| Objective:Investigate the transcription,protein expression and functione of RRM2 B c.420 G > C,and determine the pathogenicity of the mutation.Methods:Construct interference plasmid,overexpression plasmid and plasmid which carries c.420 G > C of RRM2 B.Transfect interference plasmid and plasmid which carries c.420 G > C of RRM2 B into 293 T cell at the same time to simulate the cell with c.420 G > C.Detect the content of RRM2 B mRNA by Real-time PCR in cells,detect the protein content of RRM2 B gene by Western-blot,and extract mitochondrial DNA from cells to detect the content of mitochondrial DNA.Results:1.Construct interference plasmid(pLVX-shRNA2-shRRM2B),overexpression plasmid(Plvx-IRES-ZsGreen1-RRM2B)and plasmid which carries c.420 G > C of RRM2B(Plvx-IRES-ZsGreen1-RRM2B(c.420G>C))successfully.Transfect interference plasmid and plasmid which carries c.420 G > C of RRM2 B into 293 T cell at the same time to simulate the cell with c.420 G > C.2.Real-time PCR results showed that the content of mRNA in the experimental group was significantly lower than that in the normal cells,suggesting that c.420 G > C could lead to the down-regulation of the transcription of RRM2 B gene.The results of Western-blot showed that the content of p53R2 in the experimental group was significantly lower than that in the normal cells,indicating that c.420 G > C could decrease the protein content of RRM2 B gene.And the content of mtDNA in the experimental group was significantly lower than that in the normal cells,which proved that the RRM2 B gene c.420 G > C mutation could cause the depletion of mtDNA,and verified the pathogenicity of the new mutation.Conclusion:1.The mutation of RRM2 B c.420 G > C could result in the decrease of mRNA transcription and the decrease of p53R2 expression,and thus decrease the context of mtDNA in cells.2.RRM2 B gene c.420 G > C mutation can cause mitochondrial DNA depletion in cells and mitochondrial DNA depletion syndrome clinically. |
