| Objective:Microtubules (MTs) are one of the major components of cell cytoskeleton, and are involved in many cellular active movements including cell morphological stabilization, cellular locomotion, chromosomal separation, and cell division. The different structures and functions of MTs are generated by the subcellular location of MTs or the microtubule associated protien binding them. Stathmin is a confirmed microtubule associated protien that results directly in unstabilized MTs dynamics by enhancing polymerization of MTs and sergregation of tubulin subunit. Many kinases inactivate and activate the function of stathmin to unstabilized MTs through phosphorylation and dephosphorylation. Nasopharyngeal carcinoma (NPC) is a high-incidence cancer in Chinese population and is closely associated with Epstein-Barr virus infection. The latent membrane protein 1 (LMP1) coded by EB virus is affirmed as an oncoprotein. Stathmin is a downstream molecular in LMP1 regulation network. LMP1 regulates the phosphorylation of stathmin through several pathways, which causes cell immortalization and enhances tumor cell proliferation, metastases, and invasion by destabilizing MTs.Small interfering RNA (siRNA) is a small molecule in manual RNA interference technology, which can silence the expression of the complementary mRNA sequence. It has several important characteristics such as high specificity, high efficiency, and hereditability. This study is to decrease MTs'depolymerization, enhance MTs'polymerization, thereby inducing tumor cell apoptosis, suppressing its proliferation, metastases, and invasion, through silencing the expression of stathmin by constructing the siRNA plasmid targeting to stathmin.Paclitaxel is one of the main antitumor medicine that effects on MTs. By enhanceing polymerization and preventing depolymerization of MTs, subsequently maintaining stability of MTs, paclitaxel may suppresse cell mitosis, and has remarkable radiosensitizing effect, making the similar effect as the siRNA targeting to stathmin. Thus, this study is proposed to combine both to magnify their biological effect on tumor cells.Method:In this study, we used NPC cell CNE1-LMP1 as our research model, and siRNA plasmid targeting to stathmin as our strategy, to discuss the effect of siRNA targeting to stathmin on the apoptosis, proliferation, invasion, and metastases of NPC cell by suppressing its protein expression, and also to evaluate the corporate effect of the siRNA targeting to stathmin and anti-microtubule chemotherapy medicine.First of all, we confirmed whether the mRNA and protein expression level of stathmin in NPC cell CNE1-LMP1 were decreased by siRNA targeting to stathmin with RT-PCR and Western blot asssy. The suppression of NPC cell growth and proliferation transfected with the siRNA targeting to stathmin was verified by MTT assay.The effects of the siRNA on inducing NPC cell apoptosis, changing mitotic cycle and mitochondrial potential was determined by flow cytometry (FCM) assay, and then the cell apoptosis was confirmed further by AO/EB stain, TUNEL assay and Western blot for detecting the protein marker of apoptosis caspase. The apoptosis pathway of NPC cells with the siRNA was determiated by evaluating the change of caspase protein and mitochondrial potential.Secondly, the rate change of solubilized microtubules to polymeric microtubules in NPC cells with siRNA targeting to stathmin was detected by Western blot. Regulating effect on microtubule polymerization resulted from the siRNA was determined by indirect fluorescent assay. The motility, on two dimensional floor, of NPC cells with the siRNA was detected by wound-healing assay. However, the migration potential and invasiveness of NPC cells with the siRNA, in three-dimensional matrices were assay by Transwell assay.Thirdly, we used MTT assay to detect the combination effect of transfected the siRNA targeting to stathmin and paclitaxel on NPC cell growth and proliferation. The FCM assay was performed to detect the combination apoptosis effect on cells with both paclitaxe and the siRNA. The indirect fluorescence and Western blot assay was used to identify the combination effect on MTs in NPC cells with both paclitaxel and the siRNA.Last, we used RT-PCR and Western blot to detect the generating effect of paclitaxel on NPC cells and other higher stathmin expression cells A375, MGC, and Hela on mRNA and protein level.Results:First of all, that the siRNA targeting to stathmin may obviously suppress the expression level of stathmin mRNA and protein in NPC cells was confirmed by RT-PCR and Western blot. MTT assay and cell growth curvature revealed the suppressing effect of the siRNA on NPC cell proliferation. FCM analysis showed the siRNA can remarkably induce NPC cell apoptosis (reach 30.9%) and inhibit tumor cells in G2/M phase (16.3%). AO/EB stain, TUNEL assay and Western blot assay for detecting of caspase-3,8,9 also showed the further evidence of apoptosis. Morever, the change of caspase and mitochondrial potential indicated that the siRNA induced cells apoptosis through the mitochondria pathway.Secondly, Western blot assay confirmed the siRNA targeting to stathmin increased obviously polymeric MTs, reduced soluble MTs, and also increased the rate of polymeric MTs to solubilized MTs (P/S=3.43) in NPC cells. The MTs in cells transfected the siRNA are showed long and bunchy, and fluorescence is remarkably intensed by indirect immunofluorescence assay. Transwell test showed cells with the siRNA had increased metastasis potential and invasiveness, although migration of NPC cells with the siRNA weren't revealed obvious change by wound-healing assay.Thirdly, a combination of the siRNA plasmids targeting to stathmin with paclitaxel was applied to NPC cells CNE1-LMP1. MTT assay revealed the cooperative application inhibitory effect of the two treatments on cell proliferation is much stronger than the effect of independent application individually. In addition, the proliferation suppression extent of cells with the siRNA is the paclitaxel dose-dependent in certain concentration range. Indirect immunofluorescent assay also showed individual treatment of each could make the cell MTs thicker and longer and the fluorescent intensity stronger, but the cooperative application of both treatments showed a better effect. Similarly, Western blot assay revealed that cooperative application of double treatment revealed a stronger effect although individual treatment of each can increase polymeric MTs, reduce solubilized MTs, and increase the rate of polymeric MTs to solubilized MTs (P/S=2.05).Last, we also found recently in NPC cells and other higher stathmin expression carcinoma cell lines, paclictaxel obviously inhibited the expression of stathmin, which also showed paclitaxel dose-dependent.Conclusion:The siRNA targeting to stathmin could suppresses NPC cell proliferation, metastasis and invasion through silencing stathmin protein expression, and it induces cell apoptosis by the pathway through mitochondria. The regulation on cell apoptosis, proliferation, and invasion was achieved by strengthening MTs polymerization, reducing its degradation, and enhancing its polymerization and stability. Pclitaxel could lower the expression of stathmin. The siRNA targeting to stathmin and chemotherapy medicine paclitaxel would be a combined treatment on NPC cells, which will be a novel candidate for clinical chemotherapy to NPC. |
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