Preparation of Circular RNA In Vitro

Abe, Naoko; Kodama, Ayumi; Abe, Hiroshi

doi:10.1007/978-1-4939-7562-4_15

Cited by 10 publications

(10 citation statements)

References 12 publications

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“…Since most circRNAs are composed of sequences from the coding exons, the linear mRNAs can prevent their detection by interference. Therefore, we digested linear mRNAs with Ribonuclease R, a nucleic acid exonuclease, to accurately characterize the circular RNAs [14]. Since circRNAs do not have a polyA tail, we used random primers instead of oligo dT primers to amplify them by reverse transcription [15].…”

Section: Discussionmentioning

confidence: 99%

Circ_0006332 promotes growth and progression of bladder cancer by modulating MYBL2 expression via miR-143

Liu

et al. 2019

Aging

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In this study, we analyzed the role of circular RNAs in the growth and progression of bladder cancer. Direct Sanger sequencing and quantitative RT-PCR analysis showed that circ_0006332 was significantly upregulated in bladder cancer tissues. Sequencing analysis showed that circ_0006332 is generated from splicing of exons 8 and 9 of the MYBL2 transcript. Fluorescence in situ hybridization analysis showed that circ_0006332 was localized to the cytoplasm of bladder cancer cells. Dual luciferase reporter assays showed that miR-143 specifically bound to circ_0006332 and the 3’UTR of MYBL2. High expression of circ_006332 correlated with tumor-node-metastasis stages and muscular invasion in bladder cancer patients. Knockdown of circ_0006332 in bladder cancer cells decreased proliferation, colony formation and invasiveness. Circ_0006332 knockdown increased E-cadherin levels and decreased Vimentin, CCNB1 and P21 protein expression. This suggests that circ_0006332 promotes epithelial–mesenchymal transition and cell cycle progression. In vivo experiments in nude mice showed that circ_0006332 knockdown bladder cancer cells form significantly smaller tumors than the controls. Our study demonstrates that circ_0006332 promotes the growth and progression of bladder cancer by modulating MYBL2 expression by acting as a sponge for miR-143. Circ_0006332 is thus a potential early diagnostic marker of bladder cancer.

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Section: Discussionmentioning

confidence: 99%

Circ_0006332 promotes growth and progression of bladder cancer by modulating MYBL2 expression via miR-143

Liu

et al. 2019

Aging

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“…One micromolar linear RNAs were incubated with 3 μM DNA splint oligomer (complementary 10-nt regions at the 5′ and 3′ ends of the RNA), T4 DNA ligase (Thermo Fisher) in 500 μL reaction for 2 h according to the published protocol. 30 …”

Section: Methodsmentioning

confidence: 99%

A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity

Chatterjee

et al. 2022

European Heart Journal

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Aims Cardiotoxicity leading to heart failure (HF) is a growing problem in many cancer survivors. As specific treatment strategies are not available, RNA discovery pipelines were employed and a new and powerful circular RNA (circRNA)-based therapy was developed for the treatment of doxorubicin-induced HF. Methods and results The circRNA sequencing was applied and the highly species-conserved circRNA insulin receptor (Circ-INSR) was identified, which participates in HF processes, including those provoked by cardiotoxic anti-cancer treatments. Chemotherapy-provoked cardiotoxicity leads to the down-regulation of Circ-INSR in rodents and patients, which mechanistically contributes to cardiomyocyte cell death, cardiac dysfunction, and mitochondrial damage. In contrast, Circ-INSR overexpression prevented doxorubicin-mediated cardiotoxicity in both rodent and human cardiomyocytes in vitro and in a mouse model of chronic doxorubicin cardiotoxicity. Breast cancer type 1 susceptibility protein (Brca1) was identified as a regulator of Circ-INSR expression. Detailed transcriptomic and proteomic analyses revealed that Circ-INSR regulates apoptotic and metabolic pathways in cardiomyocytes. Circ-INSR physically interacts with the single-stranded DNA-binding protein (SSBP1) mediating its cardioprotective effects under doxorubicin stress. Importantly, in vitro transcribed and circularized Circ-INSR mimics also protected against doxorubicin-induced cardiotoxicity. Conclusion Circ-INSR is a highly conserved non-coding RNA which is down-regulated during cardiotoxicity and cardiac remodelling. Adeno-associated virus and circRNA mimics-based Circ-INSR overexpression prevent and reverse doxorubicin-mediated cardiomyocyte death and improve cardiac function. The results of this study highlight a novel and translationally important Circ-INSR-based therapeutic approach for doxorubicin-induced cardiac dysfunction.

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“…Next, enzymatic ligation using DNA/RNA ligases can circularize linear RNAs. Oligonucleotides with complementary sequences spanning the 5′ and 3′ ends of linear RNAs work as splints to physically bring each end of a linear RNA into proximity; ligases can covalently connect both ends ( Abe et al, 2018 ). A caveat of this approach is the possibility of concatemer generation, reducing the overall circular RNA yield.…”

Section: Synthetic Circular Rnas As Therapeutic Moleculesmentioning

confidence: 99%

Circular RNAs in and out of Cells: Therapeutic Usages of Circular RNAs

Son

Han

2023

Molecules and Cells

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RNAs are versatile molecules that are primarily involved in gene regulation and can thus be widely used to advance the fields of therapeutics and diagnostics. In particular, circular RNAs which are highly stable, have emerged as strong candidates for use on next-generation therapeutic platforms. Endogenous circular RNAs control gene regulatory networks by interacting with other biomolecules or through translation into polypeptides. Circular RNAs exhibit cell-type specific expression patterns, which can be altered in tissues and body fluids depending on pathophysiological conditions. Circular RNAs that are aberrantly expressed in diseases can function as biomarkers or therapeutic targets. Moreover, exogenous circular RNAs synthesized in vitro can be introduced into cells as therapeutic molecules to modulate gene expression networks in vivo. Depending on the purpose, synthetic circular RNA sequences can either be identical to endogenous circular RNA sequences or artificially designed. In this review, we introduce the life cycle and known functions of intracellular circular RNAs. The current stage of endogenous circular RNAs as biomarkers and therapeutic targets is also described. Finally, approaches and considerations that are important for applying the available knowledge on endogenous circular RNAs to design exogenous circular RNAs for therapeutic purposes are presented.

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Preparation of Circular RNA In Vitro

Cited by 10 publications

References 12 publications

Circ_0006332 promotes growth and progression of bladder cancer by modulating MYBL2 expression via miR-143

Circ_0006332 promotes growth and progression of bladder cancer by modulating MYBL2 expression via miR-143

A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity

Circular RNAs in and out of Cells: Therapeutic Usages of Circular RNAs

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