靶向Survivin的RNAi治疗垂体腺瘤的研究
摘要
RNA干扰技术是目前新兴且日益成熟的的基因沉默技术,目前被认为是基因工程学领域的非常有效的研究工具。用此技术可以高效稳定特异地阻止目标基因的表达,从而达到较为理想的沉默效果。目前,dsRNA,特别是siRNA已被广泛而成功地应用于诸如基因功能研究以及疾病病因与治疗研究等多个领域。
Survivin是耶鲁大学Ambrosini等于1997年发现的,是凋亡抑制蛋白家族新成员,具有分子量小、功能强大等特点。它是迄今发现的最强的凋亡抑制因子,主要分布于胚胎及分化未成熟的组织。在成人体内除胸腺及胎盘中有微量表达外,所有分化成熟的组织,包括外周血白细胞、淋巴结、脾、胰、肾、骨骼肌、心、肝、肺、脑组织中均无表达,而在人类肿瘤中几乎都表达。并且Survivin与肿瘤的侵袭性密切相关。
本研究方法是:首先,检测Survivin在垂体腺瘤中的蛋白表达,探讨Survivin与垂体腺瘤侵袭性发生的相关性以及它们之间的内在联系。然后,通过设计在垂体瘤细胞中稳定高效表达的Survivin-siRNA质粒载体,研究该重组载体对于垂体瘤细胞中Survivin的干扰效果,并进一步明确干扰前后垂体瘤细胞的增殖及凋亡水平变化,以期探讨Survivin是否可成为未来进行垂体瘤基因治疗研究的有效靶点。
第一部分Survivin在垂体瘤中的表达及与生物学行为关系
目的
Survivin是凋亡抑制蛋白家族非常独特的成员,其基因在肿瘤中的表达与增加肿瘤的侵袭性和降低患者的生存率关系密切。本研究目的是检测Survivin基因在垂体腺瘤中的蛋白表达,并探讨Survivin与垂体腺瘤侵袭性发生的相关性以及它们之间的内在联系。
方法
1.标本
回顾性随机收集2006年7月-2008年4月期间山东省立医院神经外科的垂体腺瘤手术标本66例,其中女性29例(年龄29-77岁,平均年龄65.3±4.3岁),男性37例(年龄34-82岁,平均年龄68.1±2.5岁)。依据MRI提示肿瘤侵犯海绵窦或有骨质破坏,和/或术中观察肿瘤对周围骨质、海绵窦有/无侵犯等情况分为侵袭性组(39例)和非侵袭性组(27例)。所有病例诊断均有术后的免疫组化病理证实。
2.免疫组化染色方法
采用免疫组化链霉菌抗生物素蛋白-过氧化物酶连接(S-P)法,按标准实验流程,检测Survivin基因在侵袭性和非侵袭性垂体瘤中的蛋白表达。
3.结果判定标准
根据半定量免疫组织化学评价Survivin反应阳性细胞显色强度。在高倍视野(×400)下,每张切片随机观察5个视野的肿瘤细胞,计算200个肿瘤细胞免疫反应阳性的细胞百分比。每张切片分别有四名实验者进行单独评价,对于有分歧的进行重复评价,直至达到一致。
Survivin表达评估:强阳性(+++):阳性细胞大于50%或显色深;中度阳性(++):阳性细胞10%至50%之间或染色略深;弱阳性(+):阳性细胞小于10%或显色浅;阴性(一):无阳性细胞染色。
4.统计分析
用卡方检验进行统计分析。SPSS(版本13.0)统计学软件进行统计。设定p＜0.05有统计学意义。
结果
Survivin在垂体瘤中以细胞质着色为主,仅有散在的细胞核着色。Survivin总的阳性率为69.7%(46/66)。在侵袭性垂体瘤中Survivin的阳性率为89.7%(35/39);而在非侵袭性垂体瘤中Survivin的阳性率仅为40.7%(11/27)。卡方检验示两组差异有统计学意义(x~2=14.309,P=0.0002＜0.05)。另外,在免疫组化反应为阳性的侵袭性垂体瘤中以强阳性和中度阳性为主(71.4%,25/35),而在非侵袭性垂体瘤中以弱阳性为主(72.7%,8/11)。
结论
1.Survivin在垂体瘤中有高表达。
2.Survivin在侵袭性与非侵袭性垂体腺瘤中的表达有显著性差异,因此Survivin与垂体瘤的侵袭性密切相关。
3.Survivin可以作为判断垂体腺瘤侵袭性生长的有效指标和用做垂体瘤基因治疗研究的靶点。
第二部分设计并筛选针对Survivin有效的siRNA载体
目的
设计并合成3组针对Survivin基因的siRNA质粒载体,并筛选出其中2组有效的载体,为下一步的研究做准备。
方法
1.设计并合成siRNA载体
本实验运用公众网站设计软件,针对大鼠的Survivin靶基因序列,设计3组siRNA载体,分别标记为1#,2#,3g;同时合成1组为阴性对照载体,标记为0#。然后进行测序比对。结果显示四组载体被成功构建,测序无误。
2.转染工具细胞株(胶质瘤C6)
培养生长状态良好的工具细胞(胶质瘤C6),转染前一天将细胞分入6-孔培养板培养,转染当天按实验设计的分组情况加入不同质粒载体进行细胞的转染实验。转染48小时后荧光显微镜下观察GFP表达情况,观测转染效率。
3.检测干扰效果,筛选有效载体。
选出转染效率为90%左右的组,采用FQ-PCR的方法检测目的基因的mRNA表达情况,进而判断不同靶点的干扰效果,筛选出2组有效载体。
结果
1.在3组siRNA载体中,1#、2#、3#转染效率分别为87.4±6.2%、92.6±4.5%、75.3±11.0%;阴性对照载体0#的转染效率为95.0±4.2%。
2.3个靶点均有敲减效果。与阴性对照载体0#对比,载体1#,2#,3#敲减效率分别为68.4%、44.4%、22.7%。综合转染效率及敲减效果,1#、2#是最佳的靶点。
结论
1.本部分实验成功构建了3组特异性针对大鼠基因Survivin的siRNA载体。
2.通过该部分实验,我们成功地从3组重组载体中筛选出高效率的靶点,为下一步选用高效率的重组载体对垂体瘤细胞GH3进行转染提供了可靠的实验基础。
第三部分RNAi沉默Survivin基因对垂体瘤细胞增殖及凋亡水平的影响
目的
利用构建好的特异性强、转染率高的携带针对Survivin的siRNA载体1#、2#,研究它们介导RNA干扰对垂体瘤细胞株GH3增殖和凋亡的影响及其意义。
方法
1.分别利用siRNA载体1#、2#转染垂体瘤细胞株(GH3细胞)
培养生长状态良好的目的细胞(即GH3细胞),转染前一天将目的细胞分入6-孔培养板培养,转染当天按实验设计的组别分别利用siRNA载体1#、2#和对照载体0#进行垂体瘤细胞的转染实验。
2.用G418筛选出稳定的转染细胞株
将细胞稀释到1000个细胞/ml,在100μg/ml-1mg/ml的G418浓度范围内进行筛选,选择出在10-14天内使细胞全部死亡的最低G418浓度来进行下一步的筛选试验。在转染24小时之后才开始加G418筛选。随着细胞的代谢G418的浓度和活性都会下降,所以每3-5天都要更换一次含有G418的筛选液。荧光显微镜下观察GFP表达情况,14天后,即可筛选出稳定转染的细胞株。
3.测定基因沉默效果
分别采用FQ-PCR的方法检测目的基因的mRNA表达情况和用Western-blot方法检测Survivin蛋白表达,测定基因沉默效果。
4.MTT检测细胞增殖水平
培养生长状态良好且已稳定转染垂体瘤细胞(GH3细胞)。按实验设计的分组(空白对照组,阴性对照组以及阳性组)情况,将细胞分入96-孔培养板培养。第2天后进行MTT分析,检测对RNAi靶点有效干扰后的垂体瘤细胞增殖水平变化。
5.PI染色流式细胞技术检测GH3细胞的细胞凋亡
培养生长状态良好且已稳定转染垂体瘤细胞(GH3细胞)。按实验设计的分组(空白对照组,阴性对照组以及阳性组)情况,进行PI染色流式细胞仪分析,检测对RNAi靶点有效干扰后的垂体瘤细胞周期及凋亡的变化。
结果
1.FQ-PCR结果显示,siRNA载体1#、2#对GH3细胞中Survivin基因的表达有显著敲减作用。它们的敲减效率分别为54.4%和54.0%。Western-blot结果显示了类似的结果。
2.MTT检测细胞增殖水平实验结果表明,实验组1#,2#的增殖抑制率(IR)分别为42.5%和38.7%,与阴性对照组和空白对照组相比差异有显著性(P＜0.05),这一结果提示在Survivin被沉默后,垂体瘤细胞的增殖能力明显减弱,肿瘤细胞的生长能力明显被抑制。
3.通过PI染色流式细胞方法检测细胞群体中各时期细胞的比例,我们发现,两个siRNA载体组与对照组比较,在Survivin被沉默以后,垂体瘤细胞群体中,凋亡细胞增加(P＜0.05)。
结论
1.Survivin基因对于垂体瘤细胞的生长和增殖能力具有重要作用;特异性沉默Survivin基因能引起大鼠垂体瘤细胞生长周期迟缓,诱发垂体瘤细胞凋亡。
2.RNA干扰技术在未来的垂体瘤基因治疗的研究方面具有应用价值。Survivin基因有望成为垂体瘤基因治疗的有效靶点。
RNA interference is a newly rising and increasing full-grown gene silencing technique, which is considered to be a very effective research tool in gene engineering fields. Operating this technique, we can stably and high-effectively block the expression of target gene to 'silencing' this gene. DsRNA and siRNA have already been widely and successfully utilized on study of gene function and disease therapy research.
Survivin is a unique member of the inhibitorof apoptosis protein family (IAP), which was found by Ambrosini et al in 1997. Its molecular weight is small, but survivin is powerful. The survivin is so far the strongest apoptosis inhibitor. Survivin is expressed in embryo and immature-differentiated tissues. In adult, it is trace expression in thymus and placenta and does not exist in differentiated tissues, including peripheral blood leukocytes, lymph nodes, spleen, pancreas, kidney, skeletal muscle, heart, liver, lung and brain. However, it becomes re-expressed in a variety of cancers. Survivin expression in tumours has been associated with increased aggressiveness and decreased patient survival.
In the present study, our programs are as follows: Firstly, we detect the protein expression of survivin in pituitary adenomas and investigate the biological relationships between survivin and the invasiveness of pituitary adenomas. Secondly, we design and synthesize plasmid vectors mediated siRNA recombinant targeting survivin that can stably and high-effectively express in rat pituitary tumor cell (GH3). After transfecting GH3 cells with those vectors, we investigate targeting gene silence effect of this recombinant. In order to investigate whether survivin will be regarded as an effective gene target for pituitary tumor gene therapy in the future, the variation of cells proliferation and cell apoptosis level because of survivin knocking down are also dectected.
Part 1. Relationship of survivin expression and biological Behavior in Pituitary Tumors
Objective
Survivin is a structurally unique member of the inhibitor of apoptosis protein (IAP) family. Survivin expression in tumours has been associated with increasing invasiveness and decreasing patient survival. The aim of the present study is to detect the protein expression of survivin in pituitary adenomas, investigate the biological relationship between survivin and the invasiveness of pituitary adenomas, and evaluate the clinical magnificance.
Methods
1. Tissues
Pituitary adenomas tissues are obtained by transsphenoidal surgery from 66 patients at the Department of Neurosurgery in Shandong Provincial Hospital from July 2006 to March 2008. The subjects include 29 women (mean age 65.3±4.3 years, range 29-77 years) and 37 men (mean age 68.1±2.5 years, range 34-82years). Parasellar invasiveness of pituitary adenomas is evaluated by preoperative magnetic resonance imaging (MRI) and verified by intraoperative inspection of the medial wall of the cavernous sinus using micrological technique: 39 cases are found to be invasive, and 27 tumours are noninvasive. All pituitary adenomas are evaluated histologically according to the WHO-classification.
2. Immunohistochemical staining procedure
The protein expression of survivin is detected by the S-P method with Ultrasensitive S-P kit.
3. Analysis of immunohistochemical results
Survivin immunoreactivity is evaluated semiquantitatively based on the intensity of staining. 5 fields at a magnification of 400×at every slice are observed at random. The percent of positive staining cells in 200 tumor cells is calculated. The slides are analysed independently by four observers blinded for expression of survivin. For the discrepancies a second evaluation course is running to reach agreement.
The survivin expression is scored as: strong (+++): if more than 50% of cells are positive or a strong diffuse reaction is seen; moderate (++): if less than 50% of cells are positive, or a moderate diffuse reaction is observed; slightly positive (+): if immunoreactions are found in less than 10% of tumour cells or the diffuse reaction is weak; and negative (-): if no survivin is stained.
4. Statistical analysis
Statistical analysis of the sample groups is accomplished using the chisquare test. SPSS Version 13.0 is used for statistical analyses. P <0.05 is considered significant value.
Results
In the pituitary adenomas, a cytoplasmic survivin reaction predominates and only scattered cells exhibited nuclear reaction. Survivin is expressed in 69. 7% (46/66) of the investigated pituitary adenomas. Among invasive pituitary adenomas survivin staining is positive 89. 7% (35/39), while only 40. 7% (11/27) specimens are positive in noninvasive tumours. The chisquare test demonstrates a statistically difference in survivin expression between invasive and noninvasive pituitary adenomas (x~2=14.309, P =0.0002<0. 05). Furthermore, the strong and moderate positives (71.4%, 25/35)are t most common in invasive pituitary adenomas, while in noninvasive pituitary adenomas is slight (72.7%, 8/11).
Conclusions
1. There is high expression of survivin in pituitary adenoma.
2. There is a statistically difference in survivin expression between invasive and noninvasive pituitary adenomas. Survivin is highly associated with invasive pituitary adenomas
3. Survivin can serve as a useful tool for confirmation of invasive pituitary adenoma and survivin gene also may be an effective target for pituitary adenoma gene therapy.
Part2. Design and screen effective siRNA vector aiming at survivin
Objective
3 siRNA vectors aiming at survivin are designed and synthesized, 2 effective ones are screened for the next research.
Methods
1. Design and synthesize siRNA vector
Operating RNAi sequence designing software is downloaded from public website. Based on RNAi structure principle, we design 3 different siRNA vectors aiming directly at survivin, which are labeled as 1#, 2#, 3# and one negative control vector, which is labeled as 0#. These different vectors are collected for RT-PCR and sequence analysis. Results show that these four vectors are successfully constructed.
2. Transfect tool cell (glioma C6)
Tool cells (glioma C6) are cultured in good condition and planted in 6-well plate one day before tansfecting. According to group designing, different vectors are separately added into the plate for tansfection. Fluorescence microscope is employed to observe GFP expression 48 hours after the transfection. Transfection efficiency is recorded.
3. Detect interference effects and screen effective vectors
Those are picked out, if their transfection efficiency is over 90%. We detect the survivin RNA level of these cells with FQ-PCR method to inspect the depressing results of different siRNA vectors and screen 2 effective ones.
Results
1. The transfection efficiency of siRNA vector 1 #, 2 #, 3 # is 87.4±6.2%, 92.6±4.5%, 75.3±11.0% respectively. The transfection efficiency of negative control vector 0# is 95.0±4.2%.
2. All these 3 siRNA vectors can decrese the gene presentation. Compared to negative control vector 0#, the silencing efficiency of 1 #, 2 #, 3 # is 68.4%, 44.4%, 22.7% respectively. Considering transfection efficiency and silencing efficiency, 1 # and 2 # are better than 3 #.
Conclusions
1. In this part, we successfully design and synthesize 3 specific targeting survivin siRNA vectors.
2. We manage to pick out 2 effective siRNA vectors of these three. These results provide us reliable evidence to carry on RNAi experiment targeting survivin in GH3 cells.
Part3. Cell proliferation and apoptosis level variation of pituitary tumor cells by RNA interference survivin gene
Objective
Using constructed the conditionally replicating vector 1#、2# which can transfect to GH3 effectually and selectively and contain siRNA to rat survivin gene, we investigate the effect of the vector silencing on expression of mRNA and protein of survivin gene in GH3 cells on propagation and apoptosis of pituitary tumor cell lines GH3.
Methods
1 .Transfect GH3 cells with siRNA vector1#、2# respectively.
Objective cells (GH3) are cultured in good condition and planted in 6-well plate one day before transfecting. According to group designing, Vector1#, 2# and 0# is separately added into the plate for transfection.
2. Screen stable transfectants with G418
Cells are trypsinized and plated into 1000 cells/ml, and concentration ranges (100μg/ml -1mg/ml) G418 is added to the culture medium. We pick out the lowest concentration G418 when all cells die in 10-14 days. G418 is add 24 hours after transfection. With Cells metabolic activity and G418 concentration decreasing, the culture medium containing G418 is changed in 3-5 days. At this time the drug concentration may be reduced to 200μg/ml. Fluorescence microscope is employed to observe GFP expression. 14 days after selection, cell colonies are isolated and expanded for testing.
3. Inspect the effects of gene silencing
The survivin mRNA and protein level of these cells are detected with FQ-PCR and western-blotting method is used to inspect the effects of gene silencing.
4. Investigation of pituitary tumor cell proliferation-ability variation by MTT
Stable tansfecion pituitary tumor cells (GH3) are cultured in good condition. According to group designing (siRNA recombinants and controls), cells are planted in 96-well plate. 1 day later, MTT method is employed to contrast the changes of cell proliferation cycle.
5. Investigation of pituitary tumor cell apoptosis level variation by PI dyeing FCM
Stable tansfecion pituitary tumor cells (GH3) are cultured in good condition. According to group designing (siRNA recombinants and controls), cells are collected. PI dyeing FCM method is employed to contrast the changes of cell apoptosis levels.
Results
1. FQ-PCR data suggests that the 2 siRNA recombinant could depress survivin mRNA level of GH3 cells obviously. Compared to negative control, vector 1#, 2# silencing efficiency is 54.4%、54.0% respectively. Similar results can be seen with western blotting method.
2. The proliferation suppressing effects of RNAi targeted to survivin are observed by MTT assay. The proliferation inhibitory rates of 1# and 2# are 42.5% and 38.7% respectively, which suggest that pituitary tumor cells' multiplication ability turns to be weak, and the growth ability of tumor cells is depressed remarkably.
3. Analyzing the proportion of all period cells in cell colony by FCM, we found that, after survivin silenced, the proportion of apoptosis cell increased in the two siRNA groups, comparing with control group.
Conclusions
1. Survivin plays an important role in the process of pituitary tumor cells' survival and proliferation indeed. After survivin silenced, growth ability of tumor cell is depressed remarkably and apoptosis cell increased obviously.
2. The RNA interference technology will be important in the application value of pituitary tumor gene therapy in the future. Survivin can probably be considered as an effective gene target for pituitary tumor gene therapy.
Survivin是耶鲁大学Ambrosini等于1997年发现的,是凋亡抑制蛋白家族新成员,具有分子量小、功能强大等特点。它是迄今发现的最强的凋亡抑制因子,主要分布于胚胎及分化未成熟的组织。在成人体内除胸腺及胎盘中有微量表达外,所有分化成熟的组织,包括外周血白细胞、淋巴结、脾、胰、肾、骨骼肌、心、肝、肺、脑组织中均无表达,而在人类肿瘤中几乎都表达。并且Survivin与肿瘤的侵袭性密切相关。
本研究方法是:首先,检测Survivin在垂体腺瘤中的蛋白表达,探讨Survivin与垂体腺瘤侵袭性发生的相关性以及它们之间的内在联系。然后,通过设计在垂体瘤细胞中稳定高效表达的Survivin-siRNA质粒载体,研究该重组载体对于垂体瘤细胞中Survivin的干扰效果,并进一步明确干扰前后垂体瘤细胞的增殖及凋亡水平变化,以期探讨Survivin是否可成为未来进行垂体瘤基因治疗研究的有效靶点。
第一部分Survivin在垂体瘤中的表达及与生物学行为关系
目的
Survivin是凋亡抑制蛋白家族非常独特的成员,其基因在肿瘤中的表达与增加肿瘤的侵袭性和降低患者的生存率关系密切。本研究目的是检测Survivin基因在垂体腺瘤中的蛋白表达,并探讨Survivin与垂体腺瘤侵袭性发生的相关性以及它们之间的内在联系。
方法
1.标本
回顾性随机收集2006年7月-2008年4月期间山东省立医院神经外科的垂体腺瘤手术标本66例,其中女性29例(年龄29-77岁,平均年龄65.3±4.3岁),男性37例(年龄34-82岁,平均年龄68.1±2.5岁)。依据MRI提示肿瘤侵犯海绵窦或有骨质破坏,和/或术中观察肿瘤对周围骨质、海绵窦有/无侵犯等情况分为侵袭性组(39例)和非侵袭性组(27例)。所有病例诊断均有术后的免疫组化病理证实。
2.免疫组化染色方法
采用免疫组化链霉菌抗生物素蛋白-过氧化物酶连接(S-P)法,按标准实验流程,检测Survivin基因在侵袭性和非侵袭性垂体瘤中的蛋白表达。
3.结果判定标准
根据半定量免疫组织化学评价Survivin反应阳性细胞显色强度。在高倍视野(×400)下,每张切片随机观察5个视野的肿瘤细胞,计算200个肿瘤细胞免疫反应阳性的细胞百分比。每张切片分别有四名实验者进行单独评价,对于有分歧的进行重复评价,直至达到一致。
Survivin表达评估:强阳性(+++):阳性细胞大于50%或显色深;中度阳性(++):阳性细胞10%至50%之间或染色略深;弱阳性(+):阳性细胞小于10%或显色浅;阴性(一):无阳性细胞染色。
4.统计分析
用卡方检验进行统计分析。SPSS(版本13.0)统计学软件进行统计。设定p＜0.05有统计学意义。
结果
Survivin在垂体瘤中以细胞质着色为主,仅有散在的细胞核着色。Survivin总的阳性率为69.7%(46/66)。在侵袭性垂体瘤中Survivin的阳性率为89.7%(35/39);而在非侵袭性垂体瘤中Survivin的阳性率仅为40.7%(11/27)。卡方检验示两组差异有统计学意义(x~2=14.309,P=0.0002＜0.05)。另外,在免疫组化反应为阳性的侵袭性垂体瘤中以强阳性和中度阳性为主(71.4%,25/35),而在非侵袭性垂体瘤中以弱阳性为主(72.7%,8/11)。
结论
1.Survivin在垂体瘤中有高表达。
2.Survivin在侵袭性与非侵袭性垂体腺瘤中的表达有显著性差异,因此Survivin与垂体瘤的侵袭性密切相关。
3.Survivin可以作为判断垂体腺瘤侵袭性生长的有效指标和用做垂体瘤基因治疗研究的靶点。
第二部分设计并筛选针对Survivin有效的siRNA载体
目的
设计并合成3组针对Survivin基因的siRNA质粒载体,并筛选出其中2组有效的载体,为下一步的研究做准备。
方法
1.设计并合成siRNA载体
本实验运用公众网站设计软件,针对大鼠的Survivin靶基因序列,设计3组siRNA载体,分别标记为1#,2#,3g;同时合成1组为阴性对照载体,标记为0#。然后进行测序比对。结果显示四组载体被成功构建,测序无误。
2.转染工具细胞株(胶质瘤C6)
培养生长状态良好的工具细胞(胶质瘤C6),转染前一天将细胞分入6-孔培养板培养,转染当天按实验设计的分组情况加入不同质粒载体进行细胞的转染实验。转染48小时后荧光显微镜下观察GFP表达情况,观测转染效率。
3.检测干扰效果,筛选有效载体。
选出转染效率为90%左右的组,采用FQ-PCR的方法检测目的基因的mRNA表达情况,进而判断不同靶点的干扰效果,筛选出2组有效载体。
结果
1.在3组siRNA载体中,1#、2#、3#转染效率分别为87.4±6.2%、92.6±4.5%、75.3±11.0%;阴性对照载体0#的转染效率为95.0±4.2%。
2.3个靶点均有敲减效果。与阴性对照载体0#对比,载体1#,2#,3#敲减效率分别为68.4%、44.4%、22.7%。综合转染效率及敲减效果,1#、2#是最佳的靶点。
结论
1.本部分实验成功构建了3组特异性针对大鼠基因Survivin的siRNA载体。
2.通过该部分实验,我们成功地从3组重组载体中筛选出高效率的靶点,为下一步选用高效率的重组载体对垂体瘤细胞GH3进行转染提供了可靠的实验基础。
第三部分RNAi沉默Survivin基因对垂体瘤细胞增殖及凋亡水平的影响
目的
利用构建好的特异性强、转染率高的携带针对Survivin的siRNA载体1#、2#,研究它们介导RNA干扰对垂体瘤细胞株GH3增殖和凋亡的影响及其意义。
方法
1.分别利用siRNA载体1#、2#转染垂体瘤细胞株(GH3细胞)
培养生长状态良好的目的细胞(即GH3细胞),转染前一天将目的细胞分入6-孔培养板培养,转染当天按实验设计的组别分别利用siRNA载体1#、2#和对照载体0#进行垂体瘤细胞的转染实验。
2.用G418筛选出稳定的转染细胞株
将细胞稀释到1000个细胞/ml,在100μg/ml-1mg/ml的G418浓度范围内进行筛选,选择出在10-14天内使细胞全部死亡的最低G418浓度来进行下一步的筛选试验。在转染24小时之后才开始加G418筛选。随着细胞的代谢G418的浓度和活性都会下降,所以每3-5天都要更换一次含有G418的筛选液。荧光显微镜下观察GFP表达情况,14天后,即可筛选出稳定转染的细胞株。
3.测定基因沉默效果
分别采用FQ-PCR的方法检测目的基因的mRNA表达情况和用Western-blot方法检测Survivin蛋白表达,测定基因沉默效果。
4.MTT检测细胞增殖水平
培养生长状态良好且已稳定转染垂体瘤细胞(GH3细胞)。按实验设计的分组(空白对照组,阴性对照组以及阳性组)情况,将细胞分入96-孔培养板培养。第2天后进行MTT分析,检测对RNAi靶点有效干扰后的垂体瘤细胞增殖水平变化。
5.PI染色流式细胞技术检测GH3细胞的细胞凋亡
培养生长状态良好且已稳定转染垂体瘤细胞(GH3细胞)。按实验设计的分组(空白对照组,阴性对照组以及阳性组)情况,进行PI染色流式细胞仪分析,检测对RNAi靶点有效干扰后的垂体瘤细胞周期及凋亡的变化。
结果
1.FQ-PCR结果显示,siRNA载体1#、2#对GH3细胞中Survivin基因的表达有显著敲减作用。它们的敲减效率分别为54.4%和54.0%。Western-blot结果显示了类似的结果。
2.MTT检测细胞增殖水平实验结果表明,实验组1#,2#的增殖抑制率(IR)分别为42.5%和38.7%,与阴性对照组和空白对照组相比差异有显著性(P＜0.05),这一结果提示在Survivin被沉默后,垂体瘤细胞的增殖能力明显减弱,肿瘤细胞的生长能力明显被抑制。
3.通过PI染色流式细胞方法检测细胞群体中各时期细胞的比例,我们发现,两个siRNA载体组与对照组比较,在Survivin被沉默以后,垂体瘤细胞群体中,凋亡细胞增加(P＜0.05)。
结论
1.Survivin基因对于垂体瘤细胞的生长和增殖能力具有重要作用;特异性沉默Survivin基因能引起大鼠垂体瘤细胞生长周期迟缓,诱发垂体瘤细胞凋亡。
2.RNA干扰技术在未来的垂体瘤基因治疗的研究方面具有应用价值。Survivin基因有望成为垂体瘤基因治疗的有效靶点。
RNA interference is a newly rising and increasing full-grown gene silencing technique, which is considered to be a very effective research tool in gene engineering fields. Operating this technique, we can stably and high-effectively block the expression of target gene to 'silencing' this gene. DsRNA and siRNA have already been widely and successfully utilized on study of gene function and disease therapy research.
Survivin is a unique member of the inhibitorof apoptosis protein family (IAP), which was found by Ambrosini et al in 1997. Its molecular weight is small, but survivin is powerful. The survivin is so far the strongest apoptosis inhibitor. Survivin is expressed in embryo and immature-differentiated tissues. In adult, it is trace expression in thymus and placenta and does not exist in differentiated tissues, including peripheral blood leukocytes, lymph nodes, spleen, pancreas, kidney, skeletal muscle, heart, liver, lung and brain. However, it becomes re-expressed in a variety of cancers. Survivin expression in tumours has been associated with increased aggressiveness and decreased patient survival.
In the present study, our programs are as follows: Firstly, we detect the protein expression of survivin in pituitary adenomas and investigate the biological relationships between survivin and the invasiveness of pituitary adenomas. Secondly, we design and synthesize plasmid vectors mediated siRNA recombinant targeting survivin that can stably and high-effectively express in rat pituitary tumor cell (GH3). After transfecting GH3 cells with those vectors, we investigate targeting gene silence effect of this recombinant. In order to investigate whether survivin will be regarded as an effective gene target for pituitary tumor gene therapy in the future, the variation of cells proliferation and cell apoptosis level because of survivin knocking down are also dectected.
Part 1. Relationship of survivin expression and biological Behavior in Pituitary Tumors
Objective
Survivin is a structurally unique member of the inhibitor of apoptosis protein (IAP) family. Survivin expression in tumours has been associated with increasing invasiveness and decreasing patient survival. The aim of the present study is to detect the protein expression of survivin in pituitary adenomas, investigate the biological relationship between survivin and the invasiveness of pituitary adenomas, and evaluate the clinical magnificance.
Methods
1. Tissues
Pituitary adenomas tissues are obtained by transsphenoidal surgery from 66 patients at the Department of Neurosurgery in Shandong Provincial Hospital from July 2006 to March 2008. The subjects include 29 women (mean age 65.3±4.3 years, range 29-77 years) and 37 men (mean age 68.1±2.5 years, range 34-82years). Parasellar invasiveness of pituitary adenomas is evaluated by preoperative magnetic resonance imaging (MRI) and verified by intraoperative inspection of the medial wall of the cavernous sinus using micrological technique: 39 cases are found to be invasive, and 27 tumours are noninvasive. All pituitary adenomas are evaluated histologically according to the WHO-classification.
2. Immunohistochemical staining procedure
The protein expression of survivin is detected by the S-P method with Ultrasensitive S-P kit.
3. Analysis of immunohistochemical results
Survivin immunoreactivity is evaluated semiquantitatively based on the intensity of staining. 5 fields at a magnification of 400×at every slice are observed at random. The percent of positive staining cells in 200 tumor cells is calculated. The slides are analysed independently by four observers blinded for expression of survivin. For the discrepancies a second evaluation course is running to reach agreement.
The survivin expression is scored as: strong (+++): if more than 50% of cells are positive or a strong diffuse reaction is seen; moderate (++): if less than 50% of cells are positive, or a moderate diffuse reaction is observed; slightly positive (+): if immunoreactions are found in less than 10% of tumour cells or the diffuse reaction is weak; and negative (-): if no survivin is stained.
4. Statistical analysis
Statistical analysis of the sample groups is accomplished using the chisquare test. SPSS Version 13.0 is used for statistical analyses. P <0.05 is considered significant value.
Results
In the pituitary adenomas, a cytoplasmic survivin reaction predominates and only scattered cells exhibited nuclear reaction. Survivin is expressed in 69. 7% (46/66) of the investigated pituitary adenomas. Among invasive pituitary adenomas survivin staining is positive 89. 7% (35/39), while only 40. 7% (11/27) specimens are positive in noninvasive tumours. The chisquare test demonstrates a statistically difference in survivin expression between invasive and noninvasive pituitary adenomas (x~2=14.309, P =0.0002<0. 05). Furthermore, the strong and moderate positives (71.4%, 25/35)are t most common in invasive pituitary adenomas, while in noninvasive pituitary adenomas is slight (72.7%, 8/11).
Conclusions
1. There is high expression of survivin in pituitary adenoma.
2. There is a statistically difference in survivin expression between invasive and noninvasive pituitary adenomas. Survivin is highly associated with invasive pituitary adenomas
3. Survivin can serve as a useful tool for confirmation of invasive pituitary adenoma and survivin gene also may be an effective target for pituitary adenoma gene therapy.
Part2. Design and screen effective siRNA vector aiming at survivin
Objective
3 siRNA vectors aiming at survivin are designed and synthesized, 2 effective ones are screened for the next research.
Methods
1. Design and synthesize siRNA vector
Operating RNAi sequence designing software is downloaded from public website. Based on RNAi structure principle, we design 3 different siRNA vectors aiming directly at survivin, which are labeled as 1#, 2#, 3# and one negative control vector, which is labeled as 0#. These different vectors are collected for RT-PCR and sequence analysis. Results show that these four vectors are successfully constructed.
2. Transfect tool cell (glioma C6)
Tool cells (glioma C6) are cultured in good condition and planted in 6-well plate one day before tansfecting. According to group designing, different vectors are separately added into the plate for tansfection. Fluorescence microscope is employed to observe GFP expression 48 hours after the transfection. Transfection efficiency is recorded.
3. Detect interference effects and screen effective vectors
Those are picked out, if their transfection efficiency is over 90%. We detect the survivin RNA level of these cells with FQ-PCR method to inspect the depressing results of different siRNA vectors and screen 2 effective ones.
Results
1. The transfection efficiency of siRNA vector 1 #, 2 #, 3 # is 87.4±6.2%, 92.6±4.5%, 75.3±11.0% respectively. The transfection efficiency of negative control vector 0# is 95.0±4.2%.
2. All these 3 siRNA vectors can decrese the gene presentation. Compared to negative control vector 0#, the silencing efficiency of 1 #, 2 #, 3 # is 68.4%, 44.4%, 22.7% respectively. Considering transfection efficiency and silencing efficiency, 1 # and 2 # are better than 3 #.
Conclusions
1. In this part, we successfully design and synthesize 3 specific targeting survivin siRNA vectors.
2. We manage to pick out 2 effective siRNA vectors of these three. These results provide us reliable evidence to carry on RNAi experiment targeting survivin in GH3 cells.
Part3. Cell proliferation and apoptosis level variation of pituitary tumor cells by RNA interference survivin gene
Objective
Using constructed the conditionally replicating vector 1#、2# which can transfect to GH3 effectually and selectively and contain siRNA to rat survivin gene, we investigate the effect of the vector silencing on expression of mRNA and protein of survivin gene in GH3 cells on propagation and apoptosis of pituitary tumor cell lines GH3.
Methods
1 .Transfect GH3 cells with siRNA vector1#、2# respectively.
Objective cells (GH3) are cultured in good condition and planted in 6-well plate one day before transfecting. According to group designing, Vector1#, 2# and 0# is separately added into the plate for transfection.
2. Screen stable transfectants with G418
Cells are trypsinized and plated into 1000 cells/ml, and concentration ranges (100μg/ml -1mg/ml) G418 is added to the culture medium. We pick out the lowest concentration G418 when all cells die in 10-14 days. G418 is add 24 hours after transfection. With Cells metabolic activity and G418 concentration decreasing, the culture medium containing G418 is changed in 3-5 days. At this time the drug concentration may be reduced to 200μg/ml. Fluorescence microscope is employed to observe GFP expression. 14 days after selection, cell colonies are isolated and expanded for testing.
3. Inspect the effects of gene silencing
The survivin mRNA and protein level of these cells are detected with FQ-PCR and western-blotting method is used to inspect the effects of gene silencing.
4. Investigation of pituitary tumor cell proliferation-ability variation by MTT
Stable tansfecion pituitary tumor cells (GH3) are cultured in good condition. According to group designing (siRNA recombinants and controls), cells are planted in 96-well plate. 1 day later, MTT method is employed to contrast the changes of cell proliferation cycle.
5. Investigation of pituitary tumor cell apoptosis level variation by PI dyeing FCM
Stable tansfecion pituitary tumor cells (GH3) are cultured in good condition. According to group designing (siRNA recombinants and controls), cells are collected. PI dyeing FCM method is employed to contrast the changes of cell apoptosis levels.
Results
1. FQ-PCR data suggests that the 2 siRNA recombinant could depress survivin mRNA level of GH3 cells obviously. Compared to negative control, vector 1#, 2# silencing efficiency is 54.4%、54.0% respectively. Similar results can be seen with western blotting method.
2. The proliferation suppressing effects of RNAi targeted to survivin are observed by MTT assay. The proliferation inhibitory rates of 1# and 2# are 42.5% and 38.7% respectively, which suggest that pituitary tumor cells' multiplication ability turns to be weak, and the growth ability of tumor cells is depressed remarkably.
3. Analyzing the proportion of all period cells in cell colony by FCM, we found that, after survivin silenced, the proportion of apoptosis cell increased in the two siRNA groups, comparing with control group.
Conclusions
1. Survivin plays an important role in the process of pituitary tumor cells' survival and proliferation indeed. After survivin silenced, growth ability of tumor cell is depressed remarkably and apoptosis cell increased obviously.
2. The RNA interference technology will be important in the application value of pituitary tumor gene therapy in the future. Survivin can probably be considered as an effective gene target for pituitary tumor gene therapy.
引文
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[1]. Kovacs K, Horvath E, Vidal S. Classification of pituitary adenomas [J]. J Neurooncol, 2001, 54: 121-127.
[2]. Mello C C, Conte J rD. Revealing the world of RNA interference[J]. Nature, 2004, 431 (7.006): 338-342.
[3]. Hannonl G J, Rossi J J. Unlocking the potential of the human genome with RNA interference[ J ]. Nature, 2004,431 (7006): 371-378.
[4]. Scherr M, Batmer K, Winkler T, et al. Specific inhibition Of bcr-abl gene expression by small interfering RNA[J]. Blood,2003, 101(4): 1566-1569.
[5].Alticri DC.Validating surviving as a cancer therapeutic target [J].Nat.Rev.Cancer, 2003;3:46-54.
[6]. Uren A G, Wang l.Pakusch M,et al.Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype[J].Curr Biol,2002; 10(21): 1319.
[7].Deveraux QL, Reed JC.IAP family proteins-suppressors of apoptosis [J].Genes, 1999;13(3):239-252.
[8] .Suzuki A,Hayashida M,Ito T,et al,Survivin initiates cell cycle entry by the complex activation with CDK4/p16(INK4a)and CDK2,cyclinE complex activation[J].Oncogene.2000;19(29):3225-3234.
[9].Suzuki A, Ito T,Kawano H,et al,Suevivin initiates procaspase/P21 complex formation as a result of interaction with CDK4 to resist FAS-mediated cell death[J].Oncogene,2000;19(10):1346-1353.
[10].Wheatley SP,Kandels L,Adams RR,et al.INCENP binds directly to tubulin,and requires dynamic microtubules to target to the cleavage furrow[J].Exp,Cell Res,2001b;262:122-127.
[11]Oconnor DS,Schechner JS,Adida C,et al.Angioptosis during angiogenesis by survivin express in endothelial cell[J].Am J Pathol,2000;156(6):393.
[12] .Mono M,Rosell R,Felip E,et al. A novel anti-apoptosis gene Rexpression of Survivin messenger RNA as a prognosis marker in nonsmall-cell lung cancers[J].J Clin Oncol. 1999; 17:2100-2104.
[13].Hou Yan,Hu Qun,Liu Aiguo,Zhang Liuqing,et al.Expression of survivin and its location in cell and clinical significance in pediatric acute leukemia[J]. Journal Of China PediaTric Blood And Cancer,2006;l :6-8.
[14]. Thomas S, Muralidharan A, Shah GV. Knock-down of Calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells [J]. Int J Oncol 2007; 31: 1425-1437.
[15]. Wasko R, Jankowska A, Waligorska-Stachura J, Andrusiewicz MJaskula M, Sowinski J. Survivin expression in pituitary adenomas[J].Neuro Endocrinol Lett 2005; 26: 209-212.
[16]. Zhang YC, Gao J, Xin T, Zheng ZM, Teng LZ.Expression of survivin in invasive pituitary adenoma. [J] Saudi Med J. 2008 Nov;29(11):1589-1592.
1. Laws ER Jr, Scheithauer BW, Carpenter S, Randall RV, Abboud CF. The pathogenesis of acromegaly: clinical and immunocytochemical analysis in 75 patients. J Neurosurg. 1985;63(1):35-38.
2. Randall RV, Laws ER Jr, Abboud CF, Ebersold MJ, Kao PC, Scheithauer BW. Transsphenoidal microsurgical treatment of prolactin-producing pituitary adenomas: Results in 100 patients.Mayo Clin Proc. 1983;58(2):108-121.
3. Gürlek A, Karavitaki N, Ansorge O, Wass JA. What are the markers of aggressiveness in prolactinomas? Changes in cell biology, extracellular matrix components, angiogenesis and genetics. Eur J Endocrinol. 2007;156(2):143-153.
4. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death's door. Nat Rev Mol Cell Biol. 2002;3(6):401-410.
5. Takai N, Miyazaki T, Nishida M, Nasu K, Miyakawa I. Survivin expression correlates with clinical stage, histological grade, invasive behavior, and survival rate in endometrial carcinoma. Cancer Lett 2002;184(1):105-116.
6. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998; 396(6711):580-584.
7. Adida C, Crotty PL, McGrath J, Berrebi D, Diebold J, Altieri DC. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol. 1998;152(1):43-49.
8. Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res. 1998;58(9):1808-1812.
9. Montorsi M, Maggioni M, Falleni M, Pellegrini C, Donadon M, Torzilli G, et al. Survivin gene expression in chronic liver disease and hepatocellular carcinoma. Hepatogastroenterology. 2007;54(79):2040-2044.
10. Shariat SF, Lotan Y, Saboorian H, Khoddami SM, Roehrborn CG, Slawin KM, et al. Survivin expression is associated with features of biologically aggressive prostate carcinoma. Cancer. 2004; 100 (4):751-757.
11. Thomas S, Muralidharan A, Shah GV. Knock-down of Calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells. Int J Oncol. 2007;31(6):1425-1437.
12. Wasko R, Jankowska A, Waligorska-Stachura J, Andrusiewicz M, Jaskula M, Sowinski J.Survivin expression in pituitary adenomas. Neuro Endocrinol Lett. 2005;26(3):209-212.
13. Abe T, Lüdecke DK. Effects of preoperative octreotide treatment on different subtypes of 90 GH-secreting pituitary adenomas and outcome in one surgical centre. Eur J Endocrinol. 2001;145(2):137-145.
14. Lüdecke DK, Flitsch J, Knappe UJ, Saeger W. Cushing's disease: a surgical view. J Neurooncol. 2001;54(2):151-166.
15. Saeger W, Schroder S, Kloppel G . Pathology of important diseases of endocrine organs (excluding the thyroid).Pathologe. 2001;22(5):296-309.
16. Solcia E, Kloppel G, Sobin LH, Capella C, Lells RA de, Heitz PU, et al. (2000) Histological typing of endocrine tumors,2nd edn. Springer, Berlin Heidelberg New York, pp 1-149.
17. LaCasse EC, Baird S, Korneluk RG, MacKenzie AE. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 1998; 17(25):3247-3259.
18. Reed JC. The survivin saga goes in vivo. J Clin Inves.2001; 108(7):965-969.
19. Geske FJ, Gerschenson LE. The biology of apoptosis. Hum Pathol.2001; 32(10):1029-1038.
20. Saikumar P, Dong Z, Mikhailov V, Denton M, Weinberg JM, Venkatachalam MA. Apoptosis: definition, mechanisms, and relevance to disease. Am J Med 1999;107(5):489-506.
21. Deveraux QL, Reed JC. IAP family proteins-suppressors of apoptosis. Genes Dev 1999;13(3):239-252.
22. Pennati M, Folini M, Zaffaroni N. Targeting survivin in cancer therapy. Expert Opin Ther Targets. 2008;12(4):463-476.
23. Kappler M, Rot S, Taubert H, Greither T, Bartel F, Delias K, et al. The effects of knockdown of wild-type survivin, survivin-2B or survivin-delta3 on the radiosensitization in a soft tissue sarcoma cells in vitro under different oxygen conditions. Cancer Gene Ther. 2007;14(12):994-1001.
24. Takizawa BT, Uchio EM, Cohen JJ, Wheeler MA, Weiss RM.Downregulation of survivin is associated with reductions in TNF receptors' mRNA and protein and alterations in nuclear factor kappa B signaling in urothelial cancer cells. Cancer Invest. 2007;25(8):678-684.
25. Zhen HN, Li LW, Zhang W, Fei Z, Shi CH, Yang TT, et al. Short hairpin RNA targeting survivin inhibits growth and angiogenesis of glioma U251 cells.int J Oncol. 2007;31(5):1111-1117.
1. Abe T, Lüdecke DK. Effects of preoperative octreotide treatment on different subtypes of 90 GH-secreting pituitary adenomas and outcome in one surgical centre. Eur J Endocrinol. 2001;145(2):137-145.
2. Adida C, Crotty PL, McGrath J, Berrebi D, Diebold J, Altieri DC. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol. 1998; 152(1 ):43-49.
3. 2. Bradshaw C, Kakar SS. Pituitary tumor transforming gene: an important gene in normal cellular functions and tumorigenesis. Histol Histopathol. 2007;22(2):219-226.
4. Chesnokova V, Kovacs K, Castro AV, Zonis S, Melmed S. Pituitary hypoplasia in PTTG-/- mice is protective for Rb+/- pituitary tumorigenesis. Mol Endocrinol. 2005; 19(9):2371-2379.
5. Donangelo I, Gutman S, Horvath E, Kovacs K, Wawrowsky K, Mount M, Melmed S. Pituitary tumor transforming gene overexpression facilitates pituitary tumor development. Endocrinology. 2006; 147(10):4781 -4791.
6. Deveraux QL, Reed JC. IAP family proteins-suppressors of apoptosis. Genes Dev 1999;13(3):239-252.
7. Geske FJ, Gerschenson LE. The biology of apoptosis. Hum Pathol.2001; 32(10):1029-1038.
8. Gürlek A, Karavitaki N, Ansorge O, Wass JA. What are the markers of aggressiveness in prolactinomas? Changes in cell biology, extracellular matrix components, angiogenesis and genetics. Eur J Endocrinol. 2007; 156(2): 143-153.
9. Kim DS, Fong J, Read ML, McCabe CJ. The emerging role of pituitary tumour transforming gene (PTTG) in endocrine tumourigenesis. Mol Cell Endocrinol. 2007;278(1-2):1-6.
10. Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res. 1998;58(9):1808-1812.
11. Lüdecke DK, Flitsch J, Knappe UJ, Saeger W. Cushing's disease: a surgical view. J Neurooncol. 2001;54(2):151-166.
12. McCabe C. Genetic targets for the treatment of pituitary adenomas: focus on the pituitary tumor transforming gene. Curr Opin Pharmacol.2001 ;1(6):620-625.
13. McCabe CJ, Khaira JS, Boelaert K, Heaney AP, Tannahill LA, Hussain S, Mitchell R, Olliff J, Sheppard MC, Franklyn JA, Gittoes NJ. Expression of pituitary tumour transforming gene (PTTG) and fibroblast growth factor-2 (FGF-2) in human pituitary adenomas: relationships to clinical tumour behaviour. Clin Endocrinol (Oxf). 2003;58(2):141-150.
14. Montorsi M, Maggioni M, Falleni M, Pellegrini C, Donadon M, Torzilli G, Santambrogio R, Spinelli A, Coggi G, Bosari S. Survivin gene expression in chronic liver disease and hepatocellular carcinoma. Hepatogastroenterology. 2007;54(79):2040-2044.
15. Related Articles, Links Cui J, Hopper JL, Harrap SB.Genes and family environment explain correlations between blood pressure and body mass index. Hypertension. 2002;40(1):7-12.
16. Saikumar P, Dong Z, Mikhailov V, Denton M, Weinberg JM, Venkatachalam MA. Apoptosis: definition, mechanisms, and relevance to disease. Am J Med 1999;107(5):489-506.
17. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death's door. Nat Rev Mol Cell Biol. 2002;3(6):401-410.
18. Shariat SF, Lotan Y, Saboorian H, Khoddami SM, Roehrborn CG, Slawin KM, Ashfaq R. Survivin expression is associated with features of biologically aggressive prostate carcinoma. Cancer. 2004;100 (4):751-757.
19. Thomas S, Muralidharan A, Shah GV. Knock-down of Calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells. Int J Oncol. 2007;31(6):1425-1437.
20. Zhang X, Horwitz GA, Heaney AP, Nakashima M, Prezant TR, Bronstein MD, Melmed S. Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas. J Clin Endocrinol Metab. 1999;84(2):761-767.
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[20].Uren A G,Wang 1.Pakusch M,et al.Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype[J].Curr Biol,2002,10(21):1319
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[22]. Rosa J, Canovas P, Islam A et al. Survivin modulates microtubule dynamics and nucleation throughout the cell cycle. Mol Biol Cell. 2006; 17(3): 1483-93.
[23]. Barnes N, Haywood P, Flint P et al. Survivin expression in in situ and invasive breast cancer relates to COX-2 expression and DCIS recurrence. Br J Cancer. 2006; 94(2):253-8.
[24]. Carelli S, Lesma E, Paratore S et al. Survivin expression in tuberous sclerosis complex cells. Mol Med. 2007; 13(3-4): 166-77.
[1]. Kovacs K, Horvath E, Vidal S. Classification of pituitary adenomas [J]. J Neurooncol, 2001, 54: 121-127.
[2]. Mello C C, Conte J rD. Revealing the world of RNA interference[J]. Nature, 2004, 431 (7.006): 338-342.
[3]. Hannonl G J, Rossi J J. Unlocking the potential of the human genome with RNA interference[ J ]. Nature, 2004,431 (7006): 371-378.
[4]. Scherr M, Batmer K, Winkler T, et al. Specific inhibition Of bcr-abl gene expression by small interfering RNA[J]. Blood,2003, 101(4): 1566-1569.
[5].Alticri DC.Validating surviving as a cancer therapeutic target [J].Nat.Rev.Cancer, 2003;3:46-54.
[6]. Uren A G, Wang l.Pakusch M,et al.Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype[J].Curr Biol,2002; 10(21): 1319.
[7].Deveraux QL, Reed JC.IAP family proteins-suppressors of apoptosis [J].Genes, 1999;13(3):239-252.
[8] .Suzuki A,Hayashida M,Ito T,et al,Survivin initiates cell cycle entry by the complex activation with CDK4/p16(INK4a)and CDK2,cyclinE complex activation[J].Oncogene.2000;19(29):3225-3234.
[9].Suzuki A, Ito T,Kawano H,et al,Suevivin initiates procaspase/P21 complex formation as a result of interaction with CDK4 to resist FAS-mediated cell death[J].Oncogene,2000;19(10):1346-1353.
[10].Wheatley SP,Kandels L,Adams RR,et al.INCENP binds directly to tubulin,and requires dynamic microtubules to target to the cleavage furrow[J].Exp,Cell Res,2001b;262:122-127.
[11]Oconnor DS,Schechner JS,Adida C,et al.Angioptosis during angiogenesis by survivin express in endothelial cell[J].Am J Pathol,2000;156(6):393.
[12] .Mono M,Rosell R,Felip E,et al. A novel anti-apoptosis gene Rexpression of Survivin messenger RNA as a prognosis marker in nonsmall-cell lung cancers[J].J Clin Oncol. 1999; 17:2100-2104.
[13].Hou Yan,Hu Qun,Liu Aiguo,Zhang Liuqing,et al.Expression of survivin and its location in cell and clinical significance in pediatric acute leukemia[J]. Journal Of China PediaTric Blood And Cancer,2006;l :6-8.
[14]. Thomas S, Muralidharan A, Shah GV. Knock-down of Calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells [J]. Int J Oncol 2007; 31: 1425-1437.
[15]. Wasko R, Jankowska A, Waligorska-Stachura J, Andrusiewicz MJaskula M, Sowinski J. Survivin expression in pituitary adenomas[J].Neuro Endocrinol Lett 2005; 26: 209-212.
[16]. Zhang YC, Gao J, Xin T, Zheng ZM, Teng LZ.Expression of survivin in invasive pituitary adenoma. [J] Saudi Med J. 2008 Nov;29(11):1589-1592.
1. Laws ER Jr, Scheithauer BW, Carpenter S, Randall RV, Abboud CF. The pathogenesis of acromegaly: clinical and immunocytochemical analysis in 75 patients. J Neurosurg. 1985;63(1):35-38.
2. Randall RV, Laws ER Jr, Abboud CF, Ebersold MJ, Kao PC, Scheithauer BW. Transsphenoidal microsurgical treatment of prolactin-producing pituitary adenomas: Results in 100 patients.Mayo Clin Proc. 1983;58(2):108-121.
3. Gürlek A, Karavitaki N, Ansorge O, Wass JA. What are the markers of aggressiveness in prolactinomas? Changes in cell biology, extracellular matrix components, angiogenesis and genetics. Eur J Endocrinol. 2007;156(2):143-153.
4. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death's door. Nat Rev Mol Cell Biol. 2002;3(6):401-410.
5. Takai N, Miyazaki T, Nishida M, Nasu K, Miyakawa I. Survivin expression correlates with clinical stage, histological grade, invasive behavior, and survival rate in endometrial carcinoma. Cancer Lett 2002;184(1):105-116.
6. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998; 396(6711):580-584.
7. Adida C, Crotty PL, McGrath J, Berrebi D, Diebold J, Altieri DC. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol. 1998;152(1):43-49.
8. Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res. 1998;58(9):1808-1812.
9. Montorsi M, Maggioni M, Falleni M, Pellegrini C, Donadon M, Torzilli G, et al. Survivin gene expression in chronic liver disease and hepatocellular carcinoma. Hepatogastroenterology. 2007;54(79):2040-2044.
10. Shariat SF, Lotan Y, Saboorian H, Khoddami SM, Roehrborn CG, Slawin KM, et al. Survivin expression is associated with features of biologically aggressive prostate carcinoma. Cancer. 2004; 100 (4):751-757.
11. Thomas S, Muralidharan A, Shah GV. Knock-down of Calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells. Int J Oncol. 2007;31(6):1425-1437.
12. Wasko R, Jankowska A, Waligorska-Stachura J, Andrusiewicz M, Jaskula M, Sowinski J.Survivin expression in pituitary adenomas. Neuro Endocrinol Lett. 2005;26(3):209-212.
13. Abe T, Lüdecke DK. Effects of preoperative octreotide treatment on different subtypes of 90 GH-secreting pituitary adenomas and outcome in one surgical centre. Eur J Endocrinol. 2001;145(2):137-145.
14. Lüdecke DK, Flitsch J, Knappe UJ, Saeger W. Cushing's disease: a surgical view. J Neurooncol. 2001;54(2):151-166.
15. Saeger W, Schroder S, Kloppel G . Pathology of important diseases of endocrine organs (excluding the thyroid).Pathologe. 2001;22(5):296-309.
16. Solcia E, Kloppel G, Sobin LH, Capella C, Lells RA de, Heitz PU, et al. (2000) Histological typing of endocrine tumors,2nd edn. Springer, Berlin Heidelberg New York, pp 1-149.
17. LaCasse EC, Baird S, Korneluk RG, MacKenzie AE. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 1998; 17(25):3247-3259.
18. Reed JC. The survivin saga goes in vivo. J Clin Inves.2001; 108(7):965-969.
19. Geske FJ, Gerschenson LE. The biology of apoptosis. Hum Pathol.2001; 32(10):1029-1038.
20. Saikumar P, Dong Z, Mikhailov V, Denton M, Weinberg JM, Venkatachalam MA. Apoptosis: definition, mechanisms, and relevance to disease. Am J Med 1999;107(5):489-506.
21. Deveraux QL, Reed JC. IAP family proteins-suppressors of apoptosis. Genes Dev 1999;13(3):239-252.
22. Pennati M, Folini M, Zaffaroni N. Targeting survivin in cancer therapy. Expert Opin Ther Targets. 2008;12(4):463-476.
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