RNA Interference IASPP Enhance Susceptibility Of Non-small Cell Lung Cancer A549for Cisplayin

Objective: The ASPP (Apoptosis-stimulating protein of P53) protein family is aclosely associated with the function of p53gene. This protein family includes threemembers: ASPP1, ASPP2and iASPP. ASPP1and ASPP2can enhance the tumor-suppressor function of p53gene, while the iASPP is a special member of this tumorsuppressor gene family. Because iASPP has a similar domain with ASPP1and ASPP2,it could competitively combine with p53, thus plays an opposite effect comparing toASPP1and ASPP2. This may be one of the reasons why wild type p53gene did notplay its role in some tumors. c-jun N-terminal kinase (c-jun N-terminal kinase, JNK)pathway is one of the MAPK signal transduction pathway, and plays an importantrole. JNK could be activated by a variety of factors, such as cytokines (TNF-alpha,IL-1), growth factor (EGF) and stress stimulation (such as ultraviolet light, ionizingradiation, heat shock, high osmotic stimuli and oxidative damage, etc.), and certainG protein-coupled receptors. It plays an important role in many cellular regulations,including cell proliferation and differentiation, maintenance of cell shape, cell apoptosisand malignant transformation, and stress response. We silenced iASPP gene innon-small cell lung cancer cell line A549(p53wild type) using RNA interferencetechnology, and observed the changes of proliferation or apoptosis in non-transfectednon-small cell lung cancer cells an transfected with non-small cell lung cancer cellsafter cisplatin（CDDP）treatment. We also studied the effect of transfection and CDDPon the activity of JNK and the expression of c-Jun in non-small cell lung cancer cells.Our experiment is to lay a foundation to explore the iASPP mediated RNA interferencetherapy.Methods: iASPP interference plasmid and negative control plasmids weretransfected into A549cells. Pictures of transfected cell were took using fluorescencemicroscopy to observe the effect of transfection; We used MTT assay to screen the minimum effective concentration of chemotherapy drug CDDP, and to initially detectedof the growth inhibition of CDDP in transfected non-small cell lung cancer cells anduntransfected non-small cell lung cancer cell.24hours after the minimum effectiveconcentration therapy (CDDP) on these cells, Immunoprecipitation and Western blotwere used to analysis the activity of JNK/SAPK and the expression ofc-Jun. Experimental data were handled by software SPSS13.0, and mean comparationwere by analysis of variance (ANOVA). A value of P <0.05was considered to besignificant.Results:1. Fluorescence microscopy photo shows that: positive plasmid (si1and si2) andnegative plasmid (sin) have been successfully transferred into A549cells.2. MTT assay results showed that: CDDP inhibite the proliferation of bothuntransfected and transfected A549cells. When the concentration of CDDP was lessthan3μg/ml, it didn’t affect the proliferation of untransfected and transfected A549cell. When CDDP concentration was greater than3μg/ml, with increasing drugconcentration, the inhibition rate of A549cells was also increased (P <0.05). And theinhibition effect in positive plasmid (si1and si2) transfected A549cells was higher thanin untransfected cells, only transfection reagent handled A549cells and negativeplasmid (sin) transfected A549cells.3. JNK immunoprecipitation results and Western blot analysis showed that: JNK/SPAK in normal A549cells had basic activity. The expression of phosphorylated c-Junin Transfected A549cells was higher than untransfected A549cells. After treated withCDDP (6μg/ml), the expression of phosphorylated c-Jun in untransfected A549cellsand transfected A549cells was higher than which were not treated with CDDP.Conclusion:1. Chemotherapeutic drugs CDDP played an inhibition effect in bothuntransfected and transfected A549cells. Transfected A549cells were more sensitive toCDDP.2. The JNK/SPAK of normal A549cells is low expression phenomenon. iASPPgene silenced in A549cells could enhance the activity of JNK/SAPK and increase theexpression of phosphorylated c-Jun. CDDP could activate the JNK/SAPK andupregulate the expression of phosphorylation of c-Jun in both untransfected andtransfected A549cells，and the positive role was enhance by RNAi iASPP gene.