miR-568通过下调AKR1B10表达影响胶质母细胞瘤细胞的增殖和凋亡

miR-568 affects the proliferation and apoptosis of glioblastoma cells by downregulating AKR1B10 expression

目的 探讨微小RNA-568(miR-568)对胶质母细胞瘤细胞增殖和凋亡的影响及作用机制。方法 培养正常星形胶质细胞HA1800及胶质母细胞瘤细胞系(U251、T98G和SHG44)购自中国科学院上海细胞库,qRT-PCR检测细胞中miR-568和醛酮还原酶家族1成员B10(AKR1B10)mRNA表达,蛋白质印迹法(Western blotting)检测AKR1B10蛋白表达。以U251细胞为研究对象,构建上调miR-568或下调AKR1B10的U251细胞,MTT、流式细胞仪和Western blotting实验检测细胞增殖、凋亡及细胞中CyclinD1、p21、Bcl-2和Bax蛋白表达。双荧光素酶报告基因实验验证miR-568与AKR1B10调控关系。结果 胶质母细胞瘤细胞系(U251、T98G和SHG44)及HA1800细胞中miR-568表达分别为(1.01±0.09)、(0.39±0.03)、(0.58±0.05)及(0.46±0.04),AKR1B10 mRNA表达分别为(0.98±0.08)、(2.45±0.2)、(2.16±0.21)及(2.37±0.23),AKR1B10蛋白表达分别为(0.12±0.03)、(0.61±0.05)、(0.51±0.04)及(0.59±0.05),胶质母细胞瘤细胞系(U251、T98G和SHG44)中miR-568表达低于HA1800细胞(P<0.05),AKR1B10 mRNA和蛋白表达高于HA1800细胞(P<0.05)。上调miR-568或下调AKR1B10后,U251细胞增殖活性[(0.61±0.06)、(0.69±0.06)]、CyclinD1[(0.32±0.03)、(0.35±0.03)]和Bcl-2蛋白表达[(0.29±0.03)、(0.32±0.03)]降低(P<0.05),U251细胞凋亡率[(22.41±2.15)%、(21.26±2.14)%]、p21[(0.58±0.05)、(0.56±0.05)]和Bax蛋白表达[(0.72±0.07)、(0.67±0.07)]升高(P<0.05)。miR-568靶向结合并负调控AKR1B10。上调AKR1B10逆转了上调miR-568对U251细胞增殖和凋亡的影响。结论 上调miR-568通过靶向负调控AKR1B10抑制胶质母细胞瘤细胞增殖,并促进其凋亡。

Objective To investigate the effect and mechanism of microRNA-568 (miR-568) on the proliferation and apoptosis of glioblastoma cells.Methods Cultured normal HA1800 astrocytes and glioblastoma cell lines (U251,T98G and SHG44) were purchased from Shanghai Cell Bank,Chinese Academy of Sciences.The mRNA expression of miR-568 and aldosterone reductase family 1member B10 (AKR1B10) was detected by qRT-PCR,and the protein expression of AKR1B10 was detected by Western blotting.Taking U251 cells as the research objects,U251 cells with upregulated miR-568 or downregulated AKR1B10 were constructed.MTT,flow cytometry and Western blotting assays were used to detect cell proliferation,apoptosis and the expression of CyclinD1,p21,Bcl-2 and Bax proteins in cells.The dual-luciferase reporter gene experiment verified the regulatory relationship between miR-568 and AKR1B10.Results The expression of miR-568 in glioblastoma cell lines (U251,T98G and SHG44) and HA1800 cells was (1.01±0.09),(0.39±0.03),(0.58±0.05) and (0.46±0.04),respectively.The expression of AKR1B10 mRNA was (0.98±0.08),(2.45±0.2),(2.16±0.21) and (2.37±0.23),respectively,and the AKR1B10 protein expression was (0.12±0.03),(0.61±0.05),(0.51±0.04) and (0.59±0.05),respectively.The expression of miR-568 in glioblastoma cell lines (U251,T98G and SHG44) was lower than that in HA1800 cells (P<0.05),and the mRNA and protein expression levels of AKR1B10 were higher than those in HA1800 cells (P<0.05).After upregulating miR-568 or downregulating AKR1B10,U251 cell proliferation activity[(0.61±0.06),(0.69±0.06)],CyclinD1[(0.32±0.03),(0.35±0.03)]and Bcl-2 protein expression[(0.29±0.03),(0.32±0.03)]decreased (P<0.05),U251 cell apoptosis rate[(22.41±2.15)%,(21.26±2.14)%],p21[(0.58±0.05),(0.56±0.05)]and Bax protein expression[(0.72±0.07),(0.67±0.07)]increased (P<0.05).miR-568 targets and negatively regulates AKR1B10.Upregulation of AKR1B10 reverses the effects of upregulation of miR-568 on U251 cell proliferation and apoptosis.Conclusion Upregulation of miR-568 inhibits glioblastoma cell proliferation and promotes apoptosis by targeting and negatively regulating AKR1B10.