HOTAIR Accelerate Proteasomal Degradation of ...cancerres.aacrjournals.org/content/canres/75/11/2363.full.pdf · PLK1 and HOTAIR Accelerate Proteasomal Degradation of SUZ12

Bora and Aurora-A continue to activate Plk1 in mitosisJournalofCellScience RESEARCH ARTICLE Bora and Aurora-A continue to activate Plk1 in mitosis Wytse Bruinsma1,2, Libor Macu˚rek1,3,

Plk1 Inhibition Enhances the Efficacy of BET Epigenetic Reader … · Cancer Biology and Translational Studies Plk1 Inhibition Enhances the Efficacy of BET Epigenetic Reader Blockade

PLK1 Down-Regulates Parainfluenza Virus 5 Gene Expression

JLP forms a ternary complex with PLK1 and FOXK1 JNK associated

Research Article TBX3 Knockdown Decreases Reprogramming ...downloads.hindawi.com/journals/sci/2016/6759343.pdfResearch Article TBX3 Knockdown Decreases Reprogramming Efficiency of