The Role Of Novel Tumor Suppressor Gene NPRL2 In Inhibition Growth And Reverse Drug Resistance Of Lung Cancer Cell Lines In Vitro Study

BACKGROUNDLung cancer is a serious common diseases which hazard to human life and health,at the morbidity and mortality of lung cancer showed an upward trend at present.Since the majority of patients with lung cancer is already advanced when diagnosed,of which more than 50 percent of patients without opportunities surgery,and the overwhelming majority of lung cancer patients(lung cancer accounted for 75%to 80%,including squamous cell carcinoma and adenocarcinoma etc.),however,existing radiotherapy and chemotherapy is not sensitive to lung cancer.Despite the combination of surgery,radiotherapy,chemotherapy,biological therapy and other means is application in treatment,but there is no clear prognosis improved,with the development of molecular biology characteristics of lung cancer early diagnosis of lung cancer,treatment and is of great significance to improve the prognosis of lung cancer treatment therefore become a major clinical problem.Gene therapy and chemotherapy resistance in lung cancer has become a hot topic at present.NPRL2 is a novle discovered tumor suppressor gene is plays an important role in the tumor occurrence and development,particularly the expression of the gene deletion resulted in tumor cells resistant to cisplatin occurred,which is reason to believe that NPRL2 tumor suppressor gene transfection with the import value will inhibit tumor cells, apoptosis induction and reversal of cisplatin resistance so that the treatment of lung cancer may enter a new realmWe took the lead in NPRL2 gene clinical study,using immunohistochemical technique on China's non-small cell lung cancer (NSCLC)patients with retrospective study found that the gene in NSCLC and the pathological tissue in a variety of lung cancer cell lines with a higher loss rate.It laid the foundation for us to the next work.This study attempted to use molecular biology and cell biology method construction NPRL2 gene vector,and establish a stable protein expression NPRL2 of lung cancer cell lines,using plasmid-successfully transfected-stability cultured cell line to study gene transfection NPRL2 lung cancer cell growth inhibition and its mechanism.We will make use of cisplatin-resistant human lung adenocarcinoma cell line(A549/CDDP) in vitro experimental studies.NPRL2 gene transfection can be reversed in the A549/CDDP cell line expression NPRL2 gene deletion,and can inhibit cell proliferation and induce apoptosis and reverse of cisplatin resistance thereby inhibit the growth of tumor cells,above all,the study will provide a theoretical basis for gene therapy in lung cancer. ChapterⅠThe clinical significance of NPRL2 express in NSCLC tissueObjective:To detect the expression of NPRL2 gene in non-small cell lung cancer(NSCLC),and evaluate its clinical significance.Methods:Using immunohistochemistry in 80 cases of non-small cell lung cancer and 13 patients with normal lung tissues NPRL2 protein expression,the expression of pathological and clinical follow-up as well as the relevance of information.Multivariate Cox analysises NPRL2 expression on the survival rate of the NSCLC.Results:The expression of lung cancer in 35 patients(43.8%), normal lung tissue expression in 12 cases(92.35%),the difference was significant(P＜0.05).Positive expression of lung squamous cell carcinoma in 14 patients(33.33%),pulmonary adenocarcinoma positive in 21 cases(55.26%),the difference was significant(P＜0.05);NPRL2 protein in the positive rate for theⅠperiod of 69.24%(9/13);Ⅱperiod of 46.15%(18/39),ⅢA period of 45.32%(26/47),NPRL2 protein in non-small cell lung cancer with the TNM staging of the increase(P＜0.05).NPRL2 protein in lymph node metastasis in NSCLC (N1-2)expression in the rate of 27.28%(9/33),in the absence of lymph node metastasis in NSCLC(N0),the positive rate to 45.32%(26/47) significantly(P＜0.05).Lack NPRL2 expression with age,sex,smoking and other factors unrelated.NPRL2 protein expression group of patients disease-free survival time was significantly higher than the negative group(P=0.026).Multivariate Cox analysis showed that UICC stage, there NPRL2 expression and recurrence or metastasis three factors on the survival rate of the impact of statistical significance.Conclusion:The new tumor suppressor gene NPRL2 in lung cancer missing,development and invasion and metastasis may play an important role;NPRL2 is a new tumor markers in prognosis for lung cancer.ChapterⅡThe transfection of NPRL2 gene affects the biological behaviors of human lung cancer A549/CDDP cellsObjective:Discusses the transfection of NPRL2 gene to the lung cancer cell line A549/CDDP tumor biology behavior influence.Method:Constructs reorganization material particle pcDNA3.1 -NPRL2 using the gene recombination technology,Liposomeslife2000 lies between leads the extension to dye in vitro raise lung cancer A549/CDDP cell line,the G418 screening obtains stably expresses NPRL2 in A549/CDDP cells,the MTT method is delimits the mark to heal experimental and the attack cab experiment examines the extension to dye the A549/CDDP tumor biology behavior.Results:A549/CDDP cells in the gene transfection NPRL2, reducing the number of colony formation,cloning smaller,the number and arranged more casual,and colony formation rate was 12.6%; transfected with empty vector cloning group formed a few more,the relatively large size of Cloning,and the number of close order,cloning formation rates were 19.5%,The results showed that expression NPRL2 the A549/CDDP cell proliferation rate was significantly lower than empty plasmid transfection group and the non-transfected cells(P＜0.05);The number of A549/CDDP cells transfected cells NPRL2 was transferred 14.09±4.23,significantly lower than pcDNA3.1 Group 34.18±9.77, A549/CDDP Section 36.14±8.50(P＜0.05).It showed that A549/CDDP cells transfected NPRL2 decreased significantly it's mobility.The number of permeation in A549/CDDP cells transfected NPRL2 was 74.09±4.48, was significantly lower than pcDNA3.1 group 124.18±6.43, A549/CDDP Section 120.14±7.25(P＜0.05).It showed that A549/CDDP that NPRL2 transfected cells decrease significantly its invasion.The difference was significant(P＜0.05).Conclusion:The transfection NPRL2 gene suppresses the proliferous and invasive ability of A549/CDDP cells. ChapterⅢStudy of NPRL2 gene in inducing apoptosis and reversing drug resistant of A549/CDDP cellObjective:to study the functionary mechanism of NPRL2 gene in reversing drug resistant and inducing apoptosis of A549/CDDP cells.Methods:A549/CDDP cells were administrated with CDDP in the manner of gradual concentrations(0,10,40,160,320umol/mL).Then, cell proliferation and cisplatin resistance index was measured by MTT; cell cycle was evaluated by flow cytometry;the effect NPRL2 transfection of the activity of Caspase-3 in A549/CDDP cells was deteced by Kinase assay;Bax,P53,Blc-2 and LRP protein expression were measured by immunocytochemistry.Results:The mortality of cells transfected with pcDNA3.1- NPRL2 increased with increasing concentration of cisplatin,and the difference was significant.There is no statistical difference between cells transfected with blank plasmid pcDNA3.1 and cells without transfection.It indicated cells transfection with NPRL2 gene had enhanced sensitivity towards cisplatin.NPRL2 transfected with pcDNA3.1-cisplatin group of the 50%inhibitory concentration IC50 for 156.91±3.02μmol/L,of cisplatin resistance index for 7.62.Control group with pcDNA3.1 interim A549/CDDP to cisplatin and the IC50 for the 50%inhibitory concentration of 226.53±2.31 and 226.71±0.01μmol/L,of cisplatin resistance index for 11.17 and 11.39,the difference was significant(P＜0.05).The results suggested that NPRL2 transfected cells can be reversed A549/CDDP resistance.Cells transfected with pcDNA3.1-NPRL2 had lower half inhibit concentration and drug resistant index compared to cells transfected with blank plasmid pcDNA3.1 and cells without transfection.It indicated NPRL2 gene could reverse drug resistant of A549/CDDP cells.Cell livability was inhibited with a time-dependency mode after transfection with pcDNA3.1-NPRL2, and statistical difference was obviously compared to control group. Lower growth rate and more G0-G1 period cells were observed in A549/CDDP cells transfected with pcDNA3.1-NPRL2,and statistical difference was obviously compared to control group.NPRL2 transfected with pcDNA3.1-group at different time points can be made A549/CDDP cells Caspase-3 activity increased to 48h the most notable,and the relative activity of 1.1298±0.2502,compared with the control group there were significant difference(P＜0.05),NPRL2 decreased significantly over-expression of LRP protein and bcl-2 protein,increased bax protein expression compared with the control group there were significant differences(P＜0.05).statistical difference was obviously compared to control group.Conclusion:The transfection NPRL2 gene can be induced apoptosis A549/CDDP and reversed its resistance,NPRL2 promoted activity of caspas3,reduced expression of LRP protein and bcl-2 protein expression as well as increased bax expression in the induction of apoptosis play an important role.