Effect Of RNAi-mediated Genesilencing Of ADAR1 Expression On Mixed Mouse Lymphocyte Culture And Changes Of Gene Expression Profile Of Mouse Lymphocyte

RNA editing is a process to remodify the mRNA coding the genes and the enzyme which has the modifying function is called RNA editing enzyme. After transcribing DNA into RNA , RNA editing enzyme changes the gene sequences and the genetic information by deleting, adding or editing the nucleotide in pre-RNA, regulating the receptor-mediated signal transduction and then produces extensive physiological effect.RNA editase transforms or modifies the nucleotide of pre-RNA by deaminization, and hydrolytic deaminize C6 amino group of the adenosine at the specific RNA site, changing adenosine to inosine or changing cytosine to uracil,but inosine was recognized as guanine in mRNA. These substitutions transform the original heredity codon or shearing site, and change the structure and function of the protein they coded. In previous work we have observed that the expression of ADAR1 increases during rejection.But the role ADAR1 plays in rejection and the ways how ADAR1 involves in rejection are still unknown.In the present study we describe experiments aimed at the changes of lymphocyte proliferation and genes expression profile of lymphocyte when ADAR1 expression was degraded in mixed lymphocyte culture.Part 1AIM: To investigate the influence of ADAR1 on lymphocyte proliferation.METHODS: Primary lymphocyte was isolated from BALB/c and C57 male mice, weighed 18-25g. ADAR1 siRNA was transferred into lymphocyte using Lipofectamine 2000 before two-source lymphocytes were mixed and cultured .The cell proliferation was checked using cell counting,trypan blue staining cell viability and MTT assay. The ADAR1 mRNA was detected by RT-PCR.RESULTS: When the ADAR1 expression was suppressed by the specific siRNA, the lymphocyte proliferation was inhibited in mixed lymphocyte culture test . CONCLUSION:When the ADAR1 expression was suppressed by the specific siRNA, the lymphocyte proliferation was inhibited in mixed lymphocyte culture test ,which indicated ADAR1 has influence on mouse lymphocyte proliferation. Part 2AIM:To compare the changes of gene expression profile of mouse lymphocyte before and after 24-hours when ADAR1 expression was degraded in mixed lymphocyte culture.METHODS: Primary lymphocyte was isolated from BALB/c and C57 mice, weighed 18-25g. ADAR1 siRNA is transferred into lymphocyte after two source lymphocyte was mixed and cultured by Electroporation. The cell proliferation was checked using cell counting,trypan blue staining cell viability and MTT assay.The genes expression differences between the transferred cells and the non-transferred cells were tested by DNA microarray.RESULTS: Among 44291 genes,the expression of 393 genes were altered significantly on mRNA level. The expression of 78 genes were up-regμlated while the expression of 315 genes were down-regμlated.CONCLUSION:1.The effect of ADAR1 expression on mouse lymphocyte is related to changing the expression levels of a number of genes.2.Most of the down-regulated genes are related to cell cycle regulation and signal transduction ,indicated ADAR1 may play a key role in regulation of lymphocyte function.