MCUR1对K562细胞系增殖、周期和凋亡的影响
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摘要
目的探讨线粒体钙单向转运体调节因子1(mitochondrial calcium uniporter regulator 1,MCUR1)对慢性髓系白血病细胞系K562细胞的增殖、周期和凋亡的影响以及可能的分子机制。方法通过慢病毒包装将MCUR1敲低重组质粒转染K562细胞系,筛选出稳定低表达MCUR1蛋白的K562细胞系。实时定量PCR(quantitative realtime polymerase chain reaction,qRT-PCR)实验检测细胞中MCUR1基因的表达水平;蛋白质免疫印迹(Western blotting,WB)实验检测细胞中MCUR1蛋白及凋亡相关蛋白P53、BAX和BCL2的表达水平;细胞增殖实验(cell counting kit-8,CCK-8)检测细胞增殖能力;流式细胞术检测细胞周期和细胞凋亡。结果 MCUR1敲低重组质粒转染K562细胞后,经筛选获得了稳定低表达MCUR1蛋白的细胞系;敲低MCUR1可显著抑制K562细胞增殖,诱导K562细胞凋亡,但不影响K562细胞周期;同时,敲低MCUR1能增加P53蛋白的表达,提高蛋白BAX/BCL2的比例。结论敲低MCUR1可显著抑制K562细胞增殖,促进其凋亡。
Objective To investigate the effect of mitochondrial calcium uniporter(MCU) regulator 1(MCUR1) on proliferation,cell cycle and apoptosis of K562 cells and the possible molecular mechanism. Methods Recombinant plasmid vectors containing short hairpin RNAs(shRNAs) targeting MCUR1 were transfected into K562 cells,before the K562 cells stably expressing low MCUR1 were selected with G418. The expression of MCUR1 mRNA was detected by quantitative real-time polymerase chain reaction(qRT-PCR) assays. Western blotting(WB) assays were used to detect the expressions of MCUR1,P53,BAX and BCL2. The proliferation,cell cycle and apoptosis of K562 cells were detected by cell counting kit-8(CCK-8) assays and flow cytometry,respectively. Results The results of qRT-PCR and WB assays revealed that MCUR1 was stably down-regulated at mRNA and protein levels in the K562 cells transfected with shRNAs targeting MCUR1. Knockdown of MCUR1 significantly inhibited the cell proliferation,induced the cell apoptosis,but did not influence the cells cycle. Meanwhile,knockdown of MCUR1 increased the expression of P53 protein and the ratio of protein BAX/BCL2 in K562 cells. Conclusion MCUR1 promotes cell proliferation and inhibits cell apoptosis in K562 cells.
Objective To investigate the effect of mitochondrial calcium uniporter(MCU) regulator 1(MCUR1) on proliferation,cell cycle and apoptosis of K562 cells and the possible molecular mechanism. Methods Recombinant plasmid vectors containing short hairpin RNAs(shRNAs) targeting MCUR1 were transfected into K562 cells,before the K562 cells stably expressing low MCUR1 were selected with G418. The expression of MCUR1 mRNA was detected by quantitative real-time polymerase chain reaction(qRT-PCR) assays. Western blotting(WB) assays were used to detect the expressions of MCUR1,P53,BAX and BCL2. The proliferation,cell cycle and apoptosis of K562 cells were detected by cell counting kit-8(CCK-8) assays and flow cytometry,respectively. Results The results of qRT-PCR and WB assays revealed that MCUR1 was stably down-regulated at mRNA and protein levels in the K562 cells transfected with shRNAs targeting MCUR1. Knockdown of MCUR1 significantly inhibited the cell proliferation,induced the cell apoptosis,but did not influence the cells cycle. Meanwhile,knockdown of MCUR1 increased the expression of P53 protein and the ratio of protein BAX/BCL2 in K562 cells. Conclusion MCUR1 promotes cell proliferation and inhibits cell apoptosis in K562 cells.
引文
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[2]O'Brien S,Abboud CN,Akhtari M,et al.Chronic myelogenous leukemia[J].J Natl Compr Canc Netw,2012,10(1):64-110.
[3]Huang X,Cortes J,Kantarjian H.Estimations of the increasing prevalence and plateau prevalence of chronic myeloid leukemia in the era of tyrosine kinase inhibitor therapy[J].Cancer,2012,118(12):3123-3127.
[4]Samanta A,Perazzona B,Chakraborty S,et al.Janus kinase 2regulates Bcr-Abl signaling in chronic myeloid leukemia[J].Leukemia,2011,25(3):463-472.
[5]Larson RA.Is there a best TKI for chronic phase CML[J]?Blood,2015,126(21):2370-2375.
[6]Pan S,Ryu SY,Sheu SS.Distinctive characteristics and functions of multiple mitochondrial Ca2+influx mechanisms[J].Sci Chin Life Sci,2011,54(8):763-769.
[7]Drago I,Pizzo P,Pozzan T.After half a century mitochondrial calcium in-and efflux machineries reveal themselves[J].Embo J,2011,30(20):4119-4125.
[8]Li H,Xu J,Zhou Y,et al.PLSCR1/IP3R1/Ca2+axis contributes to differentiation of primary AML cells induced by wogonoside[J].Cell Death Dis,2017,8(5):e2768.上接第页
[9]Forchap SL,Pirmohamed M,Clark RE.Release of intracellular calcium primes chronic myeloid leukaemia cells for tyrosine kinase inhibitor-induced apoptosis[J].Leukemia,2012,26(3):490-498.
[10]Paupe V,Prudent J,Dassa EP,et al.CCDC90A(MCUR1)is a cytochrome c oxidase assembly factor and not a regulator of the mitochondrial calcium uniporter[J].Cell Metab,2015,21(1):109-116.
[11]Vais H,Tanis JE,Müller M,et al.MCUR1,CCDC90A,is a regulator of the mitochondrial calcium uniporter[J].Cell Metab,2015,22(4):533-535.
[12]Mallilankaraman K,Cárdenas C,Doonan P,et al.MCUR1 is an essential component of mitochondrial Ca2+uptake that regulates cellular metabolism[J].Nat Cell Biol,2012,14(12):1336-1343.
[13]Ren T,Wang J,Zhang H,et al.MCUR1-mediated mitochondrial calcium signaling facilitates cell survival of hepatocellular carcinoma via reactive oxygen species-dependent P53 degradation[J].Antioxid Redox Signal,2018,28(12):1120-1136.
[14]陈萍,胥莉,王敏,等.Th22细胞在慢性粒细胞白血病患者中的临床研究[J].临床血液学杂志,2016,29(3):228-231.
[15]Hanahan D,Weinberg RA.Hallmarks of cancer:the next generation[J].Cell,2011,144(5):646-674.
[16]Csordás G,Golenár T,Seifert EL,et al.MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca2+uniporter[J].Cell Metab,2013,17(6):976-987.
[17]Williams GS,Boyman L,Lederer WJ.Mitochondrial calcium and the regulation of metabolism in the heart[J].J Mol Cell Cardiol,2015,78:35-45.
[18]Hoppe UC.Mitochondrial calcium channels[J].FEBS Lett,2010,584(10):1975-1981.
[19]Yoon MJ,Kim EH,Kwon TK,et al.Simultaneous mitochondrial Ca2+overload and proteasomal inhibition are responsible for the induction of paraptosis in malignant breast cancer cells[J].Cancer Lett,2012,324(2):197-209.
[20]Tang S,Wang X,Shen Q,et al.Mitochondrial Ca2+uniporter is critical for store-operated Ca2+entry-dependent breast cancer cell migration[J].Biochem Biophys Res Commun,2015,458(1):186-193.