Effects Of Enhanced Expression Level Of RPS14on Cell Growth Of Human Myelodysplastic Syndrome Cell Line MUTZ-8

Objectives: Myelodysplastic Syndrome are a group of hematopoieticstem cell clonal bone marrow disorders, characterized by cytopenia due toineffective hematopoiesis and an increased risk of leukemic evolution. Itspathogenesis has not been fully clarified, lack of effective treatment. So itis crucial to address the mechanisms of MDS disease in order to explorenew approach for the treatment. The deletion of the long arm ofchromosome5is considered as one of the most common chromosomalabnormalities of MDS. The deleted region includes5q31-5q32, researchersdelineated it as the common deleted region(CDR). RPS14located in theCDR are proved to be significatly down regulated in patients with5q-MDS.Our previous study data had shown that RPS14was involved in MDSdisease progress. It plays a critical role for the MDS.In this study, we first constructed lentiviral vector carrying RPS14gene,which is capable of delivering the target gene RPS14into human MDS cellline MUTZ-8for increasing its expression. Then, we investigated its effectof enhanced expression level of RPS14on cell growth and apotosis of MDS cell line MUTZ-8.Methods:1. Construction and identification of lentiviral vector carrying humanRPS14gene: First, the foreign PCR production of RPS14was inserted intoa plasmid named the vector pGC-FU-EGFP, the obtained plasmidspGC-FU-RPS14-GFP were cotransfected with packing plasmid into293Tcells to acquire the recombinant lentiviral vector pGC-FU-RPS14-GFP.The recombinant lentiviral vector’titers were measured by means ofdilution-by-hole. The infection efficiency was detected by flow cytometry.RPS14mRNA was detected by reverse transcription-polymerase (RT-PCR),and western blot analysis was used to assess the protein expression ofRPS14.2. Study of effect on cell growth of MUTZ-8infected with recombinantlentiviral vector pGC-FU-RPS14: The cells were divided into three groups:MUTZ-8, negative control group, infected group. The absorbance values ofof each group cells was performed by MTT analysis.3. Study of effect on cell apotosis of MUTZ-8infected withrecombinant lentiviral vector pGC-FU-RPS14-GFP: The cells were dividedinto three groups: MUTZ-8, negative control group, infected group. FACSanalysis was used to assess cell apoptotic rate.Results:1. The recombinant lentiviral vector carrying human RPS14(pGC-FU-RPS14-GFP) was constructed successfully. The titer was2.0×109Tu/mL. pGC-FU-RPS14-GFP can be infected cells with highefficiency. The efficiency of100multiplicity of infection (MOI) toMUTZ-8cells was (51.303±3.908)%. The expression levels of RPS14mRNA and protein of MUTZ-8cells infected with pGC-FU-RPS14-GFPenhanced, compared with either negative control group or uninfected cellsgroup.2. The proliferation of MUTZ-8cells infected with recombinantlentiviral vector pGC-FU-RPS14-GFP was suppressed: Cells wereharvested at2d、3d、4d、5d post infection, absorbance values of cells wasmeasuered by MTT. Viability of cells infected with pGC-FU-RPS14-GFPwas less than the cells infected with negative control group and uninfectedMUTZ-8cells (P <0.05).3. The apoptotic rate of MUTZ-8cells infected with recombinantlentiviral vector pGC-FU-RPS14-GFP was increased: FACS analysis wasused to assess apoptosis. Levels of apoptosis were (26.233±3.095)%at5dpost infection with pGC-FU-RPS14-GFP. The apoptotic rate significallyincreased, and had a statistical significance, compared with uninfectedMUTZ-8cells and negative control group.Conclusion:1. Lentiviral vector is more suitable for hematological malignancy genetherapy because of its significantly higher infection efficiency to cells. 2. The proliferation of MUTZ-8cells with enhanced expression level ofRPS14was suppressed.3. The apoptosis rate of MUTZ-8cells with enhanced expression levelof RPS14was increased.4. RPS14would be involved in tumorigenesis and progression ofMyelodysplastic Syndrome, furthermore our study would provideexperimental data for RPS14as molecular target for MDS.