Differential MiRNA Expression In The Regulatory T Cells From Primary Graves’ Disease Patients And The Mechanism Study Of The Non-specific Binding Of The Fluorescent Labled MiRNA On Cell Surface

BackgroundMicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. Graves disease(GD) is one of the most common autoimmune diseases, and regulatory T cells (Tregs) are pivotal in the modulation of immune activities. Many studies have suggested that miRNAs can predict the occurrence and development of disease. However, so far there is little definite knowledge about the role of miRNA in GD, not to mention that in the Tregs of GD patients. In addition, the fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAsObjectiveIn this study, we aimed to describe the altered expression of microRNAs in the peripheral Tregs (pTregs) of primary GD and compare the number alteration of pTregs between primary GD and healthy controls (HC).Apart from this, some difficulties in studying the differently expressed microRNA’s function and its regulation mechanism in pTregs were pointed out. What’s more, the mechanism of nonspecific adherence of fluorescently labeled microRNA on cells were studied.Materials and methods45 newly diagnosed GD patients and 61 healthy controls were recruited. CD4+CD25high Treg cells were isolated from their peripheral blood by flow cytometry(FCM) and its percentage was compared between GD and HC. Some miRNAs which were expressed abnormally in Tregs were identified through real-time polymerase chain reaction(RT-PCR).Then, one of the differential expressed microRNAs was chosen to study its roles in normal Tregs by transfecting the fluorescence labled microRNA into cells with lipofectamine reagent. However Cy5 labled miRNA was found to adhere to cells nonspecifically with or without RNAiMAX. Therefore,its mechanism and university were investigated further. miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were detected by flow cytometer and laser confocal microscopy(LCM).ResultsThere is no difference of the percentage of CD4+CD25high Tregs accounted for peripheral blood mononuclear cells(PBMC) between GD and HC,so as the number of CD4+CD25high Tregs per volume of peripheral blood between GD and HC. While RT-PCR suggested that miR28,miR363 and miR191 expressed differently in CD4+CD25high Tregs from initial GD compared with HC group.miR28 and miR363 were upregulated while miR191 downregulated .But some microRNAs such as miR195,miR519e miR374a and so on which may be low in Tregs cells haven’t been detected in this study.Cy5 labled microRNA was proved to adhere on the surface of CD4+T cell under the detection of FCM and LCM.Following the further study, results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K.562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.ConclusionsOur results implied that the proportion of CD4+CD25high Tregs from primary GD did not changed significantly compared with HC groups. However, there still were some microRNAs expressed differently in CD4+CD25high Tregs from primary GD and these miRNAs may be related to the immune disorder in GD patients. However, limited to the small number of Treg in peripheral blood from per individual and the primary and suspension characteristic of newly isolated Treg, the functional study of microRNA in Tregs were difficult to perform for modified technology required in experiment. That flourescence labeled miRNA transfected into cells to investigate its influence on cell is convenient and wideused. But fluorescent dye will bind to the cell membrane nonspecifically. Its mechanism study demonstrated that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.