Effect Of PSMD1and ATG3Genes On Myelodysplastic Syndrome To Leukemic Transformation

Part Ⅰ. Effect of PSMD1gene silencing by small interfering RNA on SKM-1cell lineObjective:A nested case-control study cohort in435primary and untreated myelodysplastic syndrome (MDS) patients was established. Untill the end of follow-up,52patients (12%) was lost. In the rest of383patients followed,41patients progressed to acute leukemia. We analyzed the gene expression profile of patients in leukemia group at diagnosis and after transformation (self-control study). Besides, we analyzed the gene expression profile of paired patients at the initial diagnosis. Subsequently a subset of6genes was picked up to distinguishe the leukemia group and control group stably. They are TUBB、PSMD1、SLC7A5、ATG3、TUBB2C and TIMM10. TUBB gene was analyzed by our group member. So we choose the second distinguished gene, PSMD1gene, to further study.To investigate the effect of small interfering RNA (siRNA) against PSMD1gene on SKM-1cell line and PSMD1gene on myelodysplastic syndrome to leukemic transformation.Methods:PSMD1gene specific siRNAs (PSMD1-siRNAs) were synthesized chemically. PSMD1-siRNAs were transfected with LipofectamineTM2000for delivery into SKM-1cell line (a malignant cell line established in the MDS leukemic phase). Harvest cells after24hours of transfection. Figure out the transfection efficiency and PSMD1gene expression by Realtime PCR in each group. Observe and compare the changes on growth curve, cell cycle, apoptosis and nod/scid mice model.Result:Satisfactory transfection efficiency (>75%) was observed with liposome. After24hours of transfection, Realtime PCR was performed. It suggested that the expression of PSMD1gene was decreased (P<0.05). Compared with the negative control group, the PSMD1-siRNA group was inhibited in proliferation (P<0.05). The cell cycle analysis by flow cytometer demonstrated the reduction of G2phase cells and increase of S phase cells in PSMD1-siRNA group (P<0.05). We can conclude that cells of PSMD1-siRNA group were obstructed at S/G2restriction point. The cell apoptosis analysis by flow cytometer demonstrated that proportions of early apoptosis and late apoptosis were both increased (P<0.05). Symptoms that may be caused by MDS/AML were not observed on either group after15days.Conclusion:Silenced PSMD1gene, the SKM-1cell lines were inhibited in proliferation and cell cycle. They got more tendencies to apoptosis and death. Effect on nod/scid mice was not clear. In conclusion, the malignancy of SKM-1cell lines was decrease after transfection. PSMD1gene and its gene family ubiquitin-proteasome system may play an important role in transformation of MDS. Part Ⅱ. Effect of ATG3gene over expression by lentivirus on SKM-1cell lineObjective:Based on the nested case-control study cohort and gene expression profile, we have picked up a subset of6genes to distinguishe the leukemia group and control group stably. Five of them were up regulated, while only one of them was down regulated. It was ATG3. To investigate the effect of ATG3gene over expression by lentivirus on SKM-1cell line and myelodysplastic syndrome to leukemic transformation.Methods:The lentivirus vector was chosen for delivery of ATG3gene plasmid. Infect the SKM-1cell line with lentivirus vectors. Western blot was performed to detect the ATG3protein. Realtime PCR was performed to calculate the titer of viruses. Observe and compare the changes on growth curve, cell vitality, cell apoptosis and nod/scid mice model.Result:Construct lentivirus vectors carrying ATG3gene plasmid first. Results of DNA sequencing and western blot showed that the construction succeeded. Realtime PCR was performed to calculate the titer of viruses. Its concentration was2×108TU/ml. After72hours of infection by ATG3over expression lentiviruses, satisfactory transfection efficiency (>90%) was observed. Compared with the negative control group, the ATG3over expression group was inhibited in proliferation (P<0.05). The cell vitality was tested by Trypan Blue. The cell vitality of ATG3over expression was65%, less than79%in the negative control (P<0.05). The cell apoptosis analysis by flow cytometer demonstrated decreased proportion of early apoptosis and increased proportion of late apoptosis and death (P<0.05). Symptoms that may be caused by MDS/AML were not observed on either group after15days.Conclusion:Over expressed ATG3gene, the SKM-1cell lines were inhibited in proliferation and cell vitality. They were prompted from early apoptosis to late apoptosis and death. Effect on nod/scid mice was not clear. In conclusion, the malignancy of SKM-1cell lines was decrease after infection. ATG3gene and its gene family may play an important role in transformation of MDS.