Experimental Study On MMP-2 Silencing By RNA Interference In Esophageal Carcinoma Cell Line

Background and objective:Esophageal carcinoma is one of main malignancies inChina,and its invasion and metastasis are the leading cause of poor prognosis.Forthis reason,studying molecular mechanisms of invasion and metastasis in esophagealcarcinoma became one of the hotspots.Previous studies indicated that matrixmetalloproteinase(MMP)s,which can degrade and destruct the tissue around tumorsuch as basement membrane and extracellular matrix,play important roles in theprocess of tumor invasion and metastasis in several types of the solid tumors.Among all of the MMPs discovered recently,MMP-2 is one of MMPs closelyassociated with tumor invasion.The recent studies have demonstrated that MMP-2overexpersses in many kinds of malignant tumor,and the expression levels of MMP-2associated with the degree of tumor invasion and poor prognosis.RNAinterference(RNAi)is the process of sequence-specific post transcriptional genesilencing(PTGS)triggered by double stranded RNA(dsRNA).Now,RNAi is veryuseful technique and has been applied in gene function studies.In this study,wesuppressed MMP-2 expression in esophageal carcinoma cell line KYSE150 withRNA interference and then observed the behavior of KYSE150 in invasion andmigration after MMP-2 gene expression was silenced.We further analysized thealteration of downstream genes of MMP-2.Methods:1.Four esophageal carcinoma cell lines(EC9706,KYSE510,KYSE150and TE13)were cultured using common methods.Western blot and real-timequantitative PCR were used to verify the expression level of MMP-2.Therelationship between MMP-2 expression level and the malignant degree ofesophageal carcinoma cell was analyzed.The MMP-2 over-expressed cell line wasselected.2.Three MMP-2-targeting siRNAs were designed and synthesized.ThesesiRNAs were transiently transfected into KYSE150 via cationic liposomeLipofectamine 2000.Western Blot and real-time quantitative PCR were used to verifythe interference efficiency of MMP-2 protein and mRNA expression levels.ThesiRNA with high interference efficiency and the eligible siRNA density were selected.3.After MMP-2 was down-regulated in esophageal carcinoma cell lineKYSE150 by siRNA,the ability of cell invasion was measured by Boyden Chamber,the ability of cell migration was evaluated by wound healing assay.4.After silencing MMP-2 expression,the alteration of gene expression profiles was investigated through Affymetrix HU133 plus 2 gene chip,and the bioinformaticswas used to analysize data.Results:1.MMP-2 expressed in all 4 esophageal carcinoma cell lines,the expressionlevel has a correlation with cell malignant degree.The MMP-2 was significantlyexpressed in KYSE150 as compared to the other cell lines(P＜0.01).2.The levels MMP-2 mRNA and protein in KYSE150 were significantlydecreased by RNA interference(P＜0.01).The siRNA-1 is the most effective siRNA,and 50nM is the most eligible density.3.After MMP-2 expression was down-regulated in KYSE150 by siRNA,bothcell invasion and migration ability were significantly inhibited(P＜0.01).4.Microarray assay revealed that expression of 330 probes were altered inresponse to MMP-2 gene silencing,including 235 genes and 53 ESTs.Among the 235genes,127 genes were up-regulated and 108 were down-regulated.These genes areinvolved in cell cycle,apoptosis,cell signal transduction,cell adhesion,cell migrationand aging.Conclusion:1.The expression level of MMP-2 in esophageal carcinoma cell lineshas a correlation with cell malignant degree.As the MMP-2 overexpression cell line,KYSE150 is an ideal material for follow-up research.2.siRNA can efficiently down-regulate the MMP-2 gene in esophagealcarcinoma cell line KYSE150,siRNA should be a method for gene therapy.3.The ability of migration and invasion of esophageal carcinoma cell lineKYSE150 can be inhibited by MMP-2 silencing.MMP-2 may be considered as atarget gene for therapy of esophageal carcinoma.4.MMP-2 is an important gene to regulate adhesion and invasion in tumorcells.Silencing MMP-2 gene led to a significant regulation in the downstream genes.These genes are involved in cell cycle,apoptosis,cell signal transduction,celladhesion,cell migration and aging.However,the present study still can notcompletely reveal the MMP-2 signal pathways and the molecular mechanism.Furtherstudies are needed to be carried out.