An enhanced hTERT promoter-driven CRISPR/Cas9 system selectively inhibits the progression of bladder cancer cells†

IF 3.743 Q2 Biochemistry, Genetics and Molecular Biology
Xinbo Huang, Chengle Zhuang, Changshui Zhuang, Tiefu Xiong, Yawen Li and Yaoting Gui
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引用次数: 8

Abstract

The current therapies for treating tumors are lacking in efficacy and specificity. Synthetic biology principles may bring some new possible methods for curing cancer. Here we present a synthetic logic circuit based on the CRISPR/Cas9 system. The CRISPR/Cas9 technology has been applied in many biological fields, including cancer research. In this study, the expression of Cas9 nuclease was controlled indirectly by an enhanced hTERT promoter using the GAL4/upstream activating sequence (UAS) binding system. Cas9 was driven by 5XUAS, single guide RNA (sgRNA) was used to target mutant or wild-type HRAS, and the fusion gene GAL4-P65 was driven by the enhanced hTERT promoter. The system was tested in bladder cancer cells (T24 and 5637) and the results showed that the enhanced hTERT promoter could drive the expression of GAL4-P65 in these bladder cancer cell lines. Then all these devices were packed into lentivirus and the results of quantitative real-time PCR showed that the mRNA expression level of HRAS was selectively inhibited in the T24 and 5637 cells. The results of functional experiments suggested that the proliferation, cell migration and invasion were selectively suppressed, and that the apoptosis rate was increased in bladder cancer cells but not in human foreskin fibroblasts (HFF). In conclusion, we successfully constructed an enhanced hTERT promoter-driven CRISPR/Cas9 system and data showed that it could selectively suppress the progression of bladder cancer cells.

Abstract Image

一种增强的hTERT启动子驱动的CRISPR/Cas9系统选择性抑制膀胱癌细胞的进展†
目前治疗肿瘤的方法缺乏疗效和特异性。合成生物学原理可能为癌症的治疗带来一些新的可能方法。本文提出了一种基于CRISPR/Cas9系统的合成逻辑电路。CRISPR/Cas9技术已经应用于许多生物领域,包括癌症研究。本研究利用GAL4/上游激活序列(UAS)结合系统,通过增强hTERT启动子间接控制Cas9核酸酶的表达。Cas9由5XUAS驱动,单导RNA (sgRNA)靶向突变型或野生型HRAS,融合基因GAL4-P65由增强型hTERT启动子驱动。该系统在膀胱癌细胞(T24和5637)中进行了实验,结果表明,增强的hTERT启动子可以驱动这些膀胱癌细胞系中GAL4-P65的表达。然后将这些器件封装到慢病毒中,实时荧光定量PCR结果显示,在T24和5637细胞中,HRAS mRNA的表达水平被选择性抑制。功能实验结果表明,选择性抑制膀胱癌细胞的增殖、细胞迁移和侵袭,增加膀胱癌细胞的凋亡率,而在人包皮成纤维细胞(HFF)中没有。综上所述,我们成功构建了一个增强型hTERT启动子驱动的CRISPR/Cas9系统,数据显示其能够选择性抑制膀胱癌细胞的进展。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Molecular BioSystems
Molecular BioSystems 生物-生化与分子生物学
CiteScore
2.94
自引率
0.00%
发文量
0
审稿时长
2.6 months
期刊介绍: Molecular Omics publishes molecular level experimental and bioinformatics research in the -omics sciences, including genomics, proteomics, transcriptomics and metabolomics. We will also welcome multidisciplinary papers presenting studies combining different types of omics, or the interface of omics and other fields such as systems biology or chemical biology.
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