Effect Of SPAG6 Gene On Cell Proliferation Of Myelodysplastic Syndrome And Its Mechanism

Objective:By detecting the expression of SPAG6 gene in bone marrow specimens of patients with MDS and MDS-AML,and constructing SPAG6-targeting short hairpin RNA(SPAG6-shRNA)lentiviral vector knockdown of SPAG6 gene,to explore the effect of downregulation of SPAG6 on proliferation of myelodysplastic syndrome(MDS)cell line SKM-1 and its possible mechanism.Methods:A total of 21 cases bone marrow samples were collected from patients with MDS or MDS-AML(11 cases with MDS,10 cases with MDS-AML),and 7 cases of cancer-free individuals were collected as negative control.The total RNAs were isolated from mononuclear cells being extracted from fresh bone marrow samples,and the relative expression of SPAG6 mRNA was detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR).The expression of SPAG6 mRNA and the survival curve of Kaplan-Meier in bone marrow specimens of patients with AML from TCGA Database were analyzed.The experiment was divided into three groups:Blank group,NC-LV group and SPAG6-LV group.The designed negative control recombinant lentiviral vector NC-shRNA and target gene recombinant lentivirus vector SPAG6-shRNA were transfected into logarithmic SKM-1 cells.The transfection efficiency was evaluated by the inverted fluorescence microscope and the flow cytometry.The interference effect of the expression of SPAG6 gene was detected by qRT-PCR and Western blotting assay.The effect of SPAG6 gene expression downregulation on the proliferation of SKM-1 cells was detected by CCK-8 assay.Cell cycle and positive rate of cell surface differentiation antigens of each group were detected by flow cytometry.The qRT-PCR and Western blotting methods were used to observe the expression of each group related genes and proteins in cell cycle and its possible AKT/FOXO pathway.Results:Compared with cancer-free individuals control group,the SPAG6 mRNA expression was upregulated in patients with MDS or MDS-AML.Upon analysis of SPAG6 mRNA expression bone marrow specimens of patients with AML from TCGA Database,there was a significant difference in the expression of SPAG6 in bone marrow from these patients.Kaplan-Meier survival curves were further analyzed and demonstrated that the overall survival of patients with high SPAG6expression was significantly shorter than that in patients with low SPAG6expression.Compared with the Blank group,the inverted fluorescence microscope showed that a large amount of green fluorescence was observed in the SPAG6-LV group and NC-LV group,the flow cytometry showed the transfection rate was more than 80%,and the results of qRT-PCR and Western blotting assay further showed that SPAG6 gene expression decreased,all of which proved that SPAG6 gene expression was successfully constructed.Compared with the Blank and NC-LV groups,the SPAG6-LV group slowed slower cell growth and proliferation.There was cell cycle arrest at G0/G1 phase in SPAG6-LV group compared with the Blank and NC-LV group by flow cytometry.Compared with the Blank and NC-LV groups,the SPAG6-LV group showed increased expression of p27Kip1 as indicated by the qRT-PCR and Western blotting analyses.Conversely,Cyclin E1 and CDK2 expression were decreased.Moreover,qRT-PCR analysis revealed that there was no statistical effect on the expression of CyclinD1,CDK4 and CDK6.In addition,compared with the NC-LV group,the positive expression rate of CD14 on CD45~+cells in SPAG6-LV group was increased by flow cytometry.Finally,Western blotting results showed that there was no significant difference in the expression of t-AKT and t-FOXO1 in SPAG6-LV group,and the expression of p-AKT and p-FOXO1 was decreased.Conclusion:SPAG6 might be involved in the occurrence and evolution of MDS and that SPAG6 over-expression was associated with total survival time.SPAG6 knockdown has potential therapeutic value in MDS,as it inhibited SKM-1 cell proliferation and affected the cell cycle and differentiation;this antiproliferative activity might be mediated through the AKT/FOXO/p27Kip1 pathway.Thus,our study suggested a potential utility in SPAG6-targeting strategies to deliver an antiproliferative therapy against MDS and MDS-AML via the AKT/FOXO/p27Kip1 pathway.