MiRNA-103b Downregulates ITGB3 and Mediates Apoptosis in Ex Vivo Stored Human Platelets

Abstract

Blood bank-stored human platelets are one of the life-saving transfusion products to prevent bleeding in multiple clinical settings. In ex vivo storage, platelets undergo apoptosis and it is highly desirable to prevent this process to preserve platelet quality. However, underlying mechanisms of apoptosis are not well understood in stored platelets. Integrin beta 3 (ITGB3) glycoprotein plays multiple roles in platelet physiological processes, and it was reported in other cell types that downregulation of ITGB3 induces apoptosis. Small noncoding regulatory RNAs known as microRNAs (miRNAs), some of which are abundant in platelets such as miR-103b that belongs to miR-103 family of miRNAs, known to play key roles in platelet functions both in vivo and during storage; Cellular miR-103 downregulates certain genes in other cell types and promotes apoptosis. However, whether miR-103b can target and downregulate ITGB3 in stored platelets and such miRNA regulation promotes apoptosis is not known. Here we tested this working hypothesis. Our objective of this study is to validate the abundance of miR-103b in stored platelets and identify whether ITGB3 is a target of miR-103b for downregulation and this interaction promotes apoptosis. RT-qPCR validation of miR-103b was performed in 11 donor samples at 3 different storage time points. In-silico analysis was performed to identify predicted targets of the miR-103b. The miRNA and messenger RNA interactions were confirmed using different biochemical approaches such as qRT-PCR, western blotting and, suppression of luciferase reporter gene expression by ectopic expression of miR-103b in HeLa cells. Final validation of functional role of miR-103b in ITGB3 downregulation and resulting induction of apoptosis was assessed in stored platelets by FACS analysis following ectopic expression of miR-103b. Using Target Scan Vert algorithm, we identified several integrin subunit-encoding mRNAs as potential targets of miR-103b. While ITGB3 and ITGB6 found to have two targeting sites for miR-103b, since ITGB3 is known to play a role in apoptosis, we chose this for further validation in this study. Ectopic expression of miR-103b decreased the luciferase reporter activity in HeLa cells and decreased ITGB3 mRNA and protein levels in platelets, concomitant with increase in apoptosis. The results demonstrate that in stored platelets miR-103b is highly expressed and can interact with and downregulate ITGB3 and promote apoptosis in stored platelets.