Effect Of RNA Interferencing Targeting SPAG6 On The Growth Of Human MDS Cell Line In Itro And In Vivo

Objective To construct the pGC-shRNA-SPAG6 (short hairpin RNA) lentivirus targeting silence SPAG6 gene, and investigate the effect of SPAG6 gene silencing on the growth of myelodysplastic syndrome (MDS) cell lines SKM-1 in vitro and in vivo.Methods1?Construction and identification of shRNA lentiviral vector targeting human SPAG6 gene:Three shRNA lentiviral vector to interfere SPAG6 gene were designed and constructed. After transfected into SKM-1 cells, the infection efficiency were detected by flow cytometry and the interference efficiency of SPAG6 gene were determined by quantitative reverse transcription PCR and Western blot assay to screening the effective shRNA plasmid.2?Study of effect on cell growth of SKM-1 infected with recombinant shRNA-SPAG6 lentiviral vector:The expression of the SPAG6 mRNA in SKM-1 cells was measured by qRT-PCR. Cell proliferation was examined by CCK-8 assay?cell apoptosis rate and cell cycle was detected by flow cytometry. And the expression of genes known to be involved apoptosis related proteins were measured by qRT-PCR and Western blot.3% Study of effect on cell growth of SKM-1 infected with recombinant shRNA-SPAG6 lentiviral vector in vivo:The SKM-1 cells that were stably infected with a lentivirus vector carrying SPAG6-shRNA or NC-shRNA were subcutaneously inoculated into NOD/SCID mice to establish xenograft models. The tumor volume was calculated and the cell apoptosis was detected using TUNEL assays to observe the functional effect of knocking down SPAG6 on cell growth in vivo.Results1?SPAG6-shRNA lentiviral vector were successfully constructed, the infection efficiency detected by flow cytometry were more than 60%. Quantitative reverse transcription PCR and western blot results show SPAG6-shRNA3 lentiviral vector knockdowned the expression of SPAG6 most effectively, described as the best lentiviral vector.2?Quantitative RT-PCR showed significantly higher expression of SPAG6 mRNA in the hematologic malignant cell lines than in the common solid tumor cell lines, and highly expressed in SKM-1 cells. With SPAG6 gene interference, cell proliferation was significantly suppressed in SKM-1 cells infected with the SPAG6-shRNA lentivirus compared with control cells (P<0.05), and the percentage of apoptotic cells in the SPAG6 shRNA lentiviral vector infected SKM-1 cells was significantly higher than in control cells (NC-shRNA 4.12±0.52% vs. SPAG6-shRNA 16.42±3.27%, P=0.024). But the effect of SPAG6 knockdown on the cell cycle analysed by flow cytometry showed no significant difference in the proportion of cells in S, G2 or G1 phase when SKM-1 cells were infected with the SPAG6-shRNA lentivirus compared to the controls (P>0.05). Western blot analysis showed that the protein levels of caspase3, caspase9 and caspase8 were significantly higher in SKM-1 cells infected with the SPAG6 shRNA lentivirus than the control group. In addition, quantitative RT-PCR and western blot assays showed the expression of p53 at both the mRNA and protein levels was obviously higher in the SPAG6-shRNA lentivirus infected cells.3?Mice injected with SKM-1 cells transduced by the SPAG6-shRNA lentivirus developed xenografts that were significantly smaller than those in mice inoculated with cells transduced by the NC-shRNA lentivirus, histological examination of xenografts from SPAG6-shRNA lentivirus treated animals showed a significant increase in apoptosis compared with the NC-shRNA lentivirus group (NC-shRNA 18.33±3.51% vs. SPAG6-shRNA 48.33±2.52%, P=0.000).ConclusionThe present study demonstrates that SPAG6 is highly expressed in malignant hematologic cell lines. Knockdown of SPAG6 using SPAG6-shRNA lentivirus potently inhibits viability and promotes arrest of apoptosis. SPAG6 silencing may be associated with apoptosis induction via active caspases and increasing p53 expression in SKM-1. Our study would provide experimental data and theoretical basis for SPAG6 as molecular target for MDS.