【摘要】：第一部分上調表達PCK1對黑色素瘤再生細胞糖酵解側枝反應的促進作用研究目的：腫瘤干細胞在腫瘤的發(fā)生、發(fā)展和治療后復發(fā)過程中發(fā)揮重要作用。關于腫瘤細胞代謝的研究主要集中在混合細胞群體的層面,缺乏特異性針對腫瘤干細胞代謝特征及其分子調控機制的研究。本研究采用三維軟纖維蛋白基質膠(3D fibrin)分離篩選的腫瘤干細胞(定義為腫瘤再生細胞,TRCs)為模型,探究糖異生的關鍵酶之一的胞漿型磷酸烯醇式丙酮酸羧激酶(PCK1)在黑色素瘤再生細胞中的表達及其發(fā)揮的代謝功能。方法：(1)以3D fibrin分離小鼠黑色素瘤B16細胞、肝癌H22細胞、淋巴瘤EL4細胞中的TRCs,然后使用RT-PCR、Real time PCR和Western blot檢測這些TRCs和相應對照細胞以及小鼠未分化的間充質干細胞(mMSCs)和胚胎干細胞(mESCs)中PCK1的表達。(2)使用癌癥基因數據庫cbioportal中的基因表達數據分析PCK1與CD133、ALDH1A1和ABCG5等常用腫瘤干細胞標志在多細胞中的共表達關系；并采用Real time PCR檢測PCK1在CD133+與CD133-B16細胞亞群中的表達差異。(3)免疫組化檢測PCK1在9例人黑色素瘤臨床標本中的表達；分離人原代黑色素瘤新鮮標本中的TRCs, real time PCR檢測PCK 1的表達情況。(4) RT-PCR和Western blot檢測B16TRCs中糖異生的另外兩個關鍵酶FBP1和G6Pase的表達情況,以探討PCK1是否介導糖異生的反應。(5)以siRNA沉默PCK1的表達后,觀察B16 TRCs體外生長、葡萄糖消耗、乳酸釋放以及中間代謝產物(如絲氨酸／甘氨酸,三磷酸甘油)等的變化,以探究PCK1發(fā)揮的代謝功能。(6)以siRNA沉默B16或H22 TRCs中PCK1的表達,觀察其在動物體內成瘤能力。(7)以siRNA沉默B16 TRCs中PCK1的表達后,補充相關中間代謝產物,以觀察對克隆生長的影響。(8)觀察PCK1過表達對B16 TRCs體外生長的影響。(9)構建PCK1啟動子控制的EGFP熒光表達B16細胞,分離出該細胞中的TRCs并置于普通硬質環(huán)境下連續(xù)培養(yǎng),采用熒光顯微鏡動態(tài)觀察熒光變化；將B16 TRCs置于普通硬質環(huán)境下連續(xù)培養(yǎng),以RT-PCR、Real time PCR和Western blot檢測PCK1的表達變化。(10)以小分子抑制劑或封閉抗體阻斷B16 TRCs中相關信號通路位點后以Real time PCR檢測PCK1的表達,初步探究PCK 1的表達調控機制。(11)亞硫酸氫鹽測序法檢測B16 TRCs中PCK1啟動子區(qū)的甲基化水平,探討DNA甲基化對PCK1表達調控的影響。結果：(1)B16細胞、H22細胞、EL4細胞TRCs相對于分化的腫瘤細胞上調表達PCK1,未分化的小鼠間充質干細胞和胚胎干細胞高表達PCK1。(2)PCK1與常用腫瘤干細胞標志CD133、ALDH1A1和ABCG5在多細胞中的表達呈正相關；PCK1在CD133+B16亞群細胞中的表達高于CD133-亞群細胞。(3)約1/3的黑色素瘤臨床標本中檢測到PCK1的高表達,分離自人原代黑色素瘤的TRCs上調表達PCK1。(4)B16 TRCs不表達糖異生的另外兩個關鍵酶FBP1和G6Pase。(5) siRNA沉默PCK1表達后,B16 TRCs體外生長明顯受抑,葡萄糖消耗和乳酸釋放減少,甘氨酸和三磷酸甘油水平下降。(6) siRNA沉默PCK1表達后,B16和H22 TRCs的小鼠體內成瘤能力均減弱。(7)補充甘氨酸和(或)三磷酸甘油可部分逆轉PCK1沉默后B16 TRCs受抑制的生長。(8)PCK1過表達可促進B16 TRCs的體外生長。(9)硬基質環(huán)境培養(yǎng)可誘導B16 TRCs中PCK1的下調表達。(10)抑制整合素αVβ3或P13K信號可誘導B16 TRCs中PCK1的表達下調,H3K9去甲基化可導致PCK1表達增加。(11)B16 TRCs和對照細胞中PCK1啟動子均呈高度甲基化。結論：黑色素瘤再生細胞相對于分化的腫瘤細胞上調表達PCK1,因缺乏FBP1和G6Pase的表達,PCK1在腫瘤再生細胞中并不介導完整的糖異生,而是通過促進糖酵解的側枝反應以增強腫瘤再生細胞中間代謝產物的合成。干擾PCK1的表達可以抑制黑色素瘤再生細胞的體外生長和體內成瘤能力。這些研究揭示PCK1上調表達是黑色素瘤再生細胞重要的代謝標志之一,有可能作為黑色素瘤治療的一個新的靶點。第二部分PCK1、PCK2在乳腺癌腫瘤再生細胞中的表達研究目的：探究PCK1、PCK2在乳腺癌腫瘤再生細胞(TRCs)中的表達情況。方法：(1)采用三維軟纖維蛋白基質膠(3D Fibrin)分離篩選MCF-7、T47D以及MDA-MB-231等人乳腺癌細胞株中的腫瘤再生細胞(TRCs),以RT-PCR檢測PCK1在這些TRCs以及對照細胞中的基因表達。(2)以Western blot檢測PCK1, PCK2在MCF-7、T47D、MDA-MB-231等來源TRCs以及相應的對照細胞中的表達變化。結果：(1)在MCF-7、T47D、MDA-MB-231等乳腺癌來源的TRCs和對照細胞中檢測不到PCK1的基因表達。(2) MCF-7、T47D、MDA-MB-231等乳腺癌來源的TRCs相對于對照細胞上調表達PCK2。結論：乳腺癌再生細胞不表達PCK1而是上調表達其線粒體型的同工酶PCK2。PCK2上調表達可能是乳腺癌再生細胞重要的代謝特征之一,可為設計特異性針對乳腺癌腫瘤干細胞的抑制策略提供靶點。然而,關于PCK2的具體代謝功能還有待于進一步闡明。第三部分 PCK2偶聯(lián)谷氨酰胺代謝途徑調控乳腺癌腫瘤再生細胞的增殖研究目的：以乳腺癌MCF-7細胞為模型,探究丙酮酸羧化酶(PC)對PCK2上調表達的腫瘤再生細胞(TRCs)的生物學意義,并從葡萄糖和谷氨酰胺代謝偶聯(lián)的角度對乳腺癌TRCs的細胞代謝特征進行新的詮釋和探索。同時,我們嘗試通過聯(lián)合干擾PCK2和PC或谷氨酰胺裂解途徑來抑制TRCs的腫瘤治療新策略,為進一步研究腫瘤再生細胞的脂質代謝打下基礎。方法：(1)使用三維軟纖維蛋白基質膠篩選分離乳腺癌MCF-7細胞中的TRCs,以酶學方法和高效液相色譜分別檢測MCF-7 TRCs和對照細胞葡萄糖和谷氨酰胺的利用效率,探究葡萄糖代謝與谷氨酰胺代謝的偶聯(lián)。(2)觀察無谷氨酰胺培養(yǎng)基培養(yǎng)的MCF-7 TRCs體外生長情況以研究TRCs對谷氨酰胺的依賴程度。(3)以Western blot檢測丙酮酸羧化酶(PC)、谷氨酰胺轉運蛋白(SLC1A5)、谷氨酰胺裂解酶(GLS)以及異檸檬酸脫氫酶2(IDH2)在MCF-7 TRCs和普通MCF-7細胞中的表達。(4)分別以siRNA聯(lián)合干擾PCK2與PC、PCK2與GLS、PCK2與IDH2在MCF-7 TRCs中的表達,觀察并統(tǒng)訓MCF-7 TRCs克隆的體外生長情況。結果：(1)MCF-7 TRCs目對于普通MCF-7細胞消耗更多的葡萄糖和谷氨酰胺。(2)當剝奪了培養(yǎng)基中的谷氨酰胺,MCF-7 TRCs的克隆大小和數量急劇下降。(3)MCF-7 TRCs相對于對照細胞除上調表達PCK2外,還同時上調表達PC、SLC1A5和IDH2。(4)聯(lián)合干擾PCK2與PC、PCK2與GLS或PCK2與IDH2較沉默單一位點對TRCs的抑制效果更明顯。結論：在PCK2上調表達的乳腺癌MCF-7 TRCs中,為了補充線粒體內草酰乙酸的消耗以維持檸檬酸的合成,TRCs通過上調表達丙酮酸羧化酶以增加丙酮酸生成草酰乙酸的量。另外,TRCs還可通過增加谷氨酰胺的代謝來直接補充檸檬酸。通過PCK2上調可能介導的甘油骨架的合成增多以及谷氨酰胺裂解途徑增強的脂肪酸代謝,TRCs可合成更多的脂類物質以滿足其旺盛的代謝需求。聯(lián)合靶向PCK2和PC或谷氨酰胺裂解途徑可能是非常有潛在應用價值的針對腫瘤再生細胞的乳腺癌治療新策略。
[Abstract]:Part 1 Upregulation of PCK1 expression in melanoma regenerating cells promotes glycolytic collateral reaction. Objective: Tumor stem cells play an important role in the occurrence, development and recurrence of melanoma. In this study, three-dimensional soft fibrin matrix glue (3D fibrin) was used to isolate and screen tumor stem cells (defined as tumor regeneration cells, TRCs) as a model to investigate the expression of cytoplasmic phosphoenolpyruvate carboxykinase 1 (PCK1), one of the key enzymes in glyconeogenesis, in melanoma regeneration cells. Methods: (1) TRCs were isolated from murine melanoma B16 cells, hepatoma H22 cells and lymphoma EL4 cells by 3D fibrin, and then detected by RT-PCR, Real-time PCR and Western blot for the expression of P in these TRCs and the corresponding control cells, as well as the undifferentiated mesenchymal stem cells (mMSCs) and embryonic stem cells (mESCs). CK1 expression. (2) The co-expression of PCK1 with CD133, ALDH1A1 and ABCG5 was analyzed by gene expression data from cbioportal, a cancer gene database. The expression of PCK1 in CD133 + and CD133-B16 cell subsets was detected by Real-time PCR. (3) PCK1 was detected by immunohistochemistry in 9 Black patients. The expression of the other two key enzymes FBP1 and G6Pase in B16TRCs was detected by RT-PCR and Western blot to investigate whether PCK1 mediated glyconeogenesis. (5) Silencing PCK1 with siRNA. After expression, the growth of B16 TRCs in vitro, glucose consumption, lactic acid release and intermediate metabolites (such as serine/glycine, glycerol triphosphate) were observed to explore the metabolic function of PCK1. (6) The expression of PCK1 in B16 or H22 TRCs was silenced by siRNA, and the tumorigenic ability of PCK1 in B16 TRCs was observed. (7) The expression of PCK1 in B16 TRCs was silenced by siRNA. (8) To observe the effect of overexpression of PCK1 on the growth of B16 TRCs in vitro. (9) To construct EGFP fluorescent expression B16 cells controlled by PCK1 promoter, TRCs were isolated from the cells and cultured continuously in normal hard environment. Fluorescence microscopy was used to dynamically observe the fluorescence. The expression of PCK1 was detected by RT-PCR, Real-time PCR and Western blot. (10) The expression of PCK1 was detected by Real-time PCR after blocking the signal pathway sites of B16 TRCs with small molecular inhibitors or blocking antibodies. Results: (1) The expression of PCK1 was up-regulated in B16 cells, H22 cells and EL4 cells compared with differentiated tumor cells, and the expression of PCK1 was up-regulated in undifferentiated mouse mesenchymal stem cells and embryonic stem cells. (2) PCK1 was over-expressed in undifferentiated mouse mesenchymal stem cells and embryonic stem cells. The expression of CD133, ALDH1A1 and ABCG5 was positively correlated in multicellular cells, and the expression of PCK1 in CD133+B16 subgroup was higher than that in CD133-subgroup. (3) The overexpression of PCK1 was detected in about one third of melanoma clinical specimens, and the expression of PCK1 was up-regulated in TRCs isolated from human primary melanoma. After the key enzymes FBP1 and G6Pase. (5) siRNA silenced the expression of PCK1, the growth of B16 TRCs was significantly inhibited, glucose consumption and lactic acid release were reduced, and the levels of glycine and triphosphate were decreased. (6) After the expression of PCK1 was silenced by siRNA, the tumorigenic ability of B16 and H22 TRCs in mice was weakened. (7) Glycine supplementation and/or triphosphate glycerol partially reversed P After CK1 silencing, the growth of B16 TRCs was inhibited. (8) Overexpression of PCK1 promoted the growth of B16 TRCs in vitro. (9) Hard substrate culture could induce the down-regulation of PCK1 expression in B16 TRCs. (10) Inhibition of integrin alpha V beta 3 or P13K signal could induce the down-regulation of PCK1 expression in B16 TRCs, and H3K9 demethylation could induce the increase of PCK1 expression in B16 TRCs and control cells. Conclusion: Compared with differentiated tumor cells, melanoma regenerated cells up-regulate the expression of PCK1. Due to the lack of FBP1 and G6Pase expression, PCK1 does not mediate complete Glyconeogenesis in tumor regenerated cells, but enhances the intermediate metabolites in tumor regenerated cells by promoting the lateral branching reaction of glycolysis. Synthesis. Interference with the expression of PCK1 inhibits the growth of melanoma regenerating cells in vitro and in vivo tumorigenicity. These studies suggest that the up-regulation of PCK1 is one of the important metabolic markers of melanoma regenerating cells and may be a new target for melanoma treatment. Part II PCK1, PCK2 in breast cancer regenerating cells Objective: To investigate the expression of PCK1 and PCK2 in tumor regeneration cells (TRCs) of breast cancer. Methods: (1) The tumor regeneration cells (TRCs) of human breast cancer cell lines MCF-7, T47D and MDA-MB-231 were isolated and screened by three-dimensional soft fibrin matrix glue (3D Fibrin), and detected by RT-PCR. The expression of PCK1 and PCK2 was detected by Western blot. Results: (1) The expression of PCK1 was not detected in the TRCs from MCF-7, T47D, MDA-MB-231 and the control cells. (2) The expression of PCK1 was not detected in the TRCs from MCF-7, T47D, MDA-MB-231 and other breast cancer sources. CONCLUSION: The up-regulation of PCK2 expression in breast cancer regeneration cells may be one of the important metabolic characteristics of breast cancer regeneration cells, and may provide a target for designing specific inhibitory strategies against breast cancer stem cells. The specific metabolic functions of PCK2-glutamine pathway in regulating the proliferation of breast cancer regeneration cells are to be further elucidated. Part III. Objective: To investigate the biological significance of pyruvate carboxylase (PC) on PCK2-up-regulated tumor regeneration cells (TRCs) using breast cancer MCF-7 cells as a model, and from glucose and glucose. Glutamine metabolic coupling provides a new interpretation and exploration of the cellular metabolic characteristics of TRCs in breast cancer. At the same time, we attempted to inhibit TRCs by interfering with PCK2 and PC or glutamine cleavage pathways in combination to lay a foundation for further study of lipid metabolism in tumor regenerated cells. TRCs isolated from breast cancer MCF-7 cells were screened by soft fibrin matrix glue. The utilization efficiency of glucose and glutamine in MCF-7 TRCs and control cells were detected by enzyme method and high performance liquid chromatography respectively. The coupling of glucose metabolism and glutamine metabolism was explored. (2) The growth of MCF-7 TRCs cultured in glutamine-free medium was observed in vitro. To study the dependence of TRCs on glutamine. (3) The expressions of pyruvate carboxylase (PC), glutamine transporter (SLC1A5), glutamine lyase (GLS) and isocitrate dehydrogenase 2 (IDH2) in MCF-7 TRCs and MCF-7 cells were detected by Western blot. (4) The expressions of PCK2 and PC, PCK2 and GLS, PCK2 and IDH2 were interfered with by siRNA, respectively. Results: (1) MCF-7 TRCs consumed more glucose and glutamine for normal MCF-7 cells. (2) When glutamine was deprived of the culture medium, the clonal size and number of MCF-7 TRCs decreased sharply. (3) MCF-7 TRCs increased the expression of glucose and glutamine in normal MCF-7 cells. In addition to PCK2, the expression of PC, SLC1A5 and IDH2 was up-regulated at the same time. (4) Combined interference of PCK2 with PC, PCK2 with GLS or PCK2 with IDH2 was more effective than silencing a single site on TRCs. CONCLUSION: In breast cancer MCF-7 TRCs up-regulated by PCK2, TRCs can maintain citric acid synthesis by supplementing the consumption of oxaloacetic acid in mitochondria. In addition, TRCs can directly supplement citric acid by increasing the metabolism of glutamine. By up-regulating the synthesis of Glycerol Skeleton and the metabolism of fatty acids by the cleavage pathway of glutamine, TRCs can synthesize more lipids to meet their needs. Strong metabolic demands. Targeting PCK2 and PC or glutamine cleavage pathways together may be a promising new strategy for breast cancer treatment targeting tumor regeneration cells.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R730.2
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本文編號：2202206