Study On The Extraction And High-throughput Sequencing Of MiRNA From Glycyrrhiza Uralensis Fisch Decoction And The Effect Of It On Immune Cells

PurposeTo explore whether there are microRNAs(miRNAs) in Glycyrrhiza uralensis Fisch(liquorice) decoction by agarose gel electrophoresie and high-throughout sequencing. After confirming, the miRNAs will be used to treat the peripheral blood mononuclear cells(PBMC) isolated from healthy person. With cell morphological observation cell, cell proliferation count, cell phenotypic detection, immune genes expression measurement, the effect of miRNAs of liquorice on PBMC will be evaluated. MethodsOne, the water decoction of licorice were by high temperature steaming and in freeze drying processing after high temperature treatment. The small RNAs were extracted through the general plant microRNAs rapid extraction kit. Then electrophoresis detected the extracted microRNAs, and tested the concentration and purity by the Dnase Ⅰ digestion. Second, the use of HiSeq high-throughput sequencing technologies, we sequenced analysis of licorice microRNAs, and predicted the presence of new miroRNAs. The raw data was processed to get Clean sequence alignment, base bias analysis and classification of small RNA, then the new microRNAs were forecasted from the small RNA which could not compared to the database and no comment. Three, the licorice miRNAs were compared human genes to analysis the target genes, preliminary screening the regulation candidate human target genes for licorice microRNAs. And using the GO we classified analysis to the function of the target genes and annotations. Four, the licorice microRNAs used the peripheral blood mononuclear cells from healthy person in the peripheral blood, and we observed the cell morphology, counted the cells number and detected the cell phenotypic changes by flow. Using RT-PCR for part of the expression of cytokines and signalling molecules and transcription factors change were analyzed, and we were preliminary exploring of liquorice microRNAs effects on human immune cells. ResultThe microRNAs were effectively from the Licorice extract which by high temperature steaming and in freeze drying processing after high temperature treatment. It show that the structure of the plant miRNA is very stable and not easy to be destroyed, as well as it can provide evidence that the traditional Chinese medicine miRNA enters the body through the digestive tract. We sequenced licorice microRNAs and analysized the known microRNAs and predicted new microRNAs according to the result of sequencing. Clean sequence was comparative analysis.There is an important discovery that the licorice which contain 310 family of microRNAs. It is not common in the research field of current micro RNAs, because the model species arabidopsis also found only 529 family of micrornas. Another finding is forecast to seven new microRNAs.After target gene prediction, we found the part of licorice microRNAs highly match to human genes. For example, these three microRNAs MIR155、 MIR5303、 MIR8016 respectively match to IL-6、STAT1、BCL-2. The predicted target genes which involved in the immune system is a huge number, nearly 1100 or so. These results explained that licorice miRNA may have an effect on the immune function from a certain degree. It makes a possibility that micro RNAs regulated cross-species in theory.Licorice microRNAs not only can enhance the proliferation of PBMC, also can enhance the immune ability of antigen presenting cells. flow analysis, through the miRNA processing of HLA- DR + cells in PBMC happened very significant proliferation, and the increase of HLA- DR + cells means increasing ability of the antigen commission. This may partly explain the effect of licorice micrornas regulate the body’s immune function. RT- PCR results show that the licorice miRNA expression of signalling molecules and transcription factors cause obvious changes, and signaling molecules and transcription factor expression change cause the activation of signaling pathways or suppressed. This may be able to further explain the immune adjustment function. Conclusion:The existence of licorice microRNAs and the foundition that liquorice microRNAs regulating human immune cells provide us a new way of understanding and research of liquorice. Further study of its function of microRNAs bring us new ideas such as the research on the mechanism of action of licorice, clinical application of licorice, dosage form development and new drug development.