Screening The Lung Cancer For Metastasis-related Genes And RNAi Of ABCE1 Inhibited Growth And Up-regulated Adhesion Molecules Of 95-D Cells

ObjectiveBased on part of known cDNA sequences of the lung cancer metastasis related genes C1,L1 and L2,to test and to obtain full-length sequences of them.To construct siRNAs expressing plasmid aimed at ABCE1gene and study the function of ABCE1withRNA interference technology.To determine the inhibitory effect of the vector generated small interfering RNAs(siRNAs) on the expression of the ABCE1 gene in established 95-D lung carcinoma cells,and to investigate the effect of the ABCE1 siRNAs on cell proliferation and apoptosis in 95-D cells.MethodsPCR and RACE(Rapid Amplification c DNA End) are used to test and to clone 3'/5'terminal of cDNA In NCI-H446and 95-D cells.A target sequence in the middle of ABCE1 gene was selected,then according to the sequence,two complementary 71 met oligonucleotideswere synthesized with 5 single-stranded over-hangs according to the kit manual,which was ligated with the linearized RNAi-Ready pSIREN-DNR-DsRed-Express.The plasmid was transformed into DH5ot bacteria to amplifyand then purifyed.The purified plasmid was identified by gel electrophoresis and sequencing.Three siRNA-expressing vectors targeting different sites of the ABCE1gene were constructed from RNAi-Ready pSIREN-DNR-DsRed-Express vector. Cultured 95-D cells were transfected with the siRNA-expressing vector(or the negative control vector) using FuGENE 6,selected Cells by culturing the cells in the presence of G418.ResultNo EST of C1,L1 is cloned in NCI-H446 and 95-D cells;while amplification the 3'terminal of know cDNA sequence of L2 is 1169 bp in NCI-H446and 95-D,and 5'terminal of known cDNA sequence is178 bp in NCI-H446,145bp in 95-D.BLAST results shows that the full-length sequence of L2 has a sequence homology with Homo sapiens ATP-binding cassette sub-family E(OABP) member 1(chromosome 4q21),with expect=0.0 and identities=100%.The results of gel electrophoresis and sequencing showed that the plasmid was identical with the positive control,and the sequence was identical with what we have inserted in,and there was no aberrations such as mutation,deletion,or insertion.ABCE1 expression was determined with red fluorescent protein by the inverted fluorescence microscopy.Among the three siRNA expressing vectors,Si-1was the most highly functional vector and its effect on cell growth,adhesion by E-cadherin andβ-catenin expression and apoptosis in 95-D cells was further analyzed.In the study,treatment of 95-D cells with RNAi-Ready pSIREN-DNR-DsRed-Express vector Si-1 resulted in inhibition of cell proliferation, up-regulation adhesion and induction of apoptosis.Conclusions1.L2 is the ABCE1 gene.RACE is one technique used to generate full-length c DNA efficiently and rapidly.2.The plasmid which can express siRNAs aimed at ABCE1 gene has been constructed successfully.3.It can be concluded that ABCE1 is probably a target metastasis-related gene.