Objective: To investigate the impact of all-trans retinoic acid (ATRA) on MDM2 gene expression in astrocytoma cell line SHG-44, and to provide basic data for further research on the progression mechanism and gene therapy of human astrocytoma.
Methods: The differential expressions of MDM2 gene and protein in SHG-44 cells were detected by cDNA microarray and Western blot, respectively, before and after treatment of ATRA. The expressions of MDM2 protein in WHO grade II and grade IV astrocytomas were determined by immunohistochemical streptavidin-peroxidase method. Some differentially expressed genes were selected randomly for Northern blot analysis.
Results: The intensity ratio of ATRA-treated to untreated SHG-44 cell was 0.37 in the cDNA microarray, suggesting that the expression of MDM2 gene was down-regulated in SHG-44 cells after treatment with ATRA. Some genes differentially expressed in the microarray were confirmed by Northern blot. Western blot demonstrated that the optical density ratios of MDM2 to beta-actin in ATRA-treated and untreated SHG-44 were 14.02+/-0.35 and 21.40+/-0.58 (t = 24.728, P = 0.000), respectively, suggesting that the expression of MDM2 protein was inhibited in ATRA-treated SHG-44 cells. Moreover, the percentages of MDM2-positive protein were 24.00% (6/25) and 56.52% (13/23) (chi(2) = 5.298, P = 0.021) in WHO grade II and grade IV astrocytomas, respectively, suggesting that the expression of MDM2 protein may increase along with the elevation of astrocytoma malignancy.
Conclusion: ATRA can inhibit MDM2 gene expression in SHG-44 cells, and MDM2 is related to astrocytoma progression.
目的: 探讨全反式维甲酸对胶质瘤细胞SHG-44中MDM2基因表达的影响, 为进一步研究脑胶质瘤的进展机制及基因治疗提供依据。
方法: 分别利用cDNA微阵列与Western blot技术分析在10 μmol/L 全反式维甲酸(all-trans retinoic acid, ATRA)处理前后的胶质瘤SHG-44细胞中MDM2基因和蛋白的差异表达;应用免疫组化链霉菌抗生物素蛋白-过氧化酶(Streptavidin-Peroxidase, SP)法检测II级与IV级胶质瘤标本MDM2蛋白的表达。 随机选择数个差异基因进行Northern杂交实验, 以验证cDNA微阵列的结果。
结果: 应用cDNA微阵列检测发现, MDM2基因在ATRA处理与未处理的SHG-44细胞之间表达量的比值为0.37, 提示ATRA可抑制MDM2基因在SHG-44中的表达。 该结果进一步得到Northern杂交实验结果的支持。 Western blot分析结果显示10 μmol/L ATRA处理前后胶质瘤SHG-44细胞之间MDM2蛋白的相对表达量分别为21.40±0.58和14.02±0.35 (t = 24.728, P = 0.000), 提示MDM2蛋白在SHG-44中的表达受到ATRA抑制。 II级和IV级胶质瘤标本MDM2蛋白的阳性表达率分别为24.00%(6/25)和56.52%(13/23)(χ 2 = 5.298, P = 0.021), MDM2蛋白的表达随胶质瘤恶性程度的增高而增加。
结论: ATRA可抑制SHG-44胶质瘤细胞中MDM2基因的表达, MDM2基因的表达水平与胶质瘤的演进有关。