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
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