Proteomics Research Of Tubal Ectopic Pregnancy And Associated Human Tissue Sample And Target Proteins Of Chinese And Western Medicine

ObjectiveTubal ectopic pregnancy(TEP)is the most serious and common of ectopic pregnancy(EP)disease types.It is also one of the dangerous diseaseswho has a high maternal mortality rate,and it usually happens in the early stage of pregnancy.Theclinical pathogenesis of TEP is still unclear.There is not any unique and sensitive biomarkers for its diagnosis yet.This study is aiming toobservethe proteinexpression profile of human fallopian tube(hFT)and their function in different cells and tissues.The comparative proteomic is approach to investigate the candidate proteins in the tissues of TEP from relevanttissues.Investigate the candidate proteins between the TEP tissues which from being treated or not with combination of Chinese and western medicine,in order to find out potentialtarget proteins monitoring curative effect of TEP disease.The studies all above wish might become references of further study of potential target proteins monitoring curative effect of TEP and the pathogenesis of TEP disease.Low abundant proteins seem to be potential Biomarker of TEP disease and it could be valued in new references from this study.Methods1.In the study,comparative proteomic analysis in human tissues of TEP was performed,using 8-plex iTRAQ technology coupled with LC-MS/MS and strategy of pool method.Experimental groups:Planted tubal(PT),Unplanted tubal(UPT),Villus(V),Planted tubal with Medicine treatment1(MPT1,M1),MPT2(M2),Control group:Normal tubal(NT),Normal villus(NV),Normal decidua(ND)and PT.Design scheme:PTvsNT,PTvsUPT,PTvsUPTvsNT,VvsNV,PTvsND,MlvsPT and M2vsPT.Screencandidateproteins for TEP by usingSignificance Analysis of Microarrays(SAM)strategy.2.Observe the proteins expression profile of human fallopian tube(hFT)and their functions and features by using label free technology.Aiming to find out unique proteins that mightinterpret the pathogenesis of TEP disease,from the perspective of tubal physiological mechanism study.3.6 candidate proteins which was synthesized within terms of the clinical experiences,hadbeen verified by using immunohistochemistry technique(IHC).Results1.All above tests of comparative proteomic study wererepeated tags twice,andmass spectrometry four times.The results as follow:PT vs.NT:Positive Proteins 416,Negative Proteins 103;PT vs.UPT:Positive Proteins 216,Negative Proteins 79;PT vs.UPT vs.NT:Positive Proteins 601;V vs.NV:Positive Proteins 168,Negative Proteins 85;PT vs.ND:Positive Proteins 360,Negative Proteins 151;M 1 vs.PT:Positive Proteins 120,Negative Proteins 145;M 2 vs.PT:Positive Proteins 166,Negative Proteins 198.2.There were only 30 candidates showed in the text respectively(include groups of PTvsNT,PTvsUPT,PTvsUPTvsNT,VvsNV and PTvsND).And groups of MlvsPT and M2vsPT were showed fully in order to helping the furtherclinical research.12 candidates were selected,such as KRT1,FN1,HSPG2,KRT9,KRT7,FGA,HBB,HBA1,FGB and FGG.Only finished the verification of 6 proteins(KRT1,FN1,HSPG2,KRT9,KRT7 and FGA)by IHC.There were 3 type of pathway be rich in low-abundant proteins area which might be useful in interpretationof theTEP disease' s pathogenesis,e.g.mTOR signaling is related to growth factor;IL-8 signaling is related to inflammatory factor;RhoGDI signalingis related to invasion factor.3.There were twice technological repetition during the NT protein expression profile test.Results were analyzed by DAVID enrichment analysis,Pathway mapping and Referenced identification datasets.Replicate 1(rep.1)and replicate 2(rep.2)were with 5177 and3712 identified proteins respectively,and were totally summedup to 5416 with 3473 overlapped.This hFT proteome comprises 660 high-,3605 medium-and 1181 low-abundant proteins.Endocytosis pathways including clathrin and caveolar mediated endocytosis signaling,besidesactin cytoskeleton signaling pathway showed obvious tendency to be higher abundance.The extraordinary high coverage of mesenchymal stem cells(MSCs)associated proteins((Benjamini = 6.8 ×10-118))were identified in this hFT proteome.A plenty of hFT markers had been identified in our dataset includingmucins(MUC1),oviduct specific glycoprotein 1(OVGP1),beta-tubulin IV(TUBB4B),progesterone receptor(PGR),membrane-associated progesterone receptorcomponent-1(PGRMC1)and-2(PGRMC2),estrogenrelatedreceptor gamma(ESRRG),inhibinalphachain(INHA),beta A chain(INHBA),endothelial nitric oxide synthase(NOS3),nitric oxide synthase-interacting protein(NOSIP)and epidermal growth factor receptor(EGFR).Seven markers,PGRMC1,PGRMC2,ESRRG,INHA,INHBA,NOS3 and NOSIPwere newly identified by our dataset comparedwith Rungruang data.4.The results of verification of 6 proteins(KRTl,FN1,HSPG2,KRT9,KRT7 and FGA)showed:There were significant differences(P<0.5)between PTvsNT among KRT9,FGA and KRT1.There were not any significant differences among all the 6 proteins in PTvsUPT group.Conclusion1.KRT9,FGA and KRT1 might become potential target marker for clinicalcurative effectobservation(with HUO XUE HUA YU XIAO ZHENGcompound and Mifepristone/MTX),or even might serve in the pathogenesis of TEP disease.Still need multiple verification tests of those proteins in larger clinical samples.HBB,HBA1,FGB,and FGG need furtherverification tests to help discovering their valuable information in clinical research.2.The IHC and RNAseq data of 6 candidates had been searched in Human Protein Atlas(HPA)data.KRT1 and FGA were newly discovery in their proteins and transcription level in hFT expression.