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1、Using anti-atherosclerosis microRNA for gene therapy醫學遺傳學科 臨床醫學研究部AtherosclerosisAtherosclerosis causes a restricted blood flow to vital organs.The cause of atherosclerosis:lack of physical activitysmokingunhealthy eatingage family history of heart diseaseU.S.Department of health&human Services.Nati
2、onal Institutes health 95年台灣十大死因統計Drug discovery for atherosclerosisNotoginseng or sanchi,has a long history as a remedy in Oriental traditional medicine.treating trauma and bleeding from internal or external injurypromoting blood circulationreducing blood clottingalleviating pain,especially cancer
3、painTherapy for AtherosclerosisScreening model for atherosclerosis(1)Cell proliferation assay(MTT method)Screening model for atherosclerosis(2)NotoginsengSecond screenDrug:Three different extracts from notoginseng crude extractSearching for new mechanisms for 三七 anti-atherosclerosis effect10miRNAs f
4、unctionsMore than 1/3 of human genome are regulated by miRNA(Life science news,2005)One miR can target many(hundreds)genesOne gene can be regulated by several miRCurrently 700 human miR are foundIn vitro and In vivo study for atherosclerosismicroRNA:which miRNAs are influenced by 三七microRNA anti-ath
5、erosclerosis effect cell model,biomarker and human samplesmicroRNA gene therapymiR-2xxmiR-1xxbmiR-2xxmiR-4xxmiR-4xxmiR-4xxmiR-5xxmiR-4xxmiR-1xxmiR-2xx2.181.811.761.641.461.411.391.231.141.10Fold changeTop 10UpregulationMicroRNA expression pro HASMC after Notoginseng treatmentmiR-1xx miR-3xx miR-2xam
6、iR-1xb miR-1xx Let-x miR-9xa miR-4x1miR-2x1miR-3xb5.124.342.352.342.292.212.171.991.961.94Fold changeDownregulationTop 10Results from microRNAs arraymicroRNA-1xx anti-atherosclerosis effect(anti-proliferation)1xxCell proliferationmicroRNA-1xx anti-atherosclerosis effect anti-migrationox-LDL(40ng/ml)
7、-+miR-1xx precursor(100nM)-+-miR-1xx inhibitor(100nM)-+Predict miRNAs target genesWeb tool:miRanda,Pictar,TargetscanmiR-1xx 5.12 CCNE1 CCLn CXCLnmiR-3xx 4.34 CXCL11miR-2xa 2.35 -miR-1xb 2.34 CCNE1 CCLn CXCLnmiR-1xx 2.29 CXCL11Let-x 2.21 -miR-9xa 2.17 -miR-4x1 1.99 -miR-2x1 1.96 -miR-3xb 1.94 CXCL11
8、Down FoldRegulation changePredicted target geneTop 101.Based on the bioinformatic prediction,CXCLn and Cyclin E1 gene are two of miR-1xx target gene.2.We transfected with pre-miR-1xx to the VSMC cells can reduce CXCLn and Cyclin E1 expression with a dose dependent manner.Validating miRNA-1xx target
9、genes(CCNE1 and CXCLn)CCNE1CXCLnBiomarker screening for atherosclerosisMCP-1 mRNA expression has been detected in endothelial cells,macrophages and vascular smooth muscle cells in atherosclerotic arteries of patients undergoing bypass revascularization.Control oxLDL oxLDL oxLDL +statin +SanqiMCP-1 ELISABiomarker screening for atherosclerosismiR-1xx in human serumcasecontrolT testMW testAdj age,sexAdj 5 varsMir 1xx11.993.72(n=25)8.063.31(n=75)0.00010.00010.00020.0005microRNAs gene therapyThanks for your attention