血清反应因子N端片段对鼻咽癌细胞增殖及迁移能力的影响  

Effects of N-terminal fragment of serum response factor on proliferation and migration of nasopharyngeal carcinoma cells

作　　者：袁建玲 邵钟铭 邹园 伍彩霞 郑阿秀 邢敬慈 白建荣 揭伟 申志华 YUAN Jian-ling;SHAO Zhong-ming;ZOU Yuan;WU Cai-xia;ZHENG A-xiu;XING Jing-ci;BAI Jian-rong;JIE Wei;SHEN Zhi-hua(Department of Pathophysiology,School of Basic Medicine Sciences,Guangdong Medical University,Zhanjiang 524023,China;Key Laboratory of Emergency and Trauma,Ministry of Education,Hainan Medical University,Haikou 571199,China;Department of Pathology,Shenzhen Hospital of Peking University,Shenzhen 518036,China)

机构地区：[1]广东医科大学基础医学院病理生理学教研室,广东湛江524023 [2]海南医学院急救与创伤研究教育部重点实验室,海南海口571199 [3]北京大学深圳医院病理科,广东深圳518036

出　　处：《中国病理生理杂志》2022年第3期535-542,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81402415);广东省扬帆计划高层次人才项目(No.4YF16007G)。

摘　　要：目的:探讨血清反应因子N端剪切片段(SRF-N)对鼻咽癌(NPC)细胞增殖和迁移能力的影响与机制。方法:应用SRF全长(SRF-Full,1~508 aa)、SRF-N(1~254 aa)及阴性对照(NC)慢病毒颗粒感染人NPC细胞系6-10B,经嘌呤霉素抗性筛选结合Western blot检测Flag标签化融合蛋白的表达获得单克隆细胞株,CCK-8实验分析细胞活力的改变,划痕损伤修复实验和Transwell实验分析细胞迁移能力,细胞-基质黏附实验分析细胞黏附能力,Western blot检测细胞增殖相关蛋白增殖细胞核抗原(PCNA)和上皮-间充质转化(EMT)相关指标的表达,双萤光素酶报告基因实验检测SRF-Full和SRF-N对Snail1启动子的调节效应。结果:成功用SRF-Full、SRF-N及NC慢病毒感染6-10B细胞,经嘌呤霉素抗性筛选结合Flag标签蛋白的表达获得相应的单克隆细胞株,分别标记为6-10B^(SRF-Full)、6-10B^(SRF-N)和6-10B^(NC)。与6-10B^(NC)细胞相比,6-10B^(SRF-Full)细胞活力、迁移能力和与基质的黏附能力均显著增强(P<0.01),而6-10B^(SRF-N)细胞较6-10B^(SRF-Full)细胞活力、迁移能力及黏附能力均显著下降(P<0.01)。6-10B^(SRF-Full)细胞中波形蛋白(vimentin)、神经钙黏素(N-cadherin)和Snail1的表达水平较6-10B^(NC)细胞显著升高(P<0.01),上皮钙黏素(Ecadherin)表达水平显著下降(P<0.01);而6-10B^(SRF-N)细胞中vimentin、N-cadherin和Snail1的表达水平较6-10B^(SRF-Full)细胞显著下降(P<0.05),E-cadherin表达水平显著上升(P<0.05)。双萤光素酶活性实验结果表明,与NC相比,SRFFull显著激活Snail1启动子(P<0.001);与SRF-Full相比,SRF-N显著抑制Snail1启动子活性(P<0.01)。结论:SRF-Full促进而SRF-N抑制NPC细胞的增殖和迁移;SRF-N抑制Snail1启动子活性而介导NPC的EMT;SRF-N具有潜在的抗NPC作用。AIM:To explore the effects of the N-terminal fragment of serum response factor(SRF-N)on the proliferation and migration of nasopharyngeal carcinoma(NPC)cells and its mechanisms.METHODS:The full-length SRF(SRF-Full,1 to 508 aa)overexpression,SRF-N(1 to 254 aa)overexpression and negative control(NC)lentiviral particles were used to infect human NPC 6-10B cells,and the monoclonal cells were obtained by puromycin resistance screening.Western blot was applied to detect the expression of Flag-tagged fusion protein.The cell viability,migration and adhesion were analyzed by CCK-8 assay,wound healing assay,Transwell assay and matrix-adhesion assay.The changes in proliferation-associated protein proliferating cell nuclear antigen(PCNA)and epithelial-mesenchymal transition(EMT)-related factors were assessed by Western blot.Finally,dual-luciferase reporter assay was used to detect the regulatory effects of SRF-Full and SRF-N on Snail1 promoter.RESULTS:The 6-10B cells were successfully infected with SRF-Full,SRF-N and NC lentiviruses.The corresponding monoclonal cell lines,which were obtained by puromycin resistance screening combined with the expression of Flag tag protein,were labeled as 6-10B^(SRF-Full),6-10B^(SRF-N) and 6-10B^(NC),respectively.Compared with 6-10BNCcells,increased viability,migration and adhesion to the matrix were observed in 6-10B^(SRF-Full) cells(P<0.01),while these capabilities of 6-10B^(SRF-N) cells were decreased compared with 6-10B^(SRF-Full) cells(P<0.01).Compared with 6-10B^(NC) cells,the expression of vimentin,N-cadherin and Snail1 in 6-10B^(SRF-Full) cells were significantly increased(P<0.01),while the expression of E-cadherin was significantly decreased(P<0.01).Compared with 6-10B^(SRF-Full) cells,the expression of vimentin,N-cadherin and Snail1 in 6-10BSRF-Ncells was significantly decreased(P<0.05),while the expression of E-cadherin was significantly increased(P<0.05).The results of dual-luciferase reporter assay showed that SRF-Full significantly activated Snail1 promoter compared with NC(P<0

关 键 词：鼻咽癌 血清反应因子N端片段 细胞增殖 细胞迁移 上皮-间充质转化 

分 类 号：R363.2[医药卫生—病理学] R739.6[医药卫生—基础医学]