【作者】 朱江；

【导师】 洪苏玲；

【作者基本信息】 重庆医科大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 Plk1(Polo-like kinase 1)是一个从酵母到人类高度保守的真核生物丝氨酸/苏氨酸蛋白激酶家族成员。大量研究已经证明,其主要参与细胞有丝分裂过程的调节和肿瘤的发生发展,并被公认是一个有良好应用前景的恶性肿瘤治疗新靶点。然而,关于Plk1与在鼻咽癌和喉癌中的作用及其靶位治疗价值,国内外仍鲜有报道。本论文主要采用RNA干扰技术首次对此进行相对系统的研究,结果分述汇总如下:(1)高效Plk1 siRNA序列的快速筛选:首先设计并合成了四组新的候选Plk1 siRNA序列,然后主要采用人喉癌Hep2细胞系和常用肿瘤细胞系HeLa,结合细胞表型检测和基因表达水平分析,对这些候选siRNA序列进行了筛选。细胞生长分裂表型观察结果表明,这四组Plk1 siRNA均能够有效抑制Hep2和HeLa细胞的生长分裂。流式细胞仪分析结果表明,在转染后72 h,与对照组相比,各Plk1 siRNA转染组均出现明显的有丝分裂期阻滞效应,大量细胞被阻滞于M期。同时,与对照组相比,在各Plk1 siRNA转染组中,位于sub-G1期的细胞比例显著升高,强烈提示Plk1 siRNA诱发细胞发生凋亡。RT-PCR分析结果表明,各组Plk1 siRNA均能有效抑制Plk1在mRNA水平上的表达。Western印迹分析结果表明,各组Plk1 siRNA均能有效抑制Plk1在蛋白质水平上的表达。综合上述结果,我们发现:Plk1-siRNA-607、Plk1-siRNA-838和Plk1-siRNA-484这三组序列的效果更强,不仅能显著抑制内源性Plk1的表达,而且也具有很强抑制肿瘤细胞生长、诱发大规模细胞凋亡的作用,可用于后续的以Plk1为靶点的抗肿瘤基因治疗药物研发。另外,我们也尝试了逆向筛选思路,即首先直接检测不同Plk1 siRNA候选序列对细胞生长表型的影响,最后再确证Plk1 siRNA对细胞内源性Plk1表达的抑制效应。结果表明,这种筛选策略和传统的筛选策略相比,具有简易、快速和高效的特点,也非常适用于对Plk1 siRNA和小分子抑制剂的大规模筛选。(2)Plk1 siRNA-607对鼻咽癌和喉癌细胞生长增殖的影响:本部分研究则在前一部分研究的基础上,使用筛选出的高效Plk1-siRNA-607序列,在转染后分别设置了24h和48h两个检测时间点,相对系统地研究其对人喉癌细胞系Hep2和人鼻咽癌细胞系HNE1生长、细胞周期和凋亡的影响,由此初步评价以Plk1为靶点开展头颈癌治疗的可行性和潜在应用价值。细胞生长分析结果表明:Plk1 siRNA-607能够通过有效的抑制Plk1表达而抑制人喉癌细胞系Hep2和人鼻咽癌细胞系HNE1的生长增殖和诱发细胞凋亡,且以在Hep2的生长增殖抑制效应要明显强于HNE1。流式细胞仪分析结果表明, Plk1 siRNA-607转染组的Hep2和HNE1细胞均出现明显的有丝分裂期阻滞效应,大量细胞被阻滞于有丝分裂期,磷酸化Histone H3表达水平均显著提高;且位于sub-G1期的细胞比例显著升高。Annexin V-FITC细胞凋亡分析结果表明,Plk1 siRNA-607转染组的Hep2和HNE1细胞均出现大量FITC+/PI―凋亡细胞,表明Plk1 siRNA-607可诱发Hep2和HNE1细胞发生细胞凋亡。这些研究结果表明,siRNA介导的Plk1表达沉默是一种有效的鼻咽癌和喉癌治疗策略,提示Plk1可做为一个新的分子靶点用于鼻咽癌和喉癌的治疗。(3)siRNA介导的Plk1表达沉默对COS7细胞生长增殖的影响:在本部分研究中,我们观察了siRNA介导的Plk1表达沉默对体外转化细胞系COS-7细胞生长增殖的影响,主要目的在于探讨Plk1作为分子靶点用于肿瘤治疗的普遍性。结果表明,Plk1表达被抑制后,细胞生长显著受到抑制,大致大规模的细胞凋亡,大量细胞进入sub-G1期。该结果和前述第一部分和第二部分中使用HeLa、Hep2和HNE1等肿瘤细胞系获得的研究结果类似,说明Plk1可作为分子靶点用于多种肿瘤的治疗。综上,本论文对于Plk1在鼻咽癌和喉癌中的作用及其靶向治疗价值进行了相对系统的初步研究,为进一步研究和开发以Plk1为靶点的抗肿瘤治疗药物尤其是鼻咽癌和喉癌的抗肿瘤治疗药物奠定了坚实的基础,具有积极的理论和现实意义。更多还原

【Abstract】 The polo-like kinases (Plks) belongs to a family of serine/threonine kinases and are highly conserved from yeasts to mammals. Plk1 is the best-characterized member of the human Plk family. Numerous studies have shown that Plk1 plays critical roles during mitosis and regulates many mitotic events. Moreover, Plk1 was also found to be overexpressed in a broad range of human tumors and serves as a novel therapeutic target in cancer field. In the present study, we systematically investigate the role of Plk1 and its therapeutic potential in nasopharyngeal and larynx cancer. The results are summarized as following.(1) Quick screening of efficient Plk1 siRNA candidates.We designed and synthesized four different Plk1 siRNA candidates which were then are subjected to quick screening by detection of cell growth phenotype and gene expression analysis in Hep2 and HeLa cells. Cell growth analysis revealed that, all of these four Plk1 siRNA candidates efficiently inhibit the growth of Hep2 and HeLa cells. Cell cycle analyis indicated that, compared with control siRNA, Plk1 siRNA cause a significant mitotic cell cycle arrest 72-hours after transfection. In addition, Plk1 siRNA transfection also caused a significant increase in sub-G1 populations. RT-PCR and immunoblotting analysis revealed that all of these four Plk1 siRNA candidates efficiently resulted into the inhibition of Plk1 expression in both Hep2 and HeLa cells. Notably, among these four candidates, Plk1-siRNA-607, Plk1-siRNA-838, and Plk1-siRNA-484 have the strongest activity on cell growth inhibition and apoptosis induction. Moreover, we also tried to mainly use cell growth phenotype for Plk1 siRNA screening. Our results suggested that, compared with the traditional strategy, this method obviously has the advantages such as simplicity and high efficiency, and thus can be used for the high throughout screening of Plk1 siRNA and small molecule inhibitors.(2) The effects of Plk1 siRNA-607 on cell growth and proliferation in human nasopharygeal and larynx carcinoma cells.Furthermore, we investigated the effects of Plk1 siRNA-607 on cell growth and proliferation in human nasopharygeal and larynx carcinoma cells including Hep2 and HNE1. Cell growth analysis indicated that Plk1 siRNA-607 significantly inhibited the cellular growth in both Hep2 and HNE1 cells. FACS analysis indicated that Plk1 siRNA-607 caused a remarkable mitosis cell cyle arrest, the expression level of phosphor-histone H3 remarkably increased. Additionally, sub-G1 population cells also significantly increased after Plk1 siRNA-607 transfection suggestion the occurance of apoptosis. Annexin V-FITC apoptosis assay indicated that Plk1 siRNA-607 did resulted into apoptosis in both Hep2 and HNE1 cells. These results suggested that Plk1 may serve as an efficient therapeutic target in both nasopharygeal and larynx cancer.(3) The effects of siRNA-mediated Plk1 depletion on COS7 cells.The study of this part was designed to investigate the effects of siRNA-mediated Plk1 depletion on COS7 cells and the therapeutic potential of Plk1 in cancer treatment. Synthetic siRNA cocktail duplexes against Plk1 were introduced into COS7 cells. RT-PCR and immunoblotting analysis showed that Plk1 siRNA transfection resulted in a significant inhibition in Plk1 expression in the cells. Cell growth assay indicated that siRNA-mediated Plk1 depletion significantly inhibited the proliferation of COS7 cells. FACS analysis revealed that Plk1 depletion caused cell cycle arrest, resulting into large population of sub-G1 cells. These results are consistent with those obtained from Part one and two in HeLa, Hep2, and HNE1 cells, strongly suggesting that Plk1 may serve as an efficient therapeutic target in many types of cancers.Taken together, in the present study, we systematically investigate the role of Plk1 and its therapeutic potential in nasopharyngeal and larynx cancer. The results indicated that Plk1 might serve as an efficient therapeutic target in both nasopharygeal and larynx cancer.更多还原