缺氧诱导因子-3α(HIF-3α)对肝癌细胞生物钟基因表达的影响
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摘要
目的:研究缺氧诱导因子-3α(HIF-3α)对肝癌细胞生物钟基因表达的影响。方法:将HIF-3α过表达的慢病毒载体感染HepG2细胞,构建稳定过表达HIF-3α肝癌细胞系,同时通过siRNA干扰HepG2细胞中HIF-3α的表达。然后通过Real-time PCR和Western Blot检测HIF-3α感染前后肝癌细胞中生物钟基因Clock、Bmal1、NPAS2、Per1、Per2、Per3、Timeless、Cry1、Cry2、REV-ERBA、Rora、CKIε的表达水平。结果:Real-time PCR结果显示HIF-3α使Per3、CKIεmRNA表达升高(P均<0.05),Bmal1、Per1、Per2、Cry1、REV-ERBA、Rora mRNA表达降低(P均<0.05),HIF-3α对Clock、NPAS2、Timeless、Cry2 mRNA的表达无明显影响(P均>0.05)。Western Blot结果与PCR结果相一致。结论:HIF-3α可引起肝癌细胞生物钟基因的紊乱,可能是肝癌生物钟基因表达异常的原因之一。
Objective:To investigate the effects of hypoxia inducible factor-3α(HIF-3α) on the expressions of clock genes in hepatocellular carcinoma cells(HCC).Methods:HepG2 cells were infected by lentiviruses which were overexpressed HIF-3α genes,aiming at building stable overexpressions of HIF-3α in hepatocellular carcinoma cells.Meanwhile,knocking down the expression of HIF-3α in HepG2 cells by using HIF-3α siRNA.Then,the real-time polymerase chain reaction(real-time PCR) was applied to detect the expression levels of circadian clock genes(Clock,Bmal1,NPAS2,Per1,Per2,Per3,Timeless,Cry1,Cry2,REV-ERBΑ,Rora,CKIε).Results:Compared with the group which was uninfected,the expression levels of Per3,CKIε genes were increased under the influences of HIF-3α(P<0.05). However,the expression of Bmal1,Per1,Per2,Cry1,REV-ERBΑ, Rora decreased(P<0.05),and Clock,NPAS2,Timeless,Cry2 seems no difference(P>0.05).The results of Western Blot were consistent with those of PCR.Conclusion:HIF-3α can significantly disturb the expression levels of some circadian clock genes in the hepatoma cells,which may be one of the reasons for abnormal expressions of clock genes in HCC.
Objective:To investigate the effects of hypoxia inducible factor-3α(HIF-3α) on the expressions of clock genes in hepatocellular carcinoma cells(HCC).Methods:HepG2 cells were infected by lentiviruses which were overexpressed HIF-3α genes,aiming at building stable overexpressions of HIF-3α in hepatocellular carcinoma cells.Meanwhile,knocking down the expression of HIF-3α in HepG2 cells by using HIF-3α siRNA.Then,the real-time polymerase chain reaction(real-time PCR) was applied to detect the expression levels of circadian clock genes(Clock,Bmal1,NPAS2,Per1,Per2,Per3,Timeless,Cry1,Cry2,REV-ERBΑ,Rora,CKIε).Results:Compared with the group which was uninfected,the expression levels of Per3,CKIε genes were increased under the influences of HIF-3α(P<0.05). However,the expression of Bmal1,Per1,Per2,Cry1,REV-ERBΑ, Rora decreased(P<0.05),and Clock,NPAS2,Timeless,Cry2 seems no difference(P>0.05).The results of Western Blot were consistent with those of PCR.Conclusion:HIF-3α can significantly disturb the expression levels of some circadian clock genes in the hepatoma cells,which may be one of the reasons for abnormal expressions of clock genes in HCC.
引文
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[22] Noreen S,Pegoraro M,Nouroz F,et al.Interspecific studies of circadian genes period and timeless in drosophila [J].Gene,2018,648:106-114.
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[3] Yang SL,Ren QG,Wen L,et al.Research progress on circadian clock genes in common abdominal malignant tumors [J].Oncology Letters,2017,14(5):5091-5098.
[4] Takahashi JS.Transcriptional architecture of the mammalian circadian clock [J].Nat Rev Genet,2017,18(3):164-179.
[5] Yang SL,Yu C,Jiang JX,et al.Hepatitis B virus X protein disrupts the balance of the expression of circadian rhythm genes in hepatocellular carcinoma [J].Oncology Letters,2014,8(6):2715-2720.
[6] Lin YM,Chang JH,Yeh KT,et al.Disturbance of circadian gene expression in hepatocellular carcinoma [J].Molecular Carcinogenesis,2008,47(12):925-933.
[7] Yuan P,Li J,Zhou F,et al.NPAS2 promotes cell survival of hepatocellular carcinoma by transactivating CDC25A [J].Cell Death Dis,2017,8(3):e2704.
[8] Takeda N,Maemura K.The role of clock genes and circadian rhythm in the development of cardiovascular diseases [J].Cellular and Molecular Life Sciences:CMLS,2015,72(17):3225-3234.
[9] Holmquist-Mengelbier L,Fredlund E,Lofstedt T,et al.Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma:HIF-2alpha promotes an aggressive phenotype [J].Cancer Cell,2006,10(5):413-423.
[10] Yang SL,Wu C,Xiong ZF,et al.Progress on hypoxia-inducible factor-3:Its structure,gene regulation and biological function (review)[J].Molecular Medicine Reports,2015,12(2):2411-2416.
[11] Tanaka T,Wiesener M,Bernhardt W,et al.The human HIF (hypoxia-inducible factor)-3alpha gene is a HIF-1 target gene and may modulate hypoxic gene induction [J].Biochem J,2009,424(1):143-151.
[12] Levy O,Kaniewska P,Alon S,et al.Complex diel cycles of gene expression in coral-algal symbiosis [J].Science,2011,331(6014):175.
[13] Lee S,Donehower LA,Herron AJ,et al.Disrupting circadian homeostasis of sympathetic signaling promotes tumor development in mice [J].PloS One,2010,5(6):e10995.
[14] Wu Y,Tang D,Liu N,et al.Reciprocal regulation between the circadian clock and hypoxia signaling at the genome level in mammals [J].Cell Metabolism,2017,25(1):73-85.
[15] Cheng QS,Liu P,Liu LP,et al.CoCl2 simulates the change of biological clock gene expression in hepatocellular carcinoma cells in an anoxic environment [J].Chinese Journal of General Surgery,2014,23(1):48-52.[程全胜,刘平,刘利平,等.CoCl2模拟缺氧环境下肝癌细胞生物钟基因表达的变化[J].中国普通外科杂志,2014,23(1):48-52.]
[16] Makino Y,Kanopka A,Wilson WJ,et al.Inhibitory PAS domain protein (IPAS) is a hypoxia-inducible splicing variant of the hypoxia-inducible factor-3alpha locus [J].J Biol Chem,2002,277(36):32405-32408.
[17] Gupta S,Haldar C,Singh S.Daily variations in plasma melatonin and melatonin receptor (MT1),PER1 and CRY1 expression in suprachiasmatic nuclei of tropical squirrel,funambulus pennanti [J].J Comp Physiol A Neuroethol Sens Neural Behav Physiol,2013,199(9):763-773.
[18] Mteyrek A,Filipski E,Guettier C,et al.Clock gene Per2 as a controller of liver carcinogenesis [J].Oncotarget,2016,7(52):85832-85847.
[19] Zhao B,Lu J,Yin J,et al.A functional polymorphism in PER3 gene is associated with prognosis in hepatocellular carcinoma [J].Liver International,2012,32(9):1451-1459.
[20] Zhao H,Zeng ZL,Yang J,et al.Prognostic relevance of Period1 (Per1) and Period2 (Per2) expression in human gastric cancer [J].International Journal of Clinical and Experimental Pathology,2014,7(2):619-630.
[21] Elgohary N,Pellegrino R,Neumann O,et al.Protumorigenic role of timeless in hepatocellular carcinoma [J].Int J Oncol,2015,46(2):597-606.
[22] Noreen S,Pegoraro M,Nouroz F,et al.Interspecific studies of circadian genes period and timeless in drosophila [J].Gene,2018,648:106-114.