阻断Notch信号通路对肝癌细胞迁移和COX-2蛋白表达的影响
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摘要
目的探讨阻断Notch信号通路对肝癌细胞迁移的和环氧化酶-2(COX-2)蛋白表达的影响。方法在体外培养肝癌细胞系Hep G2、Huh-7和正常非肿瘤细胞系HL-7702,在Transwell小室检测细胞的迁移能力,对不同肝癌细胞系及正常非肿瘤肝细胞系的侵袭迁移能力进行比较;采用Western Blot的方法检测COX-2、E-cadher-in、Snaill的蛋白表达;采用5μmol/L的γ-分泌酶抑制剂(DAPT)阻断Notch信号通路,通过对肝癌细胞系Hep G2、Huh-7和对照组(空培养基)细胞迁移侵袭的比较得出DAPT和NS-398阻断Notch信号通路对肝癌细胞迁移能力的影响。结果不同肝癌细胞系及正常非肿瘤肝细胞系的侵袭迁移能力比较结果显示,HL-7702细胞系细胞的迁移和侵袭数量分别为(128.23±10.34)、(112.34±9.02)个,低于Hep G2、Huh-7细胞系细胞迁移、侵袭数量,差异有统计学意义(P<0.05);DAPT和NS-398处理对肝癌细胞迁移能力的影响结果显示,加5μmol/L的DAPT后Hep G2的细胞迁移侵袭数量分别为(213.36±9.02)和(197.53±11.27)个,Huh-7的细胞迁移侵袭数量分别为(201.74±11.23)和(179.63±9.26)个,采用50μmol/L NS-398后,Hep G2的细胞迁移侵袭数量分别为(203.26±11.25)和(187.54±11.37)个,Huh-7的细胞迁移侵袭数量分别为(187.63±8.87)和(165.64±8.45)个。与对照组相比,使用DAPT或NS-398处理后的肝癌细胞在Transwell小室中迁移侵袭能力均显著降低,两者比较差异有统计学意义(P<0.05);Hep G2和Huh-7两细胞系在5μmol/L DAPT干预后,COX-2、Snail蛋白表达明显出现下调,而E-cadher-inl表达明显出现上调现象。结论 Notch信号通路在肝癌细胞侵袭迁移过程中起着非常重要的作用,阻断后细胞的迁移能力下降,Notch调控COX-2后其蛋白表达下调,然后调控Snail/E-cadherin的表达,从而改变肿瘤细胞的迁移过程。
Objective To explore effects of blocking Notch signaling pathway on cell migration and expression of COX-2 protein in hepatocellular carcinoma cells.Methods The hepatocellular carcinoma cell line Hep G2,Huh-7 and normal non tumor cell line HL-7702 were in vitro cultured,Transwell cell migration ability of hepatoma cell lines and normal non tumor liver cell line invasion and migration ability were compared.Western blot method was performed for the detection of snail and E-cadherin and cyclooxygenase 2(COX-2),E-cadher-in,Snaillprotein expression;using 5 μmol/L of dapt gamma secretase inhibitor blocking Notch signaling pathway,based on human hepatoma cell line Hep G2,Huh-7 and control group(empty medium) cell migration and invasion of dapt.Results The invasion and migration of hepatoma cell lines and normal non tumor liver cell line comparison results showed that the number of migration and invasion of HL-7702 cells were(128.23 ±10.34),(112.34 ± 9.02),Hep G2 and Huh-7 cell lines,cell migration,invasion was statistically significant compared to the number difference(P < 0.05 DAPT;NS) and the effect of-398 treatment on liver cancer cell migration results showed that adding 5 μmol/L DAPT after Hep G2 cell migration and invasion were(213.36 ± 9.02) and(197.53 ± 11.27),Huh-7 cell migration and invasion were(201.74 ± 11.23) and(179.63 ± 9.26),using 50 μmol/L NS-398,Hep G2 cell migration and invasion were(203.26 ± 11.25) and(187.54 ± 11.37),Huh-7 cell migration and invasion were(187.63 ± 8.87) and(165.64 ± 8.45),compared with the control group,the dapt or ns-398 treatment of hepatoma cells in Transwell migration invasion were significantly lower,and the difference was statistically significant(P < 0.05).Hep G2,Huh-7 cells in 5 μmol/L dapt intervention,snail and COX-2 protein expression was significantly down regulated,and E-cadher-inl expression significantly increased phenomenon.Conclusion Notch signaling pathway in hepatocellular carcinoma cell invasion plays a very important role in the migration process,after the occlusion of the decline in the ability of cell migration.Notch can regulate COX-2 and protein down-regulated,and regulate Snail/E-cadherin expression,so as to change the tumor cell migration.
Objective To explore effects of blocking Notch signaling pathway on cell migration and expression of COX-2 protein in hepatocellular carcinoma cells.Methods The hepatocellular carcinoma cell line Hep G2,Huh-7 and normal non tumor cell line HL-7702 were in vitro cultured,Transwell cell migration ability of hepatoma cell lines and normal non tumor liver cell line invasion and migration ability were compared.Western blot method was performed for the detection of snail and E-cadherin and cyclooxygenase 2(COX-2),E-cadher-in,Snaillprotein expression;using 5 μmol/L of dapt gamma secretase inhibitor blocking Notch signaling pathway,based on human hepatoma cell line Hep G2,Huh-7 and control group(empty medium) cell migration and invasion of dapt.Results The invasion and migration of hepatoma cell lines and normal non tumor liver cell line comparison results showed that the number of migration and invasion of HL-7702 cells were(128.23 ±10.34),(112.34 ± 9.02),Hep G2 and Huh-7 cell lines,cell migration,invasion was statistically significant compared to the number difference(P < 0.05 DAPT;NS) and the effect of-398 treatment on liver cancer cell migration results showed that adding 5 μmol/L DAPT after Hep G2 cell migration and invasion were(213.36 ± 9.02) and(197.53 ± 11.27),Huh-7 cell migration and invasion were(201.74 ± 11.23) and(179.63 ± 9.26),using 50 μmol/L NS-398,Hep G2 cell migration and invasion were(203.26 ± 11.25) and(187.54 ± 11.37),Huh-7 cell migration and invasion were(187.63 ± 8.87) and(165.64 ± 8.45),compared with the control group,the dapt or ns-398 treatment of hepatoma cells in Transwell migration invasion were significantly lower,and the difference was statistically significant(P < 0.05).Hep G2,Huh-7 cells in 5 μmol/L dapt intervention,snail and COX-2 protein expression was significantly down regulated,and E-cadher-inl expression significantly increased phenomenon.Conclusion Notch signaling pathway in hepatocellular carcinoma cell invasion plays a very important role in the migration process,after the occlusion of the decline in the ability of cell migration.Notch can regulate COX-2 and protein down-regulated,and regulate Snail/E-cadherin expression,so as to change the tumor cell migration.
引文
[1]Artavanis-Tsakonas S,Rand MD,Lake RJ.Notch signaling:cell fatecontrol and signal integration in development[J].Science,1999,284(5415):770-776.
[2]肖莉.Notch信号通路与肝脏疾病的关系[J].中外医学研究,2010,8(23):17-19.
[3]刘兆国,朱智杰,周梁,等.Notch信号通路与肿瘤研究[J].中国药理学通报,2012,28(8):1045-1048.
[4]裘铠杰,胡彦华,吴德全,等.Notch信号通路与肿瘤侵袭、转移的研究进展[J].胃肠病学和肝病学杂志,2014,23(11):1362-1364.
[5]李大川,胡彦华,吴德全,等.Notch信号通路与恶性肿瘤侵袭、转移关系的研究进展[J].胃肠病学和肝病学杂志,2015,24(12):1531-1534.
[6]周亮.Notch信号通路参与肝癌细胞侵袭迁移的机制研究[D].西安:第四军医大学,2013.
[7]李蒙,吴玖斌,张培彤,等.E-钙黏蛋白、N-钙黏蛋白及波形蛋白在肿瘤转移中应用的研究进展[J].肿瘤研究与临床,2016,28(2):132-136.
[8]陈劲松,黄炯强,陈锡林,等.上皮钙黏蛋白和Snail在肝细胞癌中的表达及意义[J].中华实验外科杂志,2014,31(2):298-300.
[9]刘静.慢病毒介导shRNA干扰SNAIL表达对肝癌细胞上皮-间质转化以及细胞生长的研究[D].青岛:青岛大学,2014.
[10]Croquelois A,Blindenbacher A,Terracciano L,et al.Induc-ible inactivation of Notch1 causes nodular regenerative hyper-plasia[J].Hepatology,2005,41(3):487-496.
[11]胡广军,刘建中,时玲玲,等.Notch信号通路对肝癌细胞迁移能力及E-cadherin,COX-2表达的影响[J].现代生物医学进展,2017,17(27):5242-5246.
[2]肖莉.Notch信号通路与肝脏疾病的关系[J].中外医学研究,2010,8(23):17-19.
[3]刘兆国,朱智杰,周梁,等.Notch信号通路与肿瘤研究[J].中国药理学通报,2012,28(8):1045-1048.
[4]裘铠杰,胡彦华,吴德全,等.Notch信号通路与肿瘤侵袭、转移的研究进展[J].胃肠病学和肝病学杂志,2014,23(11):1362-1364.
[5]李大川,胡彦华,吴德全,等.Notch信号通路与恶性肿瘤侵袭、转移关系的研究进展[J].胃肠病学和肝病学杂志,2015,24(12):1531-1534.
[6]周亮.Notch信号通路参与肝癌细胞侵袭迁移的机制研究[D].西安:第四军医大学,2013.
[7]李蒙,吴玖斌,张培彤,等.E-钙黏蛋白、N-钙黏蛋白及波形蛋白在肿瘤转移中应用的研究进展[J].肿瘤研究与临床,2016,28(2):132-136.
[8]陈劲松,黄炯强,陈锡林,等.上皮钙黏蛋白和Snail在肝细胞癌中的表达及意义[J].中华实验外科杂志,2014,31(2):298-300.
[9]刘静.慢病毒介导shRNA干扰SNAIL表达对肝癌细胞上皮-间质转化以及细胞生长的研究[D].青岛:青岛大学,2014.
[10]Croquelois A,Blindenbacher A,Terracciano L,et al.Induc-ible inactivation of Notch1 causes nodular regenerative hyper-plasia[J].Hepatology,2005,41(3):487-496.
[11]胡广军,刘建中,时玲玲,等.Notch信号通路对肝癌细胞迁移能力及E-cadherin,COX-2表达的影响[J].现代生物医学进展,2017,17(27):5242-5246.