Rb Regulates The Development Of GABAergic Neurons In The Hippocampus Of Neonatal Mice Through Arx

Objective:The purpose of this study was to analyze the effects on GABAergic neurons in hippocampus by injecting PBS,h U6-MCS-CBh-gc GFP-IRES-puromycin,h U6-Rb-shRNA-CBh-gc GFP-IRES-puromycin,p LVX-Tight-ArxshRNA-IRES-Zs Gre en-Neo,h U6-Rb-shRNA-CBh-gc GFP-IRES-puromycin+Ubi-Dlx2-3FLAG-CBh-gc G FP-IRES-puromycin into lateral ventricle of newborn mice within 12 h,and explore whether there is a regulatory relationship between Rb and Arx,and whether Dlx2 participates in the regulatory mechanism between Rb and Arx,so as to provide new therapeutic targets for neurodevelopmental disorders(NDDs),attention deficit hyperactivity disorder(ADHD),depression,brain and other centralnervous system diseases.Method:Block-itRNAi Designer software was used to design interferingRNA sequence Rb shRNA that interferes with mouse Rb.Restriction enzymes Age I and Eco RI were added to both ends of Rb shRNA gene fragment and linked into vector GV493 plasmid to generate lentiviral vector plasmid named Lenti-Rb shRNA that interferes with Rb expression.Lentiviral vector plasmid that interferes with Arx expression named Lenti-Arx shRNA has been prepared by our research group in the early stage.Mouse Dlx2 CDS sequence was extracted from Gene Bank,and Kozak sequence was added upstream and synthesized artificially.The lentiviral vector plasmid overexpressing Dlx2 was constructed,named Lenti-Dlx2.Lenti-CON is a no-load plasmid that did not contain exogenous target genes.293 T cells were co-transfected with virus packing assistant plasmids Helper1.0,Helper2.0 and corresponding vector plasmids to package lentivirus.The experiment was divided into five groups: PBS group,CON group,Rb-shRNA group,ARX-shRNA group,Rb-shRNA+Dlx2 group.The mice were injected with lentivirus vectors in the lateral ventricular within 12 h after birth.After48 h,72h and 96 h of lentivirus vectors injection the mRNA and protein expressions of Rb,Arx and GABAergic neuron surface markers GAD65 and GAD67 in each group were detected by RT-q PCR,Western Blot and immunofluorescence.Results:1.Lentiviral vector plasmids Lenti-Con,Lenti-Rb shRNA,Lenti-Arx shRNA,and Lenti-Dlx2 were successfully synthesized,and 293 T cells were co-transfected with the vector plasmids and lentivirus-assisted packaging plasmids.The corresponding lentivirus was successfully packaged and its titer was successfully measured.2.The expression of Rb,Arx,GAD65 and GAD67 showed no significant difference between the PBS group and the CON group when lentivirus was injected into the lateral ventricle of newborn mice within 12 h,indicating that the lentivirus itself had no influence on the experimental results.3.There were no significant differences in Rb,Arx,GAD65 and GAD67 expressions among all groups after 48 h of lentivirus injection,indicating that lentivirus did not play a role at this time.4.When injected with lentivirus for 72 h and 96 h,the expression of Arx and GAD65 and GAD67 decreased while Rb was decreased by interfering Rb.Arx interference had no effect on Rb expression,but reduced the expression of GAD65 and GAD67.Compared with Lenti-Rb shRNA+Lenti-Dlx2,the expression of Arx,GAD65 and GAD67 was increased in Lenti-Rb shRNA+Lenti-Dlx2.Compared with Lenti-Arx shRNA+Lenti-Dlx2 group,the expression of Arx,GAD65 and GAD67 was increased.Conclusion:1.Rb and Arx genes positively regulate the development of hippocampal GABAergic neurons in neonatal mice.2.Dlx2 gene can save the abnormal development of GABAergic neurons caused by low expression of Arx.3.Rb regulates the expression of Arx through Dlx2 and then influences the development of GABAergic neurons in the hippocampus of newborn mice.