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

Abstract

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.

Figure 1

Deviation of NSCs from an epi-hiPSC line. (a) Characterization of an epi-hiPSC line generated with nonintegrating episomal vectors. From left to right: colony morphology, AP staining, immunostaining of Oct4, and embryoid body differentiation. (b) Characterization and in vitro neural differentiation of hiPS-NSCs. From left to right: bipolar cell morphology, immunostaining of nestin (NSC early stage marker), GFAP (astrocyte marker), and β-tubulin III (neuron marker).

Figure 2

Targeted inhibition of miR-199a/214 cluster using a CRISPRi system in NSCs. (a) Diagram showing miR-199a-5p, miR-199a-3p, and miR-214 of the miR-199a/214 cluster are targeting multiple important components in the signaling pathways that mediate hypoxia-induced migration of NSCs. (b) Schematic representation showing blockage of transcription of the miR-199a/214 cluster using a dCas9 targeting the E-box in the promoter region. The sequence of the gRNA target site is shown and the PAM sequence is underlined. The E-box sequence is highlighted in red. (c) Absolute expression levels of miR-199a-5p, miR-199a-3p, and miR-214 in NSCs are quantified by qPCR (n = 3). Error bars: SD. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 3

Inhibition of the miR-199a/214 cluster derepresses hypoxia-related targets and promotes NSC tumor tropism in vitro. (a) Relative expression levels of HIF1A, MET, MAPK1, and CXCR4 in NSCs are quantified by qPCR (n = 3). Error bars: SD. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001. (b) In vitro migration of NSCs in Boyden chamber. Only DMEM is in lower chambers of the blank group. 4T1 cells are seeded in lower chambers of other groups as attractants. NSCs are transduced with baculovirus and stained with calcein-AM and seeded in the upper chamber. After 24 h, the labelled NSCs which migrated toward the lower chamber were evaluated (n = 6). Top: percentage of migrated NSCs. Bottom: fluorescence images showing the migration of NSCs toward the lower chambers. Error bars: SD. ^∗∗∗ P < 0.001.

Figure 4

Inhibition of the miR-199a/214 cluster enhances NSC tumor tropism in vivo. (a) Diagram showing the protocol of NSC in vivo tumor tropism assay. (b) Representative ex vivo organs images showing the tumor tropism of NSCs in mice at day 8. The luminescent images (upper panels) show luc-4T1 tumor metastases in the lung. The fluorescent images (lower panels) show the organ distribution of NSCs. The organs shown in each panel from left to right: lung, liver, spleen, kidney, heart, brain, stomach, spinal cord, and femur. (c) Histogram showing the percentages of NSCs distributed in the lungs, the livers, and the spleens (n = 3). Error bars: SD. ^∗∗∗ P < 0.001.