CNOT7基因敲减通过减少HepG2细胞TGF-β1分泌影响免疫微环境
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摘要
目的研究人CCR4-NOT转录复合体亚基7 (CNOT7)基因敲减对肝母细胞瘤细胞系HepG2免疫微环境的影响并探讨其意义。方法设计细胞转染方案,将实验分为3组:靶向敲减CNOT7组、阴性对照组和过表达CNOT7组,获得各组细胞后,用倒置荧光显微镜观察细胞转染效率。Western blotting方法检测CNOT7、转化生长因子-β1 (TGF-β1)及核转录因子-κB (NF-κB) p65蛋白的表达水平,ELISA法测定细胞培养上清液中TGF-β1水平。流式细胞术检测转染后肿瘤细胞对自然杀伤(NK)细胞杀伤功能的敏感性。结果与阴性对照组相比,靶向敲减CNOT7组TGF-β1和NF-κB p65蛋白表达水平显著降低,过表达CNOT7组TGF-β1和NF-κB p65蛋白表达水平显著升高;靶向敲减CNOT7组细胞培养上清液中TGF-β1水平显著降低,过表达CNOT7组TGF-β1水平显著升高;NK细胞与肿瘤细胞共培养,靶向敲减CNOT7组细胞凋亡率显著升高,过表达CNOT7组细胞凋亡率显著降低。结论CNOT7基因参与肝细胞癌免疫微环境的形成,靶向敲减CNOT7基因可减少TGF-β1分泌,增强NK细胞对肝癌细胞的杀伤功能。
Objective To study the effect of human CCR4-NOT transcription complex subunit 7(CNOT7) gene knockdown on the immune microenvironment of HepG2 cells and explore its significance. Methods We designed a cell transfection protocol and performed the experiment with three groups:CNOT7-targeted knockdown group,control group,and CNOT7 overexpression group. The transfection efficiency was assessed using inverted fluorescence microscopy,and the expression level of CNOT7,transforming growth factor-β1(TGF-β1),and nuclear factor-kappa B(NF-κB) p65 proteins was determined by Western blotting. The concentration of TGF-β1 secreted in the cell culture supernatant was measured by ELISA. The sensitivity of tumor cells to the killing function of natural killer(NK) cells was detected by flow cytometry. Results Compared with the control group,the expression level of TGF-β1 and NF-κB p65 proteins was significantly decreased in the CNOT7-targeted knockdown group,and the TGF-β1 concentration in the culture supernatant was also significantly reduced. However,in the CNOT7 overexpression group,the expression level of the two proteins and TGF-β1 concentration were significantly increased. NK cells were co-cultured with tumor cells,and the apoptosis rate of HepG2 cells transfected with CNOT7-specific shRNA was significantly increased. However,in the CNOT7 overexpression group,the apoptosis rate was significantly decreased. Conclusion CNOT7 forms the immune microenvironment of hepatocellular carcinoma. Targeted knockdown of CNOT7 can reduce TGF-β1 secretion and enhance the killing function of NK cells toward HepG2 cells.
Objective To study the effect of human CCR4-NOT transcription complex subunit 7(CNOT7) gene knockdown on the immune microenvironment of HepG2 cells and explore its significance. Methods We designed a cell transfection protocol and performed the experiment with three groups:CNOT7-targeted knockdown group,control group,and CNOT7 overexpression group. The transfection efficiency was assessed using inverted fluorescence microscopy,and the expression level of CNOT7,transforming growth factor-β1(TGF-β1),and nuclear factor-kappa B(NF-κB) p65 proteins was determined by Western blotting. The concentration of TGF-β1 secreted in the cell culture supernatant was measured by ELISA. The sensitivity of tumor cells to the killing function of natural killer(NK) cells was detected by flow cytometry. Results Compared with the control group,the expression level of TGF-β1 and NF-κB p65 proteins was significantly decreased in the CNOT7-targeted knockdown group,and the TGF-β1 concentration in the culture supernatant was also significantly reduced. However,in the CNOT7 overexpression group,the expression level of the two proteins and TGF-β1 concentration were significantly increased. NK cells were co-cultured with tumor cells,and the apoptosis rate of HepG2 cells transfected with CNOT7-specific shRNA was significantly increased. However,in the CNOT7 overexpression group,the apoptosis rate was significantly decreased. Conclusion CNOT7 forms the immune microenvironment of hepatocellular carcinoma. Targeted knockdown of CNOT7 can reduce TGF-β1 secretion and enhance the killing function of NK cells toward HepG2 cells.
引文
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[2]中华人民共和国卫生和计划生育委员会医政医管局.原发性肝癌诊疗规范(2017年版)[J].中华消化外科杂志,2017,16(7):635-647.DOI:10.3760/cma.j.issn.1673-9752.2017.07.001.
[3]COHEN GS,BLACK M.Multidisciplinary management of hepatocellular carcinoma:a model for therapy[J].J Multidiscip Health,2013,6:189-195.DOI:10.2147/JMDH.S41206.
[4]BARTLAM M,YAMAMOTO T.The structural basis for deadenylation by the CCR4-NOT complex[J].Protein Cell,2010,1(5):443-452.DOI:10.1007/s13238-010-0060-8.
[5]CHAPAT C,KOLYTCHEFF C,LE ROMANCER M,et al.hCAF1/CNOT7 regulates interferon signalling by targeting STAT1[J].EMBOJ,2013,32(5):688-700.DOI:10.1038/emboj.2013.11.
[6]赵雷,任崇仁,贺杰峰,等.CNOT7基因参与诱导HepG2细胞对Vγ9Vδ2T细胞的免疫耐受[J].中华普通外科杂志,2017,32(1):57-60.DOI:10.3760/cma.j.issn.1007-631X.2017.01.018.
[7]HERNANDEZ-GEA V,TOFFANIN S,FRIEDMAN SL,et al.Role of the microenvironment in the pathogenesis and treatment of hepatocellular carcinoma[J].Gastroenterology,2013,144(3):512-527.DOI:10.1053/j.gastro.2013.01.002.
[8]SUN X,ZHANG J,HOU Z,et al.miR-146a is directly regulated by STAT3 in human hepatocellular carcinoma cells and involved in anti-tumor immune suppression[J].Cell Cycle,2015,14(2):243-252.DOI:10.4161/15384101.2014.977112.
[9]GIANNELLI G,MIKULITS W,DOOLEY S,et al.The rationale for targeting TGF-βin chronic liver diseases[J].Eur J Clin Invest,2016,46(4):349-361.DOI:10.1111/eci.12596.
[10]YU M,LI Z.Natural killer cells in hepatocellular carcinoma:current status and perspectives for future immunotherapeutic approaches[J].Front Med,2017,11(4):509-521.DOI:10.1007/s11684-017-0546-3.
[11]LI Z,ZHANG LJ,ZHANG HR,et al.Tumor-derived transforming growth factor-βis critical for tumor progression and evasion of immune surveillance[J].Asian Pac J Cancer Prev,2014,15(13):5181-5186.
[12]ROJAS A,ZHANG P,WANG Y,et al.A positive TGF-β/c-KITfeedback loop drives tumor progression in advanced primary liver cancer[J].Neoplasia,2016,18(6):371-386.DOI:10.1016/j.neo.2016.04.002.
[13]MASSAGU J.TGFbeta in cancer[J].Cell,2008,134(2):215-230.DOI:10.1016/j.cell.2008.07.001.