Research On The Effect Of Targeted Regulation Of Rat Hippocampal Gene CNN3 Towards Epilepsy

Objective:Calponin-3 is a dynein which regulates the dendritic spines on excitatory postsynaptic membrane. Our previous studies showed that calponin-3 and its targeted gene CNN3 were significant increased in the brain tissues and cerebrospinal fluids from epileptic patients and animals, but the exact mechanism was not clear.If we are able to prove the relationship between the change of calponin-3 and epilepsy, to reveal the relevant link, It could enrich our understanding of the biochemical changes in the brain with chronic epilepsy, deepen the understanding of the pathogenesis of drug-resistant epilepsy, and open up a new way for new drugs.Method:First,we designed and authenticated the lentiviral vectors targeted CNN3 gene which could silence or over express gene CNN3 in rat. Using stereotactic injection of packed lentiviral to the rat hippocampi which called transfection in vivo. After 8 week’s dynamic observation,we selected the most effective sequence and time point. Then we injected lithiumchloride and pilocarpine to kindl the experimental rats from the control group、the CNN3 RNA interference (CNN3-shRNA) group and the CNN3 over expression (CNN3-OE) group,Then we observed the EEG and behavior in different groups, to observe their incubation period, duration, severity of epilepsy and the success rate of the epilepsy to ignite. At last, we compared the calponin-3 expression level from different group and different time after seizure. Using patch clamp to record the field potential and whole cell potential.Results:CNN3-OE and three CNN3-shRNA lentiviral vectors had been successfully constructed, which regulated the expression level of CNN3 gene in 8 weeks after transfection. The expression of calponin-3 encoded by gene CNN3 was significant down in 8 weeks after injection by CNN3-shRNA2 lentiviral vectors, and the highest inhibition rate was 73.26%. The expression of calponin-3 raised 14 days after tranfection of CNN3-OE lentiviral vectors, the rate was 93.88%. So we chose the time when the expression of calponin-3 was significant changed to kindle the rats from different groups such as CNN3-shRNA2、CNN3-OE and their control groups. The records from EEG showed epileptic discharge in all groups, which suggested that the epilepsy model was successful construsted. Behavioral research displayed that the incubation period of seizure from the CNN3-OE group was (29.17±19.08) min, the CNN3-shRNA2 group was (40.717±28.40) min, the period of seizure from the CNN3-shRNA2 group was significant longer than the CNN3-OE group, P<0.05,it was a statistical difference.But with the change of the expression level of calponin -3, there was no significant difference between groups in the pilocarpine dose and the severity of seizure. We quickly removed the brain to slices under anesthesia 1 hour after seizue,but the epileptic discharge had not been recorded by patch clamping from field potential and whole cell potential.The expression of calponin-3 from CNN3-shRNA2 group remained at a low level after the intervention in 48 hours,it only showed a high level at 1W,and the expression of calponin-3 didn’t significantly increased in longer term.The expression of calponin-3 from CNN3-OE group remained at a high level after the intervention,there was a significantly rise at 48h,and the rise trend continued to 8W.Conclusions:Out study constructed CNN3-OE and 3 CNN3-shRNA lentiviral vectors successfully. We selected the most effective shRNA sequence:shRNA2-1: 5’-gatccGGAGAACATCGGCAACTTTATAGAGAACTTATAAAGTTGCCGATGTT CTCCTTTTTTg-3’, shRNA2-2: 5’-aattcAAAAAAGGAGAACATCGGCAACTTTATAAGTTCTCTATAAAGTTGCC GATGTTCTCCg-----3’.The regulation of the expression of CNN3 gene in local brain was realized by stereotactic injection in vivo,it up/down the expression of calponin-3 at least 50% in the rat hippocampal,and the efficient interference lasted for 8 weeks.It showed that the technology could be used in animal models for the function research of gene.It was important to clarify the function of CNN3 gene,to deppen the understanding of synaptic plasticity, the pathogenesis of epilepsy and open up a new treatment way of epilepsy.Through further intervention experiment, we found that the seizure threshold of the experiment rats increased after reducing the expression of calponin-3 at a moderated reducing level. It indicated that over expression of calponin-3 was related to the occurrence of epilepsy. Inhibition of the expression of calponin-3 could reduce epileptic susceptibility, which may have antiepileptic effect,it provided an impotant exprerimental basis for drug development.