Screening And Mechanism Study Of MicroRNA Modulating IFN-γ

The immune system represents the body’s first defending line against foreign microbial invasion and interferon is an important effecter factor within the immune system. Interferon-y (IFN-y) belongs to type Ⅱ interferons family, it is mainly produced by activated T cells, NK cells during immune response. It plays an important role in both innate and acquired immunity, especially shows potent protective effect in the case of intracellular parasite infection. microRNAs(miRNA) represents a group of recently discovered18-25bp non-coding RNAs. Over the past decade, numerous studies have revealed miRNA’s regulatory role in the immune system and important role in keeping homeostasis of immune response. The present study chose IFN-y, one of the important immuno cytokines, as target. Combine bioinformatics and biology experimental methods trying to find out and validate IFN-y regulating miRNAs. The aim of our study is to systematically interpret miRNA’s regulatory mechanisms on IFN-y expression from different levels. Hopefully, the present study will deepen our knowledge on the mechanism of miRNA-mediated regulation of IFN-y in the immune response and reveal a novel regulatory mechanism by miRNA. Furthermore, try to provide theoretical basis for developing miRNA as a diagnostic marker and theraqeutic treatment. The detailed research content includes the followings:1. The prediction of IFN-y promoter and3’UTR targeting candidate miRNAsThe sequence of IFN-y promoter region i.e.1197bp upstream of the transcription start site and the3’UTR region sequence were submitted to the online miRNA prediction software. By online software analyzing, predicting that the promoter region of IFN-y targeting50miRNAs, and the IFN-y3’UTR targeting11miRNAs. We synthesized miRNA mimics and inhibitors according to miRNA sequence from miRBase.2. Construction of reporter plasmidIFN-y promoter and3’UTR region were amplified and inserted into the pGL3-Basic and pMIR-REPORT vector respectively. Successful constructs of IFN-y promoter reporter and3’UTR reporter were termed pGL3-TSS1197and pMIR-IFNG-3’UTR.3. Screening and verification of IFN-y regulating miRNAThe candidate miRNA mimics together with IFN-y promoter reporter plasmids were co-transfected into HEK-293T cells and the reporter activity was detected by the dual luciferase assays. We found that miR-27a can significantly inhibit IFN-y promoter activity. In the other hand, after screening the candidate miRNA with3’UTR reporter, we found that miR-27a also negatively regultate IFN-y throught binding to its3’UTR region. Next, we further validated miR-27a’s effect on the IFN-y is specific by introducing miRNA inhibitors into the reporter assay. Finally, by generating mutant reporter plasmid, we showed the precise miR-27a binding site on the IFN-y and it is highly consistent with the bioinformatics predict.4. miR-27a regulate the IFN-y expression in T cellsTo further validate miR-27a’s effect on IFN-y protein expression, T-bet expression plasmid and miR-27a mimics were electroporated into EL4cells. After stimulation with PMA and ionomycin for24h, IFN-y mRNA expression was detectable by Real-Time PCR and IFN-y protein expression was detected by ELISA. Comparing with the control scrambled miRNA mimics, miR-27a significantly decrease IFN-γ mRNA level and inhibits IFN-y protein expression. Taken together, data indicates that miR-27a plays a negative regulatory function on the expression of IFN-y.5. The relationship between the expression of miR-27a and the IFN-yIn order to interpret miR-27a’s role in the regulation of IFN-γ more profoundly, we further analyzed miR-27a’s expression side by side with IFN-y. We detected the expression level of miR-27a in EL4cells at6h,12h,18h,24h post PMA and ionomycin treatment. It was shown that after the stimulation, miR-27a expression was inhibited in the first12hours but after that it gradually restored to the normal level during the following12hours. These data indicates that in the early time miR-27a was suppressed by the rapid increase expression of IFN-y in T cells, but over time, this inhibition was gradually weakened.