miR-578表达上调对胶质瘤细胞增殖和凋亡的影响及其与PREX2a的相关性分析

Effect of up-regulating miR-578 expression in glioma cell on its proliferation and apoptosis

目的探讨上调miR-578对胶质瘤细胞增殖和凋亡的影响及其与PREX2a的相关性。方法采取Western blot和qPCR两种方法检测不同级别的人脑胶质细胞瘤组织及正常脑组织标本中miR-578相对表达量。选取人恶性胶质瘤U87、U251细胞系进行miR-578对人脑恶性胶质瘤细胞增殖和凋亡影响的体外实验研究,分为miR-578类似物组及阴性序列组。qPCR及Western Blot分别检测转染后48h的胶质瘤细胞中miR-578和PREX2a的表达,平板克隆形成实验检测转染后胶质瘤细胞细胞克隆形成数目,流式细胞术检测胶质瘤细胞凋亡情况。噻唑蓝(MTT)法检测胶质瘤细胞24、48、72h时的相对生存活力。结果 qPCR结果显示胶质瘤组织标本中miR-578的相对表达量较正常脑组织标本明显降低(P<0.01)。与阴性序列组比较,miR-578类似物组U87、U251胶质瘤细胞的miR-578表达明显升高(P<0.05),miR-578的直接作用底物(PREX2a)表达则明显下降(P<0.05)。MTT实验检测转染后48h的miR-578类似物组U87、U251胶质瘤细胞在24、48、72h的生存能力较阴性序列组明显降低(P<0.05)。平板克隆形成实验显示miR-578类似物组U87、U251胶质瘤细胞克隆数较阴性序列组明显减少(P<0.05)。流式细胞术检测miR-578类似物组U87、U251胶质瘤细胞的凋亡能力较阴性序列组明显增强(P<0.05)。结论 miR-578在胶质瘤细胞增殖和凋亡中发挥了明显的负性生物学作用,该过程可能与PREX2a相关。

Objective To investigate the effect of up-regulating microRNA-578(miR-578)on the proliferation and apoptosis of glioma cells and its relationship with PREX2a.Methods The relative expression levels of miR-578 in different grades of human brain glioma cell tissue and normal brain tissue samples were detected by adopting Western blot and qRT-PCR.The human malignant glioma U87 and U251 cell lines were selected to conduct the in vitro experiment for the effect of miR-578 on the proliferation and apoptosis of human malignant glioma cells.The experiments were divided into two groups,the miR-578 analogue group and negative control group.The expression of miR-578 in glioma cells after transfection for 48 h was detected by qRT-PCR and the expression of PREX2a in glioma cells was detected by Western blot.The plate colony formation assay was used to detect the number of glioma cells clone formation in the two groups of glioma cells at 48 h after transfection.The apoptosis situation of glioma cells was detected by flow cytometry.The relative survival vitality of glioma cells at 24,48,72 h after transfection were detected by MTT assay.Results The qPCR results showed that the relative expression level of miR-578 in glioma tissue sample was significantly decreased compared with the normal brain tissue sample(P<0.01).Compared the negative sequence group,the miR-578 expression of U87 and U251 glioma cells in the miR-578 mimics group was significantly increased,but the miR-578 direct action substrate PREX2a expression was significantly declined(P<0.05).In the MTT experiment detection,the U87 and U251 glioma cells survival ability at 24,48,72 h in the miR-578 mimics group at 48 h after transfection was significantly decreased compared with the negative sequence group.The plate clone experiment showed that the cloned number of miR-578 mimics group U87 and U251 was significantly decreased compared with the negative sequence group(P<0.05).In the cytometry detection,U87 and U251 glioma cells apoptosis ability in the miR-578 mimics group was significantly increased compared with the genitive sequence group(P<0.05).Conclusion miR-578 plays a significant negative biological effect in the proliferation and apoptosis of glioma cells and this process may be associated with PREX2a.