eIF4E与膀胱癌浸润转移的关系及体内外抑制eIF4E表达的实验研究
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摘要
膀胱癌是我国最常见的恶性肿瘤之一,其发病率位居世界肿瘤第7位,最常见的膀胱癌来源于尿路上皮癌。膀胱癌的发生发展是多基因参与、多阶段性及多种因子调控的过程,因此深入研究膀胱癌发生中的基因调控的机制并且探索行之有效的防治措施和方法是膀胱癌目前诊断和治疗中的重点及难点,对于膀胱癌的防治具有非常重要的意义。
Eukaryotic initiation factor 4E(真核始动因子4E, eIF4E)为真核生物蛋白合成的重要调控因子,在蛋白合成的起始阶段有着相当重要的作用。相关研究表明,eIF4E的高表达与多种肿瘤的发生有着密切的关系。近几年来,有文献先后报道了eIF4E因子的高水平表达或活性提升与头颈部鳞癌、子宫颈癌、乳腺癌、食管癌、结肠癌等多种肿瘤发生、发展密切相关,在浸润性膀胱尿路上皮癌中eIF4E因子的表达及与肿瘤的发生和发展的关系,目前国内外尚无相关系统的研究报道。
为深入的探讨eIF4E因子与浸润性膀胱尿路上皮癌得到发生及发展的关系,并为了寻找到抑制浸润性膀胱尿路上皮癌的发生及发展的行之有效的方法,本研究首先应用免疫组化及原位杂交联合检测了浸润性膀胱尿路上皮癌、膀胱乳头状瘤及正常膀胱黏膜组织中eIF4E及相关因子肝素酶(heparanase, HPA)蛋白和mRNA的表达水平,探讨了eIF4E表达与HPA的相关性。在此基础上,采用脂质体介导方法将3条eIF4E反义寡核苷酸(antisense oligonucleotide, ASODN)序列以不同浓度、不同作用时间转染膀胱癌BIU-87细胞,采用免疫细胞化学、Western blot、原位杂交、RT-PCR方法分别检测各组BIU-87细胞中eIF4E及HPA蛋白质、mRNA基因的表达；应用3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide (MTT)法研究每组细胞的生长和抑制率；应用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)及流式细胞术(FCM)检测每组细胞的凋亡变化情况,并且筛选出来最好的转染序列。通过构建膀胱癌裸鼠移植瘤模型,用反转录PCR (RT-PCR)、原位杂交技术、免疫组织化学(IHC)的方法来检测裸鼠的膀胱癌移植瘤组织中的eIF4E基因表达。从体内角度观察eIF4E基因对裸鼠移植瘤的作用,试图阐明eIF4E的表达在浸润性膀胱尿路上皮癌发生、发展中的意义,为膀胱肿瘤的生物靶向治疗提供理论依据。这里分为三个部分来阐述。
第一部分浸润性膀胱尿路上皮癌eIF4E、HPA的表达及意义
方法
1.应用免疫组织化学(IHC)、原位杂交技术检测eIF4E蛋白及mRNA在50例浸润性膀胱尿路上皮癌、30例膀胱乳头状瘤及50例正常膀胱黏膜组织中的表达情况
2.采用免疫组织化学及原位杂交检测HPA蛋白及mRNA在50例浸润性膀胱尿路上皮癌、30例膀胱乳头状瘤及50例正常膀胱黏膜组织中表达的情况。
3.数据的统计学分析处理：确定检验水准为α=0.05,应用SPSS13.0软件处理,两样本率的比较采用χ2检验、两样本均数的比较采用t检验和方差分析,相关性分析利用Spearman相关系数进行分析。
结果
1. eIF4E蛋白及mRNA、HPA蛋白及mRNA的阳性的表达主要的定位在于膀胱乳头状瘤细胞、膀胱癌细胞及正常膀胱粘膜上皮细胞胞质内。
2.免疫组化及原位杂交检测eIF4E结果显示：在正常膀胱粘膜、膀胱乳头状瘤和浸润性膀胱尿路上皮癌组织中,eIF4E蛋白和mRNA表达的水平依次的升高,三组间比较的差异有统计学的意义(P<0.05)。且与组织学分级、浸润及淋巴结转移有关(P<0.05)。
3.免疫组化及原位杂交检测HPA结果显示：在正常膀胱粘膜、膀胱乳头状瘤和浸润性膀胱尿路上皮癌组织中,HPA蛋白和mRNA表达的水平的依次升高,三组间的比较的差异有统计学的意义(P<0.05)。且与组织学分级、浸润及淋巴结转移有关(P<0.05)
4. eIF4E的蛋白及mRNA的表达与HPA的蛋白及mRNA的表达有正相关性(r=0.504,P<0.05)
第二部分eIF4E ASODN对人膀胱癌BIU-87细胞中eIF4E及HPA表达的影响
方法
1.采用脂质体介导方法将eIF4E ASODN分别以2.5、5.0及7.5μ/ml转染膀胱癌BIU-87细胞,并设立细胞对照组(不转染)、空白对照组(转染空脂质体)及无关对照组(转染无关对照ASODN),分别转染24h、48h及72h。
2.采用免疫细胞化学、Western blot、原位杂交、RT-PCR方法分别检测各组BIU-87细胞中eIF4E及HPA蛋白及mRNA的表达。
3.细胞的生长率和抑制率采用MTT法检测。
4.各组细胞的凋亡情况变化用TUNEL法和流式细胞术来检测。
5.数据的统计分析：以α=0.05为检验水准为,(Statistical Product and Service Solutions, "统计产品与服务解决方案”软件)SPSS13.0软件,分别采用t检验、方差、和x2检验等统计学分析方法进行数据分析处理。
结果
1. eIF4E ASODN转染组细胞中eIF4E及HPA的蛋白及mRNA表达量均较对照组有所降低,与细胞对照组、空白参照组和无关参照组两者对比,差异都有统计学的意义(P<0.05),并且具有时间及浓度的依赖性。
2. eIF4E ASODN转染组细胞生长抑制率均明显高于细胞对照组、空白对照组、无关对照组(P均<0.05),以7.5μ/ml转染后72h抑制率最高。
3. eIF4E ASODN转染组细胞凋亡率与各对照组相比,差异有统计学意义,以7.5μg/ml转染72h最为明显。
第三部分eIF4E ASODN对裸鼠移植瘤的生长的影响
方法
1.分别将转染eIF4E ASODN组、转染无关序列组和未经转染处理组的膀胱癌细胞株BIU-87移植到到裸鼠的皮下,以此构建出膀胱癌的移植瘤模型,以移植瘤在不同分组中的形成、生长及大小为观察指标。
2.应用反转录PCR、ISH(原位杂交)、ICH(免疫组织化学)等方法分别检测不同分组的裸鼠膀胱癌BIU-87移植瘤中eIF4E蛋白和mRNA表达的情况。
3.数据的统计学分析处理：确定检验水准为α=0.05,应用SPSS13.0软件处理,两样本率的比较采用χ2检验、两样本均数的比较采用t检验和方差分析,相关性分析利用Spearman相关系数进行分析。
结果
1.未经转染组处理组、无关序列对照组的移植瘤的体积明显大于eIF4EASODN转染组肿瘤的体积,组间的比较存在的差异具有统计学的意义(P<0.05)。
2.未转染组、膀胱癌细胞组和无关序列参照组裸鼠移植瘤组织内,eIF4E蛋白及mRNA的表达均显著高于eIF4E ASODN转染组的表达,组间比较的差异具有统计学意义(P<0.05)。
结论
1.HPA蛋白和mRNA的表达与浸润性膀胱尿路上皮癌的发生、浸润和转移有着密切的关系,可以作为判断浸润性膀胱尿路上皮癌的淋巴结转移潜能的相关指标。
2.浸润性膀胱尿路上皮癌中eIF4E的表达与HPA有正相关,可以提示eIF4E的高表达有可能导致HPA的上调表达,促进了肿瘤细胞的侵袭和转移。
3.成功筛选了eIF4E ASODN的最佳转染序列,并将其导入浸润性膀胱尿路上皮癌细胞株BIU-87细胞中,建立了由eIF4E基因抑制的细胞株,并为下一步研究eIF4E生物学的功能及eIF4E靶向的治疗奠定基础。
4.通过体外的实验显示,eIF4E ASODN可降低膀胱癌BIU-87细胞株中eIF4E蛋白的表达水平,并可下调BIU-87细胞HPA蛋白的表达水平。
5.裸鼠的移植瘤的生长可被eIF4E ASODN明显的抑制,eIF4E蛋白、mRNA表达也受到抑制作用,为浸润性膀胱尿路上皮癌的靶向治疗提供了新的思路及理论依据。
Bladder cancer is one of the most common malignant tumors in China, the incidence rate among 7 of the World tumor, the most common cancer of the bladder from urothelial carcinoma.The development of bladder cancer is a process of multi-gene participation, multi-stage and a variety of factors regulating, Therefore, in-depth study the gene regulation mechanism of bladder cancer and explore the effective treatment measures are the priorities and difficulties in diagnosis and treatment of bladder cancer, and there is have a great significance of prevention and treatment in bladder cancer.
Eukaryotic initiation factor 4E (eIF4E) is an important regulatory factor of eukaryotic protein synthesis, and is play a very important role in protein synthesis in the initial stage. Studies have shown that, the high expression of eIF4E is closely related to the occurrence of a variety of tumor. In recent years, there have been reported in the literature the high expression of eIF4E or increased activity is closely related to the occurrence of head and neck squamous cell carcinoma, cervical cancer, breast cancer, esophageal cancer, colon cancer and other tumor. The expression of eIF4E in invasive bladder urothelial carcinoma and its relationship with the tumor occurrence and development there is no systematic study reported so far at home and abroad.
For further research on the relationship between eIF4E and invasive bladder urothelial carcinoma and find the effective way to inhibit the occurrence and development of bladder invasive urothelial carcinoma, in this study, using immunohistochemistry and in situ hybridization combined detect the expression of protein and mRNA levels of eIF4E and associated factors of heparanase(HPA) in bladder invasive urothelial carcinoma, bladder papilloma and normal bladder mucosa. On this basis, use liposome-mediated method transfected three eIF4E antisense oligonucleotides (ASODN) sequence in bladder cancer BIU-87 cells with different concentrations and different time. Then, use immunocytochemistry, Western blot, in situ hybridization and RT-PCR methods detect the protein and mRNA expression of eIF4E and HPA in each group of BIU-87 cells; Detect each group of cell growth inhibition rate by MTT; Use TUNEL and flow cytometry to detect the apoptotic cells in each group, And filter out the best transfection sequence. Finally, by the experiments of xenografts in nude mice, detect the expression of eIF4E gene in the bladder transplanted tumors of nude mice by RT-PCR, in situ hybridization and immunohistochemical, respectively Use the immunohistochemistry and in situ hybridization detect the eIF4E protein and mRNA expression in bladder cancer xenografts; and use TUNEL detect the apoptosis of bladder cancer xenograft cells. Observe the role of the eIF4E gene on xenografts in nude mice in vivo, Attempt to clarify the significance of eIF4E expression in invasive bladder urothelial carcinoma and gastric cancer occur and development. In order to provide a theoretical basis to the targeted therapy of bladder cancer. This study is divided into three parts.
Part I Expressions of eIF4E and HPA in bladder invasive urothelial carcinoma tissues and their significance
Methods
1. Using immunohistochemistry and in situ hybridization to detect the expression of eIF4E gene in 50 cases of bladder invasive urothelial carcinoma,30 cases of bladder papilloma and 50 cases of normal bladder mucosa.
2. Using immunohistochemistry and in situ hybridization to detect the expression of HPA gene in 50 cases of bladder invasive urothelial carcinoma,30 cases of bladder papilloma and 50 cases of normal bladder mucosa
3. Software of SPSS 13.0 was used to analysis the statistics, test standardα= 0.05, chi-square test,T test and variance analysis and Spearmanm were used.
Results
1. Expression of the eIF4E and HPA gene was lower or no in bladder papilloma and normal bladder mucosa membrane tissue compared to BIUC. Both the expression of eIF4E, HPA protein and mRNA were mainly detected in in cytoplast of BIUC.
2. Increased expression levels of protein and mRNA of eIF4E were seen from normal bladder mucosa membrane tissue, bladder papilloma tissue to BIUC tissue, and there were significant differences among three groups (P<0.05).
3. Gradually increased levels of the expression of HPA protein and mRNA were observed from normal bladder mucosa membrane tissue, bladder papilloma tissue to BIUC tissue, and there was significant difference among three groups (P< 0.05)
4. The expression of eIF4E protein and mRNA was positively correlated with the expression of HPA protein and mRNA(r=0.504,P<0.05)0
Part II The influence of eIF4E ASODN to the expression of eIF4E and HPA in human bladder cancer BIU-87 cells
Methods
1. Use liposome-mediated method to transfect eIF4E ASODN into bladder cancer BIU-87 cells with 2.5,5.0 and 7.5μg/ml for 24,48 and 72h, respectively. And set up cells control group (not transfected), blank control group (transfected with empty liposomes) and unrelated control group (transfected with unrelated control ASODN).
2. Use immunocytochemistry, Western blot, in situ hybridization and RT-PCR to detecte the expression of eIF4E and HPA protein and mRNA in each group BIU-87 cells.
3. Detect each group of cell growth inhibition rate by MTT.
4. Use TUNEL and flow cytometry to detect the apoptotic cells in each group.
5. Statistics analysis was performed by software of SPSS13.0, the standard of test:α=0.05, chi-square test and one-way analysis of variance were used.
Results
1. The expression of eIF4E and HPA were decreased in each eIF4E ASODN transfection group cells compared with control group. In eIF4E ASODN transfection group, there were significant differences compared with cells control group, blank control group and unrelated control group (P<0.05),which have time and concentration dependence.
2. The cell growth inhibition rate of eIF4E ASODN transfection group significantly higher than cells control group,blank control group and unrelated control group (P<0.05). The highest inhibitory rate of 7.5μg/ml transfected 72 hours
3. There was significant difference of cell apoptosis rate between eIF4E ASODN transfection group and each control groups. And the most obvious is 7.5μg/ml transfected 72 hours.
Part III Influence of eIF4E ASODN on the growth of xenografts in nude mice
Methods
1. Transplantable tumors were produced using BIU-87 cells and the transfected BIU-87 cells with eIF4E ASODN transfection group, unrelated control group and untransfected group, respectively, in nude mice. Compare the tumorigenicity and the tumor size in each group of nude mice
2. Detect the expression of eIF4E gene in the bladder transplanted tumors of nude mice by RT-PCR, in situ hybridization and immunohistochemical, respectively.
3. Use the immunohistochemistry and in situ hybridization detect the eIF4E protein and mRNA expression in bladder cancer xenografts.
4. Use TUNEL detect the apoptosis of bladder cancer xenograft cells.
5. Statistical analysis:Statistics analysis was used by SPSS13.0 software,using
chi-square test,T test and variance analysis,test standard a=0.05.
Results
1. The volume of xenografts in nude mice of unrelated control group and untransfected group was significantly greater than eIF4E ASODN transfection group. Statistically significant difference between groups (P<0.05)
2. The expression of eIF4E protein and mRNA were Significantly higher in unrelated control group, untransfected group and bladder cancer cell group compare with eIF4E ASODN transfection group. Statistically significant difference between groups (P<0.05)
3. The TUNEL method detected apoptotic cells of bladder cancer xenograft was show that the nucleus was tan, dyed shades vary, chromatin uneven distribution. Apoptotic index in unrelated control group and untransfected group was no significant difference(P> 0.05), the two groups of apoptotic index was significantly lower than the eIF4E ASODN transfection group, the differences were significant (P<0.05),
Conclusions
1. High levels of eIF4E protein and mRNA were observed in bladder invasive urothelial carcinoma,suggesting that eIF4E may be associated with the occurrence and development of bladder invasive urothelial cancer.
2. High expression of eIF4E gene can up-regulate the expression of HPA, thus contributing to tumor invasion。
3. The best transfection sequence of eIF4E ASODN was successfully screened, and introduced into BIU-87 cells. BIU-87 eIF4E gene inhibition cells were successfully established,which will lay a foundation for further studing the biological function of eIF4E and eIF4E target therapy.
4. eIF4E ASODN can reduce the expression of eIF4E in the BIU-87 cells in vitro, and down-regulate the expression of HPA protein.
5. eIF4E ASODN obviously inhibites the growth of transplantable tumors and promote apoptosis of transplanted tumor cells in vivo, which will provide the new idea and theoretical basis for target gene therapy of BIUC.
Eukaryotic initiation factor 4E(真核始动因子4E, eIF4E)为真核生物蛋白合成的重要调控因子,在蛋白合成的起始阶段有着相当重要的作用。相关研究表明,eIF4E的高表达与多种肿瘤的发生有着密切的关系。近几年来,有文献先后报道了eIF4E因子的高水平表达或活性提升与头颈部鳞癌、子宫颈癌、乳腺癌、食管癌、结肠癌等多种肿瘤发生、发展密切相关,在浸润性膀胱尿路上皮癌中eIF4E因子的表达及与肿瘤的发生和发展的关系,目前国内外尚无相关系统的研究报道。
为深入的探讨eIF4E因子与浸润性膀胱尿路上皮癌得到发生及发展的关系,并为了寻找到抑制浸润性膀胱尿路上皮癌的发生及发展的行之有效的方法,本研究首先应用免疫组化及原位杂交联合检测了浸润性膀胱尿路上皮癌、膀胱乳头状瘤及正常膀胱黏膜组织中eIF4E及相关因子肝素酶(heparanase, HPA)蛋白和mRNA的表达水平,探讨了eIF4E表达与HPA的相关性。在此基础上,采用脂质体介导方法将3条eIF4E反义寡核苷酸(antisense oligonucleotide, ASODN)序列以不同浓度、不同作用时间转染膀胱癌BIU-87细胞,采用免疫细胞化学、Western blot、原位杂交、RT-PCR方法分别检测各组BIU-87细胞中eIF4E及HPA蛋白质、mRNA基因的表达；应用3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide (MTT)法研究每组细胞的生长和抑制率；应用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)及流式细胞术(FCM)检测每组细胞的凋亡变化情况,并且筛选出来最好的转染序列。通过构建膀胱癌裸鼠移植瘤模型,用反转录PCR (RT-PCR)、原位杂交技术、免疫组织化学(IHC)的方法来检测裸鼠的膀胱癌移植瘤组织中的eIF4E基因表达。从体内角度观察eIF4E基因对裸鼠移植瘤的作用,试图阐明eIF4E的表达在浸润性膀胱尿路上皮癌发生、发展中的意义,为膀胱肿瘤的生物靶向治疗提供理论依据。这里分为三个部分来阐述。
第一部分浸润性膀胱尿路上皮癌eIF4E、HPA的表达及意义
方法
1.应用免疫组织化学(IHC)、原位杂交技术检测eIF4E蛋白及mRNA在50例浸润性膀胱尿路上皮癌、30例膀胱乳头状瘤及50例正常膀胱黏膜组织中的表达情况
2.采用免疫组织化学及原位杂交检测HPA蛋白及mRNA在50例浸润性膀胱尿路上皮癌、30例膀胱乳头状瘤及50例正常膀胱黏膜组织中表达的情况。
3.数据的统计学分析处理：确定检验水准为α=0.05,应用SPSS13.0软件处理,两样本率的比较采用χ2检验、两样本均数的比较采用t检验和方差分析,相关性分析利用Spearman相关系数进行分析。
结果
1. eIF4E蛋白及mRNA、HPA蛋白及mRNA的阳性的表达主要的定位在于膀胱乳头状瘤细胞、膀胱癌细胞及正常膀胱粘膜上皮细胞胞质内。
2.免疫组化及原位杂交检测eIF4E结果显示：在正常膀胱粘膜、膀胱乳头状瘤和浸润性膀胱尿路上皮癌组织中,eIF4E蛋白和mRNA表达的水平依次的升高,三组间比较的差异有统计学的意义(P<0.05)。且与组织学分级、浸润及淋巴结转移有关(P<0.05)。
3.免疫组化及原位杂交检测HPA结果显示：在正常膀胱粘膜、膀胱乳头状瘤和浸润性膀胱尿路上皮癌组织中,HPA蛋白和mRNA表达的水平的依次升高,三组间的比较的差异有统计学的意义(P<0.05)。且与组织学分级、浸润及淋巴结转移有关(P<0.05)
4. eIF4E的蛋白及mRNA的表达与HPA的蛋白及mRNA的表达有正相关性(r=0.504,P<0.05)
第二部分eIF4E ASODN对人膀胱癌BIU-87细胞中eIF4E及HPA表达的影响
方法
1.采用脂质体介导方法将eIF4E ASODN分别以2.5、5.0及7.5μ/ml转染膀胱癌BIU-87细胞,并设立细胞对照组(不转染)、空白对照组(转染空脂质体)及无关对照组(转染无关对照ASODN),分别转染24h、48h及72h。
2.采用免疫细胞化学、Western blot、原位杂交、RT-PCR方法分别检测各组BIU-87细胞中eIF4E及HPA蛋白及mRNA的表达。
3.细胞的生长率和抑制率采用MTT法检测。
4.各组细胞的凋亡情况变化用TUNEL法和流式细胞术来检测。
5.数据的统计分析：以α=0.05为检验水准为,(Statistical Product and Service Solutions, "统计产品与服务解决方案”软件)SPSS13.0软件,分别采用t检验、方差、和x2检验等统计学分析方法进行数据分析处理。
结果
1. eIF4E ASODN转染组细胞中eIF4E及HPA的蛋白及mRNA表达量均较对照组有所降低,与细胞对照组、空白参照组和无关参照组两者对比,差异都有统计学的意义(P<0.05),并且具有时间及浓度的依赖性。
2. eIF4E ASODN转染组细胞生长抑制率均明显高于细胞对照组、空白对照组、无关对照组(P均<0.05),以7.5μ/ml转染后72h抑制率最高。
3. eIF4E ASODN转染组细胞凋亡率与各对照组相比,差异有统计学意义,以7.5μg/ml转染72h最为明显。
第三部分eIF4E ASODN对裸鼠移植瘤的生长的影响
方法
1.分别将转染eIF4E ASODN组、转染无关序列组和未经转染处理组的膀胱癌细胞株BIU-87移植到到裸鼠的皮下,以此构建出膀胱癌的移植瘤模型,以移植瘤在不同分组中的形成、生长及大小为观察指标。
2.应用反转录PCR、ISH(原位杂交)、ICH(免疫组织化学)等方法分别检测不同分组的裸鼠膀胱癌BIU-87移植瘤中eIF4E蛋白和mRNA表达的情况。
3.数据的统计学分析处理：确定检验水准为α=0.05,应用SPSS13.0软件处理,两样本率的比较采用χ2检验、两样本均数的比较采用t检验和方差分析,相关性分析利用Spearman相关系数进行分析。
结果
1.未经转染组处理组、无关序列对照组的移植瘤的体积明显大于eIF4EASODN转染组肿瘤的体积,组间的比较存在的差异具有统计学的意义(P<0.05)。
2.未转染组、膀胱癌细胞组和无关序列参照组裸鼠移植瘤组织内,eIF4E蛋白及mRNA的表达均显著高于eIF4E ASODN转染组的表达,组间比较的差异具有统计学意义(P<0.05)。
结论
1.HPA蛋白和mRNA的表达与浸润性膀胱尿路上皮癌的发生、浸润和转移有着密切的关系,可以作为判断浸润性膀胱尿路上皮癌的淋巴结转移潜能的相关指标。
2.浸润性膀胱尿路上皮癌中eIF4E的表达与HPA有正相关,可以提示eIF4E的高表达有可能导致HPA的上调表达,促进了肿瘤细胞的侵袭和转移。
3.成功筛选了eIF4E ASODN的最佳转染序列,并将其导入浸润性膀胱尿路上皮癌细胞株BIU-87细胞中,建立了由eIF4E基因抑制的细胞株,并为下一步研究eIF4E生物学的功能及eIF4E靶向的治疗奠定基础。
4.通过体外的实验显示,eIF4E ASODN可降低膀胱癌BIU-87细胞株中eIF4E蛋白的表达水平,并可下调BIU-87细胞HPA蛋白的表达水平。
5.裸鼠的移植瘤的生长可被eIF4E ASODN明显的抑制,eIF4E蛋白、mRNA表达也受到抑制作用,为浸润性膀胱尿路上皮癌的靶向治疗提供了新的思路及理论依据。
Bladder cancer is one of the most common malignant tumors in China, the incidence rate among 7 of the World tumor, the most common cancer of the bladder from urothelial carcinoma.The development of bladder cancer is a process of multi-gene participation, multi-stage and a variety of factors regulating, Therefore, in-depth study the gene regulation mechanism of bladder cancer and explore the effective treatment measures are the priorities and difficulties in diagnosis and treatment of bladder cancer, and there is have a great significance of prevention and treatment in bladder cancer.
Eukaryotic initiation factor 4E (eIF4E) is an important regulatory factor of eukaryotic protein synthesis, and is play a very important role in protein synthesis in the initial stage. Studies have shown that, the high expression of eIF4E is closely related to the occurrence of a variety of tumor. In recent years, there have been reported in the literature the high expression of eIF4E or increased activity is closely related to the occurrence of head and neck squamous cell carcinoma, cervical cancer, breast cancer, esophageal cancer, colon cancer and other tumor. The expression of eIF4E in invasive bladder urothelial carcinoma and its relationship with the tumor occurrence and development there is no systematic study reported so far at home and abroad.
For further research on the relationship between eIF4E and invasive bladder urothelial carcinoma and find the effective way to inhibit the occurrence and development of bladder invasive urothelial carcinoma, in this study, using immunohistochemistry and in situ hybridization combined detect the expression of protein and mRNA levels of eIF4E and associated factors of heparanase(HPA) in bladder invasive urothelial carcinoma, bladder papilloma and normal bladder mucosa. On this basis, use liposome-mediated method transfected three eIF4E antisense oligonucleotides (ASODN) sequence in bladder cancer BIU-87 cells with different concentrations and different time. Then, use immunocytochemistry, Western blot, in situ hybridization and RT-PCR methods detect the protein and mRNA expression of eIF4E and HPA in each group of BIU-87 cells; Detect each group of cell growth inhibition rate by MTT; Use TUNEL and flow cytometry to detect the apoptotic cells in each group, And filter out the best transfection sequence. Finally, by the experiments of xenografts in nude mice, detect the expression of eIF4E gene in the bladder transplanted tumors of nude mice by RT-PCR, in situ hybridization and immunohistochemical, respectively Use the immunohistochemistry and in situ hybridization detect the eIF4E protein and mRNA expression in bladder cancer xenografts; and use TUNEL detect the apoptosis of bladder cancer xenograft cells. Observe the role of the eIF4E gene on xenografts in nude mice in vivo, Attempt to clarify the significance of eIF4E expression in invasive bladder urothelial carcinoma and gastric cancer occur and development. In order to provide a theoretical basis to the targeted therapy of bladder cancer. This study is divided into three parts.
Part I Expressions of eIF4E and HPA in bladder invasive urothelial carcinoma tissues and their significance
Methods
1. Using immunohistochemistry and in situ hybridization to detect the expression of eIF4E gene in 50 cases of bladder invasive urothelial carcinoma,30 cases of bladder papilloma and 50 cases of normal bladder mucosa.
2. Using immunohistochemistry and in situ hybridization to detect the expression of HPA gene in 50 cases of bladder invasive urothelial carcinoma,30 cases of bladder papilloma and 50 cases of normal bladder mucosa
3. Software of SPSS 13.0 was used to analysis the statistics, test standardα= 0.05, chi-square test,T test and variance analysis and Spearmanm were used.
Results
1. Expression of the eIF4E and HPA gene was lower or no in bladder papilloma and normal bladder mucosa membrane tissue compared to BIUC. Both the expression of eIF4E, HPA protein and mRNA were mainly detected in in cytoplast of BIUC.
2. Increased expression levels of protein and mRNA of eIF4E were seen from normal bladder mucosa membrane tissue, bladder papilloma tissue to BIUC tissue, and there were significant differences among three groups (P<0.05).
3. Gradually increased levels of the expression of HPA protein and mRNA were observed from normal bladder mucosa membrane tissue, bladder papilloma tissue to BIUC tissue, and there was significant difference among three groups (P< 0.05)
4. The expression of eIF4E protein and mRNA was positively correlated with the expression of HPA protein and mRNA(r=0.504,P<0.05)0
Part II The influence of eIF4E ASODN to the expression of eIF4E and HPA in human bladder cancer BIU-87 cells
Methods
1. Use liposome-mediated method to transfect eIF4E ASODN into bladder cancer BIU-87 cells with 2.5,5.0 and 7.5μg/ml for 24,48 and 72h, respectively. And set up cells control group (not transfected), blank control group (transfected with empty liposomes) and unrelated control group (transfected with unrelated control ASODN).
2. Use immunocytochemistry, Western blot, in situ hybridization and RT-PCR to detecte the expression of eIF4E and HPA protein and mRNA in each group BIU-87 cells.
3. Detect each group of cell growth inhibition rate by MTT.
4. Use TUNEL and flow cytometry to detect the apoptotic cells in each group.
5. Statistics analysis was performed by software of SPSS13.0, the standard of test:α=0.05, chi-square test and one-way analysis of variance were used.
Results
1. The expression of eIF4E and HPA were decreased in each eIF4E ASODN transfection group cells compared with control group. In eIF4E ASODN transfection group, there were significant differences compared with cells control group, blank control group and unrelated control group (P<0.05),which have time and concentration dependence.
2. The cell growth inhibition rate of eIF4E ASODN transfection group significantly higher than cells control group,blank control group and unrelated control group (P<0.05). The highest inhibitory rate of 7.5μg/ml transfected 72 hours
3. There was significant difference of cell apoptosis rate between eIF4E ASODN transfection group and each control groups. And the most obvious is 7.5μg/ml transfected 72 hours.
Part III Influence of eIF4E ASODN on the growth of xenografts in nude mice
Methods
1. Transplantable tumors were produced using BIU-87 cells and the transfected BIU-87 cells with eIF4E ASODN transfection group, unrelated control group and untransfected group, respectively, in nude mice. Compare the tumorigenicity and the tumor size in each group of nude mice
2. Detect the expression of eIF4E gene in the bladder transplanted tumors of nude mice by RT-PCR, in situ hybridization and immunohistochemical, respectively.
3. Use the immunohistochemistry and in situ hybridization detect the eIF4E protein and mRNA expression in bladder cancer xenografts.
4. Use TUNEL detect the apoptosis of bladder cancer xenograft cells.
5. Statistical analysis:Statistics analysis was used by SPSS13.0 software,using
chi-square test,T test and variance analysis,test standard a=0.05.
Results
1. The volume of xenografts in nude mice of unrelated control group and untransfected group was significantly greater than eIF4E ASODN transfection group. Statistically significant difference between groups (P<0.05)
2. The expression of eIF4E protein and mRNA were Significantly higher in unrelated control group, untransfected group and bladder cancer cell group compare with eIF4E ASODN transfection group. Statistically significant difference between groups (P<0.05)
3. The TUNEL method detected apoptotic cells of bladder cancer xenograft was show that the nucleus was tan, dyed shades vary, chromatin uneven distribution. Apoptotic index in unrelated control group and untransfected group was no significant difference(P> 0.05), the two groups of apoptotic index was significantly lower than the eIF4E ASODN transfection group, the differences were significant (P<0.05),
Conclusions
1. High levels of eIF4E protein and mRNA were observed in bladder invasive urothelial carcinoma,suggesting that eIF4E may be associated with the occurrence and development of bladder invasive urothelial cancer.
2. High expression of eIF4E gene can up-regulate the expression of HPA, thus contributing to tumor invasion。
3. The best transfection sequence of eIF4E ASODN was successfully screened, and introduced into BIU-87 cells. BIU-87 eIF4E gene inhibition cells were successfully established,which will lay a foundation for further studing the biological function of eIF4E and eIF4E target therapy.
4. eIF4E ASODN can reduce the expression of eIF4E in the BIU-87 cells in vitro, and down-regulate the expression of HPA protein.
5. eIF4E ASODN obviously inhibites the growth of transplantable tumors and promote apoptosis of transplanted tumor cells in vivo, which will provide the new idea and theoretical basis for target gene therapy of BIUC.
引文
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