P53 ^Mut , P16 And Smad4 In Lung Adenocarcinoma And Their Clinical And Experimental Studies

Part I Clinical Significance of Mutant P53,P16, and Smad4 Expression in Lung AdenocarcinomaObjective:The aim of this study was to clarify protein alterations of P53,P16 and Smad4 and to explore their correlations between the protein alterations and clinical outcome.Methods:We investigated associations among mutant P53 (P53^mut) expression, and loss of P16 and Smad4 expression, with clinical outcome in 120 LAC patients who underwent curative resection using immunohistochemical （IHC） methods.Results:Of the 120 patients,76 （63.3%） expressed P53^mut protein, whereas 54 （45.0%） loss of P16 expressed and 75 （62.5%） loss of Smad4 expressed. P53^mut expression was associated with tumor size （P=0.041） and pathological stage （P=0.025）. Loss of P16 expression was associated with lymph node metastasis （P=0.001） and pathological stage （P<0.001）. Loss of Smad4 expression was associated with tumor size （P=0.033）, lymph node metastasis （P=0.014）, pathological stage （P=0.017） and tumor differentiation （P=0.022）. Kaplan-Meier survival analysis showed that tumor size （P=0.031）, lymph node metastasis （P<0.001）, pathological stage （P<0.001）, P53^mut protein expression （P=0.038）, and loss of p16 or Smad4 expression （P<0.001） were significantly associated with shorter overall survival（OS）, whereas multivariate analysis indicated that lymph node metastasis （P=0.014） and loss of p16 or Smad4 expression （P<0.001） were independent prognostic factors. Analysis of protein combinations showed that patients with more alterations had poorer survival （P<0.001）. Spearman correlation analysis showed that loss of Smad4 expression inversely correlated with expression of P53^mut （r= -0.196, P=0.032） and positively with loss of P16 expression （r=0.182,P=0.047）.Conclusion:The mutant P53 expression and the loss of P16 or Smad4 expression in lung adenocarnoma was associated with malignant biological behaviors and poor prognosis.Part Ⅱ The Effects of Exogenous P53^mut, P53wt and P16 Gene Overexpression on Biological Functions and Expression of Samd4 of Lung Cancer H1299 CellsObjective:To investigate the influence of exogenous P53^mut. P53wt and P16 Gene overexpression on biological functions and expression of Samd4 of lung cancer H1299 Cells.Methods:To amplify target gene by PCR, the P53^mut. P53wt and P16 cDNA were inser-ted into effective eukaryotic expression vector pIRES2-EGFP, respectively.Restrictive enzyme EcoRI/BamHI digestion analysis was performed. These recombinant plasmid were transfected into H1299 cells with lipofectamine. The fluorescence microscope was employed to observe the transfected cells and the expression of EGFP. RT-PCR and western blot were used to validate the transfection effect.CCK-8 assay was used to detect the cell proliferation. Wound healing assay was used to measure the cell migration rate. Transwell chamber assay was used to study the invassive ability. Flow cytometry was used to detect the cell cycle and apoptosis. Western blot assay was used to detect the change of the expression of Smad4 in H1299 which were transfected with target genes.Results:The transfected H1299 cells displaying green fluorescence were observed under fluorescence microscope 72 hour post-transfection. RT-PCR and western blot indicated that P53^mut P53wt and P16 proteins were overexpressed in infected H1299 cell.There were no significant difference for the cell proliferation, migration and invasion ability between the negative control group and control group （P>0.05）. The ability of cell proliferation, migration and invasion of P53^mut transfected cell was increased than negetive control group （P<0.05）. On the contrary.the ability of the P53^mut transfected cell and P53wt. P16 cotransfected cell was decreased than negetive control group （P<0.05）. Morover, the latter inhibitory effect was stronger （P<0.05）. There were no significant difference for the cell cell cycle bwtween negetive control group and control group （P>0.05）. P53wt transfected group and P53wt、P16 co-transfected group were found G1 phase arrest compared with the negative control group（P<0.05）, moreover, the latter was more obvious （P<0.05）.There were no significant difference for the apoptosis between negative control group and control group（P>0.05）,P53wt transfected group and P53wt, P16 co-transfected group were found apoptotic index increased compared with the negative control group（P<0.05）, moreover, the latter was more obvious （P<0.05）. There were no significant difference for the expression of samd4 between negative control group and control group （P>0.05）, but the expression of samd4 of P53^mut transfected group was decreased （P<0.05）. On the contrary, the expression of smad4 was increased for the P53wt transfected group and P53wt P16 co-transfected group. Moreover, the increase was more obvious for the P53wt、 P16 co-transfected group（P<0.05）.Conclusion:Overexpressing P53^mut enhanced proliferation, migration, and invasion of the lung cancer cell line H1299, reduced Smad4 expression and reduced apoptosis of these cells. While overexpressing P53wt alone or in combination with P16 reduced proliferation, migration, and invasion of H1299 cells, elevated Smad4 protein expression, and increased apoptosis of these cells. And such effect was more potent in the P53wt+P16 co-transfected group.