CIITA M1-RNA抑制HeLa细胞表面MHC II类抗原的表达

探讨MHC II类转录激活因子(CIITA)的M1-RNA对细胞表面MHC II类分子表达的抑制。M1-RNA是核糖核酸酶P的催化活性单位,设计并克隆针对CIITA第452、629位点的M1-RNA(分别为M1-452-GS、M1-629-GS)及其相应的CIITA靶基因,分别插入pUC19、pGEM-7zf(+)载体,进行细胞外切割活性筛选。将细胞外切割作用明显的M1-629-GS亚克隆入psNAV载体(psNAV-M1-629-GS,pA629)并稳定转染HeLa细胞株,流式细胞术检测经典的MHC II类抗原(HLA-DR、-DP、-DQ)的表达,RT-PCR检测CIITA的mRNA水平。在重组人γ干扰素诱导下,pA629阳性HeLa细胞株表面HLA-DR、-DP抗原表达分别降低了83.03%及89.91%;同时CIITA的mRNA含量明显减少(P<0.05)。CIITA的M1-RNA抑制了自身mRNA含量,从而阻止其调控的MHC II类分子的表达,为移植物抗宿主病的研究提供了一种新方法。

The inhibitory effect of MHC class II transactivator M1-RNA on the expression of MHC II molecules on HeLa cells

To investigate the inhibitory effect of the M1-RNA,the catalytic activity unit of ribonuclease P(RNAase P),from MHC classs II transactivator(CIITA) on the expression of MHC class II molecules on HeLa cells,the M1-RNA with guide sequences(GS) recognizing CIITA at 452 and 629 sites(M1-452-GS and M1-629-GS) and the corresponding CIITA-target RNA(114-800) were constructed,and then cloned to vector pUC19 and pGEM-7zf(+) respectively.The recombinant M1-RNA and their target RNA were incubated in cell-free conditions.It was demonstrated that the M1-629-GS could exclusively cleave the target RNA,furthermore,it was subcloned to vector psNAV for intracellular analysis(psNAV-M1-629-GS,pA629).Stable transfectants of HeLa cells with pA629 were analyzed for the expressions of classical MHC class II molecules(HLA-DR,-DP,-DQ) by flow cytometry,and the level of CIITA mRNA was measured by RT-PCR.Through the induction with recombinant human interferon-γ(IFN-γ),the expressions of HLA-DR andDP on the surface of HeLa cells reduced to 83.03% and 89.91% respectively.Meanwhile,the content of CIITA mRNA with a reduced rate of expression up to 91.12%±7.97%(P<0.05).It is evident from the above observations that the M1-RNA inhibits CIITA,and thus the MHC class II molecules regulated by CIITA.This result would provide a new way of approach for the solution of GVHD in hematopoietic stem cell transplantation.