Investigate The Immune Regulation Mechanism Of Dendritic Cells Autophagy Mediated By Atg12 In Thymoma With Myasthenia Gravis

Objective:Compared Atg12 expression and autophagy levels of dendritic cells(DCs)in the tumor tissue between thymoma and thymoma with myasthenia gravis(MG).Figured out the regulation of Atg12 to autophagy at cellular level.Further explored the effect of thymoma on the autophagy of DCs in the microenvironment.Looked for the differentially expressed genes of thymoma subtypes.Explored the upstream key factors and pathways that may affect Atg12 expression to improve the mechanisms of Atg12-mediated autophagy.Established the animal model to explain the potential role of Atg12-mediated DCs autophagy in MG at an in vitro level.Methods: 1.43 different types of thymoma and thymoma with MG specimens were selected to make paraffin sections,and the expression of CD11 c,CD80,HLA-DR,LC3,Atg12 proteins were detected in the two groups of specimens by multicolor immunofluorescence technology.2.Atg12 expression was up-regulated or down-regulated in thymoma cell line Thy0517,and the expression of autophagy-related genes Atg12,P62,LC3,Beclin1 were detected by quantitative real-time PCR(rt-PCR)and western blot(WB).3.Mature DCs were stimulated differentiation from THP-1.After Thy0517 thymoma cells and DCs co-culturing by Transwell,the m RNA and protein of DCs were extracted to detect the expression of Atg12,P62,LC3,Beclin1 by rt-PCR and WB.4.Digging the chip information of thymoma samples in TCGA and GEO databases,differential gene expression was analyzed by bioinformatics.KIT proto-oncogene ligand(KITLG)as a potential characteristic markers of type A and AB thymoma was confirmed by PCR filtering and comparing our existing thymoma chip data.Next,KITLG-related m RNA,micro RNA and signaling pathways were further analysed.43 different types of thymoma and thymoma with MG specimens were selected,and immunohistochemistry was used to detect the expression of KITLG in the two groups and different thymoma subtypes.Transfection in Thy0517 cells changed the expression of KITLG,using rt-PCR and WB to detect the expression of key molecules in the MAPK pathway,as well as the levels of downstream Atg12 and other autophagyrelated genes P62,LC3,and Beclin1.5.Using CRISPR / Cas9 technology to knock out Atg12 gene of dendritic cell population in C57 BL / 6J mice,together with normal C57 BL / 6J mice,were purchased to establish experimental autoimmune myasthenia gravis(EAMG)model by periodically injecting Acetylcholine receptor(Ach R)polypeptide.Each week,the mice were scored for myasthenia gravis and their weight was measured.On the 63 rd day after injecting Ach R polypeptide,the peripheral blood of the mice was drawn off and DCs were isolated.The m RNA and protein of the DCs were extracted to detect the expressions of autophagy-related genes Atg12,P62,LC3,Beclin1 by rt-PCR and WB.Results: 1.Multi-color immunofluorescence through 4 color schemes showed that thymoma with MG patients had higher Atg12 expression of DCs in thymoma tissues than that in simple thymoma patients.The level of DCs autophagy in thymoma tissues was also enhanced in thymoma with MG patients.The difference was statistically significant(p <0.05).2.When the expression of Atg12 was up-regulated in the Thy0517 cell,the expression of P62 decreased,and the level of LC3Ⅱ/ LC3Ⅰ increased(p <0.05),while the expression of Beclin1 did not change.When the expression of Atg12 was down regulated in the Thy0517 cell,the changes of these autophagy factors showed the opposite trend except Beclin1.3.A stable system of THP-1 transforming mature DCs was successfully established.When the autophagy of thymoma cells increased,the levels of Atg12 and LC3Ⅱ/ LC3Ⅰ of DCs increased and the level of P62 decreased(p <0.05)in the co-culture environment,while the change of Beclin1 was not significant.When the autophagy of thymoma cells weakened,the changes of these autophagy factors(except Beclin1)of DCs showed the opposite trend to the former in the co-culture environment.4.The expression of KITLG in type A and AB thymoma was significantly up regulated compared with other subtypes,and the overall expression of KITLG in thymoma with MG was also higher than that in simple thymoma.It also triggered a series of abnormal changes in m RNA,mi RNA,and signaling pathways when KITLG was overexpressed.Altering KITLG expression in thymoma cells,it was found that GRB2,phosphorylated BRAF,phosphorylated MEK1/2 and phosphorylated ERK1/2 levels increased with KITLG overexpression or decreased with KITLG low expression(p < 0.05).At the m RNA level,GRB2 showed a similar trend,while the expression of other genes did not change significantly.In addition,when the expression of KITLG in thymoma cells increased,the expression of Atg12,Beclin1,LC3 Ⅱ /LC3 Ⅰ increased,and the expression of P62 decreased(p <0.05),and vice versa.5.After EAMG modeling,the weight of Atg12 conditional knockout mice and normol C57BL/6J mice both showed a trend of slowly decreasing after a transitory increasing.Compared with the normol C57BL/6J mice,MG appeared and progressed more slowly in Atg12 conditional knockout mice.Though in Atg12 conditional knockout mice,the clinical score was slightly higher and weight loss is smaller,the overall difference is not statistically significant between both groups.In addition,in Atg12 conditional knockout mice,Atg12 expression of DCs decreased significantly,LC3 Ⅱ /LC3 Ⅰ decreased,P62 expression increased(p <0.05),but Beclin1 expression did not change significantly.Conclusions: 1.The Atg12 expression of DCs in thymoma with MG was higher than that in simple thymoma.Altering Atg12 expression in thymoma cells could regulate the cell autophagy.Co-culture of different autophagy states thymoma cells with DCs,the Atg12 expression of DCs and DCs autophagy levels could be affected,indicating that thymoma can affect Atg12-mediated DCs autophagy through the microenvironment.2.KITLG might become a biomarker of type A and AB thymoma,and it was more highly expressed in thymoma with MG.Overexpression of KITLG promoted phosphorylation of the MAPK pathway so that downstream autophagy genes such as Beclin1,Atg12 and LC3 up-regulating and P62 down-regulating,which promotes Atg12-mediated autophagy.It was indicating that KITLG might become an important upstream target of the Atg12 autophagy pathway.3.Compared with normal C57BL/6J mice,the level of DCs autophagy in Atg12 conditional knockout mice was significantly reduced.After EAMG modeling,MG symptoms appeared later,indicating that Atg12-mediated DCs autophagy might be one of the factors of MG pathogenesis.