华支睾吸虫分泌排泄蛋白对肝癌细胞系HepG2细胞增殖、迁移和上皮间质转化的影响
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摘要
目的探讨华支睾吸虫分泌排泄蛋白(CsESP)对肝癌细胞株HepG2增殖、迁移和上皮间质转化(EMT)的影响。方法采用CCK-8实验测定不同浓度的CsESP对肝癌细胞HepG2的活力、细胞增殖能力和细胞毒性作用的影响;细胞划痕实验检测在不同浓度(1、10、20、50、100和200μg/mL)CsESP的作用下(设PBS对照组),HepG2细胞迁移的变化;荧光定量PCR法(RT-qPCR)检测HepG2细胞的癌变相关因子(MMP2和p300)以及EMT基因如E-钙黏蛋白E(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和α-连环蛋白(α-catenin)mRNA的表达水平。结果 CCK-8实验结果显示,在CsESP浓度为10和20μg/mL时,细胞增殖明显,当CsESP浓度超过20μg/mL时,细胞活力降低。细胞划痕实验结果显示,在不同浓度CsESP的作用下,在0、24、48、72 h 4个时间点分别采集照片,并对其相对迁移面积进行统计分析,发现24和48 h 2个时间点,10和20μg/mL组的迁移能力明显高于PBS组和高浓度(50μg/mL)组,差异有统计学意义(P<0.05)。RT-qPCR检测出癌变基因MMP2以及N-cadherin、Vimentin、α-catenin的mRNA水平高于PBS组,差异有统计学意义(P<0.05),而癌变因子p300和E-cadherin水平并无明显变化。结论在CsESP的最适作用浓度下,CsESP可促进癌细胞增殖、癌变、迁移和上皮间质转化。
Objective Explore the effect of Clonorchis sinensis excretory/secretory proteins(CsESP) on the proliferation,carcinogenesis and epithelial-mesenchymal transition of hepatocarcinoma HepG2 cells. Methods HepG2 cells were treated with different concentrations of CsESP( 1, 10,20,50, 100 and 200 μg/mL). The proliferation of HepG2 cells was determined by CCK-8 assay. The migration ability of HepG2 cells was assessed through the wound-healing assay. The relative mRN A levels of MMP2, p300, E-cadherin, N-cadherin, Vimentin, α-catenin were determined by RT-qPCR. Results The CsESP could significantly promote HepG2 cell proliferation and the optimal concentration was 10 and 20 μg/mL. The vitality of HepG2 cells was significantly reduced when the CsESP concentration exceeded 20 μg/mL. The wound-healing assay showed that 10 and 20μg/mL CsESP could obviously promote HepG2 cells healing in different concentrations of CsESP at 24 h, 48 h, post-treatment.The mRNA levels of MMP2, N-cadherin, Vimentin, α-catenin were increased in the experimental groups compared with the PBS group(P<0.05); however, the p300 and E-cadherin level were not increased significantly(P>0.05). Conclusion CsESP could promote HepG2 cell proliferation, carcinogenesis, migration, and epithelial-mesenchymal transition.
Objective Explore the effect of Clonorchis sinensis excretory/secretory proteins(CsESP) on the proliferation,carcinogenesis and epithelial-mesenchymal transition of hepatocarcinoma HepG2 cells. Methods HepG2 cells were treated with different concentrations of CsESP( 1, 10,20,50, 100 and 200 μg/mL). The proliferation of HepG2 cells was determined by CCK-8 assay. The migration ability of HepG2 cells was assessed through the wound-healing assay. The relative mRN A levels of MMP2, p300, E-cadherin, N-cadherin, Vimentin, α-catenin were determined by RT-qPCR. Results The CsESP could significantly promote HepG2 cell proliferation and the optimal concentration was 10 and 20 μg/mL. The vitality of HepG2 cells was significantly reduced when the CsESP concentration exceeded 20 μg/mL. The wound-healing assay showed that 10 and 20μg/mL CsESP could obviously promote HepG2 cells healing in different concentrations of CsESP at 24 h, 48 h, post-treatment.The mRNA levels of MMP2, N-cadherin, Vimentin, α-catenin were increased in the experimental groups compared with the PBS group(P<0.05); however, the p300 and E-cadherin level were not increased significantly(P>0.05). Conclusion CsESP could promote HepG2 cell proliferation, carcinogenesis, migration, and epithelial-mesenchymal transition.
引文
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[24]Lu K,Dong JL,Fan WJ.Twist1/2 activates MMP2 expression via binding to its promoter in colorectal cancer[J].Eur Rev Med Pharmacol Sci,2018,22(23):8210-8219.
[25]Zhang Z,Zheng F,Yu Z,et al.XRCC5 cooperates with p300 to promote cyclooxygenase-2 expression and tumor growth in colon cancers[J].PLoS One,2017,12(10):e0186900.
[26]Liang Y,Wu Y,Chen X,et al.A novel long noncoding RNAlinc00460 up-regulated by CBP/P300 promotes carcinogenesis in esophageal squamous cell carcinoma[J].Biosci Rep,2017,37(5).pii:BSR20171019.
[2]Li T,He S,Zhao H,et al.Major trends in human parasitic diseases in China[J].Trends Parasitol,2010,26(5):264-270.
[3]Keiser J,Utzinger J.Food-borne trematodiases[J].Clin Microbiol Rev,2009,22(3):466-483.
[4]Kim TS,Pak JH,Kim JB,et al.Clonorchis sinensis,an oriental liver fluke,as a human biological agent of cholangiocarcinoma:a brief review[J].BMB Rep,2016,49(11):590-597.
[5]Pak JH,Moon JH,Hwang SJ,et al.Proteomic analysis of differentially expressed proteins in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products[J].J Cell Biochem,2009,108(6):1376-1388.
[6]Wang X,Hu F,Hu X,et al.Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells[J].Parasitol Res,2014,113(8):3063-3071.
[7]张淑芹.原发性肝癌721例流行病学分析[A].全国疑难及重症肝病攻关协作组、吴阶平医学基金会肝病医学部、中华医学会肝病学分会重肝与人工肝学组.第7届全国疑难及重症肝病大会论文集[C].全国疑难及重症肝病攻关协作组、吴阶平医学基金会肝病医学部、中华医学会肝病学分会重肝与人工肝学组:全国重型肝病及人工肝血液净化攻关协作组,2013.
[8]Erkekoglu P,Oral D,Chao MW,et al.Hepatocellular carcinoma and possible chemical and biological causes:a review[J].JEnviron Pathol Toxicol Oncol,2017,36(2):171-190.
[9]Gos M,Mi?oszewska J,Przybyszewska M.Epithelial-mesenchymal transition in cancer progression[J].Postepy Biochem,2009,55(2):121-128..
[10]Christiansen JJ,Rajasekaran AK.Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis[J].Cancer Res,2006,66(17):8319-8326.
[11]Tse JC,Kalluri R.Mechanisms of metastasis:epithelial-tomesenchymal transition and contribution of tumor microenvironment[J].J Cell Biochem,2007,101(4):816-829.
[12]Christiansen JJ,Rajasekaran AK.Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis[J].Cancer Res,2006,66(17):8319-8326.
[13]汤昕,王彩琴,周心怡,黄艳,余新炳.华支睾吸虫分泌排泄蛋白对肝癌细胞株PLC增殖、凋亡及细胞周期的影响[J].热带医学杂志,2017,17(4):435-438.
[14]Li W,Dong H,Huang Y,et al.Clonorchis sinensis co-infection could affect the disease state and treatment response of HBVpatients[J].PLoS Negl Trop Dis,2016,10(6):e0004806.
[15]Saiki I.Cell adhesion molecules and cancer metastasis[J].Jpn JPharmacol,1997,75(3):215-242.
[16]Tse JC,Kalluri R.Mechanisms of metastasis:epithelial-tomesenchymal transition and contribution of tumor microenvironment[J].J Cell Biochem,2007,101(4):816-829.
[17]Nieman MT,Prudoff RS,Johnson KR,et al.N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression[J].J Cell Biol,1999,147(3):631-644.
[18]Hazan RB,Phillips GR,Qiao RF,et al.Exogenous expression of N-cadherin in breast cancer cells induces cell migration,invasion,and metastasis[J].J Cell Biol,2000,148(4):779-790.
[19]Shiozaki H,Oka H,Inoue M,et al.E-cadherin mediated adhesion system in cancer cells[J].Cancer,1996,77(8 Suppl):1605-1613.
[20]Bek S,Kemler R.Protein kinase CKII regulates the interaction ofβ-catenin withα-catenin and its protein stability[J].J Cell Sci,2002,115(Pt 24):4743-4753.
[21]Zhang XG,Lu XF,Jiao XM,et al.PLK1 gene suppresses cell invasion of undifferentiated thyroid carcinoma through the inhibition of CD44v6,MMP-2 and MMP-9[J].Exp Ther Med,2012,4(6):1005-1009.
[22]No?l A,Jost M,Maquoi E.Matrix metalloproteinases at cancer tumor-host interface[C].2008.
[23]Hangai M,Kitaya N,Xu J,et al.Matrix metalloproteinase-9-dependent exposure of a cryptic migratory control site in collagen is required before retinal angiogenesis[J].Am J Pathol,2002,161(4):1429-1437.
[24]Lu K,Dong JL,Fan WJ.Twist1/2 activates MMP2 expression via binding to its promoter in colorectal cancer[J].Eur Rev Med Pharmacol Sci,2018,22(23):8210-8219.
[25]Zhang Z,Zheng F,Yu Z,et al.XRCC5 cooperates with p300 to promote cyclooxygenase-2 expression and tumor growth in colon cancers[J].PLoS One,2017,12(10):e0186900.
[26]Liang Y,Wu Y,Chen X,et al.A novel long noncoding RNAlinc00460 up-regulated by CBP/P300 promotes carcinogenesis in esophageal squamous cell carcinoma[J].Biosci Rep,2017,37(5).pii:BSR20171019.