Monocarboxylate Transporter Protein Mct1 Gene Antisense Expression Of The Recombinant Vector Was Transfected Into Human Lung Adenocarcinoma Cells And Its Biological Effects

Study on Monocarboxylate Transporter-i gene anti-sense reconstructed vector transfection into lung adenocarcinomaA549 cells and its biological affectsAbstractBecause of glycolytic enzyme activity hasing enhanced, and isoenzyme spectra hasing changed , tumor cells grow up under conditions of relatively hypoxia, tumor cells often rely on glycolysis for their ATP production.This will lead to acid productions increment, and intracellular acidifying. As If efflux of lactic acid , the end product of glycolysis, from the cell does not keep pace with production, intracellular concentrations increase and cause the intracellular pH value to decrease .Usually, the transport of lactic acid and other acid products across plasma memberane in tumor cells is known to be mediated by a family of monocarboxylate transporters(MCTs), all of which catalyse the H~/lactat& cotransport,but with different tissue distribution, substrate and inhibitor specificity. Whether the effux lactic acid from tumor cells or not is essential for their survival. If it were possible to inhibit their lactic acid transport mechanism selectively, it might be a new strategy for therapy of tumor.In past study, homologous sequence and mRNA semi-quantification of MCT1 in human lung cancer cell line(A549) have been analysed and a MCT1 anti-sense reconstructed vector has been successfully constructed. On the basis of these works , the reconstructed vector was transfected into human lung cancer cell line(A549), then their biological affects were studied,by which we can investigate the function of MCT1 in human tumor cells.Methods:(1) Electroporation gene transfecting method was used to transfect the reconstructed vector into human lung cancer cell line(A549), transfection wasconfirmed to be successful by RT-PCR and PCR methods.(2) Intfacellular pH value and lactic acid content were measured withfluorescence spectrophotometer and ultraviolet spectrophotometerrespectively. In order to identify the change of transfected tUInor cells inbiological character, their groWth curves was prepared, their cell cycle and insitU aPoptosis were measured.(3) Intracellular ATP was measured with ATP bioluminescence assay tostUdy the enegy metabolism of boor cells.(4) Nude ttansplantation tumor experiment was carried ollt to investigatetUmor cel1s in vitro proliferation.Results:(1) The resu1ts of MCT1 gene ani-sense reconsmicted vector transfectionand identification: after being transfected, A549 cells were screened byG4l$,and positive cloning was obtained. Then' successful integration ofani-sense reconstructed vector and A549 cells genome DNA was identified byRT-PCR and PCR methods.(2) The result of intracellular pH value, lactic acid content and growingcharacter measurement and analysis: comparing with A549 cel1s, intrace1lularpH value of cells tfansfected anti-sense reconstructed vector was decreased,lactic acid content increased, grow velocity retarted, doubling time increased,cell cycle inhibitated, and cells aPoptosis rate increased. Difference wassignificant((P<0.00l or p<0.05 ) .Cel1s transfected pLXSN vector had the samechange, but had no significance(p>0.05).(3) The result of ATP assay and analysis: comparing with A549 cells,intrace1lu1ar ATP conteni of cells transfected aati-sense reconstructed vectorwas decreased decreased.Difference were significant(p<0.05) .Cells transfectedpLXSN vector had the sarne change,but had no significance(p>0.05 ).(4) The resu1t of nude transplantation tumor experiment measurement andIVanalysis: comparing with A549 cells, latent period of cells transfected anti-sense reconstructed vector was longer, tumor volume was smaller and weight was lighter. Difference were significant(p