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通过 CRISPR/Cas9 介导的基因敲除对前列腺癌相关 miRNA 进行功能分类。

Functional classification of prostate cancer‑associated miRNAs through CRISPR/Cas9‑mediated gene knockout.

机构信息

Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong 510180, P.R. China.

Department of Urology, Guizhou Provincial People's Hospital, The Affiliated Hospital of Guizhou Medicine University, Guiyang, Guizhou 550002, P.R. China.

出版信息

Mol Med Rep. 2020 Nov;22(5):3777-3784. doi: 10.3892/mmr.2020.11491. Epub 2020 Sep 7.

Abstract

The aim of the present study was to use the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‑associated (Cas) 9‑mediated gene knockout technology for the rapid classification of the differential function of micro (mi)RNAs screened using miRNA expression profiling by microarray. The rational design of single guide RNAs for the CRISPR/Cas9 system was verified to function in human LNCaP cells with rapid and efficient target gene editing. miRNA (miR)‑205, miR‑221, miR‑222, miR‑30c, miR‑224, miR‑455‑3p, miR‑23b and miR‑505 were downregulated in patients with prostate cancer (PCa) and were experimentally validated to function as tumor suppressors in prostate cancer cells, affecting tumor proliferation, invasion and aerobic glycolysis. In addition, the data of the present study suggested that miR‑663a and mfiR‑1225‑5p were upregulated in prostate cancer tissues and cell proliferation of miR‑663a and miR‑1225‑5p knockout PCa cells was significantly lower compared with miR‑NC cells. Furthermore, knockout of miR‑1225‑5p and miR‑663a significantly decreased the lactate production in LNCaP cells in vitro. In conclusion, the present study offered a simple and efficient method for rapidly classifying miRNA function by applying CRISPR/Cas9 in LNCaP cells. The present study suggested, for the first time to the best of the authors' knowledge, that the aberrant expression of miR‑663a and miR‑1225‑5p may be involved with the progression of prostate cancer, implying their potential as candidate markers for this type of cancer. However, the precise role of miR‑663a and miR‑1225‑5p in accelerating the development of prostate cancer and promoting tumor progression remains to be elucidated.

摘要

本研究旨在利用簇状规律间隔短回文重复序列(CRISPR)和 CRISPR 相关(Cas)9 介导的基因敲除技术,对通过微阵列 miRNA 表达谱筛选出的差异功能 microRNAs(miRNAs)进行快速分类。通过 CRISPR/Cas9 系统的单指导 RNA 的合理设计,在人 LNCaP 细胞中验证了其快速、高效的靶基因编辑功能。miR-205、miR-221、miR-222、miR-30c、miR-224、miR-455-3p、miR-23b 和 miR-505 在前列腺癌(PCa)患者中下调,并在前列腺癌细胞中被实验验证为肿瘤抑制因子,影响肿瘤增殖、侵袭和有氧糖酵解。此外,本研究的数据表明,miR-663a 和 mfiR-1225-5p 在前列腺癌组织中上调,与 miR-NC 细胞相比,miR-663a 和 miR-1225-5p 敲除 PCa 细胞的增殖显著降低。此外,miR-1225-5p 和 miR-663a 的敲除显著降低了 LNCaP 细胞在体外的乳酸产量。综上所述,本研究通过在 LNCaP 细胞中应用 CRISPR/Cas9 为快速分类 miRNA 功能提供了一种简单有效的方法。本研究首次表明,miR-663a 和 miR-1225-5p 的异常表达可能与前列腺癌的进展有关,这意味着它们可能作为该类型癌症的候选标志物。然而,miR-663a 和 miR-1225-5p 在加速前列腺癌发展和促进肿瘤进展中的确切作用仍有待阐明。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/defd/7533463/8c37a516d98f/MMR-22-05-3777-g00.jpg

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