shRNA沉默神经导向分子5A基因对A375细胞系增殖、转移和侵袭能力的影响

中华皮肤科杂志 ›› 2016, Vol. 49 ›› Issue (8): 573-577.

shRNA沉默神经导向分子5A基因对A375细胞系增殖、转移和侵袭能力的影响

张莉¹,李亚东²,陈晨²,李凌佳¹,谢玉燕¹,刘彤云³,寸韡²

1. 昆明医科大学第一附属医院皮肤科
2. 中国医学科学院&北京协和医学院医学生物学研究所
3. 昆明医科大学第一附属医院

收稿日期:2015-10-19 修回日期:2016-01-28 发布日期:2016-08-05
通讯作者: 刘彤云 E-mail:tongyun91@126.com
基金资助:
云南省科技厅-昆明医科大学应用基础研究联合专项

Effects of short hairpin RNA?mediated semaphorin 5A gene silencing on proliferation, metastasis and invasion of malignant melanoma cell line A375

Received:2015-10-19 Revised:2016-01-28 Published:2016-08-05

摘要/Abstract

摘要：

目的探讨慢病毒介导短发夹RNA（shRNA）沉默神经导向分子5A（Semaphorin 5A）基因对恶性黑素瘤A375细胞系生物活性的影响。方法针对Semaphorin 5A设计2对shRNA引物及1对阴性对照引物，构建慢病毒载体，转染至人胚肾上皮HEK293T细胞系收获慢病毒，利用慢病毒感染A?375细胞系并通过嘌呤霉素筛选，成功获得稳定转染细胞系，实验分为实验组细胞（A375?shRNA1和 A375?shRNA2）、阴性对照组细胞（A375?con）及空白对照组细胞（A375）。通过反转录PCR（RT?PCR）、Western 印迹比较实验组细胞、阴性对照组细胞及空白对照组细胞Semaphorin 5A mRNA、蛋白表达水平的差异。噻唑蓝（MTT）检测细胞生长情况；侵袭试验及划痕试验比较转染前后细胞的侵袭运动能力。结果 Semaphorin 5A成功转染A375细胞后，经嘌呤霉素筛选，成功获得稳定转染实验组A375?shRNA2细胞系及对照组A375 ?con。反转录PCR及Western 印迹检测，干扰后实验组A375?shRNA2较对照组A375?con、A375 Semaphorin 5A的转录水平及蛋白表达水平表达下调。MTT实验结果显示，A375?con与A375细胞生长差异无统计学意义（P > 0.05），A375?shRNA2细胞生长明显减慢，与A375和A375?con比较，差异均有统计学意义（P < 0.05）。划痕实验结果显示，A375?shRNA2划痕处细胞无明显迁移，划痕未得到修复，而A375及A375?con划痕处细胞最终几乎将划痕覆盖。侵袭实验结果显示，A375组与A375?con组穿过的细胞数差异无统计学意义（P > 0.05）；而A375?shRNA2通过小室的细胞明显少于A375及A375?con组，差异有统计学意义（P < 0.05）。结论慢病毒介导shRNA沉默Semaphorin 5A基因能使A375中的Semaphorin 5A有效下调，并抑制细胞的生长，降低细胞的侵袭以及运动能力。

Abstract:

Zhang Li, Li Yadong, Chen Chen, Li Lingjia, Xie Yuyan, Liu Tongyun, Cun Wei Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming 650000, China （Zhang L, Li LJ, Xie YY, Liu TY）; Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650031, China （Li YD, Chen C, Cun W） Corresponding authors: Liu Tongyun, Email: tongyun91@126.com; Cun Wei, Email: cunwei@foxmail.com 【Abstract】 Objective To study the effects of semaphorin 5A （SEMA5A） gene silencing by lentivirus?mediated short hairpin RNA （shRNA） on biological activity of malignant melanoma cell line A375. Methods Two pairs of interference sequences for SEMA5A gene （shRNA1 and A375?shRNA2）and a pair of control interference sequences were designed to build lentiviral vectors, which were then transfected into HEK293T cells to gain lentivirus. A375 cells were divided into three groups: experimental group （A375?shRNA1 and A375?shRNA2 cells） transfected with the lentivirus containing shRNA1 or shRNA2, negative control group （A375?con cells） transfected with that containing the control shRNA, and blank control group （A375 cells） receiving no transfection. The A375 cells with stable knockdown of SEMA5A gene were screened by puromycin. Subsequently, reverse transcription?PCR and Western?blot analysis were performed to detect mRNA and protein s of Semaphorin 5A in these cells, and methyl thiazolyl tetrazolium （MTT）assay was applied to evaluate the growth of cells. The scratch assay and invasion assay were conducted to estimate migration and invasion ability of cells. Results The lentivirus containing the SEMA5A?targeting shRNAs or control shRNA was successfully transfected into A375 cells, and stably transfected cells were gained after puromycin selection. The s of semaphorin 5A mRNA and protein in the A375?shRNA2 cells were significantly reduced compared with those in the A375?con and A375 cells （all P < 0.05）. MTT assay showed that the growth of A375?shRNA2 cells was significantly slower than that of A375?con and A375 cells （both P < 0.05）, while there was no significant difference in the growth rate between A375?con and A375 cells （P > 0.05）. The scratch assay showed that there was no obvious cell migration into the scratch in the experiment group, whereas the scratch was almost covered by cells in the negative control group and blank control group. The invasion assay showed that the number of A375?shRNA2 cells passing through the Transwell chamber was significantly smaller than that of A375 and A375?con cells （both P < 0.05）, while there was no significant difference between that of A375 and A375?con cells （P > 0.05）. Conclusion The silencing of SEMA5A gene by lentivirus?mediated shRNA could effectively down?regulate the of semaphorin 5A, and inhibit the growth, invasion and migration of A375 cells.

张莉李亚东陈晨李凌佳谢玉燕刘彤云寸韡. shRNA沉默神经导向分子5A基因对A375细胞系增殖、转移和侵袭能力的影响[J]. 中华皮肤科杂志, 2016,49(8):573-577. doi:

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