| MicroRNAs ( miRNA) are endogenously expressed, single-stranded non-coding RNAs, about 22 nucleotides (nt) long. The mature miRNA is derived from a 70–90-nt-long hairpin RNA structure cleaved by the RNase III enzyme Dicer. MicroRNAs regulate gene expression via perfect or imperfect complementary to their target mRNAs by cleaving homologous targets or repress their translation. miRNAs are often highly conserved among divergent species, and also the expression of microRNAs is different in distinct cells or tissues even at different developmental stages. Because of important roles microRNA plays in tissue development and other biological processes, it has been widely studied in various systems. Dendritic cells (DCs), derived from hemopoietic stem cells (HSCs), are the strongest antigen process cells (APCs) discovered up to now. The differentiation and development of DCs is distinctive in temporal and spatial, which make it a good system for microRNA regulation study in its development, proliferation, differentiation and apoptosis. Previous studies have shown that miR-200b can act as a regulator in repressing cellular malignant transformation. Although there are a few studies about microRNA expression in DCs, the effect of miR-200b on DCs development is not reported. Our study aims to explore the effect of miR-200b on the development of monocyte-derived DCs and its mechanisms.Firstly, to establish a stable peripeheral blood CD14~+ monocyte-derived DC culture system, we isolated the peripeheral blood CD14~+ monocyte by immunomagnetic beads, and induce DC differentiation with IMDM and RPMI-1640 medium. Under the same cytokines condition GM-CSF+IL-4, we identify the differences DC derived from IMDM or RPMI-1640 medium in morphology, phenotype and function. The results showed that the two medium induced DC has no significantly morphological differences. The morphology of immature DC ( iDC) is cylindrical ball, and mature DC( mDC) with LPS stimulation displayed different protruding veils, a typical morphology of DC. Endocytosis ability by flow cytometry was the same in IMDM-DC and RPMI 1640-DC. The endocytic efficiency of immature DC was more than 90%, while the mature DC was less than 50%. Moreover, there was no difference in CD14 and CD83 expression, but has a significantly decreased expression of CD1a in IMDM culture system (IMDM-iDC was 32.34%±9.02%,IMDM-DC was 21.04%±6.33%), comparing with RPMI-1640 culture system, the high expression levels of CD1a was found (RPMI 1640-iDC was 85.58%±4.26%, and RPMI 1640-mDC was 87.68%±5.83%). After LPS stimulation, as a surface marker of mature DC, CD83 expression showed significantly changes, RPMI-1640-mDC and IMDM-mDC both expressed high levels of CD83, respectively 86.62±1.37% and 64.68±5.26%. To evaluate DC immunoregulatory function, mixed leukocyte reations (MLR) was carried out. Upon stimulation of allogeneic DC, the CD3+ cell presented a strong proliferation response at a mean incorporation of 1402.6±128.07 cpm (IMDM-DC) and 5274.4±426.26 cpm (RPMI 1640-DC). However, the effect was slightly decreased by the iDC stimulation ( mean incorporation of 277.2±25.79 cpm, IMDM-iDC, 3170±454.12 cpm, RPMI 1640-iDC). These stimulatory effects were DC dose-dependent. Since cytokines play an important role in DC development and function, we used Bio-plex to test the expression of cytokines in MLR system. The inhibitor factor IL-10 secretion in the MLR by IMDM culture system was up-regulated (56.67±33.41 pg / mL), also increasing cytokines including IL-6(315.48±15.43 pg / mL ) and IL-8(17300.76±2273.89 pg / mL ), but IL-12 production was not detected. Because the secretion of IL-12 is critical for the maturation and function of DC, we next assayed IL-12 secretion in MLR by RPMI 1640 culture system. The result determined that IL-12 production was significantly increased (5658.09±540.46 pg / mL). Our data suggested that various DC could be induced by different culture system. In order to identify the mechanism of DC development, we used the RPMI-1640 culture system to explore the effect of microRNA on the DC development and differentiation.Secondly, we uncovered that miR-200b maybe effect DC development and differentiation by bioinformatics methods. miR-200 family included miR-200a/miR-141 and miR-200b/miRNA-200c/miRNA-429 subsets according to different functional areas and the"seed"sequences, which also can be divided into two gene clusters miR-200a/miR-200b/miR-429 and miR-200c/miR-141. Previous study has shown that the function of miR-200 family was diversity, and miR-200b has abnormal expression in many tumors. For example, the over-expression of miR-200b in T24 bladder cance cell lines can significantly change the cell morphology. The tumor cell was lost the presudopodia-like protrusions and the shape of cell become irregular. Further experimental results showed that the migration of tumor cells could also be significantly decreased after miR-200b transfection. Using bioinformatics websites, we predicted the target gene of miR-200b including WIPF1,ZEB1,ZEN2 and JAG2 and found they expressed in DC, whose 3`-UTR contained the target sequence of miR-200b. Combined with the similarity between morphological characteristics of DC and tumor cells, we have reason to speculate that DC was the target cell of miR-200b. Then, we used real time-PCR to detect the expression of miR-200b in DC at different culture time. The result demonstrated a decreasing trend, suggesting a relationship between miR-200b and DC development.Thirdly, according to the above results, we identified the function of miR-200b in DC development. The overexpression of miR-200b and random sequence miRNA were transfected into iDC and mDC by DOTAP transfection reagent. We observed the morphology, phenotype and function. For the transfection efficiency, flow cytometry assay showed that more than 70% cells were positive. The morphology of mDC transfected miR-200b (200b-mDC) became round and smooth rather than veiled, while mDC transfected random sequence (NC-mDC) and normal DC displayed the typical various protruding veils. The phenotype by flow cytometry almost remained unchange. The surface antigen combination is CD14-CD1a+CD83- and CD14-CD1a+CD83+ in immature DCs and mature DCs respectively. In phagocytosis experiment, the phagocytosis ability of 200b-iDC was lower than iDC and NC-iDC, suggesting miR-200b maybe inhibit the development of iDC. It has been well documented that DC can stimulated T lymphocyte proliferation in vitro. MLR assay was carried out. Upon the stimulation of mDC or NC-mDC, CD3+ T lymphocyte proliferation was not displayed the obviously difference, but the ability of stimulated T cell proliferation by 200b-mDC was significantly decreased. When the ratio of DC to T cells was 1:10, the cpm values of mDC, NC-mDC and 200b-mDC were 3623±287.7,3366±183.2, and 2250±136.2. The proliferation activity was restrained if the DC to T cells ratio was 1:50, the cpm values were 934.8±180.6, 882±123, and 546.8±75.8 respectively. These results determined that miR-200b can reduce the phagocytic activity of iDC, and down regulate the ability of mDC to stimulate T cell proliferation. In addition, the cytokine expression in MLR system displayed obviously variance. As the maturation marker, the level of IL-12 secretion was reduced in 200b-mDC MLR system, and the production of IL-2 and IL-6 also decreased, relating with T cell proliferation. Although IL-10 production was very low, it was still higher than that of mDC and NC-mDC MLR system. The data suggested that, either morphology or function, miR-200b could suppress the development of DC.In conclusion, our data suggested that miR-200b could regulate the development of DC, but the targets are still not clear. Our study provided theoretical and experimental instruction for understanding miRNA modulation of DC development and clinical therapy. |
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