Experimental Study On Inbibitory Effect Of RNA Interference On The Expression Of HMGB1 In Human Lung Cancer Cell Line

Objective:Lung cancer is a type of malignant tumor which theats human health and life.Itsmorbidity will increase dramatically in a long period.Lung cancer is the leadingcause of cancer all over the world.HMGBl(high mobility group box B 1)is anon-histone chromsone binding protein locates in the cells.It takes part in manybiological processions including gene transcription and DNA repair.HMGBloverexpression can results in cell apoptosis,cell diffrenial,cell metastisis and cellprolifration.Recent studys indicates it is not only a transciption factor but also aimportant factor that relate to tumor development,infiltration and metastasis.Thestudy of HMGB1 in lung cancer is in the beginning step around the world.There arenot literatures about systemic reseach of HMGB 1 in lung cancer.The main purpose ofthis study is detect the HMGB1 expression of 4 lung cancer cell lines to select themost suitable cell line to do the work next step.Then to suppress HMGB1 geneexpression by RNA interference in lung cancer cell,to research the invasion andmigration behavior of lung cancer ceil after HMGB l gene silence.Then we further tostudy the molecular mechanisms of the invasion of lung cancer cell by genechip.Methods:1.Four lung cancer cell lines were cultured by normal method,Western blot andreal-time quantitative PCR were used to verify the expression level of HMGB1.Select the cell line which HMGB 1 over-expressed.2.siRNA targeting HMGB1 were designed and synthesized.The siRNA wastransiently transfected into L9981 cell line which was selected that HMGB1over-epressed via cationic liposome Lipofectamine 2000,Western Blot andreal-time quantitative PCR were used to verify the interference efficiency of HMGB 1protein and mRNA expression levels.After HMGB1 was down-regulated in lungcancer cell line L9981 by RNAi,Determination and analysis of cell viability forL9981 cell line with HMGB1 gene silencing;.the ability of cell invasion was measured by Boyden chamber,3.The alteration of gene expression profiles after HMGB1 gene silencing wasinvestigated through Affymetrix HU133 plus 2 gene chip,and bioinformatics wasused to analysize data.Results:1.HMGB1 expressed in all 4 lung cancer cell lines,The cell line L9981 is themost highly expressed cell line(P＜0.01).2.HMGB1 gene mRNA and protein expression levels of lung cancer cell lineL9981 were both dramatically decreased by RNA interference(P＜0.01).AfterHMGB1 was down-regulated in lung cancer cell line L9981 by RNAi,both cellinvasion and migration ability were inhibited significantly(P＜0.01).3.Microarray assay revealed that expression of 1433 probes were altered inresponse to HMGB1 gene silencing,including 879 genes and 216 ESTs.Among 879genes there are 295 up-regulated genes and 584 down-regulated genes.These genesare involved in cell cycle,apoptosis,cell signal transduction,cell adhesion,cellmigration and aging.Conclusion:1.All 4 lung cancer cell lines expree HMGB1 gene.As the HMGB1overexpression cell line,L9981 is an ideal material for follow-up research.2.RNAi can efficiently down-regulate the HMGB 1 gene in lung cancer cell lineL9981,The ability of growth and invasion of lung cancer cell line L9981 can beinhibited by HMGB1 silencing.HMGB1 can be regarded as a target for gene therapyof lung cancer.4.HMGB1 is an important gene to regulate cells′adhesion and invasion.Afterthe silence of HMGB1 gene,many genes had a significantly differential expression.These genes are involved in cell cycle,apoptosis,cell signal transduction,celladhesion,cell migration and aging.However,the present study still can not completely reveal the HMGB1 signal pathways and molecular mechanism;morestudies are needed to carry out.