【作者】 李军；

【导师】 孔垂泽；

【作者基本信息】 中国医科大学 ， 泌尿外科， 2019， 博士

【摘要】 目的:膀胱癌是泌尿系统常见的恶性肿瘤,其发病率有逐渐上升的趋势,是严重威胁人们健康的常见病。目前膀胱癌的分子生物学机制还不完全清楚,因此,阐明其机制,有助于探寻疾病根源,及早作出诊断,针对性采取有效措施,提高治疗效果,对于临床膀胱癌的控制具有重要意义。沉默信息调节因子（silence information regulator,Sirtuin）在细胞周期控制、维持线粒体的动态平衡、自噬和细胞生长调节等过程中发挥重要作用。SIRT3是其中重要的一员,同样可以发挥上述作用,它主要存在于线粒体当中,学者们在细胞核中也发现了上述因子,但含量相对较少。它可调控多种线粒体蛋白的乙酰化修饰,从而影响细胞内的活性氧（reactive oxygen species,ROS）水平和能量代谢水平。SIRT3对于能量调节至关重要,这一点也获得了广泛共识,如果缺乏上述因子,将会对线粒体产生影响,导致过度乙酰化,进而影响功能,引发能量代谢异常,甚至会影响有丝分裂。SIRT3通过调节ROS,抑制缺氧诱导因子1α（hypoxia-inducible factor 1α,HIF-1α）的转录活性,抵抗氧化应激,达到抑制肿瘤生长的作用。上皮间质转化（Epithelial-Mesenchymal Transition,EMT）是上皮细胞向间质细胞的转换,在此过程中获取了移行能力,因而具有侵袭性。E-cadherin是EMT的关键分子,用于维持正常细胞间紧密连接的稳定性,其表达水平与EMT的发生及肿瘤的侵袭能力负相关,转录因子如锌指蛋白Snail,SIP-1,Slug,Twist等可下调E-cadherin表达进而促进EMT发生。Snail和Twist是肿瘤发生EMT的主要诱导因子,对于诱导肿瘤EMT、侵袭和转移具有独立或协同作用。研究表明,晚期膀胱癌的高侵袭性与EMT关系密切。而且,肿瘤中HIF-1α的增多表达可通过EMT使肿瘤的侵袭性增强。推测在膀胱癌中,SIRT3的表达可能通过调节HIF-1α进而影响EMT相关因子的表达来调控其生长、侵袭等生物学行为。本课题通过沉默和过表达膀胱癌T24细胞系中SIRT3的表达,利用体外实验,探讨SIRT3在膀胱癌上皮间质转化中的作用,为寻找膀胱癌治疗的新靶点提供一定的理论依据。研究方法:1、肿瘤细胞的培养:膀胱癌T24细胞株,生长在含10%胎牛血清的RPMI-1640培养液中,与37℃、5%CO₂饱和湿度条件下进行培养。2、免疫组织化学分析:收集中国医科大学附属一院泌尿科膀胱癌患者的病理标本,进行HE染色和组织化学染色,分析膀胱癌组织和癌旁组织中SIRT3、HIF-1α、E-cadherin、Snail、Slug和Vimentin的表达水平。并分析SIRT3蛋白表达水平与临床病例资料及其病理学特征之间的关系。3、质粒的构建及细胞转染:设计干扰靶点,慢病毒转染T24细胞,并用RT-PCR和Western blot检测转染细胞中沉默和过表达SIRT3基因的效率。4、Transwell小室和划痕实验主要应用于细胞能力检测,在细胞转染前后进行,可确定其侵袭、迁移能力是否发生了改变。Transwell侵袭实验中,下室加入500μl含20%胎牛血清的RPMI-1640培养液,将200μl单细胞悬液加入到上室,细胞数量为1×10⁵/孔,培养24小时候观察并计数侵袭至胶膜下的细胞数量。划痕实验中,准备6孔板,选择2×10⁵个膀胱癌细胞,要求生长良好,将其接种于孔板之上,观察融合度变化,当达到90%时,用200μl的移液器枪头在6孔板的内部底面上沿直线划痕,划痕后继续常规培养24h,置于倒置显微镜下,观察和测量单层细胞划痕的愈合情况。5、蛋白提取及Western blot检测:提取膀胱癌细胞总蛋白,对其进行检测,确定浓度大小。Western blot实验以内参抗体GAPDH,及MMP-2、MMP-9、HIF-1α、Snail、Slug、E-cadherin、Vimentin和Twist为一抗,分析其蛋白的表达。凝胶图像处理,对灰度进行分析。6、统计学分析:经三次重复实验,获取相关数据,均值±标准差表示。组间比较t检验。Pearson分析SIRT3与肿瘤分期及病理类型等的相关性。SPSS17.0及Graphpad Prism5.0分析及绘图,P<0.05有统计学差异。结果:1、SIRT3在膀胱癌组织中的表达及临床意义:免疫组化结果显示,SIRT3和E-cadherin在膀胱癌组织中的表达率明显低于癌旁组织;HIF-1α、Snail、Slug和Vimentin的水平,膀胱癌组织明显高于癌旁组织。相关性分析结果显示,T3N0M0组中SIRT3的表达水平明显低于TaN0M0组（P<0.05）;高分化膀胱癌组织中SIRT3的表达水平明显高于低分化组（P<0.05）;淋巴结转移的膀胱癌患者组织中SIRT3的水平也明显降低,与未发生淋巴结转移的患者组相比,差异有统计学意义（P<0.05）。2、沉默SIRT3基因对膀胱癌细胞侵袭转移及上皮间质转化的影响:RT-PCR和Western blot实验检测慢病毒干扰SIRT3基因的效率,pLK0.1-shRNA-SIRT3组膀胱癌T24细胞中SIRT3的表达明显低于NC组和Control组（P<0.05）。说明慢病毒沉默SIRT3基因成功。Transwell实验结果显示,pLK0.1-shRNA-SIRT3组细胞穿过基质胶的细胞数,与NC和Control组细胞相比增多,但差异无统计学意义（P>0.05）。划痕实验结果,与pLK0.1-NC和Control组细胞相比,pLK0.1-shRNA-SIRT3组细胞的迁移能力增强,具有统计学意义（P<0.05）。Western blot法检测结果显示,MMP-2、MMP-9、HIF-1α、vimentin、twist、snail和slug的表达,均是pLK0.1-shRNA-SIRT3组细胞的表达水平高于NC和Control组,但差异无统计学意义（P>0.05）;E-cadherin的表达水平,pLK0.1-shRNA-SIRT3细胞组低于NC和Control细胞组中的表达,差异也无统计学意义（P<0.05）。3、过表达SIRT3基因对膀胱癌细胞侵袭转移及上皮间质转化的影响:RT-PCR和Western blot实验检测质粒转染SIRT3基因的效率,pCDH-promoter-SIRT3组膀胱癌T24细胞中SIRT3的表达明显高于NC组和Control组（P<0.05）。说明构建过表达SIRT3基因的细胞系成功。Transwell实验结果显示,pCDH-promoter-SIRT3组细胞穿过基质数量减少,与对照组相比,统计学差异显著（P<0.01）。划痕实验结果,与NC和Control组细胞相比,过表达pCDH-promoter-SIRT3组细胞的迁移能力减弱,且差异有统计学意义（P<0.05）。Western blot法检测结果显示,MMP-2、MMP-9、HIF-1α、vimentin、twist、snail和slug的表达,均是pCDH-promoter-SIRT3组细胞的表达水平明显低于NC和Control组,且差异有统计学意义（P<0.05）;E-cadherin的表达水平,pCDH-promoter-SIRT3细胞组明显高于NC和Control细胞组中的表达,且差异有统计学意义（P<0.05）。结论:1、SIRT3在膀胱癌组织及癌旁组织中的表达存在差异;SITR3的表达水平与膀胱癌的组织分化程度、临床分期存在相关性;SIRT3的表达水平与膀胱癌的淋巴结转移显著相关。2、慢病毒载体可以有效提高shRNA的转染效率;慢病毒shRNA有效干扰了膀胱癌T24细胞系中SIRT3的表达,成功构建沉默和过表达SIRT3基因的膀胱癌细胞系。3、相关功能实验结果提示,与对照组相比,沉默SIRT3基因组膀胱癌细胞的迁移和侵袭能力增强;过表达SIRT3基因组膀胱癌细胞的迁移和侵袭能力明显减弱。沉默SIRT3的表达可以促进膀胱癌细胞中与EMT相关因子的表达;过表达SIRT3基因可以抑制与EMT相关因子的表达。揭示SIRT3基因的表达在膀胱癌细胞的侵袭转移及上皮间质转化中发挥重要作用。更多还原

【Abstract】 Objective:Bladder cancer is a common malignant tumor of urinary system,and its incidence is increasing,which threated people’s health.bThe molecular biology mechanisms of bladder cancer is not entirely clear,therefore,to clarify the molecular mechanism of its development for the early diagnosis and treatment of bladder cancer has important research significance.Silence information regulator（Sirtuin）plays an important role in cell cycle control,maintain the dynamic balance of mitochondria,autophagy,and the process of cell growth regulation,etc.SIRT3 is one of the important members of them,most exists in mitochondrial matrix,a small amount exists in the nucleus.SIRT3 affects intracellular reactive oxygen species（ROS）level and the level of energy metabolism through the regulation of mitochondrial protein acetylation modification.SIRT3 has been identified as the key to adjust the energy balance,the lack of SIRT3 leads to excessive mitochondria acetylation,changes in mitochondrial function,causing the cell energy metabolism,abnormal mitosis,etc.SIRT3,through adjusting the ROS,depresses hypoxia inducing factor alpha 1α（HIF-1α）transcription activity,resistants to oxidative stress,inhibits the growth of tumor.EMT is the process,that the polarity of epithelial cells converted into mesenchymal cells which has the capable of migration and obtained the ability of invasion and migration.E-cadherin is the key factor to the EMT,to maintain the stability of the close connection between normal cells,and the expression level of E-cadherin has negatively related to the occurrence of EMT and the invasion ability of the tumor,transcription factors,such as zinc finger protein Snail,SIP-1,Slug,Twist,which can be cut E-cadherin expression and promotes EMT.Snail and Twist are the main factors of EMT,which has an independent or synergy role in EMT for inducing cancer invasion and metastasis.Studies showed that advanced bladder cancer was closely related to high invasive and EMT.Moreover,the increased expression of HIF-1αthrough EMT enhanced the invasion of tumor.To speculate in bladder cancer,the expression of SIRT3 may affect the expression of EMT related factors to regulate the growth and invasion biology behavior of tumor by adjusting HIF-1α.This experiment using silence and overexpression of SIRT3 in bladder cancer T24cell line,through in vitro experiment,to explore the role of SIRT3 in EMT of bladder cancer,and to provide certain theoretical basis novel targets for the treatment of bladder cancer.Methods:1.The cultivation of the tumor cells:Bladder cancer T24 cell lines growled in RPMI-1640 culture medium with 10%fetal bovine serum,and under the condition of 37℃and 5%CO₂ saturated humidity were sent to culture.2.Immunohistochemical analysis:To collect China Medical University affiliated hospital urologist pathological specimens of bladder cancer patients,HE staining and Immunohistochemical staining,to analyze the expression of SIRT3,HIF-1α,E-cadherin,Slug and Vimentin in bladder cancer tissue and tissue adjacent to carcinoma.And to analyze the relationship between the expression level of SIRT3 and the clinical data and pathological characteristics.3.The construction of plasmid and cell transfection:To design interference targets,lentivirus transfected T24 cells,using RT-PCR and Western blot to detect the efficiency of SIRT3 gene in the transfection of cells.4.Transwell Chambers and Scratch test detected the changes of invasion and migration ability of each group cells before and after transfection.Transwell invasion experiment,to add500μl RPMI-1640 culture medium containing 20%fetal bovine serum to the under room,to add 200μl single cell suspension to the upper room,1×10⁵/hole of cell number,after 24 h,to observe and count the number of cells under the film.Scratch experiment,to vaccinate the 2×10⁵ growth of bladder cancer cells with good condition in inoculated orifice,when cells alignment around 90%,with 200μl liquid moving point to draw a straight line in the interior of the 6 orifices plate on the bottom scratches,after 24 h,using inverted microscope to observe and measure the healing of single cells.5.Protein extraction and Western blot detection:Application in manufacturing total protein extraction kit and BCA protein concentration extract determination of total protein and its concentration groups of bladder cancer cells.Western blot experiments to GAPDH internal antibody to MMP-2,MMP-9,HIF-1α,Slug,Snail,E-cadherin,Vimentin and Twist for a fight,analyze its protein expression.Gel image processing,analysis of gray level.6.Statistical analysis:All the experimental data obtained through at least three times repeated experiments,expressed as mean±standard deviation.The difference between group by t test.Pearson analysis SIRT3 and tumor staging and pathologic types of correlation.The result data,using SPSS17.0 and Graphpad Prism5.0 analysis and drawing,with P<0.05 as was statistically significant.Results:1.SIRT3 in bladder cancer tissue of the expression and clinical significance:（1）Immunohistochemical results showed that the expression of SIRT3and E-cadherin in bladder cancer organizations significantly lower than the tissue adjacent to carcinoma;the level of HIF-1α,Snail,Slug and Vimentin in the bladder cancer group was significantly higher than the tissue adjacent to carcinoma.（2）Correlation analysis results showed that the expression of SIRT3 in T3N0M0group was obviously lower than the TaN0M0 group（P<0.05）;the expression of SIRT3 in high differentiation of bladder cancer tissue was obviously higher than that of poorly differentiated group（P<0.05）;the level of SIRT3 in patients bladder cancer tissues with lymph node metastasis was significantly lower than patients without lymph node metastasis group,the difference was statistical significance（P<0.05）.2.Silence SIRT3 gene in bladder cancer cell invasion and metastasis,and the influence of the transformation of epithelial mesenchymal:（1）The RT-PCR and Western blot detected the efficiency of silence SIRT3 gene,the expression of SIRT3 in pLK0.1-shRNA-SIRT3 T24 bladder cancer cell was significantly lower than NC group and Control group（P<0.05）.Silence SIRT3 gene was successful.（2）The results of Transwell experiment showed that the number of cells in the pLK0.1-shRNA-SIRT3 group through the cell matrix was more,compared with NC group and the Control cells,but there was no statistically significant difference（P>0.05）.（3）The result of the scratch experiment showed,compared with NC group and the Control cells,the migration ability of cells in pLK0.1-shRNA-SIRT3 group was enhanced,and the difference was statistically significant（P<0.05）.（4）According to the results of Western blot,the expression level of MMP-2,MMP-9,HIF-1α,vimentin,twist,and slug in pLK0.1-shRNA-SIRT3 group were higher than that of NC group and the Control group,but there was no statistically significant difference（P>0.05）;the expression level of E-cadherin,the expression in pLK0.1-shRNA-SIRT3 cells group was lower than the NC and the Control group,but the difference was no statistically significant（P>0.05）.3.Overexpression of SIRT3gene in bladder cancer cell invasion and metastasis,and the influence of the epithelial mesenchymal transformation:（1）RT-PCR and Western blot experiments showed that plasmid transfection efficiency of SIRT3 in pCDH-promoter-SIRT3 bladder cancer cells was obviously higher than that of NC group and Control group（P<0.05）.The expression of SIRT3 genes in overexpressed cells was built successfully.（2）Transwell experimental results showed that the number of cells through the matrix in the pCDH-promoter-SIRT3 group was significantly reduced,and the difference was statistically significant（P<0.01）.（3）The scratches experiment results showed that compared with NC group and the Control cells,the migration ability of cells in pCDH-promoter-SIRT3 group was less,and the difference was statistically significant（P<0.05）.（4）According to the results of Western blot,the expression of MMP-2,MMP-9,HIF-1α,vimentin,twist,and slug in pCDH-promoter-SIRT3 group was obviously lower than NC group and the Control group,and the difference was statistically significant（P<0.05）;the expression level of E-cadherin in pCDH-promoter-SIRT3 group was significantly higher than the NC and the Control group,and the difference was statistically significant（P<0.05）.Conclusions:1.The expression of SIRT3 in bladder cancer tissue and tissue adjacent to carcinoma was different;the expression level of SITR3 has significantly correlation with bladder cancer pathological classification,clinical stage;the expression level of SIRT3 was significantly associated with bladder cancer lymph node metastasis.2.Lentivirus carrier can effectively improve the transfection efficiency of shRNA;Lentivirus effectively interfered with the expression of SIRT3 in bladder cancer cell line,successfully built silence and overexpression of SIRT3 genes in bladder cancer cell line.3.The relevant function experimental results indicated that the migration and invasion ability in silence SIRT3 bladder cancer cell were more increased than the blank control group;the migration and invasion ability in overexpression SIRT3bladder cancer cell was decreased significantly.Silenced the expression of SIRT3promoted the expression of related factor with EMT in bladder cancer cells;Overexpression SIRT3 can inhibited the expression related factor with EMT.The experimental results revealed that the expression of SIRT3 plays an important role in invasion and metastasis and transformation of epithelial mesenchymal in bladder cancer.更多还原