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Slowing down glioma growth by knocking out miR-21 in mice models

Original author: L. Nieland et al. (2022) (DOI: 10.1016/j.omto.2022.04.001)


Numair Arshad.jpg

Content writer – Clinical

August 05, 2022


Glioblastoma multiforme (GBM) is an aggressive form of glioma with a median survival of 15 months. Heterogeneity is one of the main characteristics of GBM. The tumor cells show variability not only from one patient to another but within the same patient. Some of this heterogeneity occurs due to dysregulated microRNAs (miRNAs). miRNAs are non-coding RNA molecules that play a role in the expression of genes. In GBM, the most important miRNA is miR-21. A higher amount of miR-21 is linked to a worse prognosis.

Studying the effect of the gain and loss of miR-21 on tumor growth in GBM can provide information about the role of miRNAs in these tumors. Previously, this has been tried using antisense oligonucleotide inhibitors. But these studies are limited due to the inherent shortcomings of antisense oligonucleotide inhibitors. The authors of this study set out to investigate the effects of miR-21 by knocking it out using clustered regulatory interspaced short palindromic repeats (CRISPR)-Cas technology.


The researchers knocked out miR-21 in mouse (GL261 and CT2A) and human (U87) glioma lines using five different CRISPR RNAs (crRNAs). Eventually, JIR327 crRNA was selected because it showed more specificity according to DNA Sanger sequencing. CRISPR-Cas12a plasmids with co-expressing fluorescent markers allowed researchers to test the efficiency of editing and track the edited cells. Successfully, edited cells were isolated and cloned to obtain identical miR-21 knock-out (KO) cells. The expression of miR-21 and disrupted genomic sequences of these cell lines were tested using qRT-PCR and CRISPResso2, respectively. RNA sequencing (RNA-seq) was used to study the effect of the miR-21 level on the expression of other genes.

Results & discussion

The expression levels of miR-21 were significantly lower in GL261, CT2A, and U87 KO cell lines as compared to their wild-type (WT) counterparts. This decrease in miR-21 expression resulted in the upregulation of 25 genes and down-regulation of 27 genes. KO cell lines had significantly more expression of anti-proliferation genes such as cell division cycle 25 homolog A (Cdc25a), C-X-C motif chemokine ligand 10 (Cxcl10), Krev interaction trapped protein 1 (Krit1), signal transducer and activator of transcription 3 (Smad7), signal transducer and activator of transcription 3 (Stat3), and cyclin-dependent kinase 6 (Cdk6). More importantly, the researchers found that KO cell lines had reduced proliferation and migration, and smaller-sized colonies in vitro. When they injected KO and WT miR-21 into the striatum of separate mice groups, the KO groups showed increased survival compared to WT groups.

Impact of the research

This study paves the way for mRNA-based interventions in the field of cancer. By knocking out miR-21 from glioma cell lines, the authors of this study have shown that glioma growth is reduced. This reduction in growth can allow the currently available glioma interventions to be more effective.

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