Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has mu...

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Main Authors: Yumei Luo, Xuehu Xu, Xiuli An, Xiaofang Sun, Shu Wang, Detu Zhu
Format: Article
Language:English
Published: Wiley 2016-01-01
Series:Stem Cells International
Online Access:http://dx.doi.org/10.1155/2016/3598542
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author Yumei Luo
Xuehu Xu
Xiuli An
Xiaofang Sun
Shu Wang
Detu Zhu
author_facet Yumei Luo
Xuehu Xu
Xiuli An
Xiaofang Sun
Shu Wang
Detu Zhu
author_sort Yumei Luo
collection DOAJ
description The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in the microRNA gene cluster. Meanwhile, the expression levels of their targets related to regulation of hypoxia-stimulated cell migration, such as HIF1A, MET, and MAPK1, were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy.
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institution Kabale University
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spelling doaj-art-8422d3090a904c179f6c73c3d148551c2025-02-03T01:29:58ZengWileyStem Cells International1687-966X1687-96782016-01-01201610.1155/2016/35985423598542Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic ConditionYumei Luo0Xuehu Xu1Xiuli An2Xiaofang Sun3Shu Wang4Detu Zhu5Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, ChinaDepartment of Gastrointestinal Surgery, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, ChinaLaboratory of Membrane Biology, New York Blood Center, New York, NY 10065, USAKey Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, ChinaDepartment of Biological Sciences, National University of Singapore, 117543, SingaporeDepartment of Genetic Medicine, Weill Cornell Medical College, New York, NY 10065, USAThe human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in the microRNA gene cluster. Meanwhile, the expression levels of their targets related to regulation of hypoxia-stimulated cell migration, such as HIF1A, MET, and MAPK1, were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy.http://dx.doi.org/10.1155/2016/3598542
spellingShingle Yumei Luo
Xuehu Xu
Xiuli An
Xiaofang Sun
Shu Wang
Detu Zhu
Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition
Stem Cells International
title Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition
title_full Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition
title_fullStr Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition
title_full_unstemmed Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition
title_short Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition
title_sort targeted inhibition of the mir 199a 214 cluster by crispr interference augments the tumor tropism of human induced pluripotent stem cell derived neural stem cells under hypoxic condition
url http://dx.doi.org/10.1155/2016/3598542
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