Hsa-miR-150过表达诱导弥漫大B淋巴瘤细胞系OCI-Ly10再分化

Over-expression of hsa-miR-150 induces re-differentiation of diffuse large B-cell lymphoma cell line OCI-Ly10

目的:探究hsa-miR-150过表达诱导弥漫大B淋巴瘤细胞系OCI-Ly10再分化的机制。方法:运用real-time PCR检测hsa-miR-150在永生化CD19+B细胞和OCI-Ly10细胞中的表达,利用Western blotting和免疫荧光细胞化学检测hsa-miR-150靶基因c-myb的表达;通过LipofectamineTM2000脂质体法将含有重组慢病毒质粒的病毒上清转染OCI-Ly10细胞(Ly10-control组和Ly10-miR-150组);对新构建的细胞亚系,通过MTT法检测细胞增殖能力,流式细胞术检测细胞周期及凋亡;运用real-time PCR、免疫荧光细胞化学和Western blotting检测Ly10-control和Ly10-miR-150的B细胞分化相关基因及c-Myb的表达;利用干扰片段干扰OCI-Ly10细胞c-myb表达后,运用real-time PCR和Western blotting检测干扰效率及干扰后转录调节因子BCL6和浆细胞分化开关蛋白PRDM1的表达。结果:(1)CD19+B淋巴细胞的hsa-miR-150表达量明显高于OCI-Ly10细胞(P<0.05);c-Myb在OCI-Ly10细胞中表达较强,而在CD19+B淋巴细胞表达较弱。(2)OCI-Ly10细胞经转染hsa-miR-150后,B细胞分化相关基因的表达都明显发生了变化,B细胞特异性激活蛋白(PAX5)和BCL6表达下调(P<0.05);干扰素调节因子4(IRF4)、PRDM1和X盒结合蛋白1(XBP1)的表达上调(P<0.05);和对照组相比,c-Myb在Ly10-miR-150细胞中表达明显下调(P<0.05)。(3)干扰OCI-Ly10细胞c-myb表达后,BCL6表达明显下调,而PRDM1表达明显上调。结论:(1)hsa-miR-150过表达对OCI-Ly10细胞抑制增殖和诱导凋亡作用非常明显。(2)过表达hsa-miR-150可诱导该瘤细胞向末端B细胞方向分化,作用机制可能与下调其靶基因c-myb的表达有关。

AIM： To investigate the mechanism that over-expression of hsa-miR-150 induces the re-differentia- tion of diffuse large B-cell lymphoma cell line OCI-Ly10. METHODS ： The expression level of hsa-miR-150 in CD19^＋ B and OCI-Ly10 cell lines was detected by real-time PCR. The expression level of c-Myb was detected by Western blotting and immunofiuorescence cytochemistry methods. Lentiviral supernatant containing recombinant plasmids was transfected in- to OCI-Ly10 cells by Lipofectamine^TM 2000 and named Ly10-control and Ly10-miR-150. The biological functions of the 2 cell sublines were identified by MTr assay. The cell cycle and apoptotic rates were detected by flow cytometry. The expres- sion levels of B-lymphocyte differentiation-related genes and c-myb in Ly10-control and Ly10-miR-150 cells were detected by real-time PCR and Western blotting. When c-myb was interfered in by interference fragment in OCI-Ly10 cells, the in- terference efficiency and the expression levels of BCL6 and PRDM1 were detected by real-time PCR and Western blotting. RESULTS： The expression level of hsa-miR-150 in CD19^＋ B cells was significantly higher than that in OCI-LyI0 cells. The expression level of c-Myb in OCI-Ly10 cells was higher than that in CD19^＋ B cells. The expression levels of B-lympho- cyte differentiation-related genes were changed significantly in OCI-Ly10 cells after transfected with hsa-miR-150. The ex- pression levels of PAXS, BCI/5 and c-Myb in Ly10-miR-150 cells were lower than those in Ly10-control cells, but the ex- pression levels of IRF4, PRDM1 and XBP1 were higher than those in Ly10-control cells. The expression level of BCL6 was lower and PRDM1 was higher after interference. CONCLUSION： Hsa-miR-150 plays a significant role in inhibiting prolif- eration and inducing apoptosis of OCI-Ly10 cells. The mechanism that over-expression of hsa-miR-150 induces OCI-Ly10 cell differentiation toward terminal B cells may be related to the down-regulation of c-myb.