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      Silencing of circHIPK3 Inhibits Pressure Overload-Induced Cardiac Hypertrophy and Dysfunction by Sponging miR-185-3p

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          Abstract

          Background

          Cardiac hypertrophy is induced by diverse patho-physiological stimuli and indicates an increase in cardiomyocyte size. Circular RNAs (circRNAs) and microRNAs (miRNAs), members of noncoding RNAs, are involved in several biological processes and cardiovascular diseases (CVD). Here, we investigated the potential role of circHIPK3, which is produced by the third exon of the HIPK3 gene in cardiac hypertrophy.

          Methods

          qRT-PCR and Sanger sequencing were conducted to identify the expression and characteristics (head-to-tail structure, stability, and location) of circHIPK3 in cardiac hypertrophy; Immunostaining of α-SMA was performed to evaluate the size of the cardiomyocytes; Transverse aortic constriction (TAC) induced hypertrophy models of mice were established to investigate the effect of circHIPK3 in vivo. Bioinformatics analysis and luciferase reporter assays, RNA immunoprecipitation, and fluorescence in situ hybridization (FISH) experiments were conducted to investigate the mechanism of circHIPK3-mediated cardiac hypertrophy.

          Results

          circHIPK3 is circular, more stable, and mainly located in the cytoplasm. Silencing of circHIPK3 inhibited the TAC induced cardiac hypertrophy, and reversed the effect of TAC on the echocardiograph parameters, such as left ventricular end-diastolic pressure (LVEDPS), left ventricular fraction shortening (LVFS), left ventricular ejection fraction (LVEF), and left ventricular systolic dysfunction (LVSD), and also the heart weight to tibial length (HW/TL). Angiotensin II (Ang II) Ang II-treated cardiomyocytes showed larger size of cardiomyocyte and upregulation of fetal genes, biomarkers of cardiac hypertrophy, peptide hormones, atrial natriuretic peptide ( ANP) and brain natriuretic peptide ( BNP), and myofilament protein, β-myosin heavy chain ( β-MHC). These effects were reversed by circHIPK3 knockdown. Mechanically, circHIPK3 sponges miR-185-3p. In addition, miR-185-3p targets CASR. The rescue experiments confirmed the interaction between circHIPK3 and miR-185-3p as well as miR-185-3p and CASR.

          Discussion

          Our data suggested that circHIPK3 serve as a miR-185-3p sponge to regulate cardiac hypertrophy revealing a potential new target for the prevention of TAC- and Ang-II induced cardiac hypertrophy.

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          Most cited references 40

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          Circular RNAs are a large class of animal RNAs with regulatory potency.

          Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.
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            Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs

            Circular RNAs (circRNAs) represent a class of widespread and diverse endogenous RNAs that may regulate gene expression in eukaryotes. However, the regulation and function of human circRNAs remain largely unknown. Here we generate ribosomal-depleted RNA sequencing data from six normal tissues and seven cancers, and detect at least 27,000 circRNA candidates. Many of these circRNAs are differently expressed between the normal and cancerous tissues. We further characterize one abundant circRNA derived from Exon2 of the HIPK3 gene, termed circHIPK3. The silencing of circHIPK3 but not HIPK3 mRNA significantly inhibits human cell growth. Via a luciferase screening assay, circHIPK3 is observed to sponge to 9 miRNAs with 18 potential binding sites. Specifically, we show that circHIPK3 directly binds to miR-124 and inhibits miR-124 activity. Our results provide evidence that circular RNA produced from precursor mRNA may have a regulatory role in human cells.
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              Regulation of circRNA biogenesis.

              Unlike linear RNAs terminated with 5' caps and 3' tails, circular RNAs are characterized by covalently closed loop structures with neither 5' to 3' polarity nor polyadenylated tail. This intrinsic characteristic has led to the general under-estimation of the existence of circular RNAs in previous polyadenylated transcriptome analyses. With the advent of specific biochemical and computational approaches, a large number of circular RNAs from back-spliced exons (circRNAs) have been identified in various cell lines and across different species. Recent studies have uncovered that back-splicing requires canonical spliceosomal machinery and can be facilitated by both complementary sequences and specific protein factors. In this review, we highlight our current understanding of the regulation of circRNA biogenesis, including both the competition between splicing and back-splicing and the previously under-appreciated alternative circularization.
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                Author and article information

                Journal
                Drug Des Devel Ther
                Drug Des Devel Ther
                dddt
                dddt
                Drug Design, Development and Therapy
                Dove
                1177-8881
                29 December 2020
                2020
                : 14
                : 5699-5710
                Affiliations
                [1 ]Department of Cardiovascular Surgery, The First Affiliated Hospital with Nanjing Medical University , Nanjing 210029, People’s Republic of China
                Author notes
                Correspondence: Xiaowei Wang Department of Cardiovascular Surgery, The First Affiliated Hospital with Nanjing Medical University , 300 Guangzhou Road, Gulou District, Nanjing210029, People’s Republic of China Email wang_xiaowei1@hotmail.com
                Article
                245199
                10.2147/DDDT.S245199
                7778681
                © 2020 Xu et al.

                This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License ( http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms ( https://www.dovepress.com/terms.php).

                Page count
                Figures: 7, References: 40, Pages: 12
                Funding
                Funded by: the National Natural Science Foundation of China;
                Funded by: National Natural Science Foundation of China, open-funder-registry 10.13039/501100001809;
                Funded by: “333” Project Of Jiangsu Province;
                This work was supported by the National Natural Science Foundation of China (grant number:81573234), National Natural Science Foundation of China (grant number:81773445) and “333” Project Of Jiangsu Province (grant number:LGY2016006).
                Categories
                Original Research

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