title: Disruption of microRNA-21 by TALEN leads to diminished cell transformation and increased expression of cell-environment interaction genes.
abstract: MicroRNA-21 is dysregulated in many cancers and fibrotic diseases. Since miR-21 suppresses several tumor suppressor and anti-apoptotic genes, it is considered a cancer therapeutic target. Antisense oligonucleotides are commonly used to inhibit a miRNA; however, blocking miRNA function via an antagomir is temporary, often only achieves a partial knock-down, and may be complicated by off-target effects. Here, we used transcription activator-like effector nucleases (TALENs) to disrupt miR-21 in cancerous cells. Individual deletion clones were screened and isolated without drug selection. Sequencing and quantitative RT-PCR identified clones with no miR-21 expression. The loss of miR-21 led to subtle but global increases of mRNAs containing miR-21 target sequences. Cells without miR-21 became more sensitive to cisplatin and less transformed in culture and in mouse xenografts. In addition to the increase of PDCD4 and PTEN protein, mRNAs for COL4A1, JAG1, SERPINB5/Maspin, SMAD7, and TGFBI - all are miR-21 targets and involved in TGFβ and fibrosis regulation - were significantly upregulated in miR-21 knockout cells. Gene ontology and pathway analysis suggested that cell-environment interactions involving extracellular matrix can be an important miR-21 pathogenic mechanism. The study also demonstrates the value of using TALEN-mediated microRNA gene disruption in human pathobiological studies.
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.|Fujian Institute of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China.
authors.forename: Buyuan
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.|Fujian Institute of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China.
authors.forename: Xinji
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Xiwei
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, USA.|Bone Marrow Transplantation Center, Department of Hematology, The First Affiliated Hospital, Zhejiang University, Hangzhou, China.
authors.forename: Yingjia
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Ting-Yu
authors.initials: TY
authors.lastname: Lin
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.|Irell & Manella Graduate School of Biological Sciences of City of Hope, Duarte, California, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Kurata
authors.affiliation: Division of Comparative Medicine, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.affiliation: Division of Comparative Medicine, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Vonderfecht
authors.affiliation: Department of Diabetes & Metabolic Diseases, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Guihua
authors.initials: G
authors.lastname: Sun
authors.affiliation: Bone Marrow Transplantation Center, Department of Hematology, The First Affiliated Hospital, Zhejiang University, Hangzhou, China.
authors.forename: He
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, USA.|Irell & Manella Graduate School of Biological Sciences of City of Hope, Duarte, California, USA.
authors.forename: Jiing-Kuan
authors.initials: JK
authors.lastname: Yee
authors.affiliation: Fujian Institute of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China.
authors.forename: Jianda
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.|Irell & Manella Graduate School of Biological Sciences of City of Hope, Duarte, California, USA.
authors.forename: Ren-Jang
authors.initials: RJ
authors.lastname: Lin
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic / Q000378:metabolism* / Q000473:pathology*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D059016:Tumor Microenvironment / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D014408:Biomarkers, Tumor; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D014157:Transcription Factors; D004720:Endonucleases
keywords: TALEN; cancer; extracellular matrix; gene editing; miR-21; microRNA