BCL3基因敲除对线粒体呼吸作用及ATP合成的影响
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摘要
B淋巴细胞瘤/白血病因子3(B cell CLL/lymphoma 3,BCL3)是一个转录辅调节因子,通过结合转录因子对基因表达起激活或抑制作用,维持细胞存活,但其机制尚不清楚。该研究采用CRISPR/Cas9技术建立BCL3基因敲除的人宫颈癌He La细胞,实时荧光定量PCR和Western blot验证敲除情况。使用特异性荧光探针法、萤火虫荧光素酶法、活细胞能量代谢动态分析法等技术手段检测BCL3基因敲除(BCL3-KO)对细胞内活性氧类(reactive oxygen species,ROS)水平、线粒体膜电位、线粒体呼吸作用以及ATP生成的影响。结果发现,BCL3-KO细胞内的相对ROS水平上升约50%,采用转染的方式恢复细胞内表达BCL3基因则可抑制ROS水平的上升;与野生型He La细胞相比,BCL3-KO细胞的线粒体膜电位明显降低(P<0.001);BCL3敲除不影响细胞基础有氧呼吸速率,但引起碳酰氰4-(三氟甲氧基)苯腙[carbomyl cyanide 4-(trifluorometyocy)phenylhydrazone,FCCP]诱导的最大(极限)呼吸速率显著上升(P<0.001);相比野生型细胞,BCL3-KO细胞中的ATP的浓度下降40%。该研究揭示了BCL3对线粒体功能的调控作用,可能是其维护癌细胞存活的原因之一。
B-cell CLL/lymphoma 3(BCL3), which acts as transcription coregulator by activating or suppressing gene expression via binding transcription factors, is required for cancer cell survival, but the underling mechanism is not yet fully known. To investigate the roles of BCL3 in mitochondria function, BCL3 knockout human cervical carcinoma He La cells(BCL3-KO cells) were generated by CRISPR/Cas9-mediated genome editing. Real-time quantitative PCR(q RT-PCR) and Western blot were used to validate gene knockout. Using the cell lines, DCFH-DA and JC-1 staining method was performed to test ROS production and mitochondria membrane potential(MMP), respectively. Cellular energy metabolism was studied using Seahorse XF24 Extracellular Flux Analyzer, and ATP production was also measured. In contrast to wild type He La cells, the relative level of ROS was decreased by nearly 50% in BCL3-KO cells; however the decline could be reversed by overexpression of BCL3. Deficiency of BCL3 led to a significant loss of MMP and a reduction in ATP production(P<0.001), which could be indication of mitochondrial dysfunction. Furthermore, BCL3 knockdown did not affect the rate of basic cellular aerobic respiration, but caused an obvious rise in the rate of carbomyl cyanide 4-(trifluorometyocy) phenylhydrazone(FCCP)-induced cellular aerobic respiration(P<0.001). ATP concentration in BCL3-KO cells was decreased about 40% compared with that of in wild type He La cells. Taken together, our results revealed a novel role for BCL3 in regulation of mitochondrial function, which might be one of the reasons for BCL3 to maintain cancer cell survival.
B-cell CLL/lymphoma 3(BCL3), which acts as transcription coregulator by activating or suppressing gene expression via binding transcription factors, is required for cancer cell survival, but the underling mechanism is not yet fully known. To investigate the roles of BCL3 in mitochondria function, BCL3 knockout human cervical carcinoma He La cells(BCL3-KO cells) were generated by CRISPR/Cas9-mediated genome editing. Real-time quantitative PCR(q RT-PCR) and Western blot were used to validate gene knockout. Using the cell lines, DCFH-DA and JC-1 staining method was performed to test ROS production and mitochondria membrane potential(MMP), respectively. Cellular energy metabolism was studied using Seahorse XF24 Extracellular Flux Analyzer, and ATP production was also measured. In contrast to wild type He La cells, the relative level of ROS was decreased by nearly 50% in BCL3-KO cells; however the decline could be reversed by overexpression of BCL3. Deficiency of BCL3 led to a significant loss of MMP and a reduction in ATP production(P<0.001), which could be indication of mitochondrial dysfunction. Furthermore, BCL3 knockdown did not affect the rate of basic cellular aerobic respiration, but caused an obvious rise in the rate of carbomyl cyanide 4-(trifluorometyocy) phenylhydrazone(FCCP)-induced cellular aerobic respiration(P<0.001). ATP concentration in BCL3-KO cells was decreased about 40% compared with that of in wild type He La cells. Taken together, our results revealed a novel role for BCL3 in regulation of mitochondrial function, which might be one of the reasons for BCL3 to maintain cancer cell survival.
引文
1 Dhingra R,Kirshenbaum LA.Regulation of mitochondrial dynamics and cell fate.Circ J 2014;78(4):803-10.
2 Loureiro R,Mesquita KA,Magalhaes-Novais S,Oliveira PJ,Vega-Naredo I.Mitochondrial biology in cancer stem cells.Semin Cancer Biol 2017;47:18-28.
3 Chen H,Chan DC.Mitochondrial dynamics in regulating the unique phenotypes of cancer and stem cells.Cell Metab 2017;26(1):39-48.
4 Vyas S,Zaganjor E,Haigis MC.Mitochondria and cancer.Cell2016;166(3):555-66.
5 Gray MW.The pre-endosymbiont hypothesis:a new perspective on the origin and evolution of mitochondria.Cold Spring Harb Perspect Bio 2014;doi:10.1101/cshperspect.a016097.
6 Kamp WM,Wang PY,Hwang PM.TP53 mutation,mitochondria and cancer.Curr Opin Gene Dev 2016;38:16-22.
7 Meier JA,Larner AC.Toward a new STATe:the role of STATs in mitochondrial function.Semin Immunol 2014;26(1):20-8.
8 Scarpulla RC.Transcriptional paradigms in mammalian mitochondrial biogenesis and function.Physiol Rev 2008;88(2):611-38.
9 Kuo SH,Tsai HJ,Lin CW,Yeh KH,Lee HW,Wei MF,et al.The B-cell-activating factor signalling pathway is associated with Helicobacter pylori independence in gastric mucosa-associated lymphoid tissue lymphoma without t(11;18)(q21;q21).J Pathol2017;241(3):420-33.
10 Alpatov R,Carstens B,Harding K,Jarrett C,Balakhani S,Lincoln J,et al.Rare double-hit with two translocations involving IGH both,with BCL2 and BCL3,in a monoclonal B-cell lymphoma/leukemia.Mol Cytogenet 2015;8:101.
11 Wu J,Li L,Jiang G,Zhan H,Wang N.B-cell CLL/lymphoma 3promotes glioma cell proliferation and inhibits apoptosis through the oncogenic STAT3 pathway.Int J Oncol 2016;49(6):2471-9.
12 Zhao H,Wang W,Zhao Q,Hu G,Deng K,Liu Y.BCL3 exerts an oncogenic function by regulating STAT3 in human cervical cancer.Onco Targets Ther 2016;9:6619-29.
13 Hatada EN,Nieters A,Wulczyn FG,Naumann M,Meyer R,Nucifora G,et al.The ankyrin repeat domains of the NF-kappa B precursor p105 and the protooncogene bcl-3 act as specific inhibitors of NF-kappa B DNA binding.Proc Natl Acad Sci USA1992;89(6):2489-93.
14 Bours V,Franzoso G,Azarenko V,Park S,Kanno T,Brown K,et al.The oncoprotein Bcl-3 directly transactivates through kappa B motifs via association with DNA-binding p50B homodimers.Cell 1993;72(5):729-39.
15 Ohno H,Takimoto G,Mc Keithan TW.The candidate protooncogene bcl-3 is related to genes implicated in cell lineage determination and cell cycle control.Cell 1990;60(6):991-7.
16 Massoumi R,Chmielarska K,Hennecke K,Pfeifer A,Fassler R.Cyld inhibits tumor cell proliferation by blocking Bcl-3-dependent NF-kappa B signaling.Cell 2006;125(4):665-77.
17 Chen CY,Lee DS,Yan YT,Shen CN,Hwang SM,Lee ST,et al.Bcl3 bridges LIF-STAT3 to Oct4 signaling in the maintenance of naive pluripotency.Stem Cells 2015;33(12):3468-80.
18 Kashatus D,Cogswell P,Baldwin AS.Expression of the Bcl-3 proto-oncogene suppresses p53 activation.Genes Dev 2006;20(2):225-35.
19 Murphy MP.Mitochondrial dysfunction indirectly elevates ROS production by the endoplasmic reticulum.Cell Metab 2013;18(2):145-6.
20 Wai T,Langer T.Mitochondrial dynamics and metabolic regulation.Trends Endocrinol Metab 2016;27(2):105-17.
21 Schrepfer E,Scorrano L.Mitofusins,from mitochondria to metabolism.Mol Cell 2016;61(5):683-94.
22 Galluzzi L,Baehrecke EH,Ballabio A,Boya P,Bravo-San Pedro JM,Cecconi F,et al.Molecular definitions of autophagy and related processes.EMBO J 2017;36(13):1811-36.
23 Fujita T,Nolan GP,Liou HC,Scott ML,Baltimore D.The candidate proto-oncogene bcl-3 encodes a transcriptional coactivator that activates through NF-kappa B p50 homodimers.Genes Dev 1993;7(7B):1354-63.
24 Na SY,Choi JE,Kim HJ,Jhun BH,Lee YC,Lee JW.Bcl3,an Ikappa B protein,stimulates activating protein-1 transactivation and cellular proliferation.J Biol Chem 1999;274(40):28491-6.
25 Georgieva E,Ivanova D,Zhelev Z,Bakalova R,Gulubova M,Aoki I.Mitochondrial dysfunction and redox imbalance as a diagnostic marker of“free radical diseases”.Anticancer Res2017;37(10):5373-81.
2 Loureiro R,Mesquita KA,Magalhaes-Novais S,Oliveira PJ,Vega-Naredo I.Mitochondrial biology in cancer stem cells.Semin Cancer Biol 2017;47:18-28.
3 Chen H,Chan DC.Mitochondrial dynamics in regulating the unique phenotypes of cancer and stem cells.Cell Metab 2017;26(1):39-48.
4 Vyas S,Zaganjor E,Haigis MC.Mitochondria and cancer.Cell2016;166(3):555-66.
5 Gray MW.The pre-endosymbiont hypothesis:a new perspective on the origin and evolution of mitochondria.Cold Spring Harb Perspect Bio 2014;doi:10.1101/cshperspect.a016097.
6 Kamp WM,Wang PY,Hwang PM.TP53 mutation,mitochondria and cancer.Curr Opin Gene Dev 2016;38:16-22.
7 Meier JA,Larner AC.Toward a new STATe:the role of STATs in mitochondrial function.Semin Immunol 2014;26(1):20-8.
8 Scarpulla RC.Transcriptional paradigms in mammalian mitochondrial biogenesis and function.Physiol Rev 2008;88(2):611-38.
9 Kuo SH,Tsai HJ,Lin CW,Yeh KH,Lee HW,Wei MF,et al.The B-cell-activating factor signalling pathway is associated with Helicobacter pylori independence in gastric mucosa-associated lymphoid tissue lymphoma without t(11;18)(q21;q21).J Pathol2017;241(3):420-33.
10 Alpatov R,Carstens B,Harding K,Jarrett C,Balakhani S,Lincoln J,et al.Rare double-hit with two translocations involving IGH both,with BCL2 and BCL3,in a monoclonal B-cell lymphoma/leukemia.Mol Cytogenet 2015;8:101.
11 Wu J,Li L,Jiang G,Zhan H,Wang N.B-cell CLL/lymphoma 3promotes glioma cell proliferation and inhibits apoptosis through the oncogenic STAT3 pathway.Int J Oncol 2016;49(6):2471-9.
12 Zhao H,Wang W,Zhao Q,Hu G,Deng K,Liu Y.BCL3 exerts an oncogenic function by regulating STAT3 in human cervical cancer.Onco Targets Ther 2016;9:6619-29.
13 Hatada EN,Nieters A,Wulczyn FG,Naumann M,Meyer R,Nucifora G,et al.The ankyrin repeat domains of the NF-kappa B precursor p105 and the protooncogene bcl-3 act as specific inhibitors of NF-kappa B DNA binding.Proc Natl Acad Sci USA1992;89(6):2489-93.
14 Bours V,Franzoso G,Azarenko V,Park S,Kanno T,Brown K,et al.The oncoprotein Bcl-3 directly transactivates through kappa B motifs via association with DNA-binding p50B homodimers.Cell 1993;72(5):729-39.
15 Ohno H,Takimoto G,Mc Keithan TW.The candidate protooncogene bcl-3 is related to genes implicated in cell lineage determination and cell cycle control.Cell 1990;60(6):991-7.
16 Massoumi R,Chmielarska K,Hennecke K,Pfeifer A,Fassler R.Cyld inhibits tumor cell proliferation by blocking Bcl-3-dependent NF-kappa B signaling.Cell 2006;125(4):665-77.
17 Chen CY,Lee DS,Yan YT,Shen CN,Hwang SM,Lee ST,et al.Bcl3 bridges LIF-STAT3 to Oct4 signaling in the maintenance of naive pluripotency.Stem Cells 2015;33(12):3468-80.
18 Kashatus D,Cogswell P,Baldwin AS.Expression of the Bcl-3 proto-oncogene suppresses p53 activation.Genes Dev 2006;20(2):225-35.
19 Murphy MP.Mitochondrial dysfunction indirectly elevates ROS production by the endoplasmic reticulum.Cell Metab 2013;18(2):145-6.
20 Wai T,Langer T.Mitochondrial dynamics and metabolic regulation.Trends Endocrinol Metab 2016;27(2):105-17.
21 Schrepfer E,Scorrano L.Mitofusins,from mitochondria to metabolism.Mol Cell 2016;61(5):683-94.
22 Galluzzi L,Baehrecke EH,Ballabio A,Boya P,Bravo-San Pedro JM,Cecconi F,et al.Molecular definitions of autophagy and related processes.EMBO J 2017;36(13):1811-36.
23 Fujita T,Nolan GP,Liou HC,Scott ML,Baltimore D.The candidate proto-oncogene bcl-3 encodes a transcriptional coactivator that activates through NF-kappa B p50 homodimers.Genes Dev 1993;7(7B):1354-63.
24 Na SY,Choi JE,Kim HJ,Jhun BH,Lee YC,Lee JW.Bcl3,an Ikappa B protein,stimulates activating protein-1 transactivation and cellular proliferation.J Biol Chem 1999;274(40):28491-6.
25 Georgieva E,Ivanova D,Zhelev Z,Bakalova R,Gulubova M,Aoki I.Mitochondrial dysfunction and redox imbalance as a diagnostic marker of“free radical diseases”.Anticancer Res2017;37(10):5373-81.