胶质瘤细胞维甲酸代谢调控机制及其对细胞增殖的影响

目的: 探讨胶质瘤细胞中的维甲酸合成调控机制,以及维甲酸对人脑胶质瘤细胞SWO-Z2的增殖抑制作用及可能的机制。方法: 运用小分子RNA干扰Krüppel 样转录因子9(KLF9);Real-time PCR和Western blotting检测 KLF9 基因RNAi沉默效率。 KLF9 基因干扰后,运用Western blotting检测醛脱氢酶1家族成员A1(ALDH1A1)蛋白表达的变化。应用CCK-8法从3种类型维甲酸药物(13-顺维甲酸、9-顺维甲酸和全反式维甲酸分别简称13- cis RA、9- cis RA和ATRA)中筛选出对SWO-Z2细胞活性抑制作用最强的药物。Western blotting检测不同浓度ATRA作用SWO-Z2细胞后增殖相关蛋白cyclin D1、Bcl-2、cleaved PARP以及胶质瘤分化标记物GFAP的表达情况。Real-time PCR检测SWO-Z2细胞的维甲酸受体表达情况。结果: 小分子RNA干扰 KLF9 基因后, 成功下调KLF9表达,引起ALDH1A1 mRNA和蛋白表达都下降。3种类型维甲酸中ATRA对SWO-Z2细胞的增殖活性抑制作用最强。ATRA作用SWO-Z2细胞72 h后cleaved PARP蛋白激活,cyclin D1和Bcl-2表达水平下降,而GFAP表达没有改变。SWO-Z2细胞维甲酸受体(RARs)表达降低。结论: KLF9作为 ALDH1A1 基因的上游调控基因,通过正调控 ALDH1A1 基因的表达促进胶质瘤细胞中的维甲酸合成;ATRA处理SWO-Z2细胞后未能促进胶质瘤分化而是明显抑制肿瘤细胞增殖能力,可能是由于胶质瘤细胞内缺乏维甲酸受体所致。

Metabolic mechanism of retinoic acid in glioma cells and its impact on cell proliferation

AIM: To investigate the mechanism for regulating the synthesis and metabolism of retinoic acid in glioma cell line SWO-Z2 and its effect on cell proliferation.METHODS: The siRNA targeting to human KLF9 mRNA was transfected into SWO-Z2 cells.The silencing efficiency was detected by real-time PCR and Western blotting.After silencing of KLF9,the protein level of ALDH1A1 was detected by Western blotting.CCK-8 colorimetric assay was used to screen the optimal concentration of retinoic acid,and the strongest inhibitory effect of retinoic acid from 3 types of chemicals,13-cis-retinoic acid(13-cis RA),9-cis-retinoic acid(9-cis RA)and all-trans retinoic acid(ATRA),on SWO-Z2 cell growth was selected.Western blotting was also applied to explore the expression levels of cyclin D1,Bcl-2,cleaved PARP and GFAP in SWO-Z2 cells with the treatment of ATRA for 72 h.Simultaneously,the mRNA levels of retinoic acid receptors(RARs) in SWO-Z2 cells were determined by real-time PCR.RESULTS: siRNA-KLF9 knocked-down the expression of KLF9 and down-regulated the expression of aldehyde dehydrogenase 1 family member A1(ALDH1A1) at mRNA and protein levels(P<0.05).Among the 3 retinoic acid drugs,ATRA was the most effective in inhibiting the proliferation of SWO-Z2 cells.After treated with ATRA on SWO-Z2 cells for 72 h,the expression of cleaved PARP was increased,Bcl-2 and cyclin D1 were decreased,and GFAP didn’t change.The mRNA level of RARs in SWO-Z2 cells was very low.CONCLUSION: KLF9 positively regulates the expression of ALDH1A1 gene to increase the synthesis of retinoic acid.ATRA inhibits proliferation but does not induce differentiation of SWO-Z2 cells,which might result from lack of retinoic acid receptors in human glioma cells.