PKCζ与Ⅱa型组蛋白去乙酰化酶相互作用调节前列腺癌细胞Warburg效应及其机制

发布时间：2018-09-08 12:42

【摘要】：一、研究背景代谢改变是肿瘤的重要特征之一,其与肿瘤的发生发展密切相关。肿瘤细胞无论在有氧或无氧条件下,均需通过糖酵解(glycolysis)途径吸收葡萄糖产生能量,满足快速生长需求,肿瘤细胞的这一代谢特点是肿瘤细胞具有的普遍现象和规律,被称为Warburg效应。Warburg效应不仅限于糖酵解和三羧酸循环的改变,脂肪酸、谷氨酰胺、丝氨酸、一碳单位等诸多代谢通路在肿瘤细胞均发生了代谢重编程(metabolic reprogramming),而且有关代谢性疾病(如肥胖、糖尿病等)的研究也显示与肿瘤的发生发展密切相关。因此,更进一步研究Warburg效应的机制及其与肿瘤发生发展的关系,不仅有助于揭示肿瘤代谢改变与肿瘤进展的内在关联,而且将为肿瘤的临床诊断寻求高度特异性代谢标志物及以肿瘤代谢为靶向治疗的新策略提供新的视野和契机。肿瘤细胞所表现出的有氧糖酵解(aerobic glycolysis),即Warburg效应使线粒体的氧化磷酸化(oxidative phosphorylation, OXPHOS)途径减弱,而有氧糖酵解及磷酸戊糖(pentose phosphate pathway, PPP)形成核苷酸等代谢途径增强。这种异常的糖代谢转变促使肿瘤细胞具有选择性生长优势,它不仅为快速增殖的肿瘤细胞提供能量(ATP)、生物大分子前体(氨基酸、核苷酸等)以及辅酶(Nicotinamide adenine dinucleotide phosphate NADPH),而且,肿瘤细胞通过Warburg效应形成的肿瘤酸化的微环境有利于肿瘤细胞的生长和浸润转移,此外,肿瘤细胞通过线粒体氧化磷酸化向糖酵解的代谢转变,减少活性氧(reactive oxygen species,ROS)的产生,从而减轻ROS对肿瘤细胞的毒性。正因如此,近年来学者们把肿瘤代谢异常与肿瘤自我增殖能力、凋亡抵抗、无限的复制潜能、对抗生长信号的不敏感性、持续的血管生成能力、组织侵袭转移能力和免疫监控逃逸共同构成了肿瘤新的八大特征性标志。多种因素导致肿瘤细胞Warburg效应的发生,主要有：肿瘤细胞中原癌基因的获得性缺失或突变,抑癌基因的缺失等；肿瘤细胞中糖酵解通路中的关键酶的活性或表达发生改变；线粒体mtDNA突变引起呼吸链功能缺失或氧化磷酸化效率降低；适应低氧微环境,肿瘤细胞中低氧诱导因子高表达,激活下游多个肿瘤代谢尤其是糖酵解相关靶基因及乳酸分泌相关的转运蛋白的表达。尽管Warburg效应是肿瘤最为重要的特征之一,但肿瘤细胞除了通过上述自身基因的突变及关键性的信号通路的激活以适应肿瘤缺氧微环境促进肿瘤的Warburg效应外,与肿瘤发生及进展密切相关的其他调控分子及关键信号通路在肿瘤Warburg效应调控中的作用仍不清楚。PKC属于一类受酪氨酸激酶受体(Receptor Tyrosine kinase,RTK)和G-蛋白偶联受体(G protein coupled receptor,GPCR)激活的丝氨酸／苏氨酸家族的蛋白激酶,包括三个亚族,即Ca2+和DAG依赖的典型PKC(PKC-α,-β,-γ)；DAG-依赖而Ca2+非依赖的PKC(PKC-δ,-ε,-η,-θ)；DAG和Ca2+非依赖的不典型的PKC(PKC-ζ,-ι).PKC家族对细胞生长代谢、分裂增殖、细胞骨架蛋白的重塑等方面有重要作用。PKCζ是PKC家族的其中一个非典型亚型,在整合细胞外信号刺激,参与调控细胞生长、代谢、细胞极性等相关的关键信号传导中起重要作用。有研究证明PKCζ可以促进肿瘤的增殖、侵袭和转移,而且,在葡萄糖供应不足时,PKCζ基因缺失能促进肿瘤细胞代谢重编程。已有的研究表明,由组蛋白去乙酰化酶(HDACs)调控的表观遗传学改变在肿瘤的增殖、迁移、基因组的稳定性、血管新生及肿瘤凋亡中扮演一个重要角色。它们主要由Class Ⅰ,ClassⅡ和ClassⅢ三型HDACs构成。最近,有关HDACs在肿瘤代谢变化中的功能作用开始受到人们的关注,但与肿瘤增殖及进展密切相关的Class Ⅱ HDACs是否参与肿瘤代谢尤其是糖代谢的调控作用至今仍不清楚。二、研究目的本研究首次探讨PKCζ和Ⅱa类HDACs在前列腺癌细胞糖酵解的功能作用及其相互作用对糖代谢和相关基因表达及细胞生长的影响,该研究不仅有助于深入了解PKCζ和Ⅱa类HDACs在前列腺癌癌的生长中的作用和分子机制,而且为进一步发现新的调控肿瘤代谢靶点奠定基础。三、研究方法本研究主要通过过表达(质粒)或干扰(si RNA)的策略探讨PKCζ或Ⅱ aHDACs在前列腺癌细胞中对有氧糖酵解途径中间产物及终产物的调节作用及其对相关蛋白的表达调节作用和分子机制；通过免疫荧光染色、免疫共沉淀等方法证实PKCζ与Ⅱa HDACs在核内的共定位以及相互作用,调节前列腺癌细胞有氧糖酵解进而最终调节肿瘤细胞的生长。四、研究结果1. PKCζ促进前列腺癌细胞DU145的生长及Warburg效应的发生PKCζ的过表达促进前列腺癌DU145细胞的生长及葡萄糖的吸收和乳酸的分泌,相反,敲低前列腺癌细胞内源性PKCζ的表达则明显降低前列腺癌DU145细胞的生长及葡萄糖的吸收和乳酸的分泌。2. PKCζ促进前列腺癌细胞中Warburg效应相关蛋白的表达通过Real time quantitative RT-PCR和Western blot检测显示,PKCζ的过表达促进前列腺癌DU145细胞中糖酵解相关蛋白、葡萄糖及乳酸转运蛋白(HKⅡ、PFKP、MCT4、CD 147)的表达,而敲低前列腺癌细胞内源性PKCζ的表达则明显降低前列腺癌DU145细胞中糖酵解相关蛋白、葡萄糖及乳酸转运蛋白(HKⅡ、PFKP、MCT4、CD 147)的表达。上述结果提示PKCζ可能通过调节前列腺癌细胞中糖酵解相关蛋白的表达而促进Warburg效应的发生及肿瘤细胞生长。3.过表达Ⅱa型HDACs (HDAC4,5,7)降低前列腺癌DU145细胞的生长及葡萄糖吸收和乳酸的分泌有研究证明,HDACs与细胞代谢之间存在着一个反馈循环效应,为了探讨Ⅱa型HDACs (HDAC4,5,7)对肿瘤细胞生长及糖酵解的影响,我们分别在前列腺癌DU145细胞中转染HA-HDAC4,5,7,首先检测其过表达对前列腺癌细胞生长的影响,结果表明,过表达HA-HDAC4,5,7均能明显降低DU145细胞的生长及存活。而敲低内源性HDAC7的表达则明显促进DU145细胞的生长,其次,在敲低内源性HDAC7表达的DU145细胞中添加乳酸转运蛋白的抑制剂a-CHCA则拮抗内源性HDAC7的敲低对细胞生长的促进作用。最后,我们进一步检测Ⅱa型HDACs对葡萄糖的吸收和乳酸分泌的影响,结果表明,过表达HA-HDAC4,5,7能以时间依赖地降低前列腺癌细胞DU145和PC-3M中葡萄糖吸收和乳酸的分泌。提示Ⅱa型HDACs可能通过负性调节糖酵解的发生而抑制肿瘤细胞的生长。4.Ⅱa型HDACs的过表达降低前列腺癌DU145细胞中Warburg效应相关蛋白的表达Real time quantitative RT-PCR显示,前列腺癌DU145细胞中HA-HDAC4,5,7的过表达明显降低前列腺癌细胞中糖酵解相关蛋白、葡萄糖及乳酸转运蛋白(HKⅡ、PFKP、MCT4、CD 147)的表达,而且Western blot更显示HA-HDAC4,5,7的过表达除了明显降低上述糖酵解相关蛋白的表达外,也明显降低糖酵解关键酶(LDHA,PDH)和缺氧诱导因子(HIF-la)的表达。5.PKC⒔与II a HDACs在核内共定位且二者相互作用,并且敲低PKCζ的表达能够明显降低HDAC出核关键性位点的磷酸化水平免疫荧光染色显示内源性PKCζ可与Ⅱa型HDACs的HDAC4,5,7在核内共定位,免疫共沉淀进一步显示HDAC4,5,7均能与PKCζ直接相互作用,此外,敲低PKCζ的表达能够明显降低HDAC出核关键性位点的磷酸化水平。提示PKCζ可能与HDAC4,5,7共同作用,通过调节Ⅱa HDACs的磷酸化出核,从而解除Ⅱa HDACs对糖酵解相关基因表达的抑制作用。6. HDAC7可拮抗PKCζ对DU145细胞生长的促进作用细胞生长检测表明,敲低内源性PKCζ的表达能明显抑制DU145细胞的生长,而敲低HDAC7的表达则明显促进DU145细胞的生长,si-HDAC7和si-PKCζ的共转染进一步显示,敲低内源性HDAC7的表达则可拮抗内源性PKCζ的敲低对DU145细胞生长的抑制作用。五、结论本研究结果表明PKCζ通过与Ⅱ a型HDACs相互作用,调节前列腺癌细胞Warburg效应相关基因的表达及乳酸的分泌,从而促进肿瘤细胞的生长,这一研究将为进一步探讨前列腺癌糖代谢改变与肿瘤生长及进展的内在关联奠定基础,并为前列腺癌的诊断和治疗提供新的潜在靶点。
[Abstract]:1. Background Metabolism is one of the most important characteristics of cancer, which is closely related to the occurrence and development of tumor. Tumor cells absorb glucose to produce energy through glycolysis pathway in both aerobic and anaerobic conditions to meet the needs of rapid growth. The Warburg effect is not only limited to changes in glycolysis and tricarboxylic acid cycles, but also to metabolic reprogramming of fatty acids, glutamine, serine, and mono-carboxylic units in tumor cells. Therefore, further study on the mechanism of Warburg effect and its relationship with tumor development will not only help to reveal the intrinsic relationship between tumor metabolic changes and tumor progression, but also seek highly specific metabolic markers for clinical diagnosis and targeted treatment of tumor metabolism. New therapeutic strategies offer new insights and opportunities. The aerobic glycolysis (Warburg effect) shown by tumor cells weakens the oxidative phosphorylation (OXPHOS) pathway in mitochondria, while the metabolic pathways such as aerobic glycolysis and pentose phosphate pathway (PPP) to form nucleotides increase. Strong. This abnormal glycometabolic transformation promotes the selective growth of tumor cells. It not only provides energy (ATP), biological macromolecular precursors (amino acids, nucleotides, etc.) and coenzymes (Nicotinamide adenine dinucleotide phosphate NADPH) for rapidly proliferating tumor cells, but also forms tumor cells through Warburg effect. Acidified microenvironment is conducive to the growth, invasion and metastasis of tumor cells. In addition, the metabolic transformation of tumor cells from mitochondrial oxidative phosphorylation to glycolysis reduces the production of reactive oxygen species (ROS) and thus reduces the toxicity of ROS to tumor cells. Proliferation, apoptosis resistance, unlimited replication potential, insensitivity to growth signals, persistent angiogenesis, tissue invasion and metastasis, and immune surveillance and escape constitute the eight new characteristic markers of tumor. Acquired deletion or mutation, loss of tumor suppressor genes, changes in the activity or expression of key enzymes in the glycolysis pathway in tumor cells, loss of respiratory chain function or decreased oxidative phosphorylation due to mitochondrial mtDNA mutation, high expression of hypoxia-inducible factors in tumor cells, activation of downstream multiple tumors, and adaptation to hypoxia microenvironment Although Warburg effect is one of the most important characteristics of tumors, tumor cells adapt to the Warburg effect of hypoxic microenvironment by mutation of these genes and activation of key signaling pathways. The role of other regulatory molecules and key signaling pathways closely related to tumorigenesis and progression in the regulation of the Warburg effect is still unclear. PKC belongs to a family of serine/threonine proteins activated by Receptor Tyrosine kinase (RTK) and G-protein coupled receptor (GPCR). Kinases, including three subgroups, namely Ca2+ and DAG-dependent typical PKC (PKC-a, -beta, -gamma); DAG-dependent but Ca2+ independent PKC (PKC-delta, -e, -_, -theta); DAG and Ca2+ independent atypical PKC (PKC-_, -_). PKC family plays an important role in cell growth and metabolism, mitosis and proliferation, cytoskeleton protein remodeling. One of the atypical subtypes plays an important role in integrating extracellular signal stimuli and regulating key signaling pathways related to cell growth, metabolism and cell polarity. Programming. Previous studies have shown that epigenetic changes regulated by histone deacetylases (HDACs) play an important role in tumor proliferation, migration, genome stability, angiogenesis and tumor apoptosis. They are mainly composed of Class I, Class II and Class III HDACs. Recently, HDACs have been involved in tumor metabolism. However, it is still unclear whether Class II HDACs, which are closely related to tumor proliferation and progression, are involved in the regulation of tumor metabolism, especially glucose metabolism. This study not only helps to understand the role and molecular mechanism of PKC_and Class II a HDACs in the growth of prostate cancer, but also lays a foundation for further discovery of new targets for regulating tumor metabolism. 3. Research methods This study mainly through overexpression (plasmid) or interference. (si RNA) strategy to investigate the role of PKC_or II a HDACs in regulating the expression of intermediate and end products of aerobic glycolysis pathway and their molecular mechanisms in prostate cancer cells; the co-location and interaction of PKC_and II a HDACs in the nucleus were confirmed by immunofluorescence staining and immunoprecipitation PKC promotes the growth of prostate cancer cell DU145 and Warburg effect. Overexpression of PKC promotes the growth of prostate cancer cell DU145 and glucose uptake and lactic acid secretion. On the contrary, it knocks down prostate cancer cells. The expression of PKC_significantly decreased the growth, glucose uptake and lactic acid secretion of prostate cancer DU145 cells. 2. PKC_promoted the expression of Warburg effect-related proteins in prostate cancer cells. Real-time quantitative RT-PCR and Western blot analysis showed that the over-expression of PKC_promoted the glycolysis of prostate cancer DU145 cells. The expression of related proteins, glucose and lactate transporters (HK II, PFKP, MCT4, CD 147) was significantly decreased by knocking down the expression of endogenous PKC_in prostate cancer DU145 cells, while the expression of glucose and lactate transporters (HK II, PFKP, MCT4, CD 147) was significantly decreased by knocking down the expression of endogenous PKC_. Overexpression of type II a HDACs (HDAC4,5,7) reduces the growth, glucose uptake and lactic acid secretion of prostate cancer DU145 cells. Studies have shown that there is a feedback loop between HDACs and cell metabolism. The effects of type II a HDACs (HDAC4,5,7) on the growth and glycolysis of tumor cells were studied. HA-HDAC4,5,7 was transfected into prostate cancer DU145 cells. The results showed that overexpression of HA-HDAC4,5,7 significantly decreased the growth and survival of DU145 cells. In addition, lactate transporter inhibitor a-CHCA was added to knock down endogenous HDAC7 expression DU145 cells to antagonize the growth-promoting effect of endogenous HDAC7 knockdown. Finally, we further examined the effects of type II a HDACs on glucose uptake and lactate secretion. Expression of HA-HDAC4,5,7 in prostate cancer cells DU145 and PC-3M decreased glucose uptake and lactic acid secretion in a time-dependent manner, suggesting that type II a HDACs may inhibit tumor cell growth by negatively regulating glycolysis. 4. Overexpression of type II a HDACs decreased the expression of Warburg effect-related proteins in prostate cancer DU145 cells. L-time quantitative RT-PCR showed that the overexpression of HA-HDAC4,5,7 in prostate cancer DU145 cells significantly decreased the expression of glycolysis-related proteins, glucose and lactate transporters (HKII, PFKP, MCT4, CD 147) in prostate cancer DU145 cells, and Western blot showed that the overexpression of HA-HDAC4,5,7 in addition to significantly reducing the above-mentioned glycolysis-related proteins. PKC_and II a HDACs were co-localized in the nucleus and interacted with each other, and knocking down the expression of PKC_could significantly reduce the phosphorylation level of the nucleus key sites of HDAC. Immunofluorescence staining showed that endogenous PKC_could be associated with type II a HDACs. HDAC4,5,7 were co-localized in the nucleus. Immunocoprecipitation further showed that HDAC4,5,7 could interact directly with PKC. In addition, knocking down the expression of PKC could significantly reduce the phosphorylation level of the key sites of HDAC exocytosis. Inhibitory effect of HDAC7 on the expression of glycolysis-related genes.6.HDAC7 could antagonize the growth-promoting effect of PKC_on DU145 cells.The results showed that knocking down the expression of endogenous PKC_significantly inhibited the growth of DU145 cells, while knocking down the expression of HDAC7 significantly promoted the growth of DU145 cells. Tapping down the expression of endogenous HDAC7 may antagonize the inhibition of endogenous PKC_on the growth of DU145 cells. 5. Conclusion PKC_can regulate the expression of Warburg-related genes and the secretion of lactic acid in prostate cancer cells by interacting with type II a HDACs. This study will promote the growth of tumor cells. It lays a foundation for further study of the relationship between the changes of glucose metabolism and the growth and progression of prostate cancer, and provides a new potential target for the diagnosis and treatment of prostate cancer.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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