The Studies On RNA Interference IASPP Gene In NSCLC Cell Lines

As one of the most important tumor suppressor genes,the products of p53 expression known as p53 protein(P53),is posttranslationally modified. In response to various cellular stresses, P53 expression is increased and resulting in either arrest of the cell cycle at G1 and G2/M,and then DNA damage repair,or commitment to death through apoptosis. The p53 is also one of the most frequently mutated genes. Mutations in this gene have been found in about 50% of human cancers. The other 50% of human tumours retain wild-type p53,but it is not known how the function of p53 protein is suppressed and, moreover, by what and how it can be reactivated in these tumors? These questions remain unclear recently, a new family of protein, apoptosis-stimulating protein of p53 (ASPPs) was discovered. This discovery may shed some light on above conundrum.RNA interference is the most effective gene silencing technology-spe- cific inhibition of target gene transcription, which reduced the level of the corresponding proteins and functions. Now,we have made a lot of progress in RNAi studies.Objective:In this experiment,we investigated the expression of iASPP in NSCLC cell lines in order to explore the relationship between p53 status and the expression of iASPP. At the same time, we aimed to investigate the inhibitory action of RNA interference to reduce the expression of iASPP,and observed the interference effect on cell proliferation and apoptosis.We studied preliminarily the expression of iASPP in different p53 genetic background of lung cancer cell,in order to provide experimental basis for treatment.Methods: Constructed the eukaryotic expression vector of iASPP, and transfected them into A549 and NCI-H157.And at the same time the mRNA and protein of iASPP were detected by RT-PCR and western blot respectively. Morphological features of apoptotic A549 and NCI-H15 cells induced by RNAi were observed with electronmicroscopy, meanwhile, imm- unofluorescence was used to show the effect of transfection.Results: The level of iASPP mRNA and protein were higher in A549 than in NCI-H157. Observed under transmission electron microscopy, untreated tumor cell shape normal, cell membrane integrity; the RNA interference groups can see an increase in apoptotic cells. Irregular nuclear membrane invagination can be seen , meanwhile,chromatin margination and the crescent-shaped body could been observed.Compared with the group without transfection,the expression of iASPP mRNA and protein were lower in the group of transfecting.Conclusion:Our experiment suggested that the construction of the recombinant expression vector iASPP-shRNA could inhibit the expression of iASPP ,which results in the cell apoptosis increased.And the level of iASPP is higher in p53 wild-type cell line than in p53 mutant-type cell lines.