Supplementary Material for: Profiling and Bioinformatics Analysis Revealing Differential Circular RNA Expression about Storage Lesion Regulatory in Stored Red Blood Cells

Introduction: Circular RNA (circRNA) plays an important role in regulating metabolism of red blood cells (RBCs) and their storage lesions, but the study of how circRNA expression changes in stored RBCs has rarely been conducted. Methods: The expression change of circRNA was systemically evaluated via high-throughput sequencing on healthy RBCs on day 0, 20, and 40. And then we confirmed the reliability of the high-throughput sequencing analysis by RT-qPCR characterization on selected circRNAs. A higher parental gene enrichment was used to explore circRNA function in pathways. In addition, we deciphered a dysregulated circRNA-related ceRNAs network, and identified three circRNA-miRNA-mRNA regulatory axes related to storage lesion. Results: We identified 2,586 known and 6,216 putative novel circRNAs, more than 100 circRNAs expression levels were shifted, and the number of downregulated circRNAs was greater with longer storage time. Furthermore, a higher parental gene enrichment related to circRNA was found in pathways, including cAMP signaling pathway, ubiquitin-mediated proteolysis, apoptosis, adhesion, MAPK signaling pathway, cystine methionine metabolism, RNA degradation, RNA transport, TGF-β, and actin regulatory pathway. hsa_circ_0007127-miR-513a-5p-SMAD4, hsa_circ_0000033-miR-19a-3p-VAMP3, and hsa_circ_0005546-miR-4720-CCND3 regulatory axes related to storage lesion was found. Conclusions: Through investigation in circRNAs profile and circRNA-miRNA-mRNA interactions, this study provides insights on stored RBC circRNA expression changes, which closely relate to the storage lesion of RBCs and their physiological functions.

引言：环形RNA（circular RNA, circRNA）在调控红细胞（red blood cells, RBCs）代谢及其储存损伤中发挥重要作用，但目前针对储存过程中红细胞内circRNA表达变化的相关研究仍较为匮乏。方法：本研究通过对第0、20、40天的健康红细胞进行高通量测序，系统评估了circRNA的表达变化情况；随后选取部分circRNA进行实时定量逆转录聚合酶链反应（RT-qPCR）验证，确认了高通量测序分析结果的可靠性。通过对circRNA的亲本基因进行富集分析，探究其参与的信号通路功能；此外，本研究解析了失调的circRNA相关内源竞争RNA（competing endogenous RNAs, ceRNAs）调控网络，并鉴定出3个与红细胞储存损伤相关的circRNA-微小RNA（microRNA, miRNA）-信使RNA（messenger RNA, mRNA）调控轴。结果：本研究共鉴定出2586个已知circRNA以及6216个潜在新circRNA；其中超过100种circRNA的表达水平发生显著改变，且随着储存时间延长，表达下调的circRNA数量更多。进一步的亲本基因富集分析显示，circRNA相关的富集通路包括cAMP信号通路、泛素介导的蛋白水解、细胞凋亡、细胞黏附、丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路、胱氨酸-蛋氨酸代谢、RNA降解、RNA转运、转化生长因子β（transforming growth factor-β, TGF-β）以及肌动蛋白调控通路。本研究鉴定出与红细胞储存损伤相关的3个调控轴：hsa_circ_0007127-miR-513a-5p-SMAD4、hsa_circ_0000033-miR-19a-3p-VAMP3以及hsa_circ_0005546-miR-4720-CCND3。结论：本研究通过分析储存红细胞的circRNA表达谱及其circRNA-miRNA-mRNA互作关系，揭示了储存过程中红细胞circRNA的表达变化特征，该变化与红细胞储存损伤及其生理功能密切相关。