【作者】 郭锋杰；

【导师】 李官成；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 目的：本研究利用前期构建的地面对照和太空诱变宫颈癌CaSki细胞株消减文库,筛选在地面对照和太空诱变宫颈癌CaSki细胞中差异表达的基因,并对其功能进行研究。方法：利用消减杂交文库制备反向Northern杂交膜,对48A9CaSki细胞和对照CaSki细胞进行同位素探针标记,而后反向Northern杂交,通过放射性自显影筛选差异性表达基因。通过RNAi方法和构建真核表达载体方法,分别抑制和增加差异性表达基因的表达,利用MTT、流式细胞术、裸鼠成瘤等实验检测CaSki细胞生物学性状的变化,进而检测细胞对抗肿瘤药物的敏感性,用RT-PCR和Western blot检验此基因作用机制。结果：1.我们筛选到33个差异表达基因,涉及细胞骨架、细胞分化凋亡、基因转录等。我们利用太空这一特殊环境得到了和肿瘤发生发展相关的基因,为研究肿瘤发生发展的机制奠定了基础。其中STC1基因是我们比较感兴趣的一个差异表达基因。2.STC1基因在宫颈癌组织中的表达弱于正常组织。MTT实验检测发现,STC1基因沉默组(CaSki/siSTC1)细胞生长速度比阴性对照组(CaSki/siNC)细胞的生长速度明显加快,集落形成实验表明集落形成率明显上升,流式细胞分析显示基因沉默组细胞G1期细胞减少,细胞迁移侵袭实验表明STC1表达减少后,CaSki细胞的侵袭迁移能力均上升。裸鼠成瘤实验表明沉默组细胞成瘤能力增强,这些表明STC1基因可能抑制CaSki细胞的生长、转移,诱导细胞凋亡。3.在CaSki细胞转染STC1真核表达载体后,MTT实验检测发现,STC1基因转染组(CaSki/STC1)细胞生长速度比阴性空载体转染对照组(CaSki/NC)细胞的生长速度明显减慢,集落形成实验表明集落形成率明显下降。流式细胞分析显示基因转染组细胞G1期细胞增加。细胞迁移侵袭实验表明STC1表达增加后,CaSki细胞的侵袭迁移能力均下降。裸鼠成瘤实验表明STC1基因转染组成瘤能力下降。以上观测表明,STC1基因转染CaSki细胞后抑制了CaSki细胞的生长、转移,诱导了细胞的凋亡。所以STC1基因转染有可能用于肿瘤的治疗。4.顺铂、毒胡萝卜素和雷帕霉素均抑制了CaSki细胞的增殖,诱导了细胞凋亡,当CaSki细胞增加STC1基因含量时,顺铂、毒胡萝卜素和雷帕霉素对细胞的抑制增强,凋亡细胞增加,这些表明STC1基因增强了CaSki细胞对顺铂、毒胡萝卜素和雷帕霉素药物的敏感性,为肿瘤的治疗提供了新的途径。5.无论在mRNA水平还是蛋白水平,当CaSki细胞内STC1表达水平下降时(RNAi),NF-κB水平随之下降；当STC1表达水平上升时(真核转染STC1-pcDNA3.1 (+)), NK-κB表达水平也随之上升,表明STC1与NF-κB在表达水平上呈正相关。当用顺铂(2μg/ml)或毒胡萝卜素(3μM)刺激CaSki和HeLa细胞时,Caspase-3 mRNA表达量逐渐增加,而STC1与NF-κB也呈现表达上升趋势,且呈正比。染色质免疫共沉淀实验发现,NF-κB可在细胞内与STC1的启动子发生结合。这些结果表明,核转录因子NF-κB在宫颈癌细胞中和STC1基因启动子结合启动了STC1的表达,从而诱导了宫颈癌细胞的凋亡。结论：通过构建抑制性消减杂交文库结合反向Northern dot blot筛选差异性表达基因的方法,是一种高通量高效率的筛选方法。STC1在宫颈癌细胞的表达可抑制细胞的增殖,诱导细胞的凋亡,并且通过核转录因子NF-κB的启动发挥作用。这为进一步探讨肿瘤的发生发展机制奠定了基础,并有可能成为临床治疗肿瘤的一条新途径。更多还原

【Abstract】 AIM:This study will use the suppression subtractive hybridization library in the ground and space cervical cancer CaSki cells to select the differentially expressed genes and investigate the functions of the genes.METHODS:The hybridization membrane was made by the library, and 48A9Caski and ground CaSki were labed P32 before the reverse Northern hybridization. The differentially expressed genes were screened by autoradiography. The gene expression was inhibited or enhanced by the method of RNA interference or constructing eukaryotic expression vector. MTT, flow cytometry and in vivo tumor growth assay were used to investigate the biologic change of CaSki cells. The sensitivity of CaSki cells to anti-tumor drugs was checked. The mechanism of the gene was further studied by RT-PCR and Western blot.RESULTS:1.We found 33 differentially expressed genes which were associated with cytoskeleton, cell differentiation, apoptosis, and gene transcription and so on. The differentially expressed genes associated with tumor established the foundation for tumorigenesis and development. STC1 was one of most significant genes. 2. The expression of STC1 in cervical cancer tissues was low compared with normal tissues. MTT assay showed that the speed of CaSki/siSTC1 cells was quick compared to negative control CaSki/siNC. The colony conforming assay showed the colony numbers of CaSki/siRNA cells were elevated. Flow cytometry demonstrated the G1 phase cells of CaSki/siRNA cells reduced. Transwell assay showed the invasion ability of CaSki/siRNA cells enhanced. In vivo tumor growth assay showed that the tumor growth of CaSki/siRNA cells enhanced.3. MTT assay showed that the speed of CaSki/STC1 cells transfected with pcDNA3.1-STC1 vector was slow compared to negative control CaSki/NC. The colony conforming assay showed the colony numbers of CaSki/STC1 cells were reduced. Flow cytometry demonstrated the G1 phase cells of CaSki/STC1 cells enhanced. Transwell assay showed the invasion ability of CaSki/STC1 cells decreased. In vivo tumor growth assay showed that the tumor growth of CaSki/STC1 cells reduced. STC1 might be used to treat the tumor.4. Cisplatin, thapsin, and rapamycin inhibited the proliferation and induced the apoptosis of CaSki cells. When the expression of STC1 gene enhanced, these drugs readily suppressed the cell proliferation and induced the cell apoptosis. These demonstrated that STC1 sensitized CaSki cells to cisplatin, thapsin, and rapamycin, and provided a new way of curing tumor.5. In mRNA and protein level, when the expression of STC1 decreased and elevated, the expression level of NF-κB accordingly changed. The expression between STC1 and NF-κB was positive correlation. When cisplatin or thapsin was added into CaSki or HeLa cells, the mRNA level of Caspase-3 increased and the expression of STC1 and NF-κB also increased. Chromatin immunoprecipitation (CHIP) showed that NF-κB combined with the promoter of STC1 gene. These results demonstrated that NF-κB combined with promoter of STC1 and evoke the gene expression, which induced the apoptosis of cervical cancer cells.CONCLUSION:The way that constructing suppression subtractive hybridization library combining with reverse Northern blot selected the differentially expressed genes is a high throughput and efficient screening. The expression in CaSki cells can inhibit the proliferation and induce the apoptosis of cells by nuclear factor NF-κB. These results establish the foundation for carcinogenesis and development and might be a new road to cure tumor.更多还原