Ultrasound microbubble-mediated CRISPR/Cas9 knockout of C-erbB-2 in HEC-1A cells
Ultrasound microbubble-mediated CRISPR/Cas9 knockout of C-erbB-2 in HEC-1A cells
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DOI：
10.1177/0300060519840890
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发表时间：
2019-05-01
影响因子：
1.6
通讯作者：
Bao, Shan
中科院分区：
文献类型：
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作者：
Cai, Junhong;Huang, Sizhe;Bao, Shan
目的表皮生长因子受体2（Epidermal growth factor receptor 2，C-erbB-2）是人类肿瘤中最常发生突变的癌基因之一。我们的目的是评估使用超声微泡转染靶向人子宫内膜癌（HEC）-1A细胞中C-erbB-2的成簇规则间隔短回文重复序列（CRISPR）技术的敲除效率。方法设计3个靶向C-erbB-2的单向导RNA（single guide RNA，sgRNA），构建CRISPR/CRISPR相关（Cas）9-C-erbB-2质粒。用超声微泡法将构建的质粒转染HEC-1A细胞。通过绿色荧光鉴定C-erbB-2敲除的克隆细胞。逆转录聚合酶链反应（RT PCR）检测C-erbB-2 mRNA表达，免疫印迹法检测C-erbB-2 mRNA蛋白表达。结果RT-PCR结果显示，转染sgRNA 1的细胞C-erbB-2 mRNA表达（0.57 ± 0.06）明显低于空白组（1.00 ± 0.09）和阴性对照组（1.02 ± 0.12）。Western blotting显示sgRNA 1转染细胞中C-erbB-2蛋白表达（0.269 +/- 0.033）显著低于空白组（0.495 +/- 0.059）和阴性对照组（1.243 +/- 0.281）。然而，与对照组相比，sgRNA 2和sgRNA 3转染的细胞中C-erbB-2蛋白和mRNA的表达无显著差异。结论超声微泡可介导质粒转染HEC-1A细胞，干扰基因表达，敲除C-erbB-2。
Objective Epidermal growth factor receptor 2 (C-erbB-2) is one of the most frequently mutated oncogenes in human tumors. We aimed to evaluate the knockout efficiency of clustered regularly interspaced short palindromic repeat (CRISPR) technology using ultrasound microbubble transfection to target C-erbB-2 in human endometrial cancer (HEC)-1A cells. Methods Three single guide RNAs (sgRNAs) targeting C-erbB-2 were designed and used to construct CRISPR/CRISPR-associated (Cas)9-C-erbB-2 plasmids. The constructed plasmids were transfected into HEC-1A cells using ultrasound microbubbles. C-erbB-2 knockout cloned cells were identified by green fluorescence. C-erbB-2 mRNA and protein expression was measured by reverse transcription (RT)-PCR and western blotting, respectively. Results RT-PCR showed that C-erbB-2 mRNA expression was significantly lower in sgRNA1-transfected cells (0.57 +/- 0.06) than in blank (1.00 +/- 0.09) and negative-control groups (1.02 +/- 0.12). Western blotting revealed C-erbB-2 protein expression to be significantly lower in sgRNA1-transfected cells (0.269 +/- 0.033) than in blank (0.495 +/- 0.059) and negative-control groups (1.243 +/- 0.281). However, there was no significant difference in C-erbB-2 protein and mRNA expression in sgRNA2- and sgRNA3-transfected cells compared with controls. Conclusion Ultrasound microbubbles can mediate plasmid transfer into HEC-1A cells to interfere with gene expression and knockout C-erbB-2.