RNA聚合酶Ⅱ转录的ALU对细胞凋亡及HBV复制的影响的开题报告.docxVIP

RNA聚合酶Ⅱ转录的ALU对细胞凋亡及HBV复制的影响的开题报告

Title:TheeffectsofALUtranscribedbyRNApolymeraseIIoncellapoptosisandHBVreplication

Introduction:

ALUisahighlyrepetitiveDNAsequencefoundinprimategenomesandaccountsformorethan10%ofthehumangenome.ItistranscribedbyRNApolymeraseIIIandprocessedintonon-codingRNAssuchasAluRNAsandsmallcytoplasmicRNAs.Recently,therehavebeenreportsofALUbeingtranscribedbyRNApolymeraseIIundercertainconditions,butthefunctionofthisprocessremainslargelyunknown.Inthisstudy,weinvestigatetheeffectsofALUtranscribedbyRNApolymeraseIIoncellapoptosisandHBVreplication.

Objectives:

Theobjectivesofthisstudyare:

1.TodeterminewhetherALUtranscribedbyRNApolymeraseIIcaninducecellapoptosis.

2.ToinvestigatethemechanismofALU-inducedcellapoptosis.

3.TodeterminewhetherALUtranscribedbyRNApolymeraseIIaffectsthereplicationofHBV.

4.ToinvestigatethemechanismofALUseffectonHBVreplication.

Methods:

1.Cellculture:HepG2cellswillbeculturedinDMEMsupplementedwith10%FBS,100U/mLpenicillin,and100μg/mLstreptomycin.

2.Transfection:ThecellswillbetransfectedwithplasmidscontainingALUtranscribedbyRNApolymeraseIIusingLipofectamine3000.

3.Cellapoptosisassay:ApoptosiswillbedetectedbyflowcytometryusingAnnexin-V/PIstaining.

4.Westernblot:Proteinexpressionwillbeanalyzedbywesternblotusingspecificantibodies.

5.HBVreplicationassay:HepG2cellswillbeinfectedwithHBVandtheamountofHBVDNAandRNAwillbequantifiedbyqPCRandRT-qPCR,respectively.

6.Statisticalanalysis:TheresultswillbeanalyzedbyStudentst-testorANOVAanalysis.

Expectedresults:

WeexpecttodemonstratethatALUtranscribedbyRNApolymeraseIIcaninducecellapoptosisinHepG2cells.Moreover,wehypothesizethatALU-inducedapoptosismayinvolveactivationofthep53pathwayand/orinhibitionoftheNF-κBpathway.In