Study On WWOX And Its Correlation To Chemoresistance

Lung cancer is a major cause of cancer death, more than a million people in the world die from the disease each year.However the pathogenesis of lung cancer is still unclear.With the development of molecular biology, people have reliazed it closely linked to the activation of proto-oncogene, the deletion of tumor suppressor gene,the DNA repair genes are abnormal,and the abnormal expression of many tumor-associated factors. As is known to all,WWOX(WW domain containing oxidoreductase ) is an essential mediator of tumor necrosis factor-alpha-induced apoptosis, suggesting a similar, important role in apoptosis for the human protein. In addition, there is evidence that this gene behaves as a suppressor of tumor growth. we study on the expression of WWOX protein in NSCLC and their correlation to chemoresistance.Part two:Study on the WWOX reverse the chemoresistance in vitroObjective:To find out a new MDR reversal medicine.To investigate the possible function and mechanism of WWOX in multidurg resistance of NSCLC in vitro.Methods:1. We detected expression levels of WWOX in A549 lines by westen blot.2. The human adenocarcinoma multi-durg resistance cell line A549/TAX was established by intermittent low dose taxol selection from the parental cell line A549, and then we detect expression levels of WWOX in A549/TAX by westen blot.3. First,through PCR and enzyme cutting,we constructed the plasmid pDC316-IRES-EGFP.Second, human WWOX gene was cloned into shuttle plasmid by enzyme cutting and connecting and formed transfer plamid of pDC316-WWOX-IRES-EGFP.And then Ad-WWOX-IRES-EGFP was constructed via integration of human WWOX cDNA into adenvirus vector-AdMAX.Finally Ad-WWOX-IRES-EGFP was used to infect A549/T after encapsulation and proliferation in 293 cells.After 48 hours culture in vitro,expression of WWOX was examined with westen-blot.4.Cells were divided into three groups, T+(A549/T infected with Ad-WWOX- IRES-EGFP),T-(A549/T infected with Ad-IRES-EGFP) and two durgs(taxol and DDP)were chosen in our study.Through CCK-8 assay, in three experimental concentrations for 72h, Control group didn't receive any drug-treatment. we get the IC50 value and the resistance index.5. we also count the Apoptosis cell through Hoechst Staining Kit.6. Statistical analysis was carried out using R2.2.0. t test was used to evaluate the statistical significance of differences in different average.Weighted linear regression was used to IC50 value.Results were considered statistically significant if P<0.05.Result:1. There was no WWOX protein in A549 cell line.2. The human adenocarcinoma multi-durg resistance cell line A549/TAX was established successfully .The resistance index to TAX was 22.43 and the resistance index to DDP was 14.18.There also was not WWOX protein in A549/TAX through westen blot.3. After the shuttle plasmid pDC316-WWOX-IRES-EGFPCR were co-transfected into 293 cells, Ad-WWOX-IRES-EGFP was finally obtained. Enzyme cutting test indicated that the recombinant Ad contained the WWOX gene.So the target gene was inserted successfully into adenovirus vector. The replication-defective adenovirus coding WWOX gene was packaged by virus recombination technology. EGFP expression could be observed on the second day after A549/TAX infected with Ad-WWOX, and WWOX prontein could be detected by westen blot.4. In vitro drug sensitivity assay after transfection with Ad-wwox showed significantly increased sensitivity, compared with T- . And the apotosis cells was on the rise in T+, compared with T-(p<0.01).Conclusions WWOX could reserve the multidrug resistance (MDR) of A549/TAX and the function of WWOX in MDR of adenocarcinoma might be associated with apotosis. The conclusion may illuminate new mechanism of multi-drug resistance (MDR), and contribute to direct monitoring disease and prognosis analysis in clinical work.