MICA基因多态性与乳腺癌细胞对NK细胞杀伤的敏感性
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摘要
目的:探讨MHC-Ⅰ类链相关分子A(MHC class I chain-related molecule A,MICA)多态性与乳腺癌细胞对NK细胞杀伤敏感性的关系。方法:测序分析乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-435S和SK-BR-3的MICA等位基因,用Western blotting和流式细胞术检测MICA重组表达载体转染293T细胞(分别命名为p MCFA5.1、p MCFA4、p231A5R、p231A9和p435A5P)MICA蛋白的表达水平,用LDH法检测NK细胞对转染MICA的293T细胞的杀伤活性,酶联免疫斑点法检测NK细胞穿孔素、颗粒酶B分泌水平。结果:MCF-7细胞表达MICA*008/A5.1和MICA*001/A4,MDA-MB-231和SK-BR-3细胞均表达MICA*019/A5和MICA*002/A9,MDA-MB-435S细胞表达MICA*010/A5。转染MICA后,p MCFA5.1组293T细胞MICA蛋白的表达水平最低(P<0.05),p435A5P组次之(P<0.05),p MCFA4组、p231A5R组和p231A9组表达水平较高(均P<0.05)。NK细胞对转染MICA的293T细胞杀伤活性及穿孔素、颗粒酶B分泌:p435A5P组对NK细胞杀伤的敏感性最低(P<0.05),穿孔素、颗粒酶B分泌水平最低(均P<0.05);p MCFA5.1、p MCFA4、p231A5R和p231A9各组之间比较差异无统计学意义(P>0.05)。结论:MICA基因多态性与乳腺癌细胞对NK细胞杀伤的敏感性密切相关。
Objective:To investigate the associations of MHC class I chain-related molecule A(MICA)gene polymorphism with susceptibility of breast cancer cells to NK cell-mediated cytotoxicity.Methods:MICA alleles of breast cancer cell lines(MCF-7,MDAMB-231,MDA-MB-435 S and SK-BR-3)were analyzed by DNA sequencing.MICA protein expression in 293 T cells transfected with MICA recombinant expression vectors(namely p MCFA5.1,p MCFA4,p231 A5 R,p231 A9 and p435 A5 P) was detected by Western blotting and Flow cytometry;the cytotoxicity of NK cells against the above 293 T cells were measured by lactate dehydrogenase(LDH)assay and the release of perforin(PFN)and granzymes B(Gzm B)were measured by ELISPOT assay.Results:DNA sequencing result showed that MICA*008/A5.1 and MICA*001/A4 were expressed in MCF-7 cells,MICA*019/A5 and MICA*002/A9 were expressed in both MDA-MB-231 and SK-BR-3 cells while MICA*010/A5 was expressed in MDA-MB-435 S cells.After the MICA recombinant expression vectors were transfected into 293 T cells,the level of MICA were the lowest in p MCFA5.1 group(P<0.05),following after p435 A5 P group(P<0.05),and highly expressed in the p MCFA4,p231 AR and p231 A9 groups(P<0.05).NK cell-mediated cytotoxicity and the release of PFN and Gzm B:NK cell-mediated cytotoxicity were the lowest in p435 A5 P group(P<0.05),and the ability of inducing NK cells to release PFN and Gzm B was also the lowest in p435 A5 P group(P<0.05),but there were no statistical difference among the other transfected groups(P>0.05).Conclusion:MICA gene polymorphism is closely associated with susceptibility of breast cancer cells to NK cell-mediated cytotoxicity.
Objective:To investigate the associations of MHC class I chain-related molecule A(MICA)gene polymorphism with susceptibility of breast cancer cells to NK cell-mediated cytotoxicity.Methods:MICA alleles of breast cancer cell lines(MCF-7,MDAMB-231,MDA-MB-435 S and SK-BR-3)were analyzed by DNA sequencing.MICA protein expression in 293 T cells transfected with MICA recombinant expression vectors(namely p MCFA5.1,p MCFA4,p231 A5 R,p231 A9 and p435 A5 P) was detected by Western blotting and Flow cytometry;the cytotoxicity of NK cells against the above 293 T cells were measured by lactate dehydrogenase(LDH)assay and the release of perforin(PFN)and granzymes B(Gzm B)were measured by ELISPOT assay.Results:DNA sequencing result showed that MICA*008/A5.1 and MICA*001/A4 were expressed in MCF-7 cells,MICA*019/A5 and MICA*002/A9 were expressed in both MDA-MB-231 and SK-BR-3 cells while MICA*010/A5 was expressed in MDA-MB-435 S cells.After the MICA recombinant expression vectors were transfected into 293 T cells,the level of MICA were the lowest in p MCFA5.1 group(P<0.05),following after p435 A5 P group(P<0.05),and highly expressed in the p MCFA4,p231 AR and p231 A9 groups(P<0.05).NK cell-mediated cytotoxicity and the release of PFN and Gzm B:NK cell-mediated cytotoxicity were the lowest in p435 A5 P group(P<0.05),and the ability of inducing NK cells to release PFN and Gzm B was also the lowest in p435 A5 P group(P<0.05),but there were no statistical difference among the other transfected groups(P>0.05).Conclusion:MICA gene polymorphism is closely associated with susceptibility of breast cancer cells to NK cell-mediated cytotoxicity.
引文
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[11]YAMANEGI K,YAMANE J,KOBAYASHI K,et al.Sodium valproate,a histone deacetylase inhibitor,augments the expression of cell-surface NKG2D ligands,MICA/B,without increasing their soluble forms to enhance susceptibility of human osteosarcoma cells to NK cell-mediated cytotoxicity[J/OL].Oncol Rep,2010,24(6):1621-1627[2017-05-02].http://www.spandidos-publications.com/or/.DOI:10.3892/or_00001026.
[12]CAMPILLO J A,LOPEZ-HERNANDEZ R,MARTINEZ-BANA-CLOCHA H,et al.MHC class I chain-related gene a diversity in patients with cutaneous malignant melanoma from southeastern Spain[J/OL].Dis Markers,2015,2015:831864[2017-05-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370202/.DOI:10.1155/2015/831864.
[13]COX S T,MADRIGAL J A,SAUDEMONT A.Diversity and characterization of polymorphic 5'promoter haplotypes of MICA and MICB genes[J/OL].Tissue Antigens,2014,84(3):293-303[2017-05-02].http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1399-0039.DOI:10.1111/tan.12400.
[14]张丽平,周智锋,李洁羽,等.调节性T细胞对NK细胞体外杀伤乳腺癌细胞的影响[J].免疫学杂志,2010,26(1):58-62.DOI:10.13431/j.cnki.immunol.j.20100014.
[15]叶韵斌,周智锋,郑蕊蕊,等.KIR不相合的NK细胞对乳腺癌细胞的体外杀伤作用[J].中国肿瘤生物治疗杂志,2007,14(1):26-30.DOI:10.3872/j.issn.1007-385X.2007.01.006.
[2]WANG J,LI C,YANG D,et al.Clinico-pathological significance of MHC-I type chain-associated protein A expression in oral squamous cell carcinoma[J/OL].Asian Pac J Cancer Prev,2012,13(2):715-718[2017-05-02].http://journal.waocp.org/article_26210_657a 546 deac420d734cff54bfd0d9f65.pdf.DOI:10.7314/APJCP.2012.13.2.715.
[3]CHAUVEAU A,AUCHER A,EISSMANN P,et al.Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells[J/OL].Proc Natl Acad Sci U S A,2010,107(12):5545-5550[2017-05-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851811/.DOI:10.1073/pnas.0910074107.
[4]RAACHE R,BELANTEUR K,AMROUN H,et al.Association of major histocompatibility complex class 1 chain-related gene a dimorphism with type 1 diabetes and latent autoimmune diabetes in adults in the Algerian population[J/OL].Clin Vaccine Immunol,2012,19(4):557-561[2017-05-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318287/.DOI:10.1128/CVI.05473-11.
[5]GARCíA G,DEL PUERTO F,PéREZ A B,et al.Association of MI-CA and MICB alleles with symptomatic dengue infection[J].Hum Immunol,2011,72(10):904-907.DOI:10.1016/j.humimm.2011.06.010.
[6]CHO W K,JUNG M H,PARK S H,et al.Association of MICA alleles with autoimmune thyroid disease in Korean children[J/OL].Int J Endocrinol,2012:235680[2017-05-20].http://www.hindawi.com/journals/ije/2012/235680.DOI:10.1155/2012/235680.
[7]周智锋,柳硕岩,郑庆丰,等.NKG2D配体在中晚期食管癌患者术后NK细胞免疫治疗中的作用[J].中国肿瘤临床,2013,40(22):1373-1377.DOI:10.3969/j.issn.1000-8179.20130898.
[8]IBANA J A,AIYAR A,QUAYLE A J,et al.Modulation of MICAon the surface of Chlamydia trachomatis-infected endocervical epithelial cells promotes NK cell-mediated killing[J/OL].FEMS Immunol Med Microbiol,2012,65(1):32-42[2017-05-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029121/.DOI:10.1111/j.1574-695X.2012.00930.x.
[9]RAULET D H.Roles of the NKG2D immunoreceptor and its ligands[J].Nat Rev Immunol,2003,3(10):781-790.DOI:10.1038/nri1199.
[10]王新利,李卿,蒋涛,等.NK细胞杀瘤活性与肿瘤细胞表面MI-CA/B表达的相关性研究[J].中国医科大学学报,2010,39(2):95-97.
[11]YAMANEGI K,YAMANE J,KOBAYASHI K,et al.Sodium valproate,a histone deacetylase inhibitor,augments the expression of cell-surface NKG2D ligands,MICA/B,without increasing their soluble forms to enhance susceptibility of human osteosarcoma cells to NK cell-mediated cytotoxicity[J/OL].Oncol Rep,2010,24(6):1621-1627[2017-05-02].http://www.spandidos-publications.com/or/.DOI:10.3892/or_00001026.
[12]CAMPILLO J A,LOPEZ-HERNANDEZ R,MARTINEZ-BANA-CLOCHA H,et al.MHC class I chain-related gene a diversity in patients with cutaneous malignant melanoma from southeastern Spain[J/OL].Dis Markers,2015,2015:831864[2017-05-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370202/.DOI:10.1155/2015/831864.
[13]COX S T,MADRIGAL J A,SAUDEMONT A.Diversity and characterization of polymorphic 5'promoter haplotypes of MICA and MICB genes[J/OL].Tissue Antigens,2014,84(3):293-303[2017-05-02].http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1399-0039.DOI:10.1111/tan.12400.
[14]张丽平,周智锋,李洁羽,等.调节性T细胞对NK细胞体外杀伤乳腺癌细胞的影响[J].免疫学杂志,2010,26(1):58-62.DOI:10.13431/j.cnki.immunol.j.20100014.
[15]叶韵斌,周智锋,郑蕊蕊,等.KIR不相合的NK细胞对乳腺癌细胞的体外杀伤作用[J].中国肿瘤生物治疗杂志,2007,14(1):26-30.DOI:10.3872/j.issn.1007-385X.2007.01.006.