title: Artificial small RNA for sequence specific cleavage of target RNA through RNase III endonuclease Dicer.
abstract: 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.
authors.affiliation: State 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.|Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Xu
authors.affiliation: Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State 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.
authors.forename: Yali
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.|Shantou University Medical College, Shantou 515041, China.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.|Shantou University Medical College, Shantou 515041, China.
authors.forename: Yonghao
authors.initials: Y
authors.lastname: Zhan
authors.affiliation: Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.|Shantou University Medical College, Shantou 515041, China.
authors.forename: Chengle
authors.initials: C
authors.lastname: Zhuang
authors.affiliation: Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.|Shantou University Medical College, Shantou 515041, China.
authors.forename: Junhao
authors.initials: J
authors.lastname: Lin
authors.affiliation: Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.
authors.forename: Mingwei
authors.initials: M
authors.lastname: Chen
authors.affiliation: Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.|Shantou University Medical College, Shantou 515041, China.
authors.forename: Jianfa
authors.initials: J
authors.lastname: Li
authors.affiliation: Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Cai
authors.affiliation: Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Shenzhen, Shenzhen 518000, China.
authors.forename: Weiren
authors.initials: W
authors.lastname: Huang
authors.affiliation: State 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.|South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510275, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D053487:DEAD-box RNA Helicases / Q000502:physiology*; D006801:Humans; D017393:RNA Editing; D034622:RNA Interference; D016372:RNA, Antisense / Q000235:genetics; D062085:RNA, Long Noncoding / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics*; D043244:Ribonuclease III / Q000502:physiology*
chemical_list: C557041:MALAT1 long non-coding RNA, human; D016372:RNA, Antisense; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases
keywords: artificial RNA; dicer; gene regulation