| There is more infiltration or metastasis in peripheral tissues when it diagnosed due to the lack of high specificity of early diagnosis techniques,which leads to the failure of treatment,loss of effective mitigation and improvement of prognosis.Therefore,it is of great clinical significance to seeking for ideal markers or molecular targets for the early diagnosis,exploring to early detection,standardized and ordered diagnosis,and prognosis improvement of lung cancer.The most important characteristic of EMT is the decrease of epithelial markers(such as E-cadherin,cytokeratin,and closed protein)and increases of interstitial markers(such as vimentin and N-cadherin).Decreased or absent expression of epithelial E-cadherin(E-cadherin,EC)may promote metastasis of tumor cells.Research has shown lung cancer.CD44 is a glycoprotein,who has many biological functions involved specific adhesion between cell to cell,cell to matrix or cell to matrix to cell,which correlates with the invasion,lymph node metastasis and prognosis of tumor.Many transcription factors have been shown to be the major regulators of E-cadherin expression,including Snail,ZEB1(zinc,finger,E,box,binding,protein-1,ZEB1),ZEB2.As the most important transcriptional repressor of EC,the expression of ZEB1 plays a key role in the induction of EMT.The increased expression of ZEB1 may directly or indirectly lead to the down-regulation of EC expression and more possible of EMT,which increases the ability of the migration,invasion and anti-apoptosisof lung cancer tumors,which is.The exact role of ZEB1 in non-small cell lung cancer is unclear though its relationship with the tumor has drawn much attention at this stage.In view of the important role of ZEB1 in regulating EMT,it may provide new therapeutic strategies to study the expression of ZEB1 for the treatment of non-small cell lung cancer.This study is of great significance to better understand the mechanism of occurrence and development and explore the effective treatment mode and guide the use of drugs of non-small cell lung cancer.In view of the above reasons,this study explored the impact of hypoxia on the expression of ZEB1 protein at first,studied the hypoxia chemosensitivity of lung adenocarcinoma cell line A549 cells then,we studied the regularity of chemotherapeutic effect of blocking ZEB1 expression by ZEB-1 specific siRNA in A549 cells at the same time which revealed that hypoxia decreased the chemosensitivity to cisplatin,and transfecting with ZEB1-specific siRNA increased sensitivity to cisplatin chemotherapy in the A549 cells.On the basis of this research,we further studied the expression of ZEB1 in 103 non-small cell lung cancer(NSCLC),and found that the expression of ZEB1 in NSCLC tissues was higher than that in paracancerous tissues.The expression of ZEB1 was correlated with clinical stage,lymph node metastasis,and the differentiation degree of tumor.Our study found that hypoxia can induce the expression of ZEB1 in A549 lung adenocarcinoma cells,while hypoxia-induced high expression of ZEB1 was the molecular basis of its resistance.It can improve the efficacy of vitro drug treatment by blocking the expression of ZEB1 with specific siRNA in lung adenocarcinoma cells.Moreover,the high expression of ZEB1 protein is associated with PFS in non-small cell lung cancer.High expression of ZEB1 protein is an important prognostic factor and an important molecular marker for the treatment of non-small cell lung cancer and potential targets.Part Ⅰ:ZEB-1 protein expression and its clinicopathological correlations in Non-small Cell Lung CancerAim:To investigate the ZEB1 protein expression pattern in non-small cell lung cancers and its correlations with classical clinicopathological factors and survival,in addition to the protein expression association with E-cadherin and CD44.Methods:1.The expressions of ZEB-1,E-cadherin and CD44 in tumor tissues of 103 non-small cell lung cancer patients were detected by immunohistochemistry.2.The expressions of E-cadherin,ZEB1 and CD44 and their relationship with clinicopathological parameters were analyzed by Pearson χ2 test,Spearman rank correlation analysis was used for association analysis.3.Kaplan-Meier analysis was used to analyze the survival data.Single factor analysis of the impact on survival rates was performed using the Log Rank(Mantel-Cox)test;Cox regression was used for multivariate analysis.The test level is α = 0.05.Results:1.The ZEB1 protein expression rate was higher in the NSCLC tissues than the adjacent normal tissues(P<0.001);Significantly higher CD44 positivity was discovered in the NSCLC tissues than the adjacent normal tissues(P<0.001);But,significantly lowere E-cadherin expression was revealed in the NSCLC tissues than the adjacent normal tissues(P<0.001);However,all of the three proteins expressions were not corerelated to smoking,age,tumor diameter and pathological type,but all significantly associated with clinical stage,lymph node metastasis,tumor differentiation degree(P<0.05),where ZEB1 and CD44 were positively associated,while E-cadherin was negative(?)y associated.2.The ZEB1 protein expression was positively associated with the CD44 protien expression(r = 0.302,P = 0.001),but negatively associated with the E-cadherin expression(r =-0.267,P = 0.015).Similarly.3.The mean PFSs in the ZEB1 negative,low level and high level expression groups were 18.66 months,14.57 months and 9.37 months,respectively,with statistically signficant difference(χ2 = 27.849,P<0.001).The mean 5-year overall survivals in the ZEB1 negative,low level and high level expression groups were 51.10 months,45.13 months and 41.34 months,respectively,with significantly greater difference among the groups(χ2 = 5.967,P = 0.015).ZEB1 expression,lymph node metastasis and tumor differentiation were all independent risk factors of PFS.Conclusions:1.Tit is discovered in our study that there is significantly higher levels of ZEB1 and CD44 exptressions in the non-small cell lung cancer tissues than that in the adjacent normal tissues,there is significantly lower levels of E-cadherin protein expression in the non-small cell lung cancer tissues.In addition,ZEB1 and CD44 protein expressions are all significantly associated with clinical stage,lymph node metastasis and tumor differentiation,and the E.-cadherin protein expression is negative ly associated with clinical stage,lymph node metastasis and tumor differentiation in these tumors.2.The protein expression of ZEB1 is positively assocated with the CD44 protien expression,but negatively associated with the protein expression of E-cadherin in the tumor tissues,verifying the role of ZEB1 in the EMT process by downregulating the expression of E-cadherin and uoregulationg the expression of CD44.3.The ZEB1 protein expression is significantly negatively associated with the PFS and 5-year overall survivals in the tumors,indicating strongly an independent risk/prognostic factor role of ZEB1 in non-small cell lung cancers.Part II:Hypoxia Influence On ZEB-1 Protien Expression And Its Effect On Proliferation And Invasion In Lung Adenocarcinoma CellsAim:To study hypoxia influence on ZEB1 protein expression and its effect on proliferation and invasion in human lung adenocarcinoma A549 cells in vitro,in addition to the correlation to CD44 and E-cadherin factors.Methods:1.Hypoxia influence experiment.Lung adenocarcinoma A549 cells were cultured under normoxia(21%)and hypoxia(3%),and their growth was observed under inverted microscope.The expression of ZEB1,E-Cadherin and CD44 in lung adenocarcinoma cell line A549 was detected by Western blot.The proliferation of cells was detected by MTT assay.The effect of ZEB1 on invasion ability of lung adenocarcinoma A549 cells was detected by Transwell chamber invasion assay.Migration ability of lung adenocarcinoma A549 cell was detected by Scratch Injury assay.2.ZEB1 expression interering experiment.Constructing siRNA targeting ZEB1 mRNA and transfecting it into lung adenocarcinoma A549 cells by Lipofectamine.The cells were cultured in normoxia and hypoxia for a certain time.The expression of ZEB1 mRNA was observed by fluorescence microscopy.(1)Western blot was used to detect the expression of E-cadherin and CD44 before and after transfection.(2)The proliferation of cells was detected by MTT assay.(3)The effect of ZEB1 on invasion ability of lung adenocarcinoma A549 cells was detected by Transwell chamber invasion assay.(4)Migration ability of lung adenocarcinoma A549 cell was detected by Scratch Injury assay.2.Statistical analyses.SPSS 17.0 statistical analysis was used to analyze experimental data;analysis of variance or LSD was used to analyze measurement data;α = 0.05 was regarded as test standard.Results:1.Western blot analysis showed that the expression of E-cadherin after 24-hours and 48-hours cultured in hypoxia was sigbnificantly reduced than that in normoxia in lung adenocarcinoma A549 cells,while the expression of ZEB1 and CD44 under hypoxia was significantly upregulated.(P<0.05).2.When ZEB1 siRNA was applied,the expression of the ZEB1 was blocked,and correspondingly,the expression of CD44 was significantly decreased,but the expression of E-cadherin was increased,both in normoxia and hypoxia culture conditions.3.The proliferation rates of the four groups was significantly lower than that of the control group(P<0.05).The proliferation rate of the cells in the hypoxia group was higher than normoxia group than hypoxia transfection group than normoxia transfection group.4.Transwell chamber invasion assay revealed that the number of penetrating cells of normoxia group,normoxia ZEB1siRNA group,hypoxia group,hypoxia ZEBI siRNAgroup were(3549±643.9653,(1568±311.6294),(3983±985.8247),and(2364±852.8745),respectively,and the difference was statistically significant(P<0.05).5.The results of the Scratch Injury assay were as follows:the migration width for the cells 24hrs in culture in the four groups of normoxia,hypoxia,ZEB1siRNA in niormoxia and ZEB1siRNA in hypoxia were(352.6±108.123),(229.4±85.267),(572.8 ±92.379)and(331.6±26.796),respectively.The migration widths of the above 4 grouops cells 36hrs in culture were(674.4±45.258),(331.8±65.279),(836.6±94.298)and(485.4±121.096),respectivey and the difference was statistically significant(P<0.05).Conclusions:Hypoxia upregulated the ZEB1 protein expression,and in parallele with the ZEB1 protein expression in the human lung adenoicarcinoma cells,the CD44 proetin exptression was conrespondingly upregulated,however,the E-cadherin protein expression in these cells was correspondingly reduced.Additionally,the cells showed significantly greater invasion ability.When the ZEB1siRNA technology was applied,the ZEB1 and CD44 protien expression was significantly reduced,while the E-Cadherin protein expression was increased,and the cell invation capability was correspondingly blocket as well.It is concluded that ZEB1 is associated with EMT and cell invasion in the human lung adenocarcinoma cells in vitro.Part III:Effects of ZEB-1 on Chemosensitivity in Lung AdenocarcinomaAim:To study the effect of ZEB1 on sisplantin chemosensitivity in human lung adenocarcinoma cell A549 by menas of ZEB1 siRNA technology.Methods:1.MTT colorimetric assay.MTT colorimetric assay was used to detect the tumor cell sensitivity to cisplatin:the above three groups of cells were seeded in 96-well culture plate at the concentration of 1× 104/well.The culture medium was changed after 24hrs in culture,and the cisplatin(final concentration of 3mg/L)was added.The cells without cisplantin were applied as controls.The experiments were performed three times independently.The absorbance of each well was measured on enzyme-linked immunosorbent assay.The proliferation rate of the cell growth was calculated as the mean value of A in the experimental group/control group A ×100%.2.Flow cytometry.Flow cytometry was used to detect the changes of cell cycle and apoptosis rate:the cells of the above three groups were cultured in the logarithmic growth phase and the cell concentration was adjusted to 2 × 105 cells/ml.The cells were inoculated with cisplatin(final concentration 3mg/L);Another negative control group(untransfected,without cisplatin)was set up.The cells were harvested to prepare into single cell suspension after culturing for 24h,and the cell cycle and apoptosis rate were measured.3.Statistical analyses.SPSS 17.0 statistical software was used for statistical analyses,and the data was analyzed by one-way ANOVA.The LSD method was used to compare the two groups,The test level is a = 0.05.Results:1.Effect of ZEB1 siRNA on the cisplatin sensitivity in A549 cellsMTT assay showed that the cisplatin sensitivity of the A549 cells after ZEB 1 siRNA was successfully applied was significantly increased.The cell proliferation rate of hypoxia group was higher than normoxia group than hypoxia transfection group than normoxia transfection group,and the difference was statistically significant(P<0.05).untransfected group and the negative plasmid group(P= 0.256).2.Changes of apoptosis rate of A549 cells after transfected with ZEB1-siRNAThe apoptosis rate of untransfected group,negative plasmid group and transfection group were significantly higher than that in the negative control group atfter the treatment of cisplatin(P<0.01).The difference between the untransfected group and the negative plasmid group was statistically significant(P<0.05).The apoptosis rate was gradullay and significantly lower following an order from the hypoxia group,normoxia group,ZEB 1 siRNA hypoxia group and ZEB 1 siRNA normoxia group(P<0.05).Conclusion:Hypoxia reduced the to cisplatin chemosensitivity because of the ZEB1 protein expression induction,and ZEB 1 siRNA interfering ZEB1 expression could sensitize the ciaplantin chemotherapy in the human lung carcinoma cells A549 in vitro,indicating a new target of ZEB1 for lung cancer treatment. |
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