Effects Of Human GSTP1Targeting Inhibited By Decoy ODN On Platinum-resistance Of Lung Cancer Cell

The incidence of lung cancer occupies forefront in all tumors, its incidence ishigh and has a tendency to rise year by year.Most advanced lung cancer patients canachieve better effect on prognosis only through chemotherapy, but individualdifference of lung cancer chemotherapy effect is very big.Therefore, it is of greatsignificance for individualized treatment for cancer patients to find some molecularmarker guiding tumor patients chemotherapy regimens.From literature reports, GSTP1is associated with tolerance of cancer cells, maybe one of the symbols of the cancer drug resistance, so it can be used as a clinicalindex predicting expression of GSTP1in tumor tissues or organs and chemotherapydrug resistance.Based on this, targeting GSTP1as platinum resistance research, through decoyoligonucleotide technology (decoy ODN), this experiment aims to inhibitexpression of GSTP1gene in human lung cancer cells, study the change ofproliferation and apoptosis of lung cancer cells under the influence of chemotherapydrug Carboplatin (CBP).Methods: MTT was used to determine A549growth inhibition rate and IC50under the action of CBP;Three ODNs were designed andsynthesized:â‘ ApSpBS-aimed at GSTP1gene transcription factor bindingsites;â‘¡RNApolBS-aimed at RNA polymerase binding sites on GSTP1gene (TATAbox)â‘¢Scrambled ODN-based on ApSpBS;The most suitable transfectionconcentration was developed;Western blot was used to detect differences of GSTP1protein expression among different groups after transfection;Immunofluorescencewas used to observe the differences of GSTP1expression among different groupsafter transfection and dosing;After the drug was determined by MTT detectiontransfection and A549inhibition rate differences between different groups;Determination of flow cytometry after transfection and dosing of apoptosisand necrosis rate differences between different groups.Results:MTT showed IC50of A549cells under the action of carboplatin is91ug/mL.The most suitable transfection concentration was100nM. Western blot resultsshowed after transfection,compared with Scrambled ODN group, GSTP1proteinexpression of ApSpBS or RNApolBS was significantly lower(p<0.01);compared withcontrol group, GSTP1protein expression of ApSpBS or RNApolBS groups wassignificantly lower(p<0.01).And between Scrambled ODN and control group therewas no significant difference (p>0.05).Immunofluorescence staining showed inApSpBS or RNApolBS group GSTP1positive expressions are significantly weakerthan Scrambled ODN and the control group;Live cells was significantly reducedunder the action of carboplatin and cell shape altered obviously.MTT results showA549growth inhibition rate of ApSpBS-CBP or RNApolBS-CBP group wasobviously higher than that of Scrambled ODN-CBP or CBP groups, with extremelysignificant difference(p<0.01);A549growth inhibition rate of ApSpBS-CBP washigher than that of RNApolBS-CBP group,with significant difference(p<0.05).Flowcytometry results showed,in ApSpBS-CBP or RNApolBS-CBP group,necrosis andapoptosis rate of A549cell was obviously higher than that of Scrambled ODN-CBPor CBP groups, with extremely significant difference (p<0.01);necrosis and apoptosisrate of A549cell in ApSpBS-CBP group was higher than that in RNApolBS-CBPgroup,with significant difference(p<0.05).Conclusion:After transfecting ApSpBS or RNApolBS, GSTP1expression levelin A549cell decreased obviously. ODNs Targeting ApSpBS and RNApolBS can bothsuppress GSTP1gene expression effectively, and enhance carboplatin chemotherapysensitivity of the A549cells.