Inhibition Of EGFR Gene Expression In A549 Cells By RNAi

Objective Using RNA interference technique,observe the effect of recombinant plasmid carry EGFR homologous gene on the expression of EGFR mRNA and EGFR protein after it be transfected to human lung cancer cell A549 cell;at the same time, we pick out the recombinant plasmid which have the best interference effect , pave the way for further research on the bionomics of the stable transfected monoclone cells.Method According to the principle of RNAi,we design and synthesize four strips of interferencing fragment targeting EGFR gene:h-EGFR1,h-EGFR2,h-EGFR3,h-EGFR4,then use pGensil-1 as vector, construct the recombinant plasmid pGensil-1-EGFR-1,pGensil-1-EGFR-2,pGensil-1-EGFR-3,pGensil-1-EGFR-4 respectively. the recombinant plasmid was transfected into human lung cancer A549 cell line mediated by Lipofectamine 2000,Then the transfected cells were selected with G418 and pick out the stable transfected monoclone cells .After collect and culture them,identify the transcription and expression of EGFR by RT-PCR and Western-blot, calculate the inhibition ratio .Result After the stable transfected monoclone cells were picked out , there is no significant difference of the level of the expression of EGFR between the un-transfected control and group pGensil-1-EGFR-1 (P>0.05), but the level of the expression of EGFR of groups pGensil-1-EGFR-2,pGensil-1-EGFR-3 and pGensil-1-EGFR-4 is significant be inhibited compared with un-transfected control (P<0.05), the inhibition ratio of mRNA is 27.6%,45.8%,51.7% respectively,there are significant difference between group pGensil-1-EGFR-2 and group pGensil-1- EGFR-4(P<0.05),and no significant difference between group pGensil-1-EGFR-3 and group pGensil-1-EGFR-4. the inhibition ratio of protein is 34.3%,50.9% and 47.1% respectively, and no significant difference among the three groups(P>0.05).there is no significant difference among blank control, negative control and un-transfected control of the expression of EGFR (P>0.05).Conclusion Use RNAi technique, stable transfect four recombinant plasmids targeting EGFR gene to adenocarcinoma of lung cell, there are three recombinant plasmids could depress the expression of EGFR mRNA and EGFR protein significantly, This provides a cue that RNA interference technique could affect the function of EGFR gene; ;at the same time , we pick out recombinant plasmids pGensil-1-EGFR-3 and pGensil-1-EGFR-4 which have the best interference effect, pave the way for further research on the bionomics of the stable transfected monoclone cells.