Flow cytometry-based high-throughput RNAi screening for miRNAs regulating MHC class II HLA-DR surface expression.

HLA-DR isotype is a MHC-II cell-surface receptor found on APCs and plays a key role in initiating immune responses. In severely immunocompromised patients with conditions like sepsis, the number of HLA-DR molecules expressed on leukocytes is considered to correlate with infectious complications and patients' probability of survival. The underlying regulatory mechanisms of HLA-DR expression remain largely unknown. One probable path to regulation is through microRNAs (miRNAs), which have been implicated as regulatory elements of both innate and adaptive immune system development and function. In our study, flow cytometry-based high-throughput miRNA screening was performed in a stable HLA-DR-expressing human melanoma cell line, MelJuSo, for either up- or downregulating miRNAs of the surface HLA-DR expression. By the end of the screening, the top ten upregulators and top five downregulators were identified, and both the HLA-DR protein and mRNA regulations were further verified and validated. In-silico approaches were applied for functional miRNA-mRNA interaction prediction. The potential underlying gene regulations of different miRNAs were proposed. Our results promote the study of miRNA-mediated HLA-DR regulation under both physiological and pathological conditions, and may pave the way for potential clinical applications.