【作者】 蔡文娟；

【导师】 孙保存；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2011， 博士

【摘要】 目的：研究滑膜肉瘤融合基因SYT-SSX的下游基因,筛选并明确其中部分基因在滑膜肉瘤发生发展中的功能与内在联系,从融合基因及下游基因的角度阐明滑膜肉瘤的发生发展机制,为临床治疗提供新的线索。方法：选取滑膜肉瘤细胞系SYO-1,构建特异性封闭融合基因SYT-SSX的siRNA真核载体,采用脂质体转染的方法,干扰并封闭融合基因的表达。利用Realtime RT-PCR的方法验证封闭效果。选取其中封闭效果良好的样本进行人类全基因组表达谱基因芯片分析。实验所得所有变化基因使用基于网络的基因分析工具,进行分类注释分析及代谢通路分析。选取其中一条与SYT-SSX密切相关的细胞代谢通路,Realtime RT-PCR验证其关键位点基因,在SYT-SSX封闭后mRNA表达水平的变化。Western Blot方法验证融合基因被特异性siRNA封闭后,细胞代谢通路关键位点的基因蛋白水平的变化。采用MTT染色活细胞数,评估细胞增殖活力,验证封闭SYT-SSX后,对于细胞的增殖的影响。PI染色,流式细胞仪检测细胞周期,同时检测细胞周期相关蛋白水平表达变化,验证SYT-SSX及所选通路对于细胞周期的影响。Annexin V-FITC和PI双染色,流式细胞仪检测细胞坏死与凋亡,初步探讨SYT-SSX与所选通路对于细胞凋亡的影响。同时,选取表达融合基因SYT-SSX及不表达融合基因的人体滑膜肉瘤及其它组织学类型肉瘤共74例,以免疫组织化学法及TUNEL染色方法,在蛋白水平验证体内环境中,SYT-SSX表达及所选通路蛋白与其下游功能蛋白之间的联系。应用统计学分析方法对所有结果进行计数资料的正态性检验、方差齐性检验,方差分析和t检验等,以P<0.05,差别具有统计学意义。结果：(1)构建的siRNA真核表达载体,在体外环境中可以特异性封闭融合基因SYT-SSX,并且在72h达到93.7%抑制率；(2)基因芯片检测结果全基因组表达谱基因芯片中375个基因上调,418个基因下调,所有变化的793个基因中653个可以进行基因功能分类,其中涉及蛋白磷酸化,细胞分裂,细胞周期,细胞转运,RNA结合,离子代谢,核蛋白代谢等。对所有变化基因进行细胞通路分析,5条通路被选中,其中ERK通路与SYT-SSX相关性最为显著(P<0.05)；(3)mRNA水平及蛋白水平验证ERK表达量在封闭融合基因SYT-SSX后,有明显下降；(4)封闭融合基因SYT-SSX后,MTT法检测活细胞数封闭组明显低于未处理组,封闭组细胞周期调控出现明显GO/G1期阻滞,处于DNA合成期(S期)细胞明显减少。Western Blot结果显示细胞周期相关蛋白,CyclinD1表达量明显下降,CDK4表达变化不明显；(5)融合基因封闭后, Annexin V-FITC和PI双染色,流式细胞仪测定,凋亡细胞数明显高于阴性对照siRNA组及仅使用脂质体处理组；(6)免疫组化染色结果显示,表达SYT-SSX融合基因患者,具有较高的ki-67染色指数,体内环境中表达较高的p-ERK蛋白及CyclinDl及CDK4蛋白,而TUNEL染色显示凋亡指数组间差别无统计学意义；(7)免疫组化结果显示,表达较高水平p-ERK的患者,同时表达较高水平的ki-67染色指数,CyclinD1和CDK4蛋白表达量高于p-ERK表达水平较低的患者,而TUNEL染色凋亡指数组间差别无统计学意义。结论：(1)构建siRNA真核载体,在体外环境中可以高效率,特异性的抑制融合基因SYT-SSX的表达；(2)融合基因可影响细胞增殖,凋亡,分化发育的多种基因,其中ERK可能为其中重要的通路之一,融合基因通过ERK通路促进细胞增殖,调控细胞周期,在一定程度上抑制细胞凋亡,从而在滑膜肉瘤的发生发展中起重要作用。(3)体内环境中,SYT-SSX可以上调p-ERK蛋白表达,进而提高细胞增殖活性,对细胞周期发挥正向调控作用,与体外实验结果一致。更多还原

【Abstract】 OBJECTIVE:This study is designed to gain more insight into the function of synovial sarcoma (SS) fusion gene. Screening part of genes on downstream of SYT-SSX, the aim was to explain the relationship of SYT-SSX and other pathways. These results would be hoped to elicit a novel path to discovery the function of the fusion genes in SS.METHODS:Two distinct siRNA duplexes for SYT-SSX carried by vector were synthesized, transfected by LipofectamineTM 2000. Total cellular RNAs were isolated using Trizol reagent. Then quantitative real-time PCR (qRT-PCR) was performed using SYBR Green Premix Ex TaqTM in order to manifest the inhibiting effect of SYT-SSX. The study evaluated the whole genome expression in SYO-1 cells inhibited as a result of specific small interfering RNA for SYT-SSX. All microarray data were submitted to the Database of Annotation, Visualization and Integrated Discovery (DAVID) 2008. Some down-stream genes of SYT-SSX chosen from the microarray analysis and involved in cell proliferation were further verified. Their expression was compared in SYT-SSX-blocked SYO-1 and control SYO-1 cells by qRT-PCR. The protein expression of some related down-stream genes was detected by western blot. Cell proliferation was measured using the methyl thiazolyl blue tetrazolium bromide colorimetric dye assay. Cell cycle analysis was conducted by fluorescence-activated cell sorting with cells stained by propidiumiodide. Apoptotic cells were measured with an Annexin V/FITC kit. and analyzed by flow cytometer after transfection. Seventy-four cases were selected into the study, including 54 SYT-SSX positive SSs tested by RT-PCR,4 SYT-SSX negative SSs (diagnosed as SS according to typical clinical context, histologic aspect and immunohistochemical profile), and 16 non-SSs (4 malignant melanoma,4 Ewing’s sarcomas,4 malignant peripheral nerve sheath tumor and 4 hemangio-peritheliomas). A tissue microarray (TMA) was constructed. Visualization of DNA fragmentation, a marker of apoptosis, was performed by the TUNEL method using the In Situ Apoptosis Detection Kit. Immunohistochemistry staining was performed on TMA. The expressions of Cyclin D1, CDK4, and p-ERK were analyzed in considering the staining intensity and area extent. Statistical comparisons were made by using ANOVA with subsequent application or t test where appropriate. P value of less than 0.05 was considered statistically significant.RESULTS:1 Compared with the cells only treated with lipofectamineTM, the expression of SYT-SSX reduced about 93.7%in the cells transfected with SYT-SSX-specific siRNA1.2. Compared with gene expression in cells transfected with negative control siRNA, the expression of 375 genes was up-regulated and of 418 genes was down-regulated in cells transfected with SYT-SSX-specific siRNAl. Among the 793 genes with significant change,653 genes could be assigned into different functional categories with DAVID gene functional category analysis. Subsequently,5 pathways hit by biocarta pathway analysis were indicated, including ERK1/2 pathway (P=0.043).3.The expression of ERK mRNA was confirmed to be significantly deceased after blocking SYT-SSX. Moreover, protein expression of ERK and p-ERK detected by western blot was equal to the results of qRT-PCR.4. The survival rate of SYO-1 cells transfected with specific siRNAl was most significantly reduced compared with that of SYO-1 cells only treated with lipofectamineTM.5. In comparison with control SYO-1 cells treated with lipofectamineTM, SYT-SSX-specific siRNAl and siRNA2 caused an increase in the percentage of G1/G0 phase cells, accompanied by a significant decrease in the percentage of S phase cells, cell cycle related proteins, Cyclin D1 and CDK4 were detected in SYO-1 cells after transfection with specific siRNAl and negative control siRNA. The expression of CyclinDl in siRNA1 transfected SYO-1 cells was significantly lower than that in negative control siRNA transcripted cells and cells untreated. But there was no notable change of CDK4 expression in SYO-1 cells treated with different siRNAs. It was shown the apoptotic rate in SYT-SSX-specific siRNAs transfected cells obviously increased.6. Cases positive for SYT-SSX showed higher ki-67 LI than SYT-SSX negative ones. In comparison of cases not detected SYT-SSX, expression of CyclinDl, CDK4 and p-ERK showed a trend to be more expressed in cases with SYT-SSX. Although the AI means in SYT-SSX positive SSs was higher than that in SYT-SSX negative cases, there were not significant difference between TUNEL AI according to expression of SYT-SSX.7. Higher ki-67 LI were shown in p-ERK positive group. Moreover, CyclinD1 and CDK4 were more expressed in p-ERK positive group than those in negative group.CONCLUSIONS:1. vector-based siRNA to block the expression of SYT-SSX had high specificity and efficiency.2. All genes changed were involved in phosphoprotein, transcription regulation, alternative splicing, direct protein sequencing, nucleotide-binding, transport, cell cycle, kinase and so on. Then pathway analysis indicated the ERK pathway were included in. SYT-SSX might play an important role via ERK pathway. Therefore activation of ERK may affect a broad array of cellular functions, including proliferation, survival, apoptosis, motility, transcription, metabolism and differentiation, and is in part responsible for oncogenesis.3. The results in vivo also suggested that the fusion gene SYT-SSX should be considered to play important role on SS cell growth maybe via ERK pathway.更多还原