Screening Of BPDE-induced Tumor Associated Gene In Mice Whole Genomes And Preliminary Study On The Role Of EFNB2 Gene Silencing In A549 Lung Cancer Cells By RNAi.

Objective The carcinogenesis mechanism of benzo[α]pyrene dihydrodiol epoxide (BPDE) is still unclear according to the known genes interaction with BPDE. So it is an urgent problem to screen more BPDE target susceptible genes. The purposes of this study were to found a new method to screen more BPDE target susceptible genes from whole genomes in mice, to make a preliminary study using RNAi to explore the role of EFNB2 gene silencing in A549 lung cancer cell lines, and to found a basis for study on carcinogenesis mechanism of BPDE.Methods Amplified fragment length polymorphism (AFLP) combination with immunomagnetic separation (IMS) were first used to screen, enrich and amplify the fragments of BPDE interaction with DNA in mice whole genomes. Differential fragments were displayed by polyacrylamide gel electrophoresis and Silver nitrate staining. The Differential fragments were collected, cloned, sequenced, and compared homology similarity with the known genes in GenBank BLAST. RNA interference (RNAi) was used to explore the role of EFNB2 gene silencing in A549 lung cancer cell lines.Results(1) A new effective and specific method, AFLP combination with IMS, was first successfully founded to screen BPDE target susceptible genes from whole genomes in mice.(2) Distinct electrophoresis patterns were obtained by denatured polyacrylamide gel electrophoresis (DPAGE) and differential bands of DNA fragments interaction with BPDE were displayed in DPAGE electrophoresis pattern. The differential bands DNA were successfully collected, cloned, sequenced, and 125 nucleotide sequences were obtained.(3) Homology similarity was compared.with the known genes in GenBank BLAST. 193 known genes sequences in GenBank mouse genomes database showed homology similarity with the differential bands DNA sequences, the function of these 193 genes is in different categories, such as signal transduction, development, angiogenesis, immune and inflammatory response, regulation of transcription, cell differentiation, etc; The function of some genes among 193 genes is still unknown today. 6 sequences showed no significant similarity in GenBank mouse genomes database, These 6 sequences were submitted to GenBank database, which assigned accession numbers are EF176681, EF473140, EF473141, EF473142, EF473143, EF473144, respectively. These genes and sequences may be BPDE-induced tumor associated genes or sequences.(4) EFNB2 mRNA and protein expressed abundantly in A549 lung cancer cell lines according to the results of RT-PCR and immunofluorescence.(5) Small interference RNA (siRNA) expressed by RNAi plasmid expression vector can inhibit the expression of EFNB2 mRNA and protein in A549 lung cancer cell lines.(6) The number of A549 lung cancer cells clone formation, A549 lung cancer cells adhesion to vascular endothelial cells, A549 lung cancer cells invasion through Matrigel micropore membrane in vitro significantly decreased when EFNB2 gene silencing compared with control groups. Silencing of EFNB2 gene expression may significantly inhibit A549 lung cancer cells' proliferation, adhesion and invasion ability in vitro.ConclusionsOur data suggest that AFLP combination with IMS as a new effective and specific method can be used to screen BPDE target susceptible genes from whole genomes in mice, that the genes and sequences abtained by AFLP combination with IMS may be BPDE-induced tumor associated genes or sequences, and that EFNB2 gene silencing may significantly inhibit A549 lung cancer cells' proliferation, adhesion and invasion ability in vitro.