Subcellular Location And Functional Study Of Cell Proliferation-related Gene NYD-SP15 In Human Retinal Pigment Epithelial Cells

Objective: To investigate cell proliferation-related gene NYD-SP15'sexpression in human retinal pigment epithelial cells (ARPE19) and itseffect on the proliferation of ARPE19, further explore the mechanism ofNYD-SP15's effect on the pathogenesis and development of proliferativevitreoretinopathy (PVR).Methods: Fluorescence expressing vector pEGFP-C1-NYD-SP15 andeukaryotic expressing vector pcDNA3.1-myc-NYD-SP15 wereconstructed by using gene recombination technique. After confirmed byPCR and DNA sequencing, the plasmids were transfected into ARPE19cells using liposome-mediated transfecting methods. The subcellularlocation of GFP-NYD-SP15 fusion proteins in ARPE19 cells wasobserved. Cell cycle distribution and apoptosis of ARPE19 cellstransfected by pcDNA3.1-myc-NYD-SP15 was determined using flowcytometry.Results: Confirmed by PCR and DNA sequencing, the fluorescenceexpressing vector pEGFP-C1-NYD-SP15 and eukaryotic expressingvector pcDNA3.1-myc-NYD-SP15 were successfully constructed. Fusion protein was lowly expressed in the cytoplasm, while highly expressed inthe nucleus of ARPE19 cells. The results of transient transfection ofpcDNA3.1-myc-NYD-SP15 into ARPE19 cells indicate that at the 24thhour after transfection, there was no change of the cells, but at the 48thhour, the average percentage of RPE cells which entered the S stage ofcell cycle was 19.37 for pcDNA3.1-myc-NYD-SP15, 10.87 forpcDNA3.1-myc and 3.33 for control. The differences among the threegroups were statistically significant (P＜0.01).Conclusion: Bioinformatic analysis indicates that NYD-SP15 protein is amember of cytidine deaminase family. It is different from otherdeaminases in that it contains two dCMP_cyt_deam domains. Evolutionalanalysis reveals that the structural domain in the N-terminus originatesfrom cytidine deaminase, while the one in the C-terminus originates fromdeoxycytidylate deaminase. Therefore, NYD-SP15 conforms to thecommon features of cytidine deaminase-related proteins. The results offlow cytometry indicate that NYD-SP15 could induce cell cycle ofARPE19 cells entering S stage from G1 stage, promote DNA synthesis,thus promote the proliferation of ARPE19 cells. The results conform tothe general function of dCMP_cyt_deam domain, suggesting thatNYD-SP15 could promote DNA synthesis and induce cell proliferation inthe pathogenesis and development of PVR. This study lays a foundationfor the functional study of NYD-SP15, and provides new pathway for the clinical application of gene therapies for PVR.