The Clinical And Basic Study On Gene That Associated To Adjuvant Chemotherapy For Non-small Cell Lung Cancer

[Background]The malignant tumor is one of the severe diseases which are harmful to the health of human beings.The investigation of the death causes shows that the death rate of lung cancer is more rapidly increasing in last twenty years,Now lung cancer is usually treated with local measures including surgery,chemotherapy,radiotherapy and immunotherapy.Chemotherapy as a systemic therapy means can not be replaced by surgery and radiotherapy as for the therapies of malignant tumors.Several randomized trials now have confirmed the survival benefit with adjuvant platinum-based chemotherapy for patients with late-stage NSCLC.The role of adjuvant chemotherapy in patients with completely resected Early-Stage NSCLC is unclear.A recent meta-analysis of all randomised trials showed that the absolute risk of death was reduced by 3%at 3 years and 5%at 5 years for patients who were treated with postoperative cisplatin-containing regimens compared to patients who were treated with surgery alone.On these bases,some institutions,in spite of drug toxicity,proposed the introduction of adjuvant chemotherapy even after presumed radical surgery at stage I NSCLC.Nevertheless,according to another multicentric study,there was no significant effect on survival;hence the toxicity induced by chemotherapy appeared to radically operated patients,especially for those at stage I NSCLC,to be too high cost to pay.our researches focused on the molecular mechnism of adjuvant chemotherapy in patients with completely resected Early-Stage NSCLC.we try to develop methods to judge the effect of adjuvant chemotherapy and guide the treatment to this patient.No documents has been published on such a study till now.This provided vacuum for our expriment.So the present study proceed five parts.[Study]PartⅠClinical Analysis of adjuvant chemotherapy in patients with completely resected NSCLC in 10 Years and meta-analysis of Adjuvant Chemotherapy with Complete Resection for Early-Stage NSCLCObjective.To evaluate the effect of adjuvant chemotherapy after radical surgery for NSCLC and analyze the factor that effect the adjuvant chemotherapy.Methods:we analysed clinical data in 542 cases of NSCLC undergone resectional surgery in our hospitol.268 patients isⅢNSCLC and 274 patients isⅠ,ⅡNSCLC.Domestic and overseas literatures were reviewed to collecte correlative study data of adjuvant chemotherapy or solitary opration for Early-Stage NSCLC.3-year survival times of the two treatment methods were meta analyzed with Review manager 4.2 according to the data of literatures.Results:1\To stageⅢNSCLC patients administer adjuvant chemotherapy with platinum-based regimen for 3 or 4 cycles can improve survival rate.2\ To stageⅠ,ⅡNSCLC patients administer adjuvant chemotherapy is not a recommended therapy methods.3\ The effectiveness of some adjuvant chemotherapy regimens with complete resection in patients with NSCLC has been improved.Conclusion:it is a important work to looking for molecular marker guide the treatment the patient with completely resected Early-Stage NSCLC.PartⅡStudy on the Relationship between Combined Multigene Detection and Response to Chemotherapy and Prognosis in Early-Stage non-small cell lung cancer tissuesobjective:Many studies have show that 30 genes expression associated to chemotherapy in cancer.in this study we detect the 30 genes expression in the patient with completely resected Early-Stage NSCLC follow by adjuvant chemotherapy.in order to fine the molecular marker relation to adjuvant chemotherapy Methods:The tissue microarray technique was used to prepare for tissue microarray in 86 cases of Early-Stage NSCLC that received adjuvant chemotherapy after undergone radical surgery.The expressions of Caspase-3,Fas,Bax,Bcl-2,Survivin,PCNA,Ki67,MGMT,P53,P63,P73,P16,P27,VEGF,nm23,P-gp,MRP,LRP,GST-π,TopoⅡ,C-myc,Cyclin-D1,Her-2,Cox-2,Ku70,Ku80,DNA-PKcs,ERCC1,MSH2,BCRP proteins were detected using immunohistochemical two-step method.Results:The positive rate of 30 genes in lung cancer tissue were 27.9%-91.9%respectively.There are 8 genes expression was related to NSCLC adjuvant chemotherapy.The higher expressing intensity of Survivin,P-gp,LRP,Ki67,P53 and ERCC1,the lower 3-year survival rate in NSCLC,there was significant discrepancy(P＞0.05). The higher expressing intensity of Bax and VEGF,the higher 3-year survival rate in NSCLC,there was significant discrepancy(P＞0.05).by logistic regression analysis the ERCC1,Survivin,Bax and VEGF are good group for predict the effective of adjuvant chemotherapy in Early-Stage NSCLC.Conclusion.ERCC1 is the most sensitivity marker to judge effect of adjuvant chemotherapy and it is ideal marker to be deeply research. Survivin,ERCC1,Bax and VEGF are ideal group factor that effect the adjuvant chemotherapy and their must be comfirn by deeply research.PartⅢA study on the expression of ERCC1 gene in human non-small cell lung cancer tissues and cell linesobjective:The aim of this study is to study the mRNA expression of ERCC1 in human lung cancer tissues and normal lung tissues.and to investigate the relationship between ERCC1 protein expression and the effect of chemotherapy to NSCLC in vivo or vitro.Methods:1\ Semi-quantification expression analysis of ERCC1 mRNA in human lung cancer and corresponding normal lung tissues were performed by RT-PCR and electrophoresis band opacity density comparison analysis.2\ Establishment of an DDP-resistant human NSCLC cell line A549/DDP by high does interval inactivated methods.3\ Semi-quantification expression analysis of ERCC1 mRNA in A549/DDP and corresponding A549 were performed by RT-PCR.4\ The expressions of ERCC1 protein in 64 cases of NSCLC that received chemotherapy alone were detected using immunohistochemical two-step method.Results: 1\ The mRNA expression of ERCC1 gene decreased significantly in human lung cancer tissue compared withthat in normal lung tissue.2\ DDP-resistant human NSCLC cell line A549/DDP were established and the values of IC50 of resistant cells to relative drugs were elevated 4-5 times.3\ The mRNA expression of ERCC1 gene increased significantly in A549/DDP compared with A549.4\ The protein expression of ERCC1 gene in good effect group of NSCLC is significantly lower than bed effect group patients.Conclusion:the ERCC1 is lower expression gene in lung cancer compared to lung tissue,the ERCC1 protein expression is relationship to the effect of chemotherapy to NSCLC in vivo or vitro.PartⅣStudy on biological effects of ERCC1 in lung cancer cellsobjective:Study on biological effects of ERCC1 in lung cancer cells by gene clone technich.Methods:1\ the ERCC1 expression vector were established and introduced into H1299 cells by liposome transfection reagent.Positive clones were screened with G418.RT-PCR was used to confirm whether the recombinant vector DNA integrated with the genomic DNA of H1299 cells.Western-blot was used to confirm whether the clone gene expression in Positive clones cells.2\ The cell growth curves,clonogenicity efficiency in plating and the cell cycle were measured to observe the changes in cell proliferation.3\The characteristic of drug resistance in stable transfection cell were measured by MTT assay.4\ Intracellular concentrations of chemotherapy drugs were detected using high-performance liquid chromatography.5\The expression of resistant gene P-pg and MRP in stable transfection cell were measured by Flow Cytometry method.Results:1\ the ERCC1 expression vector were identified by the sequence analysis.2\the Positive clones H1299/ERCC1 and H1299/vecter were confirm by RT-PCR and Western-blot.3\ The characteristic of cell proliferation was not change in H1299/ERCC1 and H1299/vecter.4\ the values of IC50 of H1299/ERCC1 to chemotherapy drugs were elevated 4.23 times than H1299/vecter.5\ Intracellular concentrations of chemotherapy drugs and The expression of resistant gene P-pg and MRP were not different between H1299/ERCC1 and H1299/vecter..Conclusion:the ERCC1 function is not relactive to cell proliferation.The expression of ERCC1 can improve capitent of resistant chemotherapy drugs in lung cancer cell.The mechnism of resistant chemotherapy drugs in ERCC1 expression cell not relative to change of Intracellular concentrations of chemotherapy drugs and change of expression of resistant gene P-pg and MRP.PartⅤ:Effects of RNA interference on ERCC1 expression in non-small-cell lung cancer cells in vitroobjective.To investigate whether RNA interference(RNAi)could induce gene silencing in non-small-cell lung cancer(NSCLC)cells as well as assess the degree of ERCC1 gene silencing and its effect on functional outcome..Methods:1\ The methods of detect expression of ERCC1 gene by Fluorescence real-time quantitative PCR(FQ-PCR)are constructed satisfactorily.2\the small interference RNA(siRNA)of ERCC1 expression vector were established and introduced into lung cancer cells(A549)by liposome transfection reagent. RT-PCR and Western-blot were used to confirm whether the siRNA silencing the expression of ERCC1 in A549 cell.3\ The characteristic of drug resistance in instantaneous transfection cell were measured by MTT assay.Results:1\ the FQ-PCR can be applied to detect expression of ERCC1 gene accurately.2\ the siRNA of ERCC1 expression vector were identified by the sequence analysis.3\ RT-PCR and Western-blot assay show that the expression of ERCC1 in instantaneous transfection cell were significantly lower than not siRNA of ERCC1 transfection cell.4\ the values of IC50 of A549/siRNA to chemotherapy drugs were decrease 2.93 times than A549/vecter.Conclusion.the RNA interference to ERCC1 gene is a good method to silence expression of ERCC1 in lung cancer cell.inhibit the expression of ERCC1 can enhance sensitivity to chemotherapy drugs in lung cancer cell.it may be a available therapeutic modalities in the treatment of lung cancer.