FAT10调控survivin泛素化降解在膀胱癌增殖中的作用及机制研究

发布时间：2018-05-13 11:12

本文选题：膀胱肿瘤 + FAT10　；参考：《南昌大学》2016年博士论文

【摘要】：研究背景和目的:膀胱癌(Bladder cancer,BC)是我国最常见的泌尿系统肿瘤,其发病率和死亡率均占泌尿系统肿瘤的首位。泛素-蛋白酶体系统(Ubiquitin-proteasome system,UPS)是细胞内蛋白质降解的一个重要途径,FAT10(the human HLA-F adjacent transcript 10,FAT10)是唯一能直接介导蛋白酶体降解途径的类泛素蛋白,在调控细胞生物学功能方面发挥重要作用。Survivin属于凋亡抑制蛋白家族(inhibitor of apoptosis proteins,IAPs)的成员,具有抑制细胞凋亡、促进细胞有丝分裂、调节细胞周期及增加细胞增殖的能力。本研究主要探讨膀胱癌组织及膀胱癌细胞中FAT10和survivin的表达,分析两者表达水平之间的关系及其临床意义;研究FAT10通过调控survivin的表达对膀胱癌细胞增殖的影响及其具体机制。方法:1、选取随访5年以上资料完整的膀胱癌患者133例,免疫组化检测FAT10和survivin在膀胱癌组织石蜡块中的表达情况,分析FAT10和survivin表达的相关性、并分析其与患者的临床病理资料及预后的关系。2、收集50例膀胱癌患者的膀胱癌组织及癌旁组织,通过荧光定量PCR、Western blot检测膀胱癌组织和癌旁组织中FAT10和survivin的表达情况,并分析两者表达是否存在相关性。3、通过荧光定量PCR和Western blot检测膀胱癌细胞T24、J82、5637、UM-UC-3及正常细胞株人输尿管上皮永生化细胞SV-HUC-1中FAT10和survivin的m RNA和蛋白表达情况。4、我们构建了4个sh FAT10干扰质粒,并将我们构建的4干扰质粒转染至膀胱癌细胞,利用荧光定量PCR和Western blot检测转染48h后FAT10的表达变化,筛选出sh FAT10最佳干扰质粒;将筛选出的最佳sh FAT10干扰质粒转染到膀胱癌5637细胞中,运用Western blot检测转染48h后细胞中FAT10和survivin蛋白表达变化,运用Ed U实验观察干扰FAT10后膀胱癌细胞增殖能力的变化;将过表达FAT10质粒转染到膀胱癌UM-UC-3细胞中,运用Western blot检测转染48h后细胞中FAT10和survivin蛋白表达变化,运用Ed U实验观察过表达FAT10后膀胱癌细胞增殖能力的变化;利用稳定转染sh FAT10的膀胱癌细胞株建立人膀胱癌裸鼠皮下瘤模型,观察裸鼠肿瘤的生长情况。5、我们构建了4个shsurvivin干扰质粒,并将我们构建的4个干扰质粒转染至膀胱癌细胞,利用荧光定量PCR和Western blot检测转染48h后survivin蛋白和m RNA的表达变化,筛选出shsurvivin最佳干扰质粒;在膀胱癌细胞中降低FAT10的同时增加survivin的表达及在膀胱癌细胞中增加FAT10的同时干扰survivin的表达,利用Ed U实验观察膀胱癌细胞增殖能力的变化。6、利用免疫共沉淀、激光共聚焦及体外泛素化实验等方法明确FAT10通过调控survivin的泛素化降解稳定survivin表达的具体机制。结果:1、免疫组化显示133例膀胱癌组织中FAT10的阳性率为52.6%,Survivin的阳性率为56.4%,且两者的表达呈正相关(P0.001);荧光定量PCR及Western blot检测50例膀胱癌组织及癌旁组织中FAT10及survivin的表达,结果发现癌组织中FAT10和survivin m RNA及蛋白的表达明显高于癌旁组织(P0.05),通过散点图分析发现FAT10与survivin呈正相关(p0.05);通过荧光定量PCR和Western blot检测四株膀胱癌细胞和正常细胞株人输尿管上皮永生化细胞SV-HUC-1中FAT10和survivin的表达情况,结果发现所有膀胱癌细胞株中的FAT10和survivin的表达明显高于正常细胞株人输尿管上皮永生化细胞SV-HUC-1,且其表达趋势较一致;FAT10和survivin的表达与患者的肿瘤分期有关(P0.05),FAT10阳性表达组和survivin阳性表达组患者的总生存期及无疾病生存期5年生存率低于阴性表达组,两者比较差异有统计学意义(P0.05);将患者FAT10和survivin的表达分为四类:FAT10(-)survivin(-),FAT10(+)survivin(-),FAT10(-)survivin(+),FAT10(+)survivin(+),结果显示FAT10(+)survivin(+)组对应的平均生存时间最短(P0.05);多因素Cox回归分析显示肿瘤分级为影响预后独立的影响因素。2、稳定转染sh FAT10质粒的膀胱癌细胞5637中,FAT10蛋白表达明显下降(p0.05),同时survivin蛋白的表达也随之减少(p0.05),Ed U实验发现膀胱癌细胞5637中sh FAT10组的增殖能力比sh NC组明显降低(P0.05);稳定转染过表达FAT10质粒的膀胱癌细胞UM-UC-3中,FAT10蛋白表达明显升高(p0.05),同时survivin蛋白的表达也随之增加(p0.05),Ed U实验发现过表达FAT10可以明显增加UM-UC-3细胞的增殖能力(P0.05);利用人膀胱癌裸鼠皮下肿瘤模型发现sh FAT10组的裸鼠成瘤体积明显小于sh NC组,瘤体的重量也明显小于空载质粒对照组(p0.05)。3、转染sh FAT10至5637细胞后发现survivin表达下降,而转染GV141-Survivin至稳定干扰FAT10的5637细胞中可以恢复survivin的表达,增殖能力明显升高;转染pc DNA3.1(-)myc-His-FAT10至UM-UC-3细胞后,Survivin表达升高,而转染shsurvivin至稳定高表达FAT10的UM-UC-3细胞中发现survivin的表达下降,且增殖能力明显下降。表明survivin是FAT10调节膀胱癌增殖的关键效应分子。4、免疫共沉淀及激光共聚焦发现survivin和Ub直接结合,在膀胱癌细胞中survivin蛋白经过泛素蛋白酶体降解;免疫共沉淀及激光共聚焦发现survivin和FAT10直接结合;FAT10稳定survivin是通过拮抗survivin的泛素化降解。结论:FAT10通过拮抗survivin泛素化降解从而稳定survivin的表达促进膀胱癌的增殖。
[Abstract]:Background and purpose: Bladder cancer (BC) is the most common urological tumor in our country. The incidence and mortality of Ubiquitin-proteasome system (Ubiquitin-proteasome system, UPS) are an important pathway for the degradation of protein in cells, FAT10 (the human HLA-F adjacent transcript 10) FAT10) is the only ubiquitin protein that directly mediates proteasome degradation pathway and plays an important role in regulating cell biological functions..Survivin belongs to the members of the inhibitor of apoptosis proteins (IAPs) family, which inhibits apoptosis, promotes cell mitosis, regulates cell cycle and increases cells This study mainly discussed the expression of FAT10 and Survivin in bladder cancer cells and bladder cancer cells, analyzed the relationship between the two expressions and their clinical significance, and studied the effect of FAT10 on the proliferation of bladder cancer cells by regulating the expression of survivin. Methods: 1, select the complete bladder for more than 5 years. The expression of FAT10 and Survivin in the paraffin block of bladder cancer tissue was detected by immunohistochemistry in 133 patients. The correlation between FAT10 and survivin expression was analyzed, and the relationship with the clinicopathological data and prognosis of the patients was analyzed.2. 50 cases of bladder cancer were collected and the bladder cancer tissues and para cancerous tissues were collected by fluorescence quantitative PCR, Western blot. To detect the expression of FAT10 and Survivin in bladder cancer tissues and adjacent tissues and to analyze whether there is a correlation between the expression of.3 and the expression of T24, J825637, UM-UC-3 and normal cell lines of human uretero epithelial immortalized cells by fluorescence quantitative PCR and Western blot, and the expression of protein expression in the SV-HUC-1 of the ureteral immortalized cells of human ureteral epithelial cells. .4, we constructed 4 sh FAT10 interference plasmids and transfected the 4 interference plasmids constructed to bladder cancer cells. The expression of FAT10 in 48h after 48h was detected by fluorescence quantitative PCR and Western blot, and the optimal interference plasmid of SH FAT10 was screened, and the best sh FAT10 interference plasmid was transfected into 5637 cells of bladder cancer. The expression of FAT10 and survivin protein in cells transfected with 48h was detected by ern blot. The changes of proliferation ability of bladder cancer cells after FAT10 were observed by Ed U test. The overexpressed FAT10 plasmids were transfected into UM-UC-3 cells of bladder cancer. The proliferation ability of bladder cancer cells was observed after expression of FAT10; the tumor cell model of human bladder cancer was established by using SH FAT10 stable transfected bladder cancer cell line to observe the growth of tumor in nude mice.5. We constructed 4 shsurvivin interference plasmids and transfected our 4 interfering plasmids into bladder cancer cells and use fluorescence. Quantitative PCR and Western blot were used to detect the changes in the expression of survivin protein and m RNA after transfection of 48h, and the optimal interference plasmids of shsurvivin were screened. The expression of survivin was increased at the same time in bladder cancer cells and FAT10 was added to the bladder cancer cells, and the proliferation ability of bladder cancer cells was observed. .6, by using immuno coprecipitation, confocal laser confocal and in vitro ubiquitination, the specific mechanism of FAT10 to stabilize survivin expression by regulating the ubiquitination of survivin was determined. Results: 1, immunohistochemistry showed that the positive rate of FAT10 in 133 bladder cancer tissues was 52.6%, and the positive rate of Survivin was 56.4%, and the expression of both of them was positive. Correlation (P0.001), fluorescence quantitative PCR and Western blot were used to detect the expression of FAT10 and Survivin in 50 cases of bladder cancer tissues and adjacent tissues. The results showed that the expression of FAT10 and Survivin m RNA and protein in the cancer tissues was significantly higher than that in the paracancerous tissues (P0.05). The expression of FAT10 and Survivin in the ureteral immortalized cell SV-HUC-1 of four bladder cancer cells and normal cell lines was detected by estern blot. The results showed that the expression of FAT10 and Survivin in all the bladder cancer cell lines was significantly higher than that of the normal cell human ureteral immortalized cell SV-HUC-1, and the expression trend was the same. The expression of 10 and Survivin was related to the tumor staging of the patients (P0.05). The total survival time and the 5 year survival rate in the FAT10 positive and Survivin positive groups were lower than those of the negative expression group, and the difference was statistically significant (P0.05); the expression of FAT10 and Survivin in patients was divided into four categories: FAT10 (-) survivin (-), FAT10. (+) survivin (-), FAT10 (-) survivin (+), FAT10 (+) survivin (+), the results showed that the average survival time of FAT10 (+) survivin (+) group was the shortest (P0.05). Multiple factor Cox regression analysis showed that the tumor classification was the influence factor of the independent prognosis of.2, and the stable transfection of the bladder cancer cell of SH FAT10 plasmids was 5637. At the same time, the expression of survivin protein decreased (P0.05). The Ed U experiment found that the proliferation ability of SH FAT10 group was significantly lower than that of the SH NC group (P0.05), and the expression of the FAT10 protein expression was significantly increased in the UM-UC-3 transfected bladder cancer cells expressing FAT10 plasmids, and the expression of the protein was also increased. The D U experiment found that the expression of FAT10 could significantly increase the proliferation ability of UM-UC-3 cells (P0.05). Using the subcutaneous tumor model of human bladder cancer nude mice, it was found that the tumor volume of nude mice in the SH FAT10 group was significantly smaller than that of the SH NC group, and the weight of the tumor body was significantly smaller than that of the empty plasmid control group (P0.05).3. In 5637 cells transfected with GV141-Survivin to stable interference FAT10, the expression of survivin could be restored and the proliferation ability was significantly increased. The expression of Survivin increased after transfection of PC DNA3.1 (-) myc-His-FAT10 to UM-UC-3 cells, and the expression of survivin was decreased and the proliferation ability was obvious in the cells transfected from shsurvivin to stable high expression FAT10. It showed that survivin was the key effect molecule of FAT10 to regulate the proliferation of bladder cancer,.4. Immunoprecipitation and laser confocal direct combination of Survivin and Ub showed that survivin protein was degraded by ubiquitin proteasome in bladder cancer cells; immunoprecipitation and laser confocal microscopy found direct combination of Survivin and FAT10; FAT10 stable survivin was Conclusion: FAT10 can promote the proliferation of bladder cancer by inhibiting the ubiquitination of Survivin and stabilizing the expression of survivin by inhibiting the ubiquitination of survivin.

【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.14

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