Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

Kluiver, Joost; Gibcus, Johan H.; Hettinga, Chris; Adema, Annelies; Richter, Mareike K. S.; Halsema, Nancy; Ślęzak-Prochazka, Izabella; Ding, Ye; Kroesen, Bart-Jan; Berg, Anke van den

doi:10.1371/journal.pone.0029275

Cited by 124 publications

(115 citation statements)

References 32 publications

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“…Luciferase reporter plasmids were transfected into HCT116 cells seeded in 12-well plates using Lipofectamine 2000 (Invitrogen) per the manufacturer's suggestions. MiR-215 sponge was constructed as described previously (34) and as shown in SI Appendix, Fig. S7.…”

Section: Methodsmentioning

confidence: 99%

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The CDX1–microRNA-215 axis regulates colorectal cancer stem cell differentiation

Jones

Hara²,

Francis

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using highthroughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP-PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC.T he caudal-type homeobox 1 (CDX1) transcription factor controls enterocyte differentiation in the colon, where its expression is excluded from the crypt-base stem cell compartment. CDX1 is also central to the capacity of a colorectal cancer (CRC) cell line to differentiate, and it is a negative marker of CRC stem cells (1-3). In 19% of CRC cell lines assayed in a large study, CDX1 expression was completely lost due to promoter methylation and was down-regulated in a further 13% as a result of hemimethylation of the promoter (4). Comparison of CDX1 expression in colorectal adenocarcinoma versus matched normal tissue showed downregulation of CDX1 in 73% of tumors, which was also attributable to promoter methylation (5). Expression of CDX1 also correlates inversely with that of the polycomb complex protein BMI1, which is necessary for the maintenance of quiescent injury-inducible stem cells in the normal crypt and is expressed in cancer stem cells (2, 6-8). The mechanism underlying this inverse correlation has not yet been elucidated.In addition to these correlative data, CDX1 has also been shown to promote directly the expression of structural proteins important for epithelial differentiation including cytokeratin 20 (KRT20) (1), villin (VIL) (9), and FABP1 (10). Introduction...

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Section: Methodsmentioning

confidence: 99%

“…S7 A and B). The sponge was created as described previously (34) and was validated on the basis of its ability to de-repress a synthetic miR-215 target reporter construct (SI Appendix, Fig. S7 C and D).…”

Section: Mir-215mentioning

confidence: 99%

The CDX1–microRNA-215 axis regulates colorectal cancer stem cell differentiation

Jones

Hara²,

Francis

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Therefore, the absorption of such overexpressed miRNAs by synthetic circRNAs should be investigated for their potential as cancer suppressors. Studies describing a new sponge technology indicated that circRNAs can be ef-fective in de-repression of miRNA target genes in cellular systems [39]- [41]. In one such study, Kluiver and colleagues have developed a fast and efficient method to generate sponges that can be applied to inhibit one or more miRNAs.…”

Section: Health Relevance and Potential Uses In Biomedicinementioning

confidence: 99%

“…Using different vector to oligo duplex ratios, the number of the miRNA binding sites can be controlled. The authors used several different approaches to demonstrate that the sponges are efficient inhibitors of miRNAs [39]. They performed luciferase assays to confirm that miRNAs can bind to the sponges in a dose dependent manner.…”

Section: Health Relevance and Potential Uses In Biomedicinementioning

confidence: 99%

Circular RNAs: New Players in Gene Regulation

Andreeva¹,

Cooper²

2015

ABB

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The existence of circular RNAs (circRNAs) was demonstrated over 30 years ago. They did not gain much interest at the time because they appeared to be relatively rare when compared to the abundance of the canonical linear RNAs. However, more recent evidence suggests that circRNAs are abundant in cells and tissues and possess intriguing biological properties. These recent developments have renewed our interest in this novel class of molecules. This report will provide an overview of circRNAs, discuss how they may modify our understanding of gene regulation and indicate their most likely relevance to health. The circRNAs from viruses, bacteria and archaea are not in the scope of this report, and we focused this review on circRNAs in eukaryotes.

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“…It has been shown that adeno-associated virus-delivered TuDs induced long-term miRNA inhibition in mice [27,28]. TuDs and sponges are both more effective when the miRNA binding sites are non-complementary to the target miRNA at position 10–11, presumably because Ago2-dependent cleavage of the TuD is prevented [23,25,29]. Whether TuD activity is mediated by miRNA sequestration alone, or also by miRNA degradation remains unclear, since several studies have reported conflicting results [20,23,27].…”

Section: Introductionmentioning

confidence: 99%

RNA accessibility impacts potency of Tough Decoy microRNA inhibitors

et al. 2018

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MicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression through silencing of complementary target mRNAs. miRNAs are involved in many biological processes, including cell proliferation, differentiation, cell signaling and cellular defense responses to infection. Strategies that allow for strong and stable suppression of specific microRNA activity are needed to study miRNA functions and to develop therapeutic intervention strategies aimed at interfering with miRNA activity in vivo. One of these classes of miRNA inhibitors are Tough Decoys (TuD) RNAs, which comprise of an imperfect RNA hairpin structure that harbors two opposing miRNA binding sites. Upon developing TuDs targeting Epstein-Barr virus miRNAs, we observed a strong variation in inhibitory potential between different TuD RNAs targeting the same miRNA. We show that the composition of the ‘bulge’ sequence in the miRNA binding sites has a strong impact on the inhibitory potency of the TuD. Our data implies that miRNA inhibition correlates with the thermodynamic properties of the TuD and that design aimed at lowering the TuD opening energy increases TuD potency. Our study provides specific guidelines for the design and construction of potent decoy-based miRNA inhibitors, which may be used for future therapeutic intervention strategies.

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Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

Cited by 124 publications

References 32 publications

The CDX1–microRNA-215 axis regulates colorectal cancer stem cell differentiation

The CDX1–microRNA-215 axis regulates colorectal cancer stem cell differentiation

Circular RNAs: New Players in Gene Regulation

RNA accessibility impacts potency of Tough Decoy microRNA inhibitors

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