Clin Res Cardiol (2021). 10.1007/s00392-021-01933-9 |
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Hans-Jürgen-Bretschneider-Abstract-Preis: |
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S. Cushman1, K. Xiao1, D. LU1, C. Bär1, T. Thum1 | ||
1Institut für Molekulare und Translationale Therapiestrategien, OE-8886, Medizinische Hochschule Hannover, Hannover; | ||
Introduction: Cardiovascular diseases are the leading cause of death worldwide however, there is increasing evidence that a subtype of non-coding RNA, circular RNAs (circRNAs), formed through backsplicing, are emerging as important regulators of cardiac development and cardiovascular diseases. CircRNAs are known to regulate gene expression through a multitude of functions such as; acting as miRNA sponges, transcription enhancers, interacting with RNA binding proteins, or interfering with the expression of the linear mRNA host. Prevalent circRNAs involved in cardiac development and potential regeneration can be determined by focusing on their differential expression in regeneration competent versus non-competent neonatal mouse hearts.
Methods and Results: RNA-seq
was performed to determine the expression levels of the circRNA candidate in
day 1 and day 7 mouse hearts. Several candidates underwent further tests for
circRNA validation until a potential candidate for regeneration and
proliferation, circREGEN, was found to be downregulated on day 7 both in
RNA-seq analysis as well as in neonatal mouse tissue samples using RT-PCR. Tissue distribution was
measured for circREGEN, showing high expression levels in the heart,
specifically, neonatal cardiomyocytes. Importantly, the function of circRNAs
can be partially determined by their localization. Through subcellular
fractionation as well as CRISPR/dCas13 live cell tracking studies, we showed
that circREGEN is localized in the cytoplasm.
Further experiments were performed to functionally validate circREGEN using siRNAs, to specifically target its unique backsplice sequence in neonatal mouse and rat CMs. Since we found a decreased level of circREGEN expression after regeneration, we assumed that knocking down this circRNA in neonatal cells would decrease proliferation. Using Ki67 staining, we were able to confirm this hypothesis in the neonatal mouse and rat cells after siRNA knockdown, as well as see a decrease in cell viability using a WST-1 assay.
Future
experiments will include overexpression of circREGEN to determine if
proliferation and cell viability increase, as well as to use RNA pull-down and
mass spectrometry to determine interaction partners and regulators of this
circRNA. In conclusion, the ultimate goal would be to determine in an in vivo model if circREGEN has
therapeutic potential to regenerate damaged heart tissue after a myocardial
infarct by stimulating the proliferation of CMs in the adult heart. |
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https://dgk.org/kongress_programme/ht2021/BS890.htm |