线粒体型磷酸烯醇式丙酮酸羧激酶在黑色素瘤肿瘤再生细胞中的代谢研究

发布时间：2019-05-27 10:28

【摘要】：目的：肿瘤干细胞的存在让肿瘤更具侵袭性、耐药性以及转移性。目前关于肿瘤代谢的研究集中在混合细胞群体的层面,缺乏针对肿瘤干细胞代谢特征以及其分子调控机制的研究。本研究采用三维软蛋白基质胶(3D Fibrin)培养获取的肿瘤再生细胞(TRCs)为模型,探究线粒体型磷酸烯醇式丙酮酸(PCK2)的表达变化对肿瘤干细胞代谢的影响。方法：(1)RT-PCR和Western blot检测比较小鼠黑色瘤细胞B16-F1、小鼠肝癌细胞H22、人喉癌细胞Hep-2以及人黑色素瘤细胞A875培养获得的TRCs和相应对照细胞中PCK2的表达. RT-PCR和Western blot检测比较小鼠胚胎干细胞(mESCs)在分化与未分化状态下PCK2的表达。流式分选小鼠黑色素瘤细胞B1-F1中CD133+细胞亚群,RT-PCR检测比较CD133+细胞亚群中PCK2的表达。收集两例黑色素瘤临床肿瘤病人标本,分离肿瘤细胞培养获取TRCs, RT-PCR检测两例病人标本TRCs中PCK2的表达。(2)检索TCGA数据库并分析PCK2的表达与肿瘤病人生存期是否相关。(3)构建PCK2过表达质粒载体,转染B16-F1TRCs,6小时后再将其尾静脉接种小鼠,24天后观察小鼠肺部成瘤情况；B16-F1细胞转染PCK2 siRNA,6小时后再将其尾静脉接种小鼠,24天后观察小鼠肺部成瘤情况：构建PCK2 Tet-On细胞株,将其种入3D fibrin胶中,在添加或不添加多西环素的情况下,比较克隆大小和数目,并比较TRCs对顺铂的敏感性。将PCK2 Tet-On细胞株,接种到小鼠皮下,分别用添加或不添加多西环素(Dox)的水喂养,比较该细胞在小鼠皮下的成瘤能力。(4)分析比较B16-F1 TRCs和B16-F1葡萄糖消耗以及乳酸释放量；过表达PCK2,比较B16-F1 TRCs葡萄糖消耗和乳酸释放量；比较B16-F1 TRCs和对照细胞TCA循环中间物柠檬酸的含量；过表达PCK2,检测B16-F1 TRCs柠檬酸的含量变化；siRNA沉默异柠檬酸脱氢酶IDH3A以及苹果酸脱氢酶MDH2,观察B16-F1 TRCs和对照细胞的生长情况并检测其葡萄糖消耗；siRNA沉默柠檬酸转运蛋白SLC25A1、柠檬酸裂解酶ACLY、脂肪酸合酶FASN以及苹果酸酶ME1,检测B16-F1 TRCs葡萄糖消耗；比较B16-F1 TRCs和对照细胞TCA循环代谢中间产物延胡索酸的含量；过表PCK2,检测B16-F1 TRCs中延胡索酸含量变化；Western blot检测B16-F1 TRCs和对照细胞中HIF1α和HIF2α的表达；调控PCK2表达后,Western blot检测B16-F1 TRCs中HIF1α和HIF2α的表达；用质谱检测比较B16-F1 TRCs和对照细胞中DNA的5羟甲基胞嘧啶和5甲基胞嘧啶的含量：过表达PCK2,检测B16-F1 TRCs DNA中5羟甲基胞嘧啶和5甲基胞嘧啶的含量变化；Seahorse XF24设备检测比较B16-F1 TRCs和对照细胞的耗氧量水平；过表达PCK2后,检测B16-F1 TRCs耗氧量变化：不同浓度的电子传递链复合物I抑制剂(Rotenone)、复合物V抑制剂寡霉素(Oligomycin)分别处理B16-F1 TRCs和对照细胞,用CCK-8试剂盒检测比较细胞增殖情况,并检测葡萄糖消耗量。结果：(1)B16-F1细胞、H22细胞、Hep-2细胞和A875细胞TRCs相对于普通肿瘤细胞,PCK2呈下调表达。mESC处于未分化状态时低表达PCK2。PCK2在B16-F1 CD133+细胞亚群中的表达低于CD133细胞亚群。两例黑色瘤病人标本TRCs中PCK2呈低表达。(2)PCK2的表达与黑色素瘤、肺癌和胃癌病人的总生存期相关。(3)B16-F1 TRCs过表达PCK2后,在小鼠肺部形成转移斑点数目减少；B16-F1转染siRNA沉默PCK2后,在小鼠肺部形成转移斑点数目增多；将PCK2 Tet-On稳定细胞株种入3D fibrin中,在添加多西环素培养下,形成克隆的体积变小、数目减少,并且对顺铂敏感性变差。PCK2 Tet-On稳定细胞株接种小鼠皮下,多西环素喂养组小鼠成瘤能力减弱。(4)与B16-F1相比,B16-F1TRCs葡萄糖消耗增多,乳酸释放量增多；过表达PCK2后,B16-F1 TRCs葡萄糖消耗减少,乳酸产生量降低；B16-F1 TRCs柠檬酸的含量增高；过表达PCK2后,B16-F1 TRCs中柠檬酸的含量减少；B16-F1 TRCs沉默异柠檬酸脱氢酶IDH3A以及苹果酸脱氢酶2后,克隆生长没有明显变化,葡萄糖消耗也没有明显差异,而B16-F1细胞生长明显受抑制,葡萄糖消耗减少；沉默柠檬酸转运蛋白SLC25A1、柠檬酸裂解酶ACLY、脂肪酸合酶FASN以及苹果酸酶ME1,B16-F1 TRCs葡萄糖消耗明显减少；B16-F1 TRCs中延胡索酸含量增多；过表达PCK2后,B16-F1TRCs中延胡索酸含量降低；在缺氧的情况下,B16-F1 TRCs中HIF蛋白的表达升高：过表达PCK2后,B16-F1 TRCs中HIF蛋白表达降低；B16-F1 TRCs细胞DNA的5-羟甲基化程度降低；过表达PCK2后,B16-F1 TRCs中DNA的5-羟甲基化程度增高；B16-F1 TRCs细胞耗氧量降低；过表达PCK2后,B16-F1 TRCs耗氧量增高：与B16-F1细胞相比,电子传递链抑制剂Rotenone和Oligomycin对B16-F1 TRCs细胞的抑制作用更差。结论：肿瘤再生细胞相对于对照细胞下调表达PCK2, PCK2低表达对维持肿瘤再生细胞的生长和成瘤至关重要,而且PCK2的表达与肿瘤病人的总生存期密切相关。过表达PCK2后,B16-F1 TRCs细胞体外生长受抑制,体内致瘤能力减弱。B16-F1 TRCs糖酵解能力增强,氧化磷酸化水平降低。B16-F1 TRCs下调表达PCK2,发挥的生物学效应的内在机制可能是：①生成更多的柠檬酸,用于合成脂肪酸；②中间代谢物延胡索酸含量增高,进而影响缺氧诱导因子的表达,对细胞糖酵解产生影响；③中间代谢物延胡索酸含量增高,进而影响DNA羟甲基化程度,维持细胞的未分化状态；④降低细胞氧化磷酸化的水平,减少氧消耗。这些发现揭示PCK2的下调表达是黑色素肿瘤再生细胞重要的代谢标志,在其代谢过程中发挥重要调节作用,有望作为黑色素瘤治疗的靶标。
[Abstract]:Objective: The presence of tumor stem cells has made the tumor more aggressive, resistant and metastatic. At present, the research on the tumor metabolism is focused on the level of the mixed cell population, and lacks the research on the metabolic characteristics of the tumor stem cells and the molecular control mechanism of the tumor stem cells. The effect of the expression of the mitochondrial phosphoenolpyruvate (PCK2) on the metabolism of the tumor stem cells was investigated by using the three-dimensional soft-protein matrix gel (3D Fibroin) culture to obtain the tumor-regeneration cells (TRCs). Methods: (1) The expression of PCK2 in mouse melanoma cells B16-F1, H22, Hep-2 and human melanoma cells A875 was compared with RT-PCR and Western blot. The expression of PCK2 in mouse embryonic stem cells (mESCs) was detected by RT-PCR and Western blot. The expression of PCK2 in CD133 + cell subpopulation was compared by RT-PCR. Two cases of melanoma clinical tumor were collected, and the expression of PCK2 in two patients with TRCs was detected by RT-PCR. (2) The TCGA database was searched and the expression of PCK2 was related to the survival of the tumor. and (3) constructing a PCK2 overexpression plasmid vector, transfecting B16-F1TRCs, and then inoculating the tail vein into a mouse after 6 hours, and observing the lung tumorigenicity of the mouse after 24 days; the B16-F1 cell is transfected with the PCK2 siRNA, and the tail vein is inoculated with the mouse after 6 hours, and the lung tumorigenicity of the mouse is observed after 24 days; and the PCK2 Tet-On cell line is constructed, In the case of adding or not adding doxycycline, the size and number of the clones were compared, and the sensitivity of the TRCs to cisplatin was compared. The PCK2 Tet-On cell line was inoculated to the subcutaneous of the mouse and was fed with water added or not added with doxycycline (Dox), respectively, and compared to the tumor-forming ability of the cell in the subcutaneous part of the mouse. (4) analyzing the consumption of B16-F1 TRCs and B16-F1 and the amount of lactic acid release; overexpressing PCK2, comparing the glucose consumption and the lactic acid release amount of B16-F1 TRCs; comparing the content of the citric acid in the B16-F1 TRCs and the control cell TCA; and detecting the content of the B16-F1 TRCs citric acid by the expression of the PCK2; The siRNA-silent isocitrate dehydrogenase IDH3A and the malic acid dehydrogenase MDH2 were used to observe the growth of B16-F1 TRCs and control cells and to detect their glucose consumption; siRNA-silent citric acid transport protein SLC25A1, citric acid lyase ACLY, fatty acid synthase FASN and malic enzyme ME1 were used to detect the glucose consumption of B16-F1 TRCs; The expression of HIF1 and HIF2 in B16-F1 TRCs and control cells was detected by Western blot, and the expression of HIF1 and HIF2 in B16-F1 TRCs was detected by Western blot. The content of 5-hydroxymethyl-1-and 5-methylpinacolin in B16-F1 TRCs and control cells was compared by mass-mass spectrometry. The content of 5-hydroxymethyl-1-and 5-methylpinacolin in B16-F1 TRCs and control cells was detected by mass-mass spectrometry. The level of oxygen consumption of B16-F1 TRCs and control cells was detected by Sehorse XF24 equipment. After the expression of PCK2, the oxygen consumption of B16-F1 TRCs was detected: the different concentrations of the electron transfer chain complex I inhibitor (Rotenone) and the compound V inhibitor, the oligonin, respectively treated B16-F1 TRCs and the control cells, and the proliferation of the cells was compared with the CCK-8 kit and the glucose consumption was detected. Results: (1) B16-F1 cells, H22 cells, Hep-2 cells and A875 cells TRCs were down-regulated with respect to normal tumor cells and PCK2. The expression of PCK2 and PCK2 in the subpopulation of B16-F1 CD133 + cells was lower than that of CD133 cells. In two cases of black tumor, PCK2 was expressed in low expression. (2) The expression of PCK2 is related to the overall survival of melanoma, lung cancer and gastric cancer. (3) After the B16-F1 TRCs overexpressing PCK2, the number of transfer spots in the lung of the mouse is reduced; after the B16-F1 transfected siRNA is silent PCK2, the number of transfer spots in the lung of the mouse is increased; the PCK2 Tet-On stable cell strain is seeded into the 3D fibrin, and the volume of the clone is reduced when the doxycycline is added, The number is reduced and the sensitivity to cisplatin is deteriorated. The tumor-forming ability of PCK2 Tet-On stable cell line was reduced under the subcutaneous and doxycycline-fed mice. (4) As compared with B16-F1, the consumption of B16-F1TRCs increased and the amount of lactic acid release increased; after the expression of PCK2, the glucose consumption of B16-F1 TRCs decreased and the amount of lactic acid production decreased; the content of the citric acid in B16-F1 TRCs was increased; after the expression of PCK2, the content of citric acid in B16-F1 TRCs decreased; When the B16-F1 TRCs were silent and the isocitrate dehydrogenase IDH3A and the malate dehydrogenase 2 were silent, the growth of the clones did not change significantly, and the consumption of glucose was not significantly different, and the B16-F1 cell growth was significantly inhibited, and the consumption of glucose decreased; the silent citric acid transport protein SLC25A1, the citric acid cleavage enzyme ACLY, In the case of hypoxia, the expression of HIF protein in B16-F1 TRCs increased: after overexpression of PCK2, the expression of HIF protein in B16-F1 TRCs increased: after overexpression of PCK2, The expression of HIF protein in B16-F1 TRCs decreased; the degree of 5-hydroxymethylation of the DNA of B16-F1 TRCs decreased; after the expression of PCK2, the degree of 5-hydroxymethylation of the DNA in B16-F1 TRCs increased; the oxygen consumption of B16-F1 TRCs decreased; after overexpression of PCK2, the oxygen consumption of B16-F1 TRCs increased: compared with B16-F1 cells, The inhibitory effect of the electron transfer chain inhibitor Rotenone and the oligonin on the B16-F1 TRCs cells is even worse. Conclusion: The low expression of PCK2 and PCK2 in tumor-regenerating cells is critical to the growth and tumorigenesis of tumor-regenerating cells, and the expression of PCK2 is closely related to the overall survival of tumor patients. After the expression of PCK2, the in vitro growth of B16-F1 TRCs cells was inhibited and the in vivo tumorigenicity decreased. The glycolysis ability of B16-F1 TRCs was enhanced and the level of oxidative phosphorylation was decreased. B16-F1 TRCs downregulated the expression of PCK2, and the intrinsic mechanism of the biological effects of B16-F1 may be: to produce more citric acid for the synthesis of fatty acids; to increase the content of the intermediate metabolite, to influence the expression of hypoxia-inducing factors, and to have an effect on the glycolysis of the cells; And the level of the oxidative phosphorylation of the cells is reduced, and the consumption of oxygen is reduced. These findings reveal that the down-regulation expression of PCK2 is an important metabolic marker in the regeneration of melanin tumor cells, plays an important role in the metabolism process, and is expected to be a target for melanoma treatment.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.5

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