ABSTRACT
OBJECTIVE@#To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.@*METHODS@#Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.@*RESULTS@#WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.@*CONCLUSION@#Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.
Subject(s)
Animals , Humans , Annexin A5 , Apoptosis , Blotting, Western , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Cell Line , Cell Proliferation , Cells, Cultured , Flow Cytometry , Genotype , In Situ Hybridization , Larva/physiology , Phenotype , RNA/isolation & purification , Real-Time Polymerase Chain Reaction/standards , Rho Guanine Nucleotide Exchange Factors/metabolism , Sincalide/analysis , Spectrophotometry/methods , Zebrafish/physiologyABSTRACT
Objective The guanine exchange factors( GEFs) of Dbl family is a major regulatory unit for the malignant transformation of Rho family proteins. It plays a role by converting Rho protein from inactive GDP form to GTP form of Rho protein. In this paper,we discuss the structure and function of a GEF molecule-ARH-GEF 10,and discuss its role in the process of tumor development. Methods The expression of ARHGEF 10 in 42 normal tissues was measured by Real-Time PCR. GST-pulldown technique was used to detect the GEF ac-tivity of ARHGEF 10 in vivo. The transcription factor activity of downstream small molecules was detected by dual-luciferase report gene assay. The high expressive effect of ARHGEF 10 on normal cytoskeleton morphology was performed by dual immunofluorescence staining labeling method. High expressive effects of ARHGEF 10 on cell proliferation,invasion and tumorigenic ability in vitro were examined using CCK8,Transwell and soft agar clony formation assays. Results ARHGEF 10 has a typical GEFs structure,which binds to RhoA in vitro and promotes the proliferation and invasion of NIH3T3 cells,and has significant ability to clone in vitro. Conclusion ARH-GEF 10 is a typical family molecule of guanosine exchange factor that activates RhoA of Rho family,which has obvious oncogene characteristics.