Artificial small RNA for sequence specific cleavage of target RNA through RNase III endonuclease Dicer
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Wen Xu1,2,*, Yuchen Liu2,*, Yali Liu1, Li Liu2,3, Yonghao Zhan2,3, Chengle Zhuang2,3, Junhao Lin2,3, Mingwei Chen2, Jianfa Li2,3, Zhiming Cai2, Weiren Huang2, Yong Zhang1,4
1State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals, and the Guangdong Province Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China
2Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China
3Shantou University Medical College, Shantou 515041, China
4South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510275, China
*These authors have contributed equally to this work
Yong Zhang, e-mail: [email protected]
Weiren Huang, e-mail: [email protected]
Zhiming Cai, e-mail: [email protected]
Keywords: artificial RNA, dicer, gene regulation
Received: January 07, 2016 Accepted: April 26, 2016 Published: May 25, 2016
CRISPR-Cas9 system uses a guide RNA which functions in conjunction with Cas9 proteins to target a DNA and cleaves double-strand DNA. This phenomenon raises a question whether an artificial small RNA (asRNA), composed of a Dicer–binding RNA element and an antisense RNA, could also be used to induce Dicer to process and degrade a specific RNA. If so, we could develop a new method which is named DICERi for gene silencing or RNA editing. To prove the feasibility of asRNA, we selected MALAT-1 as target and used Hela and MDA-MB-231 cells as experimental models. The results of qRT-PCR showed that the introduction of asRNA decreased the relative expression level of target gene significantly. Next, we analyzed cell proliferation using CCK-8 and EdU staining assays, and then cell migration using wound scratch and Transwell invasion assays. We found that cell proliferation and cell migration were both suppressed remarkably after asRNA was expressed in Hela and MDA-MB-231 cells. Cell apoptosis was also detected through Hoechst staining and ELISA assays and the data indicated that he numbers of apoptotic cell in experimental groups significantly increased compared with negative controls. In order to prove that the gene silencing effects were caused by Dicer, we co-transfected shRNA silencing Dicer and asRNA. The relative expression levels of Dicer and MALAT-1 were both detected and the results indicated that when the cleavage role of Dicer was silenced, the relative expression level of MALAT-1 was not affected after the introduction of asRNA. All the above results demonstrated that these devices directed by Dicer effectively excised target RNA and repressed the target genes, thus causing phenotypic changes. Our works adds a new dimension to gene regulating technologies and may have broad applications in construction of gene circuits.
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