GRP78异常O-糖基化对乳腺癌细胞侵袭迁移的影响

doi:10.3969/j.issn.1000-1565.2018.01.011

摘要/Abstract

摘要： 为了探索O-糖基化异常对GRP78促进人乳腺癌MCF-7细胞侵袭转移能力的影响,克隆人GRP78基因,利用点突变技术,构建了GRP78野生型及T648A、T648G 2个糖基化位点突变真核表达载体.Western blot检测外源性GRP78在MCF-7细胞中的表达.利用划痕修复实验和transwell侵袭小室实验,研究GRP78第648位Thr突变后引起的异常糖基化对其生物学功能的影响.划痕实验结果显示与野生型相比,转染突变体的MCF-7细胞的覆盖面积较少.Transwell检测结果显示突变体转染组的侵袭细胞数少于对照组,说明GRP78 O-糖基化缺失后明显降低了其促进MCF-7细胞侵袭迁移的能力.本研究为深入探讨O-糖基化在介导GRP78生物学功能中的作用机制奠定了基础.

关键词: 葡萄糖调节蛋白78(GRP78), 异常O-糖基化, 定点突变

Abstract: To investigate the effect of aberrant O-glycosylation of GRP78 on invasive and metastatic ability of breast cancer cell line MCF-7, Human GRP78 cDNA was cloned and O-linked glycosylation site of GRP78 was mutated by site-directed mutagenesis.The expressions of exogenous wild-type and mutant GRP78 in MCF-7 were confirmed by Western blot.Wound repair assays and transwell assays were performed to study the effect of GRP78 mutants on its biological functions.Wound repair assays showed that the ability of cell migration in MCF-7 cells transfected with mutant GRP78 was significantly decreased compared to that of the cells transfected with wild-type GRP78.Transwell assay showed that the invading cell number of mutant group was less than that of cells transfected with wild-type GRP78,which suggested that the deficiency in O-glycosylation of GRP-78 abolished its ability of cell invasion and migration.These findings laid a good foundation for further study on the effect of O-glycosylation on the biological functions of GRP78.- DOI:10.3969/j.issn.1000-1565.2018.01.011GRP78异常O-糖基化对乳腺癌细胞侵袭迁移的影响刘洋,谢蓉,王秀敏,赵丽娜,王茜,吴琛(河北大学生命科学学院,河北保定 071002)摘要:为了探索O-糖基化异常对GRP78促进人乳腺癌MCF-7细胞侵袭转移能力的影响,克隆人GRP78基因,利用点突变技术,构建了GRP78野生型及T648A、T648G 2个糖基化位点突变真核表达载体.Western blot检测外源性GRP78在MCF-7细胞中的表达.利用划痕修复实验和transwell侵袭小室实验,研究GRP78第648位Thr突变后引起的异常糖基化对其生物学功能的影响.划痕实验结果显示与野生型相比,转染突变体的MCF-7细胞的覆盖面积较少.Transwell检测结果显示突变体转染组的侵袭细胞数少于对照组,说明GRP78 O-糖基化缺失后明显降低了其促进MCF-7细胞侵袭迁移的能力.本研究为深入探讨O-糖基化在介导GRP78生物学功能中的作用机制奠定了基础.关键词:葡萄糖调节蛋白78(GRP78);异常O-糖基化;定点突变中图分类号:Q78 文献标志码:A 文章编号:1000-1565(2018)01-0065-08Effect of aberrant O-glycosylation of GRP78 on invasiveand metastatic ability of breast cancer cell line LIU Yang, XIE Rong, WANG Xiumin, ZHAO Lina, WANG Xi, WU Chen(College of Life Sciences, Hebei University, Baoding 071002, China)Abstract: To investigate the effect of aberrant O-glycosylation of GRP78 on invasive and metastatic ability of breast cancer cell line MCF-7, Human GRP78 cDNA was cloned and O-linked glycosylation site of GRP78 was mutated by site-directed mutagenesis.The expressions of exogenous wild-type and mutant GRP78 in MCF-7 were confirmed by Western blot.Wound repair assays and transwell assays were performed to study the effect of GRP78 mutants on its biological functions.Wound repair assays showed that the ability of cell migration in MCF-7 cells transfected with mutant GRP78 was significantly decreased compared to that of the cells transfected with wild-type GRP78.Transwell assay showed that the invading cell number of mutant group was less than that of cells transfected with wild-type GRP78,which suggested that the deficiency in O-glycosylation of GRP-78 abolished its ability of cell invasion and migration.These findings laid a good foundation for further study on the effect of O-glycosylation on the biological functions of GRP78.- 收稿日期:2017-08-02 基金项目:国家自然科学基金资助项目(31670812);河北省高层次人才资助项目(CY201602);河北大学研究生创新资助项目(X201724) 第一作者:刘洋(1988—),男,河北秦皇岛人,河北大学在读硕士研究生.E-mail:dra04@sina.com 通信作者:吴琛(1977—),女,河北保定人,河北大学教授,博士,主要从事肿瘤分子生物学研究.E-mail:dawnwuchen@l63.com第1期刘洋等:GRP78异常O-糖基化对乳腺癌细胞侵袭迁移的影响

Key words: glucose regulated protein 78(GRP78), abnormal O-glycosylation, site-directed mutagenesis

中图分类号:

刘洋,谢蓉,王秀敏,赵丽娜,王茜,吴琛. GRP78异常O-糖基化对乳腺癌细胞侵袭迁移的影响[J]. 河北大学学报(自然科学版), 2018, 38(1): 65-72.

LIU Yang, XIE Rong, WANG Xiumin, ZHAO Lina, WANG Xi, WU Chen. Effect of aberrant O-glycosylation of GRP78 on invasive and metastatic ability of breast cancer cell line[J]. Journal of Hebei University (Natural Science Edition), 2018, 38(1): 65-72.

参考文献

[1] PEGRAM M D, BORGES V F, IBRAHIM N, et al.Phase I dose escalation pharmacokinetic assessment of intravenous humanized anti-MUC1 antibody AS1402 in patients with advanced breast cancer[J].Breast Cancer Res, 2009, 11: R73.DOI: 10.1186/bcr2409.
[2] BURCHELL J M, MUNGUL A, TAYLOR-PAPADIMITRIOU J.O-Linked glycosylation in the mammary gland: changes that occur during malignancy[J].J Mammary Gland Biol Neoplasia, 2001,6(3):355-364.DOI: 10.1023/A:1011331809881.
[3] HENDERSHOT L M.The ER function BiP is a master regulator of ER function[J].Mt Sinai J Med, 2004, 71(5): 289-297.
[4] LEE E, NICHOLS P, SPICER D, et al.GRP78 as a novel predictor of responsiveness to chemotherapy in breast cancer[J].Cancer Res, 2006, 66(16): 7849-7853.DOI: 10.1158/0008-5472.CAN-06-1660.
[5] SU R, LI Z, LI H, et al.Grp78 promotes the invasion of hepatocellular carcinoma[J].BMC Cancer, 2010, 10: 20.DOI: 10.1186/1471-2407-10-20.
[6] URAMOTO H, SUGIO K, OYAMA T, et al.Expression of endoplasmic reticulum molecular chaperone Grp78 in human lung cancer and its clinical significance[J].Lung Cancer, 2005, 49(1): 55-62.DOI: 10.1016/j.lungcan.2004.12.011.
[7] FERNANDEZ P M, TABBARA S O, JACOBS L K, et al.Overexpression of the glucose-regulated stress gene GRP78 in malignant but not benign human breast lesions[J].Breast Cancer Res Treat, 2000, 59(1): 15-26.DOI: 10.1023/A:1006332011207.
[8] DELPINO A, CASTELLI M.The 78 kDa glucose-regulated protein(GRP78/BIP)is expressed on the cell membrane, is released into cell culture medium and is also present in human peripheral circulation [J].Biosci Rep, 2002, 22(3-4): 407-420.DOI: 10.1023/A:1020966008615.
[9] MISRA U K, GONZALEZ-GRONOW M, GAWDI G, et al.The role of Grp 78 in alpha 2-macroglobulin-induced signal transduction.Evidence from RNA interference that the low density lipoprotein receptor-related protein is associated with, but not necessary for, GRP 78-mediated signal transduction[J].J Biol Chem, 2002, 277(44): 42082-42087.DOI: 10.1074/jbc.M206174200.
[10] JINDADAMRONGWECH S, THEPPARIT C, SMITH D R.Identification of GRP 78(BiP)as a liver cell expressed receptor element for dengue virus serotype 2[J].Arch Virol, 2004, 149(5): 915-927.DOI: 10.1007/s00705-003-0263-x.
[11] 申浩, 张秀, 张蕾, 等.代谢性标记结合二维电泳寻找O-糖基化蛋白的研究[J].生命科学研究,2014,18(5):377-381.DOI:10.16605/j.cnki.1007-7847.2014.05.001. SHEN H, ZHANG X, ZHANG L, et al.Searching O-Glycoproteins with metabolic labeling and two dimensional electrophoresis[J].Life Science Research, 2014, 18(5): 377-381.DOI:10.16605/j.cnki.1007-7847.2014.05.001.
[12] LIN J, CHUNG S, UEDA K, et al.GALNT6 Stabilizes GRP78 Protein by O-glycosylation and enhances its activity to suppress apoptosis under stress condition[J].Neoplasia, 2017, 19(1): 43-53.DOI: 10.1016/j.neo.2016.11.007.
[13] BALDUS S E, ENGELMANN K, HANISCH F G.MUC1 and the MUCs: a family of human mucins with impact in cancer biology[J].Crit Rev Clin Lab Sci, 2004, 41(2): 189-231.DOI: 10.1080/10408360490452040.
[14] SPRINGER G F.Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy[J].J Mol Med, 1997,75(8): 594-602.DOI:10.1007/s001090050144.
[15] GETHING M J.Role and regulation of the ER chaperone BiP[J].Semin Cell Dev Biol, 1999, 10(5): 465-472.DOI: 10.1006/scdb.1999.0318.
[16] LEE A S.GRP78 induction in cancer: therapeutic and prognostic implications[J].Cancer Res, 2007, 67(8): 3496-3499.DOI: 10.1158/0008-5472.CAN-07-0325.
[17] LI Z, ZHANG L, ZHAO Y, et al.Cell-surface GRP78 facilitates colorectal cancer cell migration and invasion[J].Int J Biochem Cell Biol, 2013, 45(5): 987-994.DOI: 10.1016/j.biocel.2013.02.002.
[18] BUCHKOVICH N J, MAGUIRE T G, YU Y, et al.Human Cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone BiP/GRP78, which is required for virion assembly[J].Journal of Virology, 2008(1): 31-39.DOI: 10.1128/JVI.01881-07.
[19] LI B,GAO B,YE L,et al.Hepatitis B virus X protein(HBx)activates ATF6 and IRE1-XBP1 pathways of unfolded protein response[J].Virus Research, 2007, 124(1-2): 44-49.DOI: 10.1016/j.virusres.2006.09.011.
[20] ZHOU H, ZHANG Y, FU Y, et al.Novel mechanism of anti-apoptotic function of 78-kDa glucose-regulated protein(GRP78): endocrine resistance factor in breast cancer, through release of B-cell lymphoma 2(BCL-2)fromBCL-2-interacting killer(BIK)[J].J Biol Chem, 2011, 286(29): 25687-25696.DOI: 10.1074/jbc.M110.212944.
[21] LIN Y, WANG Z, LIU L, et al.Akt is the downstream tar-get of GRP78 in mediating cisplatin resistance in ER stress-tolerant human lung cancer cells[J].Lung Cancer, 2011, 71(3): 291-297.DOI: 10.1016/j.lungcan.2010.06.004.
[22] MISRA U K, WANG F, PIZZO S V.Transcription factor TFII-I causes transcriptional upregulation of GRP78 synthesis in prostate cancer cells[J].J Cell Biochem, 2009, 106(3): 381-389.DOI: 10.1002/jcb.22016.
[23] MISRA U K, PAYNE S, PIZZO S V.Ligation of prostate cancer cell surface GRP78 activates a proproliferative and antiapoptotic feedback loop: a role for secreted prostate-specific antigen[J].J Biol Chem, 2011, 286(2): 1248-1259.DOI: 10.1074/jbc.M110.129767.
[24] CHIU C C, LIN C Y, LEE L Y, et al.Glucose-regulated protein 78 regulates multiple malignant phenotypes in head and neck cancer and may serve as a molecular target of therapeutic intervention[J].Mol Cancer Ther, 2008, 7(9): 2788-2797.DOI: 10.1158/1535-7163.MCT-08-0172.
[25] COOK K L, SHAJAHAN A N, WÄRRI A, et al.Glucose-regulated protein 78 controls cross-talk between apoptosis and autophagy to determine antiestrogen responsiveness[J].Cancer Res, 2012, 72(13): 3337-3349.DOI: 10.1158/0008-5472.CAN-12-0269.
[26] GRKOVIC S, O'REILLY V C, HAN S, et al.IGFBP-3 binds GRP78, stimulates autophagy and promotes the survival of breast cancer cells exposed to adverse microenvironments[J].Oncogene, 2013, 32(19): 2412-2420.DOI: 10.1038/onc.2012.264.
[27] DONG D, NI M, LI J, et al.Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development[J].Cancer Res, 2008, 68(2): 498-505.DOI: 10.1158/0008-5472.CAN-07-2950.
[28] JIANG C C, YANG F, THORNE R F, et al.Human melanoma cells under endoplasmic reticulum stress acquire resistance to microtubule-targeting drugs through XBP-1-mediated activation of Akt[J].Neoplasia, 2009, 11(5): 436-447.DOI:10.1593/neo.09208.
[29] 浦龙健, 黄莹莹, 李阳, 等.siRNA沉默葡萄糖调节蛋白78可增强乳腺癌细胞对顺铂的敏感性[J].南方医科大学学报, 2013, 33(1): 44-47.DOI:10.3969/j.issn.1673-4254.2013.01.10. PU L J, HUANG Y Y, LI Y, et al.Small interfering RNA-mediated glucose-regulated protein 78 knockdown enhances chemosensitivity of breast cancer cells to cisplatin[J].Journal of Southern Medical University, 2013, 33(1): 44-47.DOI:10.3969/j.issn.1673-4254.2013.01.10.