Effect Of Antisense Peptide Nucleic Acid Of Survivin On Lung Cancer A549 Cell Line

Tumorigenesis is a complicated multi-step process.Previous researches had showed that proto-oncogenes activation and anti-oncogenes inactivation,which can induce the abnormal differentiation and proliferation of tumor cells,contribute to tumorigenesis. Recently there were reports indicating that cell apoptosis may also play a role in tumorigenesis.Cell apoptosis is an important life process which plays a key role in the homeostasis maintenance for organisms.Aberrant cell apoptosis may leads to cell transformation and subsequent tumorigenesis,inhibitor of apoptosis protein(IAP) can suppress cell apoptosis.Survivin is a newly identified member of IAP.This protein is expressed in various tumor cells,thymus and germen but not in other normal tissues and it is involved in the suppression of cell apoptosis and in the mitosis.Survivin is usually expressed at the G2-M stage in cell cycle and it competitively binds to activated Caspase-3 and Caspase-7,which disrupts the formation of super-molecular Caspase polymers and hereafter suppresses the cell apoptosis through Caspase pathway.Cell apoptosis can eliminate cells with abnormal DNA sequence and these cells usually show high potentials to undergo tumorigenesis.Disruption of cell apoptosis can help these abnormal cells to persist longer in vivo,to accumulate more gene mutations,and to escape the immune surveillance of the host,which cooperate to contribute to the tumorigenesis.Currently,there are several ways to inhibit Survivin synthesis and activity:firstly, inhibition of Survivin synthesis using nucleoside analogues;secondly,inhibition of Survivin synthesis using antisense-RNA;thirdly,inhibition of Survivin activity using differentiation inductors;fourthly,inhibition of Survivin synthesis using antagonists; fifthly,Inhibition of Survivin expression using antisense-peptides.In this study,Survivin and pl6 protein expressions were detected by immunohistochemistry methods.The promoter upstream in the coding regions(CDS) of human Survivin gene,which code the Survivin protein,was used as a target site(232- 251bp) and was inhibited using synthesized and fluorescin-labeled peptide nucleic acid (PNA).The PNA was cotransfected into lung cancer A549 cell line using liposome and the transfection results was identified under an inverted fluorescence microscope.Survivin activity was measured at different time points and different PNA concentrations.Cell survival rate was detected through MTT assay.Cell cycle ratio was assessed using flow cytometry.Expression of survivin after aPNA transfection was investigated using RT-PCR and Western Blot methods.Conclusions:1.The expression of the Survivin is higher in non-small-cell lung cancer(NSCLC) cells than that in tissues adjacent to the tumors.The relationship between Survivin overexpression in NSCLC and sex or pathology-grouping is not statistically significant.. Survivin overexpression is positively related to the differentiation,the TNM stages and the lymph node metastases of NSCLC cells.There is a positive correlation between Survivin expression and P16 gene expression in NSCLC,which indicate that these two proteins may play a role in NSCLC progression.2.Antisense-PNA was successfully transfected into lung cancer A549 cell line,and the inhibition of lung cancer cells growth was observed after transfection.PNA can suppress Survivin synthesis in lung cancer cell blocking the cell at G2/M stage and leading to apoptosis of lung cancer cell.The inhibitory effect is increased in a time- and concentration- dependent manner.3.Survivin aPNA and CDDP coadministration is more effective in the inducement of lung cancer cell apoptosis in a time-dependent manner.