The Role Of MMP1 In MSCs' Suppression On MDS Cell Proliferation And Mechanism Research

Objective:This study aims to investigate the role of MMP1 in MSCs' suppression on MDS cell proliferation,and the role of epigenetic regulation in MSCs and MDS cells.Methods:(1)MSCs were isolated from bone marrow of MDS patients and healthy donors.The celluar morphology,immunophenotype,multiple differentiation and cell senscence were analyzed.Establish the co-culure system of MSCs and MDS cells.Proliferation of MDS cells co-cultured with MDS-MSCs or normal MSCs was determined by EdU assay or cell-counting assay.(2)Both mRNA and protein levels of MMP1 were determined by qRT-PCR analysis and Western blot analysis.MSCs were transfected with siRNA targeting MMP1 or siRNA negative control.MDS cells were co-cultured with MMP-KD MSCs or negative MSCs.Proliferation rate of MDS cells in both groups was measured by EdU assay,and apoptosis was determined by Annexin V /PI dual staining.The proliferation and apoptosis proportion of MDS cells was determined when exogenous activated MMP1 was added to MDS-MSCs and MDS cells in co-culture.Phosphorylation of p38 MAPKs in response to MMP1 was evaluated by western blot analysis.PAR1 antagonist and p38 inhibitor were administrated respectively to confirm their roles in this process.(3)The mRNA and protein levels of MMP1 in AZA-treated MDS-MSCs were evaluated by qRT-PCR analysis and Western blot analysis.MDS cell proliferation was determined by EdU analysis after co-culture with AZA-treated MDS-MSCs or untreated MDS-MSCs.MDS and AML cells were treated with chidamide(HDAC inhibitor),HDAC enzyme activity,cell growth,cell cycle arrest and apoptosis were analyzed.JAK2/STAT3 signaling and its downstream genes were also evaluated.Results:(1)MSCs obtained from bone marrow of MDS patients and healthy donors conform with the characteristics of MSCs specified by the ISCT.However,the differentiation capacity differed between MDS-MSCs and normal MSCs,with decreased adipogenic differentiation capacity in general MDS-MSCs,impaired osteogenic differentiation in low-grade MDS-MSCs and decreased chondrocytic differentiation in high-grade MDS-MSCs.The proportion of senescent cells increased in MDS-MSCs.MDS-MSCs exhibited reduced capacities to restrict the proliferation of MDS cells compared with normal MSCs.(2)MMP1 was decreased in MDS-MSCs compared with normal MSCs.In addition,high-grade MDS patients possessed lower levels of MMP1 than low-grade MDS patients.The addition of FN439 to normal MSCs and SKM-1 co-culture significantly increased the proportion of SKM-1 cells in the S phase.When MDS cells were co-cultured with MMP1-KD MSCs or negative MSCs,the proliferating proportion of MDS cells was increased while the apoptotic proportion was decreased in the MMP1-KD group compared with the negative control group.MMP1-induced growth inhibition and apoptosis was blocked by the PAR1 antagonist.MMP1 induced phosphorylation of p38,which was also inhibited by PAR1 antagonist.P38 inhibitor blocked the increase of apoptotic cells and expression of pro-apoptotic proteins induced by MMP1.Thus,the mechanism may involve in the MMP1/PAR1 interaction and subsequent activation of p38 MAPK signaling.(3)The hypomethylating agent(azacitidine)could upregulate the mRNA and protein expression of MMP1 in MDS-MSCs and enhance their capacity to restrict MDS cell proliferation.The HDAC inhibitor(chidamide)efficiently inhibited the HDAC enzyme activity and cell growth of MDS and AML cells,and induced G0/G1 phase arrest and cell apoptosis.Chidamide downregulated JAK2/STAT3 signaling and its downstream targets,such as Bcl-xL,Mcl-1 and c-Myc,which were associated with cell cycle arrest and anti-apoptosis.Conclusion:MDS-MSCs have reduced capacity to restrict MDS cell proliferation compared with normal MSCs.Downregulation of MMP1 in MDS-MSCs may account for their reduced capacity to restrict MDS cell proliferation.MMP1 exerts its growth inhibition and apoptosis inducing effects on MDS cells through PAR1-p38 MAPK signaling activation.AZA could upregulate MMP1 expression in MDS-MSCs and enhance their capacity to restrict MDS cell proliferation.Chidamide could inhibit the viability of MDS cells by suppressing JAK2/STAT3 signaling.