Development of circRNA Detection Protocol for circHIPK3 and its Potential Application in Identifying Novel Biomarkers for Sepsis

Abstract

Sepsis is a severe condition that occurs when the body's response to infection becomes dysregulated, leading to organ dysfunction. Patients that survive are often left with chronical illnesses or long-term complications. Early detection of sepsis is crucial for effective treatment. However, there is a lack of useful biomarkers to improve early detection, prognosis and start of adequate treatment to prevent death and the development of chronical complications. This study focus on the field of epigenetics to find new biomarkers. Epigenetics is a field of biomedical sciences that concentrates on the changes in gene activity and gene expression levels in, among others, circular RNAs (circRNAs). CircRNAs are covalently closed loops that are derived from parts of their precursor mRNAs. They are able to regulate various cellular processes through several mechanisms and have been associated with disease development, including sepsis. This study develops a protocol to be able to detect circRNA, with a specific focus on circHIPK3, levels in vitro and eventually in vivo. Therefore, we treated human umbilical vein endothelial cells (HUVECs) with extracellular histones to mimic sepsis pathology in vitro, extracted their RNA and treated these extracts with RNase R to eliminate all linear RNA, so only the circRNAs would be left. Gene expression levels were determined with SYBRGreen RT-qPCR where household gene circGAPDH functioned as a reference. Downstream expression levels of miRNA targets of circHIPK3 were determined with TaqMan RT-qPCR. Our results show that the developed protocol is specific and sensitive for even small concentrations of circHIPK3 in HUVECs. Furthermore, the RNase R treatment application time was determined to be after RNA extraction to yield the highest RNA concentration and purity for analysis. CircHIPK3 and its downstream miRNA targets were not differentially expressed after HUVECs were treated with extracellular histones. It is possible that circHIPK3 levels are unaffected following histone-mediated cellular stress. Moreover, circHIPK3 levels were undetectable in patient plasma, probably due to the low levels of RNA in the samples. This thesis developed a protocol for the detection of circHIPK3 and potentially other circRNAs. However, it needs to be optimized for patient samples. Despite these challenges, it lays the groundwork for future research in this area. By understanding the genetic markers associated with sepsis, healthcare professionals may diagnose and treat patients more effectively, leading to improved clinical outcomes.