【作者】 王新君；

【导师】 赵晓昆；

【作者基本信息】 中南大学 ， 临床医学， 2014， 博士

【摘要】 背景：膀胱癌是人类泌尿系统最常见的恶性肿瘤,其发病率和死亡率均占泌尿系肿瘤首位,其中约90%为尿路上皮癌。近十几年来,男性和女性的膀胱癌发病率均呈上升趋势。虽经多年研究,膀胱癌的发生发展的机制目前仍不明确,因此研究膀胱癌发生、发展的分子机制对于膀胱癌的预防及治疗具有重要意义。肿瘤的特征性代谢改变对其生长和增殖具有重要作用。丙酮酸激酶M2亚型(pyruvate kinase M2isoform,PKM2)催化糖酵解最后一步反应,在多种肿瘤中高表达,且其活性受到多种代谢中间产物和细胞因子等的调控,是肿瘤细胞有氧糖酵解(Warburg效应)的关键调节因子。肿瘤可迅速生成能量并累积糖酵解中间代谢产物用于合成代谢,促进肿瘤的生长和增殖。PKM2在肿瘤代谢和生长中的特殊作用,使其成为肿瘤治疗的可能靶点,对PKM2在膀胱癌中的表达和功能进行研究将有助于阐明相关机制和并具有潜在的应用前景。目的：(1)检测PKM2在膀胱癌组织及细胞株中的表达情况,分析PKM2的表达和膀胱癌临床病理特征之间的关系。(2)构建慢病毒介导的RNA干扰以特异性的敲低PKM2基因,并经药物筛选构建稳定敲低PKM2的膀胱癌细胞株。(3)通过体外试验和体内实验研究PKM2基因敲低对膀胱癌细胞株糖代谢和生物学行为的变化。(4)探索PKM2影响人膀胱癌侵袭、转移的可能机制。方法：(1)收集36例膀胱癌组织及其配对的癌旁组织,采用Western-blot和实时荧光定量-PCR (Quantitative real-time polymerase chain reaction, Real-time PCR)及免疫组织化学的方法检测PKM2在膀胱癌组织、配对癌旁组织及膀胱癌细胞株中的表达水平,并分析其表达水平与膀胱癌临床病理特征间的关系。(2)构建靶向PKM2基因的pLV-PKM2-shRNA表达载体,包装病毒后感染膀胱癌细胞株,采用Western-blot和Real-time PCR检测shRNA干扰序列对PKM2表达的影响,筛选最优的shRNA序列进入后续试验。(3)通过药物筛选构建稳定敲低PKM2的膀胱癌细胞株。通过葡糖糖、乳酸测定、CCK8法、流式细胞术、划痕实验、Transwell试验等方法研究PKM2基因敲低对膀胱癌细胞糖代谢、增殖、凋亡、迁移、侵袭等的影响。通过裸鼠成瘤实验观察敲低PKM2对膀胱癌细胞株活体成瘤能力的影响。通过构建膀胱癌原位裸鼠模型和活体小动物发光实验观察PKM2基因敲低对膀胱癌成瘤能力及转移的影响。(4)采用Western-blot和Real-time PCR检测敲低PKM2后上皮间质转化相关标志物的表达变化,探讨PKM2影响膀胱癌细胞侵袭、转移的可能机制。结果：(1)PKM2蛋白和mRNA的表达水平在膀胱癌组织和细胞株中明显增高。PKM2的表达水平与组织学分级和TNM分期正相关。(2)成功构建了3条pLV-PKM2-shRNA和1条pLV-control-shRNA稳定表达的质粒。构建的3条PKM2-shRNA干扰序列均可显著敲低T24细胞PKM2蛋白和mRNA水平的表达,其中PKM2-shRNA-1的干扰效果最显著。(3)体外实验结果显示,敲低PKM2使T24细胞的葡萄糖消耗和乳酸生成降低、增值、凋亡、迁移和转移能力下降,ROS生成增加,对顺铂的敏感性增加。裸鼠成瘤实验提示敲低PKM2后T24细胞的成瘤重量和成瘤速度降低。原位膀胱成瘤模型提示敲低PKM2后UMUC-3细胞成瘤能力降低,转移灶的数量减少。(4)敲低PKM2后T24的E-cadherin表达上调,而Vimentin、Fibronectin表达下调。结论：(1)PKM2在膀胱癌组织和细胞中高表达,其表达与膀胱癌的组织学分级和TNM分期正相关,PKM2可能在膀胱癌的发生、发展中起重要作用。(2)体内实验和体外实验表明,PKM2基因敲低可抑制膀胱癌细胞的有氧糖酵解、增殖、迁移、侵袭和成瘤能力,并促进ROS生成和凋亡。(3)PKM2可能通过影响上皮间质转化促进膀胱癌的侵袭和转移。图26幅,表27个,参考文献82篇。更多还原

【Abstract】 Background:Bladder cancer is one of the most common malignant tumors in urogenital system, of which bladder urothelial carcinoma accounts for90%approximately.The incidence of bladder cancer is in an upward trend in the past decades. Though studied extensively, the molecular mechanism for the growth and spreading in bladder cancer remains unclear.Altered metabolism is fundamental to the growth and survival of cancer cells. The M2splice isoform of pyruvate kinase is a key regulator of Warburg effect in cancer cells.Most cancer cells has increased expression of PKM2, which positions PKM2as an attractive target for cancer therapy. However, until now there has been little information about the expression and function of PKM2in bladder cancer.Objectives:(1) To investigate the expression of PKM2in human bladder cancer tissues and cell lines with regard to related clinicopathologic significance.(2) To construst a lentivirus vector-based RNA interference of PKM2and verify the effect of RNAi vectors.(3) To analyze the alterations of glucose metabolism and biological behaviors upon the knockdown of PKM2in bladder cell lines.(4) To explore the potential role of PKM2in the spreading.of bladder cancerMethods:(1) Thirty six cases of bladder cancer tissues and matched normal adjacent tissues were collected. The expression of PKM2was detected by immunohistochemistry, Western-blot and quantitative real time polymerase chain reaction (Real-time PCR).The relationship between clinicopathologic parameters and PKM2expression was analyzed.(2) The pLV lentivirus vector with PKM2shRNA was constructed. Western-blot and real time-PCR were used to measure the interference effects of RNAi on PKM2expression.(3) The effects of PKM2knockdown on T24cell proliferation, apoptosis, ROS,migration, invasion and spreading were assessed by CCK8assay, transwell assay, flow cytometry and nude mouse xenografts and orthotopic model.(4) Western-blot and Real-time PCR were performed to measure the expression of E-cadherin,vimentin and fibronectin after the down-regulation of PKM2.Results:(1) Bladder cancer tissues and cell lines have increased expression of PKM2compared with matched normal adjacent tissue and normal bladder cell line respectively. The expression of PKM2correlate with stage and grade of bladder cancer.(2)Three PKM2shRNA and one control shRNA with pLV lentivirus vector were constructed. All the PKM2shRNA vector significantly downregulate the expression of PKM2, of which PKM2shRNA1performs best. And the control shRNA show no effects on the expression of PKM2.(3) Knockdown of PKM2decreases the glucose consumption and lactate production and suppress cell proliferation, migration, invasion while increase the ROS level and apoptosis in T24bladder cancer cells in vitro. Knockdown of PKM2leads to smaller xenografts tumor volume in nude mouse models.And in the Orthotopic models, knockdown of PKM2lead to smaller tumor volume as well as lesser metastastis formation.(4) Knockdown of PKM2decreases the expression of vimentin and fibronectin while increases E-cadherin expression.Conclusions:(1) PKM2protein and mRNA have a significantly high expression in bladder tissues and cell lines with positive corelation with tumor grade and stage. PKM2may be involved in the occurence and progression of bladder cancer.(2) Knockdown of PKM2impair the glucose metabolism and suppress cell proliferation, migration, invasion and spreading in vitro and in vivo and lead to the ROS accumulation and apoptosis. PKM2may serve as a potential therapeutic target for the treatment of bladder cancer.(3) Knockdown of PKM2decrease the expression of vimentin and fibronectin while increase the expression of E-cadherin,indicating that PKM2may play a role in the epithelial-mesenchymal transition.PKM2may promote invasion and metastasis via inducing the EMT process in bladder cancer. Figures:26; tables:27; references:85.更多还原