【作者】 张莹；

【导师】 宋敏；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的肿瘤易感基因101(tumor susceptibility gene 101, TSG101)是1996年在NIH3T3鼠成纤维细胞中发现的。TSG101蛋白介导了多种生物学功能,如调控囊泡运输、细胞周期等,在多种类型的人类恶性肿瘤中存在异常表达,但其与肿瘤发生的关系尚未完全阐明。细胞外信号调节激酶(extra cellular signal-regulated protein kinase, ERK)是丝裂原活化蛋白激酶(mitogen-actived protein kinase, MAPK)家族的经典转导通路之一,在促进细胞的增殖、分化及抑制细胞凋亡中发挥重要作用。低氧诱导因子-1a(hypoxia-inducible factor-1α, HIF-1α)是一种低氧条件下诱导产生的转录因子,在多效性低氧应答中发挥关键作用。有研究显示TSG101在转基因小鼠乳腺中的过表达导致了MAPK/ERK信号通路的异常激活。另有研究表明多种因素均可以通过激活MAPK/ERK信号途径来介导HIF-1α的产生。而迄今为止,在人类乳腺癌中,这些分子的相关调控作用如何尚不清楚。因此,本研究采用靶向TSG101的siRNA方法特异性下调了乳腺癌细胞系中TSG101蛋白的表达,观察TSG101的下调对乳腺癌细胞系生物学特性的影响,同时检测了人乳腺癌组织中三者的表达及相关性,探讨了TSG101与HIF-1α、MAPK/ERK信号通路的关系。实验材料与方法1、应用脂质体法将靶向TSG101的siRNA转染入人乳腺癌细胞系MCF-7细胞中。以未转染的MCF-7细胞及转染普适型阴性对照siRNA (siRNA NC)的MCF-7细胞作为阴性对照。MTT比色法：1×103个／孔的细胞数分别接种转染组和对照组细胞于96孔细胞培养板内,酶标仪机检前加／入MTT培养细胞4小时,DMSO溶解沉淀颗粒,连续4天检测各组细胞的吸光度值,检测细胞增殖能力。细胞周期检测：分别收集TSG101 siRNA转染后72小时的MCF-7细胞和对照组细胞,70%乙醇4℃固定,将细胞密度调整至1×106／ml,PI染色后,流式细胞仪检测细胞周期变化,Modfit LTV3.0软件分析结果。细胞凋亡检测：分别收集TSG101 siRNA转染后72小时的MCF-7细胞和对照组细胞,以binding buffer重悬细胞,调整细胞密度为1×106／ml,加入AnnexinⅤ-FITC和PI染液,流式细胞仪检测细胞凋亡率变化。细胞迁移能力检测(transwell小室法)：分别收集TSG101 siRNA转染后48小时的MCF-7细胞和对照组细胞,接种到含有微孔滤膜的transwell小室,37℃、5%C02培养24小时后,95%乙醇固定,胎盘蓝染色,显微镜下观察细胞迁移能力的改变情况。免疫荧光：分别取TSG101 siRNA转染后72小时的MCF-7细胞爬片和对照组细胞爬片,4%多聚甲醛固定,Triton X-100处理,BSA封闭后,分别滴加HIF-1α、p-ERK一抗,4℃孵育过夜。避光加FITC标记二抗,激光共聚焦扫描显微镜观察,检测TSG101下调前后HIF-1α、p-ERK蛋白的变化。Western blot:分别提取转染组和对照组细胞蛋白,采用考马斯亮蓝法进行蛋白定量。取等量蛋白,进行SDS-PAGE凝胶电泳和转印,分别加入TSG101、HIF-1α、ERK1/2、p-ERK一抗和辣根过氧化物酶(HRP)标记二抗孵育后ECL发光。以(3-actin作为内对照。检测TSG101 siRNA后MCF-7细胞中TSG101、HIF-1α、ERK1/2和p-ERK蛋白的表达。2、本研究采用54例乳腺癌组织标本,均来自中国医科大学第一临床学院2004年-2005年行外科切除手术的乳腺癌患者,所有患者术前均未接受过放疗、化疗。标本经4%甲醛固定,常规石蜡包埋,4μm厚连续切片,经脱蜡,脱苯,水化后,采用链霉菌抗生物素蛋白-过氧化物酶免疫组化法(S-P)法检测ITSG101、HIF-1α、和p-ERK蛋白表达,以PBS代替一抗作为阴性对照,以已知阳性乳腺癌切片作为阳性对照。结果判定：以细胞浆和(或)细胞核内出现棕黄色颗粒为阳性细胞。每张切片随机选取5个高倍视野,每个视野计数200个肿瘤细胞,共计1000个细胞。首先按染色强度评分：无色为0分,淡黄色为1分,棕黄色为2分,棕褐色为3分；再按阳性细胞所占百分比评分：阴性为0分,阳性细胞数≤10%为1分,11%-50%为2分,51%～75%为3分,>75%为4分；最后按二者乘积分数≥3定为阳性,<3为阴性。3、应用SPSS13.0统计分析软件进行数据处理,以P<0.05为差异有统计学意义。实验结果1、使用Western blot方法,应用抗TSG101单克隆抗体检测,在45KD处可见特异性染色条带。以β-actin作内参照,对Western blot结果进行灰度测定和分析,发现TSG101 siRNA组的细胞TSG101蛋白相对表达量(0.1338±0.0086,0.0708±0.0091)与对照组(0.5460±0.0145,0.4782±0.6778)相比明显降低(P<0.05)。2、TSG101 siRNA转染后,细胞增殖能力减弱,细胞凋亡率增加,细胞周期阻滞。MTT法检测结果表明,TSG101 siRNA组的细胞增殖能力明显低于对照组细胞(P<0.05)。流式细胞仪检测发现,TSG101 siRNA组的细胞凋亡率增加,为13.71±1.31%,与对照组相比(4.37±0.87%,6.00±0.08%),差异有统计学意义(P<0.01)。细胞周期检测显示,TSG101 siRNA组细胞Go／G1期比例(70.51±0.23%)与对照组(59.15±0.77%,59.21±0.68%)相比明显增多(P<0.01)；S期细胞(23.65±0.78%)与对照组(34.96±0.45%,34.89±0.30%)相比明显减少(P<0.01)。3、TSG101 siRNA转染后,细胞迁移能力减弱。transwell小室检测细胞迁移能力实验发现,TSG101 siRNA组的细胞迁移数(4.60±0.80)与对照组(24.47±1.90,23.80±2.20)相比差异有统计学意义(P<0.01)。4、TSG101 siRNA转染后,HIF-1α和p-ERK蛋白表达减少。免疫荧光染色结果可见,TSG101 siRNA转染后,细胞HIF-1α和p-ERK蛋白染色强度与对照组相比明显减弱。Western blot结果显示,TSG101 siRNA转染后,HIF-1α蛋白相对表达量(0.1235±0.0057)与对照组(0.6762±0.0080)相比,明显减少(P<0.01); TSG101 siRNA转染后,p-ERK蛋白相对表达量(0.1582±0.0093)与对照组(0.5078±0.0036)相比明显减少(P<0.01)。5、免疫组化结果显示,TSG101、HIF-la和p-ERK蛋白主要定位于乳腺癌细胞浆和(或)细胞核内,呈棕黄色颗粒,阳性率分别为74.07%(40／54)、77.78%(42／54)、81.48%(44／54)。统计学分析显示,TSG101和HIF-1α在乳腺癌中的蛋白表达存在正相关(P<0.05)；TSG101和p-ERK在乳腺癌中的蛋白表达存在正相关(P<0.05)。结论1、TSG101的下调能够抑制乳腺癌细胞增殖,诱导乳腺癌细胞凋亡,阻滞乳腺癌细胞周期于G1／S期,并降低其迁移能力。2、TSG101和HIF-1α在乳腺癌组织中的蛋白表达存在正相关,并且TSG101的下调能够减少乳腺癌细胞中HIF-1α蛋白的表达,提示TSG101在乳腺癌中可能对HIF-1α的基因表达和转录活性具有调节作用。3、TSG101和p-ERK在乳腺癌组织中的蛋白表达存在正相关,并且TSG101的下调能够减少乳腺癌细胞中p-ERK蛋白的表达,提示TSG101在乳腺癌中可能通过MAPK/ERK信号通路发挥生物学作用。更多还原

【Abstract】 The tumor susceptibility gene 101(TSG101)was originally found in murine NIH3T3 cells in 1996. TSG101 protein mediates a variety of biochemical and biological functions,such as the regulations of vesicle transport and cell cycle. TSG101 expressed abnormally in many kinds of human malignant tumors.But the relationship between TSG101 and tumorigenesis is not clear. Hypoxia-inducible factor-1α(HIF-1α) is a kind of transcription factors induced by hypoxic condition,play key roles in the pleiotropic response observed under hypoxic conditions. Extracellular signal-regulated protein kinase (ERK) is a classical signal transduction pathway of the mitogen-actived protein kinase(MAPK) family. ERK was thought to be associated with proliferation,differentiation and inhibiting apoptosis.A study showed that TSG101 overexpression in the mammary gland of transgenic mice led to abnormal activation of MAP kinases.Another study showed that many factors could induce the production of HIF-1αby activating MAPK/ERK signal transduction pathway.The regulations of these molecules are still unclear in human breast cancer so far. In the present study,we used small interfering RNA(siRNA) targeting TSG101 gene to transfect human breast cancer cell line MCF-7 cells,observed the effects of HIF-la and p-ERK expression and the biological traits of MCF-7 cells.Then we used immunohistochemical technique to detect the expressions of TSG101,HIF-la and p-ERK in human breast cancer tissues and to find the relationships among them.Materials and Methods1. The siRNA sequence targeted against human TSG101 gene was synthesized.The siRNA negetive control(siRNA NC),TSG101 siRNA were transfected into breast cancer MCF-7 cell line respectively via lipofection.MCF-7 cells that were transfected siRNA NC and MCF-7 cells were used as control groups.The proliferation abilities of MCF-7 cells transfected by TSG101 siRNA and control groups were tested by MTT.The cell cycle and apoptosis of MCF-7 cells before and after transfection were examined with flow cytometry. Changes of migratory capacities of MCF-7 cells before and after transfection were measured by Transwell. The expressions and locations of MCF-7 cells before and after transfection were detected with immunofluorescence.The protein expressions of TSG101,HIF-la and p-ERK in MCF-7 cells before and after transfection were assessed by Western blot.2. The expressions of TSG101,HIF-1αand p-ERK were detected with immunohistochemistry in 54 breast cancer samples.3. The SPSS 13.0 software was employed to analyze the data.P<0.05 was considered as statistical significance.Results1. The expression level of TSG101 protein in TSG101 siRNA transfected MCF-7 cells (0.1338±0.0086,0.0708±0.0091) was lower than those in the control groups (0.5460±0.0145,0.4782±0.6778) (P<0.05).2. TSG101 siRNA transfected group cells were significantly slower than the control groups in the rate of cell proliferation (P<0.05).The apoptosis rate of TSG101 siRNA transfected group cells was significantly higher(13.71±1.31%) than the control groups (4.37±0.87%,6.00±0.08%) (P<0.01).The percentage of G0/G1 stage cells in TSG101 siRNA transfected group cells was significantly higher(70.51±0.23%) than the control groups(59.15±0.77%,59.21±0.68%)(P<0.01); The percentage of S stage cells in TSG101 siRNA transfected group cells was significantly lower(23.65±0.78%) than the control groups (34.96±0.45%,34.89±0.30%) (P＜0.01)3. The migratory capacity of the TSG101 siRNA transfected group cells was lower than the control groups.The cell numbers of migratory capacity (4.60±0.80) were Significantly lower in the control groups (24.47±1.90,23.80±2.20) (P＜0.01)4. The expression of HIF-la protein was reduced significantly after TSG101 siRNA transfection (0.1235±0.0057) than before (0.6762±0.0080) (P＜0.01).The expression of p-ERK protein was reduced significantly after TSG101 siRNA transfection (0.1582±0.0093) than before (0.5078±0.0036) (P＜0.01)5. The positive expressions of TSG101, HIF-la and p-ERK were 74.07% (40/54),77.78%(42/54) and 81.48%(44/54) separately in invasive ductal carcinoma of breast.The correlations between the expression of TSG101and HIF-la and between the expression of TSG101and p-ERK in breast cancer were both significant (P<0.01)Conclusions1. Breast cancer cells were decreased proliferation and induced apoptosis with transfected TSG101 siRNA into them. The cell cycle of TSG101 siRNA transfected breast cancer cells was arrested at G1/S period.Inhibition of TSG101 protein expression by TSG101 siRNA could decrease migratory capacity of breast cancer cells.2. Maybe TSG101 is an important regulator of HIF-1αgene expression and transcriptional activation in breast cancer.3. TSG101 may play its biology effect through MAPK/ERK signal pathway in breast cancer.更多还原