利用规律间隔成簇短回文重复序列/相关蛋白9系统敲减α2δ1对人肝癌细胞系Hep-12干性的影响

何琦; 赵威; 张志谦

doi:10.16098/j.issn.0529-1356.2020.06.012

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解剖学报 ›› 2020, Vol. 51 ›› Issue (6) : 882-887. DOI: 10.16098/j.issn.0529-1356.2020.06.012

肿瘤生物学

利用规律间隔成簇短回文重复序列/相关蛋白9系统敲减α2δ1对人肝癌细胞系Hep-12干性的影响

何琦¹ 赵威² 张志谦^2*

作者信息 +

Suppression of stem cell-like properties of human hepatocellular carcinoma cell line Hep-12 using the clustered regularly interspaced short palindromic repeats/associated protein 9 system

HE Qi¹ ZHAO Wei²ZHANG Zhi-qian^2*

Author information +

文章历史 +

摘要

目的通过规律间隔成簇短回文重复序列/相关蛋白9(CRISPR/Cas9)系统敲减人肝癌细胞系Hep12中电压依赖性钙离子通道 α2δ1的表达，观察α2δ1敲减后对肝癌细胞干性的影响。方法设计3对靶向α2δ1的向导 (sgRNA)，长度为20 bp，构建到lenti CRISPRv2-puro载体上，然后在体外检测sgRNA切割活性，利用慢病毒包装系统包装含有sgRNA的重组质粒，将包装好的病毒感染Hep-12细胞，2 d后加入嘌呤霉素筛选。利用Western blotting验证α2δ1的敲减效果和干性相关基因的表达。通过成球实验检测其体外自我更新能力的变化。结果测序结果显示，sgRNA成功插入载体质粒；体外切割实验显示，3条sgRNA均有切割活性；Western blotting结果显示，α2δ1基因的表达显著降低，干性相关基因B细胞特异性莫洛尼白血病病毒插入位点1(BMI1)和Nanog的表达显著被抑制；无血清培养基成球实验结果表明，敲减α2δ1导致Hep-12细胞的体外自我更新能力减弱。结论利用CRISPR/Cas9技术成功构建敲除α2δ1基因的Hep-12细胞系；敲除α2δ1基因后能抑制Hep-12细胞肿瘤干细胞样特性。

Abstract

Objective To investigate the effects of knockdown the expression of the voltage-gated calcium channel α2δ1 subunit on the stem cell-like traits in the tumor-initiating cell enriched Hep-12 hepatocellular carcinoma cell line through clustered regularly interspaced short palindromic repeats/-associated protein 9(CRISPR/Cas9) system. Methods Three pairs of single guide RNA (sgRNA) targeting α2δ1 were constructed into the lenti CRISPRv2-puro vector using standard DNA recombinant technique, and their cleavage activities were verified in vitro. The lenti CRISPRv2-puro vector containing the sgRNA sequence was packaged into lentivirus in 293FT cells using the 3rd generation packing system. The viruses were then used to infect Hep-12 cells which were subsequently selected with puromycin. The expression of α2δ1 and stemness related genes were detected by Western blotting, and the in vitro self-renewal capacity was measured by spheroid formation assay. Results The designed sgRNAs targeting α2δ1 gene were demonstrated to cleave α2δ1 DNA fragment efficiently in vitro, and the lenti CRISPRv2-puro vectors harboring the corresponding sgRNA sequences were successfully constructed. Compared with that of control cells, the expression of α2δ1 and stem cell associated genes B-cell-specific moloney leukemia virus insertion site 1(BMI1), and Nanog were remarkably suppressed following the infection with the lentivirues harboring sgRNAs against α2δ1 in Hep-12 cells. Furthermore, the in vitro self-renewal ability of Hep-12 cells was retarded significantly as evidenced that the spheroid formation efficiency in serum free medium of these cells reduced dramatically with knockdown of α2δ1. Conclusion The voltage-gated calcium channel α2δ1 is essential for the maintanence of the self-renewal properties of the tumor-intiating cell enriched hepatocellular carcinoma Hep12 cells.

导出引用

何琦赵威张志谦. 利用规律间隔成簇短回文重复序列/相关蛋白9系统敲减α2δ1对人肝癌细胞系Hep-12干性的影响[J]. 解剖学报. 2020, 51(6): 882-887 https://doi.org/10.16098/j.issn.0529-1356.2020.06.012

HE Qi ZHAO Wei ZHANG Zhi-qian. Suppression of stem cell-like properties of human hepatocellular carcinoma cell line Hep-12 using the clustered regularly interspaced short palindromic repeats/associated protein 9 system[J]. Acta Anatomica Sinica. 2020, 51(6): 882-887 https://doi.org/10.16098/j.issn.0529-1356.2020.06.012

中图分类号： R757.7

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