射频消融亚致死性温度对肝癌干细胞的影响
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摘要
目的研究亚致死温度射频消融(RFA)对肝癌干细胞(LCSC)产生及其相关转录因子表达的影响。方法利用小鼠Hep1-6肝癌细胞株和肝细胞癌(HCC)患者临床样品,检测LCSC相关标志物和转录因子的表达。结果不同温度分别刺激Hep1-6细胞后发现,45℃是不能诱导细胞死亡的亚致死性温度。流式细胞仪(FCM)检测显示45℃处理可引起CD13~+、CD44~+、CD90~+、CD133~+Hep1-6细胞水平明显上调,提示45℃温度导致Hep1-6细胞中以上各型LCSC产生增加;实时定量聚合酶链反应(RT-q PCR)检测显示45℃温度导致CD13、CD90、CD133 m RNA水平明显上调。CD13 m RNA水平在5例HCC患者复发肝癌组织中均明显上调,CD133 m RNA在4例复发肝癌中上调,CD90 m RNA仅在1例复发肝癌中上调;FCM检测显示CD13~+LCSC水平在4例复发肝癌中明显上调,CD133~+LCSC水平仅在1例复发肝癌中上调,提示CD13~+LCSC水平上调与45℃温度关系更密切。RT-q PCR检测显示4例CD13~+LCSC上调的复发肝癌患者13个LCSC相关转录因子中Sox2、Stat1明显上调,FCM检测显示45℃处理Hep1-6细胞后Sox2、Stat1 m RNA明显上调。用Sox2、Stat1 si RNA分别沉默了Sox2、Stat1基因,表明Sox2、Stat1均参与了45℃温度诱导的CD13~+LCSC产生。结论 RFA治疗中45℃亚致死性温度所致CD13~+LCSC水平增高与Sox2、Stat1表达有关。该结果对肝癌复发研究有一定借鉴意义。
Objective To study the effect of radiofrequency ablation(RFA) with sublethal temperature on the production of liver cancer stem cells(LCSCs) and on the expression of LCSCs-related transcriptional factors. Methods Mouse hep1-6 hepatoma cell line and clinical samples of patients with hepatocellular carcinoma(HCC) were used to test the expressions of LCSCs-related markers and transcriptional factors.Results Different temperatures were used to stimulate Hep1-6 cells, and it was proved that the temperature of 45℃ was a sublethal temperature that could not induce cell death. Flow cytometry testing showed that treatment with 45℃ could obviously increase CD13~+, CD44~+, CD90 and CD133~+Hep1-6 cells, suggesting that treatment with 45℃ could increase the production of above mentioned types of LCSCs in hep1-6 cells.Real-time quantitative polymerase chain reaction(RT-q PCR) assay indicated that the temperature of 45℃could cause significant increase in CD13, CD90 and CD133 m RNA. In all 5 HCC patients, CD13 m RNA in the recurrent HCC lesions was remarkably increased, CD133 m RNA was increased in 4 patients with recurrent HCC, and CD90 m RNA was increased in only one patient with recurrent HCC. Flow cytometry testing revealed that CD13~+LCSCs were strikingly increased in 4 recurrent HCC patients, while CD133~+LCSC was increased in only one patient, suggesting that more close correlation existed between the increase of CD13~+LCSCs and the temperature of 45℃. RT-q PCR assay showed that in 4 recurrent HCC patients with increased CD13~+LCSC, the Sox2 and Stat1 among 13 LCSCs-related transcriptional factors were obviously increased. Flow cytometry testing showed that 45℃ treatment also increased the expression of Sox2 and Stat1 m RNA in Hep1-6 cells. Finally, Sox2 and Stat1 could be knockdown by si RNAs, indicating that both Sox2 and Stat1 transcriptional factors were involved in 45℃-induced production of CD13~+LCSCs in Hep1-6 cells.Conclusion In RFA therapy, the use of sublethal temperature of 45℃ can increase CD13~+LCSCs, which is related to the promotion of Sox2 and Stat1 expression. The results of this study can be used for reference in the research of liver cancer recurrence.
Objective To study the effect of radiofrequency ablation(RFA) with sublethal temperature on the production of liver cancer stem cells(LCSCs) and on the expression of LCSCs-related transcriptional factors. Methods Mouse hep1-6 hepatoma cell line and clinical samples of patients with hepatocellular carcinoma(HCC) were used to test the expressions of LCSCs-related markers and transcriptional factors.Results Different temperatures were used to stimulate Hep1-6 cells, and it was proved that the temperature of 45℃ was a sublethal temperature that could not induce cell death. Flow cytometry testing showed that treatment with 45℃ could obviously increase CD13~+, CD44~+, CD90 and CD133~+Hep1-6 cells, suggesting that treatment with 45℃ could increase the production of above mentioned types of LCSCs in hep1-6 cells.Real-time quantitative polymerase chain reaction(RT-q PCR) assay indicated that the temperature of 45℃could cause significant increase in CD13, CD90 and CD133 m RNA. In all 5 HCC patients, CD13 m RNA in the recurrent HCC lesions was remarkably increased, CD133 m RNA was increased in 4 patients with recurrent HCC, and CD90 m RNA was increased in only one patient with recurrent HCC. Flow cytometry testing revealed that CD13~+LCSCs were strikingly increased in 4 recurrent HCC patients, while CD133~+LCSC was increased in only one patient, suggesting that more close correlation existed between the increase of CD13~+LCSCs and the temperature of 45℃. RT-q PCR assay showed that in 4 recurrent HCC patients with increased CD13~+LCSC, the Sox2 and Stat1 among 13 LCSCs-related transcriptional factors were obviously increased. Flow cytometry testing showed that 45℃ treatment also increased the expression of Sox2 and Stat1 m RNA in Hep1-6 cells. Finally, Sox2 and Stat1 could be knockdown by si RNAs, indicating that both Sox2 and Stat1 transcriptional factors were involved in 45℃-induced production of CD13~+LCSCs in Hep1-6 cells.Conclusion In RFA therapy, the use of sublethal temperature of 45℃ can increase CD13~+LCSCs, which is related to the promotion of Sox2 and Stat1 expression. The results of this study can be used for reference in the research of liver cancer recurrence.
引文
[1]左婷婷,郑荣寿,曾红梅,等.中国肝癌发病状况与趋势分析[J].中华肿瘤杂志,2015,37:691-696.
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[10]Yamashita T,Wang XW.Cancer stem cells in the development of liver cancer[J].J Clin Invest,2013,123:1911-1918.
[11]Haraguchi N,Ishii H,Mimori K,et al.CD13 is a therapeutic target in human liver cancer stem cells[J].J Clin Invest,2010,120:3326-3339.
[12]Liu K,Jiang T,Ouyang Y,et al.Nuclear EGFR impairs ASPP2-p53 complex-induced apoptosis by inducing SOS1 expression in hepatocellular carcinoma[J].Oncotarget,2015,6:16507-16516.
[13]Solazzo SA,Ahmed M,Schor-Bardach R,et al.Liposomal doxorubicin increases radiofrequency ablation-induced tumor destruction by increasing cellular oxidative and nitrative stress and accelerating apoptotic pathways[J].Radiology,2010,255:62-74.
[14]Carpino G,Renzi A,Franchitto A,et al.Stem/progenitor cell niches involved in hepatic and biliary regeneration[J].Stem Cells Int,2016,2016:3658013.
[15]Huang P,Qiu J,Li B,et al.Role of Sox2 and Oct4 in predicting survival of hepatocellular carcinoma patients after hepatectomy[J].Clin Biochem,2011,44:582-589.
[16]Yin X,Li YW,Zhang BH,et al.Coexpression of stemness factors Oct4 and nanog predict liver resection[J].Ann Surg Oncol,2012,19:2877-2887.
[17]Pan C,Jia W,Lu B,et al.Expression of TAT recombinant Oct4,Sox2,Lin28,and Nanog proteins from baculovirus-infected Sf9 insect cells[J].Gene,2015,556:245-248.
[18]Torres-Padilla ME,Chambers I.Transcription factor heterogeneity in pluripotent stem cells:a stochastic advantage[J].Development,2014,141:2173-2181.
[19]Calo V,Migliavacca M,Bazan V,et al.STAT proteins:from normal control of cellular events to tumorigenesis[J].J Cell Physiol,2003,197:157-168.
[20]Ishihara J,Umemoto T,Yamato M,et al.Nov/CCN3 regulates long-term repopulating activity of murine hematopoietic stem cells via integrinαvβ3[J].Int J Hematol,2014,99:393-406.
[2]李晓燕,翟博,刘晟.多电极组合穿刺在较大肝癌射频消融中的应用[J].介入放射学杂志,2009,18:348-352.
[3]El-Serag HB.Hepatocellular carcinoma[J].N Engl J Med,2011,365:1118-1127.
[4]Dodd GD 3rd,Soulen MC,Kane RA,et al.Minimally invasive treatment of malignant hepatic tumors:at the threshold of a major breakthrough[J].Radiographics,2000,20:9-27.
[5]Lam VW,Ng KK,Chok KS,et al.Incomplete ablation after radiofrequency ablation of hepatocellular carcinoma:analysis of risk factors and prognostic factors[J].Ann Surg Oncol,2008,15:782-790.
[6]Raut CP,Izzo F,Marra P,et al.Significant long-term survival after radiofrequency ablation of unresectable hepatocellular carcinoma in patients with cirrhosis[J].Ann Surg Oncol,2005,12:616-628.
[7]Kim YS,Rhim H,Cho OK,et al.Intrahepatic recurrence after percutaneous radiofrequency ablation of hepatocellular carcinoma:analysis of the pattern and risk factors[J].Eur J Radiol,2006,59:432-441.
[8]Vivarelli M,Guglielmi A,Ruzzenente A,et al.Surgical resection versus percutaneous radiofrequency ablation in the treatment of hepatocellular carcinoma on cirrhotic liver[J].Ann Surg,2004,240:102-107.
[9]Nguyen LV,Vanner R,Dirks P,et al.Cancer stem cells:an evolving concept[J].Nat Rev Cancer,2012,12:133-143.
[10]Yamashita T,Wang XW.Cancer stem cells in the development of liver cancer[J].J Clin Invest,2013,123:1911-1918.
[11]Haraguchi N,Ishii H,Mimori K,et al.CD13 is a therapeutic target in human liver cancer stem cells[J].J Clin Invest,2010,120:3326-3339.
[12]Liu K,Jiang T,Ouyang Y,et al.Nuclear EGFR impairs ASPP2-p53 complex-induced apoptosis by inducing SOS1 expression in hepatocellular carcinoma[J].Oncotarget,2015,6:16507-16516.
[13]Solazzo SA,Ahmed M,Schor-Bardach R,et al.Liposomal doxorubicin increases radiofrequency ablation-induced tumor destruction by increasing cellular oxidative and nitrative stress and accelerating apoptotic pathways[J].Radiology,2010,255:62-74.
[14]Carpino G,Renzi A,Franchitto A,et al.Stem/progenitor cell niches involved in hepatic and biliary regeneration[J].Stem Cells Int,2016,2016:3658013.
[15]Huang P,Qiu J,Li B,et al.Role of Sox2 and Oct4 in predicting survival of hepatocellular carcinoma patients after hepatectomy[J].Clin Biochem,2011,44:582-589.
[16]Yin X,Li YW,Zhang BH,et al.Coexpression of stemness factors Oct4 and nanog predict liver resection[J].Ann Surg Oncol,2012,19:2877-2887.
[17]Pan C,Jia W,Lu B,et al.Expression of TAT recombinant Oct4,Sox2,Lin28,and Nanog proteins from baculovirus-infected Sf9 insect cells[J].Gene,2015,556:245-248.
[18]Torres-Padilla ME,Chambers I.Transcription factor heterogeneity in pluripotent stem cells:a stochastic advantage[J].Development,2014,141:2173-2181.
[19]Calo V,Migliavacca M,Bazan V,et al.STAT proteins:from normal control of cellular events to tumorigenesis[J].J Cell Physiol,2003,197:157-168.
[20]Ishihara J,Umemoto T,Yamato M,et al.Nov/CCN3 regulates long-term repopulating activity of murine hematopoietic stem cells via integrinαvβ3[J].Int J Hematol,2014,99:393-406.