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 patte...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.展开更多
基金supported by the National Natural Science Foundation of China[NSFCno.81741021]the National Key Research and Development Program of China[no.2016YFC1000501,2016YFC1000500]。
文摘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.
