A Study On Changes Of Gene Expression Profile In Human Large Cell Lung Cancer Cell Lines With Different Metastatic Potential

Background and object Lung cancer is the most lethalmalignangy that threatens human heath and lives nowadays in the world, andmeanwhile is also the one with worst clinical outcome. Invasion andmetastasis of lung cancer is the leading cause of treatment failure andpatients' deaths. Therefore the study of the molecular mechanisms of tumorinvasion and metastasis has an important theoretical meaning and clinicalapplication prospect. Invasion and metastasis of lung cancer is a complicatedprocedure with. interactions among tumor cells, human body and targettissues,which is controlled by multiple genes and takes multiple steps todevelop. A series of tumor associated genes and signal transduction pathhwaysare also involved in this procedure. As for this large number of genes,traditional approaches such as Southern blot, North blot and RT-PCR havetheir limitations that can only study one or few genes and the simpleinteractions among them, so we can not systematically view the implicatedinteractions among the genes which are involved in the tumorigenesis,invasion and metastasis of lung cancer. But the genechip/DNA microarraytechniques which develops with thee enforcement of Human Genome Projecthas characteristics of high-throughput, large-scale, high sensitivity, high automation and reproducibility. It is able to detect the changes of wholegenome of the tumor, which helps investigators to study the molecularmechanism of tumorigenesis, progression, invasion and metastasissystematically and roundly in a level of genome. NL9980 and L9981 arehuman large cell lung cancer cell lines with same genetic background anddifferent metastasic potentials. They are highly comparable and are goodmodels to study the mechanism of invasion and metastasis. Through thecomparison of cell morphology, biologic characteristics, gene expression andproteome, we can study their relationship with invasion and metastasis andtheir mechanisms. The aim of this study is to compare the changes of geneexpression profiles of cell lines NL9980 and L9981 , study the expressioncharacteristics of human large cell lung cancer with different metastaticpotentials and screen out genes that is associated with metastasis, providebio-marker for lung cancer diagnosis and prognosis judgement, andultimately try preliminarily studying the mechanisms of lung cancer invasionand metastasis from the level of whole genome.Methods Extract and purify total RNA of NL9980 and L9981,retro-transcript to cDNA with Oligo dT Primer, and in vitro transcript tocRNA with biotin labeling. Purify cRNA and fragmentate into small pieceswith length between 35 and 200bp, then hybridize to Affymetrix HG U133Plus 2.0 array. After wash,stain and scan, we use GCOS software to comparethe different probes between the expression profiles of the two cell lines.Annotate,classify the different probes and screen out the genes and signalpathways associated with metastasis.Results1. Through the comparison of gene expression profiles of NL9980 andL9981 cell lines, we have found that the expressions and distributions of themajority of genes in the genome are identical, so in this point we have provedthat these two cell lines are from the same origin and have same heredity background.2. There are 1274 probes that are different between the gene expressionprofiles of NL9980 and L9981, 970 genes with definite gene name arescreened out from them, with 686 genes up-regulated, 283 genesdown-regulated, and 1 gene both up- and down-regulated. There are 90probes without annotations. Among them, 88 probes are ESTs(ExpressedSequence Tags).3. Among 970 genes with definite name, 686 genes are reported inPubmed, among which 181 genes are associated with lung cancer, 240genes are associated with invasion and metastasis. After searching GRIF, wefound 518 genes out of 970 genes which have reports about their functions.Among them, 17 genes are associated with lung cancer, 49 genes areassociated with invasion and metastasis, 22 genes are associated withangiogenesis, 10 genes are associated with cytoskeleton, 56 genes areassociated with adhesion, 32 genes are associated with oncogene, 98 genesare associated with apoptosis. The main gene ontology categories thechanged genes involved are, binding, catalytic activity, signal transduceractivityå’Œtranscription regulator activity.4. Up-regulation of u-PA,MT1-MMP,CXCR4 and mtsl andde-regulation of PAI-1 and PAI-2, are found in high metastatic cell lineL9981, which proved that L9981 cell lines is highly metastatic from the levelof genome.5. There are 242 genes from the different expression genes which havedefinite pathways, and 132 pathways are involved, These pathways aremainly involved in cell adhesion, cell growth,differentiation andtumorigenesis.Conclusion1. Human large cell lines NL9980 and L9981 with different metastaticpotential have different expression profiles. 2. Metastasis promotion genes are over expressed and metastasisinhibiton genes are insufficient expressed in human large cell line L9981 withhigh metastatic potential. These differential genes are mainly involved ininvasion and metastasis, adhesion, angiogenesis, cytoskeleton,tumorigenesis, cell proliferation and differentiation,apoptosis and pathwaysassociated with invasion and metastasis.3. Further investigations are needed in the differential expression genes inL9981 cell line.