Mechanism Exploration Of TLR7/MiR-15b Signal Axis In Regulating CCND3 Expression Of Lupus B Cells

Systemic lupus erythematosus (SLE) is a typical human autoimmune disease which is characterized by abnormal activation of B and T cells. And the abnormal of B cells plays center role in the pathogenesis of SLE; however the mechanism behind this is still a puzzle. In recent years, it has become apparent that TLR7 which senses single-stranded RNA plays a pivotal role in the development of SLE. Our previous research has indicated that IFN-α, TLR7 and BCR signal pathway participating the abnormality of B cells in SLE. CCND3 is one of the three D-type (D1, D2, and D3) cyclins that regulate the G1/S phase transition of the cell cycle. Experiments from CCND3-/-mice demonstrated that CCND3 is required for the proliferation, development and differentiation. And abnormal expression of CCND3 is associated with many diseases. To date, there is no evidence to confirm the abnormality of CCND3 in SLE.In this study, we have done a B cell profiling chip and identified that CCND3 is a hub node in SLE B cell gene-net-work. We also found higher CCND3 and lower miR-15b expression level in SLE patients and lupus mice B cells. Activation of TLR7 increased CCND3 expression but decreased miR-15b level in B cells in vitro. Furthermore, we identified CCND3 is a direct target gene of miR-15b. Consistently, we found that treatment B6 mice with TLR7 agonists imiquimod significantly downregulated the expression of miR-15b while upregulateed CCND3 in mice B cells. Together, our results showed that activation of TLR7 increased CCND3 expression via downregulating miR-15b in B cells, suggesting that extrinsic factor-induced CCND3 expression may contributes to B cell abnormality in SLE.1. Bioinformatics analysis of DEGs in B cells from active SLE patients.To explore candidate genes related to SLE in B cells, a microarray-based gene expression profiling of CD19+ B cells between five active SLE patients and five healthy donors were conducted. And 1812 genes were significantly different in the SLE groups compared to the healthy groups. Of those,958 genes were upregulated while 854 genes were downregulated. To verify the data obtained from the microarray, 8 selected DEGs (4 upregulated and 4 downregulated) were examined by real-time PCR, based on their involvement in different functional groups and/or pathways. Our results suggested that the investigated genes had congruent results between real-time PCR and the microarray assays. Further analysis revealed that many of these DEGs were enriched in inflammation (e.g. type I interferon-mediated signal pathway and cytokine-mediated signaling pathway) and cell cycle (e.g. M phase of mitotic cell cycle and mitotic cell cycle). GO Trees analysis found that those upregulated genes mainly influence the cell cycle. Gene interaction network showed that CCND3 was a hub gene among upregulated genes in the network map, whereas other DEGs in the network were indirectly or directly associated with it, suggesting it probably plays important roles in the development of SLE. At last, qPCR results showed the mRNA levels of CCND3 were significantly higher in both the SLE patient samples and B6.MRL-Faslpr/J mice compared to the normal samples and control C57BL/6 mice.2. Activation of TLR7 increases CCND3 expression via downregulating miR-15b in B cellsGene profiling results suggested that CCND3 is involvement in the B cell abnormity and some researches demonstrated that CCND3 plays pivotal roles in B cell differentiation and formation GC B cells. So, next, we explored what caused the deregulation of CCND3. Interestingly, miR-15b, a miRNA that could regulate CCND3 as predicted by Targetscan software has an opposite expression level as compared to CCND3. In vitro experiments demonstrated that CCND3 is increased while miR-15b was decreased after stimulation with TLR7 ligand-R848. Then, we found that CCND3 is a direct target of miR-15b. In order to confirm the relationship between CCND3 and miR-15b in vivo, we established an imiquimod-induced lupus-like mice model. Expectedly, topical treatment with imiquimod also significantly increased CCND3 and decreased miR-15b in B6 mice B cells.In summary, we found elevated CCND3 and decreased miR-15b in SLE patients and lupus mice B cells. Furthermore, activation of TLR7 decreased miR-15b and increased CCND3 in mice B cells both in vitro and in vivo and CCND3 is a direct target of miR-15b. These results indicated activation of TLR7 by extrinsic factor may contribute to the abnormality of B cells in SLE.