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.

Clustered miRNAs and their role in biological functions and diseases

“…Tandem duplications followed by nucleotide substitutions can also generate miRNA clusters (Zhang et al, 2007). Glazov et al (2008) proposed that the miR-379/656 cluster originated by amplification of an ancient precursor sequence, while Chen et al (2015a) suggested that insertions, deletions of existing miRNAs, and duplication of the whole cluster were possible mechanisms for the origin of miRNA clusters in actively evolving clusters such as miR-302/367. A functional co-adaptation model was proposed to explain the formation and adaptation of miRNA clusters by Wang et al (2016).…”

MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs

“…NHEJ often results in insertions or deletions (indels) at the cleavage site, which can cause frame shift mutations in protein-coding genes. MicroRNA genes can also be inactivated by the formation of indels in the mature miRNA sequence or in regions necessary for the biogenesis of the miRNA (Jiang et al 2014;Chen et al 2015a;Chang et al 2016).…”

Non-Coding RNA Roles in Ruminant Mammary Gland Development and Lactation

“…Targeted miRNA editing will enable revelation of the complex regulatory circuits governed by miRNAs and realization, in the long term, of their full diagnostic and therapeutic potentials. For instance, Chen et al [164] successfully used TALEN to disrupt the function of miR-21 in cancerous cells. A transgenic calf engineered to express miRNA-4 and miR-6 showed an absence of β-lactoglobulin and a concurrent increase in casein proteins in milk [165].…”