ADAM12基因表达沉默对CD133阳性胶质瘤细胞自我更新能力的影响

摘要

Objective To investigate the inhibitory effect of a disintegrin and metalloprotease 12 silenced by shR⁃NA on self-renewal capacity of CD133 positive giloma cells. Methods The shRNA recombinant lentivirus aimed at si⁃lencing ADAM12 was prepared. Human glioma cells U87 were employed in this study and assigned into three groups:shRNA-ADAM12, shRNA-NCandshRNA-C. ADAM12 expression was detected at mRNA and protein level using Re⁃al-time quantitative-PCR and western bloting, respectively. U87 cells were cultured with stem cell culture medium, to obtain cell sphere formation in which CD133 positive glioma cells were enriched. Immunofluorescence was employed to detect the expression of ADAM12 and CD133 in cell spheres and U87 cells; Self-renewal was tested by using tumor sphere formation assay. Molecular markers for differentiated or undifferentiated cells (CD133,GFAP and Tuj1) were de⁃tected at protein using western blotting. Western blotting was employed to test protein expression of HES1. Results AD⁃AM12 shRNA significantly down-regulated the mRNA and protein expression levels of ADAM12. Compared with shRNA–C group, the relative expression levels of mRNA in shRNA-ADAM12 group and shRNA-NC group were 0.22 ± 0.03 and 0.98 ± 0.06 (F=425.37,P<0.01). The relative expression levels of protein in shRNA-ADAM12 group, shRNA-NC group and shRNA-C group were 28.72%±2.36%, 69.21%±3.92%and 69.04%±3.57%, respectively (F=145.42,P<0.01). Immunofluorescence staining showed that expression levels of ADAM12 and CD133 in cell spheres were significantly higher than those in normal cells. The number of spheres in three groups were 45.5±2.3、104.2±5.8 and 109.6±6.2, tumor sphere formation ability of shRNA-ADAM12 group was lower than that of shRNA-NC group and shRNA-C group (F=147.03,P<0.01). Compared with the shRNA-NC group and shRNA-C group, the protain expression of GFAP and Tuj1 were increased up to 166% and 146% (P<0.01) whereas the protein expression levels of CD133 and HES1 were down-regulated by 54% and 50% (P<0.01). Conclusion Knockdown of ADAM12 may suppress self-renewal ability of CD133 positive glioma cells by inhibiting the Notch pathway activity.%目的：研究解整合素-金属蛋白酶12(a disintegrin and metalloprotease 12,ADAM12)基因表达沉默抑制CD133阳性（CD133+）胶质瘤细胞的自我更新能力。方法采用shRNA重组慢病毒转染技术沉默胶质瘤U87细胞系ADAM12基因表达分为ADAM12基因表达干扰序列组（shRNA-ADAM12）、阴性对照组（shRNA-NC）及空白对照（shRNA-C）组。通过Western blotting以及Real-time PCR验证各组细胞ADAM12表达情况；采用悬浮培养得到胶质瘤细胞球以富集CD133+的胶质瘤细胞；并通过免疫荧光染色技术检测ADAM12与CD133在细胞球与贴壁细胞的表达情况；通过神经肿瘤球形成实验检测三组细胞的自我更新能力；利用Western blotting分别检测三组细胞成球后未分化或分化相关蛋白CD133、GFAP及TUBB3以及Notch通路靶基因Hes1的蛋白表达情况。结果慢病毒转染技术可显著下调U87胶质瘤细胞ADAM12的mRNA及蛋白的表达量，shRNA-ADAM12组与shRNA-NC组较shRNA-C组mRNA的相对表达量为0.22±0.03与0.98±0.06（F=425.37，P＜0.01）；三组AD⁃AM12蛋白的相对表达量分别为28.72%±2.36%、69.21%±3.92%及69.04%±3.57%（F=145.42, P＜0.01）；免疫荧光染色显示细胞球中ADAM12与CD133表达量明显高于普通细胞；神经肿瘤球形成实验结果显示，三组成球数分别为45.5±2.3、104.2±5.8以及109.6±6.2，与shRNA-NC组及shRNA-C组相比，shRNA-ADAM12组的成球能力明显降低，差异具有统计学意义（ F=147.03，P＜0.01）。shRNA-ADAM12组与shRNA-C组相比，GFAP与TUBB3蛋白表达量分别上调约166%与146%，CD133与HES1的蛋白表达量分别下调了54%与50%，差异均具有统计学意义（P＜0.01）。结论 ADAM12基因表达沉默可能通过抑制Notch通路活性降低CD133+胶质瘤细胞的自我更新能力。