Lin28B/let-7通路与肝癌细胞紫杉醇耐药的相关性研究
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摘要
为证明Lin28B/let-7通路与肝癌紫杉醇耐药密切相关和采用浓度梯度间歇刺激法建立Hep3B/PTX耐药细胞系。通过四甲基偶氮唑盐比色(methyl thiazolyl tetrazolium,MTT)分析Guava微流式分析对Hep3B/PTX耐药细胞系进行鉴定,realtime PCR检测耐药细胞中Lin28B及let-7表达水平。经过6个月的诱导,Hep3B/PTX耐药细胞系对紫杉醇的耐药指数为13.55;与亲本细胞相比,Hep3B/PTX处于G0/G1期的细胞比例较高,S期的细胞比例较低;0.2μmol/L紫杉醇处理亲本细胞24h后,有(41.73±1.80)%细胞发生凋亡,而Hep3B/PTX细胞凋亡率只有(17.21±3.60)%;real-time PCR发现,Hep3B/PTX与亲本细胞相比,Lin28B基因表达水平升高了49倍,而Lin28B调控的let-7家族成员表达水平均有不同程度的降低。Hep3B/PTX细胞中Lin28B基因和let-7家族成员表达量的变化揭示了肝癌细胞对紫杉醇的耐药与Lin28B/let-7通路相关,Lin28B基因可能通过影响let-7家族miRNA生物发生过程而改变肝癌细胞对紫杉醇的敏感性。
To determine whether the Lin28B/let-7 pathway is tightly related to paclitaxel resistance in hepatocellular carcinoma cells,apaclitaxel-resistant cell line Hep3B/PTX was established by intermissive and stepwise exposure to paclitaxel.The Hep3B/PTX cell line was identified by methyl thiazolyl tetrazolium(MTT)assay and Guava easy Cyte Flow Cytometers assay.The expressions of Lin28B and let-7 were detected by real-time PCR.After 6 months induction,the Hep3B/PTX cell line was established,and the drug resistance index was 13.55.Compared with parental cells,the Hep3B/PTX cells had a higher percentage of G_0/G_1 phase and a lower percentage of S phase.After treatment by 0.2μmol/L paclitaxel for 24 h,the apoptosis rate of parental cells was(41.73±1.80)% but that of Hep3B/PTX was only(17.21±3.60)%.The real-time PCR analysis showed that the expression of Lin28B in Hep3B/PTX cells was 49 times more than parental cells,but the expressions of let-7 miRNAs which were regulated by Lin28B were all decreased in different degree.Aberrant expression of Lin28B and let-7 in Hep3B/PTX cells suggested that paclitaxel resistance in hepatocellular carcinoma cells was related to the Lin28B/let-7 pathway,Lin28B might change the chemosensitivity of human hepatocellular carcinoma by affecting let-7 processing.
To determine whether the Lin28B/let-7 pathway is tightly related to paclitaxel resistance in hepatocellular carcinoma cells,apaclitaxel-resistant cell line Hep3B/PTX was established by intermissive and stepwise exposure to paclitaxel.The Hep3B/PTX cell line was identified by methyl thiazolyl tetrazolium(MTT)assay and Guava easy Cyte Flow Cytometers assay.The expressions of Lin28B and let-7 were detected by real-time PCR.After 6 months induction,the Hep3B/PTX cell line was established,and the drug resistance index was 13.55.Compared with parental cells,the Hep3B/PTX cells had a higher percentage of G_0/G_1 phase and a lower percentage of S phase.After treatment by 0.2μmol/L paclitaxel for 24 h,the apoptosis rate of parental cells was(41.73±1.80)% but that of Hep3B/PTX was only(17.21±3.60)%.The real-time PCR analysis showed that the expression of Lin28B in Hep3B/PTX cells was 49 times more than parental cells,but the expressions of let-7 miRNAs which were regulated by Lin28B were all decreased in different degree.Aberrant expression of Lin28B and let-7 in Hep3B/PTX cells suggested that paclitaxel resistance in hepatocellular carcinoma cells was related to the Lin28B/let-7 pathway,Lin28B might change the chemosensitivity of human hepatocellular carcinoma by affecting let-7 processing.
引文
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[3]PAN Z,GOLLAHON L.Taxol directly induces endoplasmic reticulum-associated calcium changes that promote apoptosis in breast cancer cells[J].Breast Journal,2011,17(1):56-70.
[4]LIU Q,WU Y,YOSHIZAWA T,et al.Basic helixloop-helix transcription factor DEC2functions as an anti-apoptotic factor during paclitaxel-induced apoptosis in human prostate cancer cells[J].International Journal of Molecular Medicine,2016,38(6):1727-1733.
[5]WU G,HUANG P,JU X,et al.Lin28Bover-expression mediates the repression of let-7by hepatitis B virus X protein in hepatoma cells[J].International Journal of Clinical and Experimental Medicine,2015,8(9):15108-15116.
[6]RAHKONEN N,STUBB A,MALONZO M,et al.Mature Let-7 miRNAs fine tune expression of LIN28Bin pluripotent human embryonic stem cells[J].Stem Cell Research,2016,17(3):498-503.
[7]RYBAK A,FUCHS H,SMIRNOVA L,et al.A feedback loop comprising Lin-28and let-7controls pre-let-7maturation during neural stem-cell commitment[J].Nature Cell Biology,2008,10(8):987-993.
[8]ZHOU M,LI Z,HAN Z,et al.Paclitaxel-sensitization enhanced by curcumin involves down-regulation of nuclear factor-κB and Lin28in Hep3Bcells[J].Journal of Receptor and Signal Transduction Research,2015,35(6):618-25.
[9]FERLAY J,SOERJOMATARAM I,DIKSHIT R,et al.Cancer incidence and mortality worldwide:sources,methods and major patternsin GLOBOCAN 2012[J].International Journal of Cancer,2015,136(5):E359-86.
[10]CHAO Y,CHAN W K,BIRKHOFER M J,et al.Phase II and pharmacokinetic study of paclitaxel therapy for unresectable hepatocellular carcinoma patients[J].British Journal of Cancer,1998,78(1):34-39.
[11]MEENA A S,SHARMA A,KUMARI R,et al.Inherent and acquired resistance to paclitaxel in hepatocellular carcinoma:molecular events involved[J].PLoSOne,2013,8(4):e61524.
[12]付翠群,沈国栋,沈干,等.人胃癌SGC-7901/TCF7L2细胞株的建立及其生物学特性观察[J].安徽医科大学学报,2016,51(11):1579-1583.FU Cuiqun,SHEN Guodong,SHEN Gan,et al.Establishment of the human gastric cancer SGC-7901/TCF7L2cell line and observing its biological characteristics[J].Acta Universitatis Medicinalis Anhui,2016,51(11):1579-1583.(in Chinese)
[13]颜晨,余德才,江勇.癌细胞代谢相关的耐药干预方法的研究进展[J].中国药理学与毒理学杂志,2015,29(6):986-992.YAN Chen,YU Decai,JIANG Yong.Intervention with drug resistance related to metabolism of cancer cells:advances in research[J].Chinese Journal of Pharmacology and Toxicology,2015,29(6):986-992.(in Chinese)
[14]延冰,刘国勤,姜永胜,等.高剂量法诱导胃癌铂类耐药细胞株的建立及其生物学特性[J].山东大学学报(医学版),2014,52(4):49-53.YAN Bing,LIU Guoqin,JIANG Yongsheng,et al.Establishment of cisplatin-resistant gastric cancer cell line and the biological characteristics[J].Journal of Shandong University(Health Sciences),2014,52(4):49-53.(in Chinese)
[15]HAMEIRI-GROSSMAN M,PORAT-KLEIN A,YA-NIV I,et al.The association between let-7,RAS and HIF-1αin Ewing Sarcoma tumor growth[J].Oncotarget,2015,6(32):33834-33848.
[16]ROWE R G,WANG L D,COMA S,et al.Developmental regulation of myeloerythroid progenitor function by the Lin28b-let-7-Hmga2axis[J].Journal of Experimental Medicine,2016,213(8):1497-1512.
[17]ZHANG W F,XIONG Y W,ZHU T T,et al.MicroRNA let-7g inhibited hypoxia-induced proliferation of PASMCs via G0/G1cell cycle arrest by targeting c-myc[J].Life Sciences,2017,170:9-15.