The Effect Of GABRG2 Gene Expression Reduction On Matrix Metalloproteinase 3(MMP3)

ObjectiveWe used the Cre/loxp recombinase system to construct GABAA receptorγ2 subunit（GABRG2）gene conditional knockout mouse model and constructed the GABRG2 gene conditional knockout neuronal cells（HT22）by CRISPR/Cas9 technology.We first investigated the possible effects of GABRG2 gene deletion on matrix metalloproteinase 3（MMP3）in cortical and hippocampal regions and neurons in hippocampal regions through in vivo animal models,and then explored the effects of GABRG2 gene deletion on MMP3 and the possible relationship between them through in vitro cellular experiments.Methods1.First,we identified the mice with loxp locus expression and Cre enzyme expression,and by mating and breeding,the offspring were re-identified to obtain the offspring genotypes of GABRG2^fl/wtCre+mice,which were the GABRG2 conditional knockout mouse models required for the experiments.We obtained brain tissue proteins from the cortex and hippocampus of wild-type mice and GABRG2 conditional knockout mice to detect the expression of GABRG2,MMP3 and other proteins by Western blot;then we obtained intact mouse brain tissues and obtained tissue sections by using frozen sectioning.We detected the expression of GABRG2 and MMP3 proteins by immunofluorescence and the morphological and structural changes of neurons by Nissler staining.2.We used the method of rectal temperature probe to record the EEG of GABRG2 gene knockout mice and wild-type mice at 37 degrees.3.We cultured GABRG2 gene conditional knockout and normal neuronal cells（HT22）and obtained cellular proteins to detect the protein expression of GABRG2,MMP3,protein kinase A（PKA）,phosphorylated protein kinase A（p-PKA）,and nuclear transcription factor（NF-κB）by Western blot.We detected the GABRG2 and MMP3 protein expression by cellular immunofluorescence.Results1.We obtained the whole genomic DNA of mice and obtained the offspring genotype of GABRG2^fl/wtCre+mice by agarose gel electrophoresis as the model mice used in the experiment.Western blot results showed that GABRG2 conditional knockout mice had significantly lower GABRG2 expression than wild-type mice（P<0.05）,while the expression of MMP3 was significantly higher than that of wild-type mice（P>0.05）.Immunofluorescence results showed that in hippocampal and cortical tissues,GABRG2 expression was significantly lower in GABRG2 conditional knockout mice compared with wild-type mice,whereas there was an elevated expression of MMP3,especially in the CA3 region of the hippocampus.Nissler staining showed that neurons in GABRG2 conditional knockout mice had loose neuronal structure and increased neuronal disassociation compared to wild-type mice.2.The EEG waveforms of GABRG2 conditional knockout mice showed that the amplitude of their brain waves was higher than that of wild-type mice.3.Western blot results of cells showed that the protein expression of MMP3,p-PKA,and NF-κB was significantly increased in GABRG2 conditional knockout neuronal cells compared with normal neuronal cells（P<0.05）,the protein expression of PKA was not significantly changed（P>0.05）,and the expression of GABRG2 was extremely low expression.Cellular immunofluorescence results showed that GABRG2 conditional knockdown neuronal cells had extremely low GABRG2 protein expression and significantly increased MMP3 protein expression compared to normal neuronal cells.Conclusion1.The decrease of GABRG2 gene expression will cause the increase of MMP3 gene expression.2.The expression of MMP3 gene is may regulated by proteins in the PKA/NF-κB signaling pathway.