The Study Of DKK1Gene Expression And Biological Function In Lung Cancer

Objective: Lung cancer is considered as one of the most commonmalignant tumors in the world, which seriously endanger human health.Recent studies show that mutations in oncogenes or tumor suppressor genesare directly associated with lung cancer of which DKK1gene can be involvedin the development and progression. However, the specific role of DKK1inlung cancer is still unclear. The aim of this study was to evaluate the biologicalfunction of DKK1in lung cancer cell line.In this study, DKK1expression vectors were built with molecular cloningtechniques and expressed in eukaryotic cells. Stable cell line was establishedsubsequently, thus we measured whether DKK1transfected into95-C cellshad impact on the migration or the invasion of cancer cell line95-C.Methods:1The pCMV-Tag-2b-DKK1was constructed by molecular cloningtechnique.2Western Blot and RT-PCR were used to verify DKK1protein andmRNA expression levels, and95C cells were transfected with empty vectorand non-transfected plasmid as a negative control;3The migration level of95-C cell lines after transfected withpCMV-Tag-2b-DKK1was examined by wound healing assay.4The invasion level of transfected95-C cell lines were examined byBoyden Chamber method compared to the cells transfected with empatyvector.Results:1Plasmid pCMV-Tag-2b-DKK1was successfully constructed andconfirmed by enzyme restriction and DNA sequencing2DKK1gene was expressed in pCMV-Tag-2b-DKK1transfected95-C cells conformed by western blot and RT-PCR.395-C cell were transfected with pCMV-Tag-2b-DKK1and empty vector.The inhibiton rates were measured by scarification test after transfection for48h. While the cells transfected with PCMV-TAG-2b and the blank groupshowed inhibition rates of62%and55%, respectively, cells transfected withpCMV-Tag-2b-DKK1had an inhibition rate of16%, significantly lower thanthe former two groups(P=0.000);495-C cell were transfected with pCMV-Tag-2b-DKK1and empty vector.Boyden chamber invasion models showed that the average invasive quantityof cells transfected with pCMV-Tag-2b-DKK1was85.92, significantly higherthan the cells transfected with PCMV-Tag to-2b group and control group, ofwhich the average invasive quantity of cells were22.52and38.37,respectively (P=0.000).Conclusion:1Successfully construct plasmid pCMV-Tag-2b-DKK1, establish stablytransfected DKK1lung cancer cell lines.;2We found that after transfection of pCMV-Tag-2b-DKK1to95-C celllines, cell migration and invasion level have been increased, suggesting thatDKK1may be associated with lung cancer invasion and metastasis.