The Mechanism Of Deubiquitinase MYSM1 Regulating The Immunomodulatory Funciton Of Adipose-derived Mesenchymal Stem Cell

Mesenchymal stem cells (MSCs) are a class of adult stem cells with self-renewal and multilineage differentiation capacity . In recent years,another ability of MSCs,namely,immunomodulatory function, has become a hot topic. MSCs can inhibit the functions of various lymphocytes and are widely studied in many immune-related diseases, but the immunomodulatory mechanism of MSCs is still not very clear.Ubiquitination and deubiquitination of histone modification are important fields in epigenetics. One of the histone H2A deubiquitination enzymes, MYSM1, plays a critical role in the developments and functions of various immune cells in mice. We hypothesize that whether MYSM1 will regulate the immunomodulatory function of MSCs. This is helpful to provide a new idea and foundation for widely used of MSCs in clinical practice of. So we proceed the following three parts in this study.Part 1: Isolation, culture and identification of murine adipose - derived mesenchymal stem cellsMethods:1. MSCs derived from inguinal adipose tissue (ADSCs) of wild type (WT) and knockout(KO) mice were isolated and cultured. The morphology, proliferation, phenotype and differentiation ability were checked.Results:1. Mysml knockout in ADSCs didn't affect the morphology,phenotype and proliferation of ADSCs. WT and KO ADSCs were positive expression of Sca-1, CD29, CD44 and negative expression of CD86, CD45, CD11b, CD31 and MHC class ? molecules.2. Both WT and KO ADSCs have osteogenic and adipogenic differentiation. The results of qRT-PCR showed that the expressions of osteogenesis related genes Runx2,Osterix and Ocn and adipose related genes Ppar-y, Cebp-a and aP2 were up-regulated under different induction conditions.Part 2: MYSM1 plays an important role in regulating the immunomodulatory function of ADSCs in vivo and in vitroMethod:1. The expression of Mysm1 in inflammatory environment was detected by qRT-PCR.2. The effects of WT ADSCs and KO ADSCs on the proliferation of T cells were compared by co-culture with CD3+T cells. Beforehand, CD3+T cells were labled with CFSE and stimulated by PMA and ionomycin and at last the CFSE signal changes were analyzed via flow cytometry.3. Immune-related cytokines and immunophenotype of ADSCs in the inflammatory environment were examined by qRT-PCR and flow cytometry.4. IBD model was used to detect the different immunomodulatory function of WT ADSCs and KO ADSCs in vivo.Results:1. Mysml was expressed highest under the stimulation of TNFa + IFNy, and increased with the concentration and time.2. Both WT ADSCs and KO ADSCs could inhibit T cells proliferation, but the immunosuppression capacity of WT ADSCs was significantly stronger than KO ADSCs.3. WT ADSCs expressed a higher anti-inflammatory cytokine iNOS and IL10, while KO ADSCs expressed higher inflammatory cytokine IL1?, TNF?, IFN? and IL6. The expression of PD-L1 increased significantly at a low stimulated concentration,but there was no difference between the two ADSCs. The expression of ICAM-1 and VCAM-1 also showed no difference.4. IBD model also confirmed that WT ADSCs have a stronger immunosuppressive capacity, and contribute to the recovery of inflammation in mice.Part 3: MYSM1 regulates immunoregulatory function of ADSCs via miR-150Method:1. The expression of miR-150 in WT and KO ADSCs was detected by qRT-PCR.2. The expression of miR-150 was detected once more in KO ADSCs which was transfected recombinant lentiviral vectors of LV-MYSM1.3. The effect of MYSMI on miR-150 was verified by Chromatin Immunoprecipitation (CHIP).4. C3H10T1/2 were transfected recombinant lentiviral vectors of LV-miR-150 (C3H LV-miR-150) and LV-GFP(C3H LV-GFP) , and the phenotype and proliferation capacity of the two cells were detected.5. C3H LV-miR-150 and C3H LV-GFP were co-cultured with T cells to compare their different capacity on the proliferation of T cells. Beforehand, CD3+ T cells were labled with CFSE and stimulated by PMA and ionomycin and at last the CFSE signal changes were analyzed via flow cytometry.6. Immune-related cytokines and immunophenotype of C3H LV-miR-150 and C3H LV-GFP were dected by qRT-PCR and flow cytometry .Results:1. miR-150 expression of KO ADSCs was significantly lower than that of WT ADSCs.2. The expression of miR-150 in KO ADSCs increased when KO ADSCs overexpressed MYSM1.3. CHIP showed that MYSM1 could bind to the 1200-1000 region upstream of the miR-150 promoter and promoted the expression of miR-150.4. C3H LV-miR-150 and C3H LV-GFP were positive expression of Sca-1, CD29, CD44 and negative expression of CD45, CD11b and MHC class ? molecules. C3H LV-miR-150 had an increased proliferation ability than C3H LV-GFP5. Overexpressed miR-150 showed an increased immunosuppression capacity through detecting the effect on T cells proliferation.6. C3H LV-miR-150 expressed a lower IFN? and SOCS1 but a higher anti-inflammatory factor iNOS and IL10.7. VCAM-1 and PD-L1 were upregulated in C3H10T1/2 stimulated by TNFa+IFNy, the higher concentration of stimuli, the higher expression they exhibited, but no significant difference was detected between C3H LV-miR-150 and C3H LV-GFP. The expression of ICAM-1 showed no significant change with or without stimulationConclusion:MYSM1 plays a critical role in the immunomodulatory function of ADSCs,Mysml knockout attenuates the immunosuppressive ability of ADSCs. MYSM1 could bind to the promoter of miR-150 and promoted the expression of miR-150, which accordingly affect the immunomodulatory function of ADSCs. However, the downstream of miR-150 still need to be further studied.