Objective:This study aimed to investigate the crucial mutant gene of Chinese patients with retinoblastoma (Rb) by whole-exome sequencing (WES), and explore the functional role of this genetic variant in the development of Rb using biological experiments.Methods:Blood samples of 82 Rb patients with negative RB1 gene mutation (RB1-/-) in China were screened for rare variations via WES analysis. Moreover, the expression of protein encoded by selected rare variants was detected by Western blot. We further observed the effect of selected gene knockdown or overexpression on the proliferation, migration of Rb cells and xenograft tumor size in vitro and in vivo.Results:We identified a rare variation Y318H of PLK3 gene in RB1-/- patients by WES and SKAT analysis, and Y318H substitution was a pathogenic mutation. The PLK3 protein of Rb cell lines transfected with Y318H mutation plasmids was reduced compared to the control. PLK3 knockdown promoted the proliferation and migration potentials of Rb cells, while PLK3 overexpression inhibited the proliferation and migration potentials of Rb cells. Additionally, the transition of PLK3 expression regulated Rb growth of mouse subcutaneous xenograft model in vivo.Conclusions:Our findings provide evidence that the rare variation Y318H of PLK3 gene is an significant pathogenic factor for Rb progression in Chinese patients. The mutation of Y318H can reduce PLK3 protein expression. PLK3 knockdown promoted the proliferation and migration of Rb cells, thereby resulting in the growth of Rb. The study suggests that PLK3 rare variation Y318H can be used as a new molecular marker for the early genetic screening of Rb, and may provide a novel target for diagnosis and treatment of the disease.

Objective To explore the feasibility and efficacy of an MRI-visible,targeted,nano-vector which is synthesized by attaching a targeting ligand,the GD2 single chain antibody (scAb GD2),to the distal ends of PEG-g-PEI-SPION as a carrier for gene delivery into human bone marrow mesenchymal stem cells (hBMSCs) and in vitro cellular MR imaging.Methods scAbGD2-PEG-g-PEI-SPION was synthesized as previously reported.Gel electrophoresis was performed to assess the pDNA condensation ability of scAbGD2-PEG-g-PEI-SPION.The particle size and Zeta potential of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes were observed by dynamic light scattering.Cytotoxicity of scAbGD2-PEG-g-PEI-SPI-ON was evaluated by CCK-8 assay using hBMSCs.Gene transfection efficiency of scAbGD2-PEG-g-PEI-SPION in hBMSCs was quantified by flow cytometry,PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION+ free AbGD2 and scAbIgG2a-PEG-g-PEI-SPION group was established.The cellular internalization of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes was observed by confocal laser scanning microscopy and Prussian blue staining.MRI of scAbGD2-PEG-g-PEI-SPION was performed by cellular MRI scanning in vitro.Results scAbGD2-PEG-g-PEI-SPION condensed pDNA to form stable nanocomplexes of 80-100 nm in diameter and showed low cytotoxicity to hBMSCs.At the same N/P ratio,the transfection efficiency of scAbGD2-PEG-g-PEI-SPION group was significantly higher than those of other groups (P＜0.001).At the optimal N/P ratio of 20,scAbGD2-PEG-g-PEI-SPION/pDNA obtained the highest transfection efficiency of (59.60 ± 4.50) ％ in hBMSCs.Furthermore,hBMSCs labeled with scAbGD2-PEG-g-PEI-SPION showed sensitive low signal intensity on MRI T2/T2 *-weighted images in vitro.Conclusion scAbGD2-PEG-g-PEI-SPION is an efficient MRL visible targeted nano vector for gene delivery into hBMSCs.