IDH1-R132H突变体通过miR-191-5p/p53轴调控U87MG胶质母细胞瘤干细胞增殖及凋亡

IDH1-R132H Mutant Regulates Proliferation and Stem Transformation of U87 MG Glioblastoma Stem Cells Via the miR-191-5p/p53 Axis

目的 探索IDH1-R132H突变抑制U87 MG胶质母细胞瘤干细胞增殖及诱导其凋亡的可能机制。方法 (1)以U87 MG细胞为研究对象,采用慢病毒转染构建IDH 1-R132H突变型及野生型细胞株,并诱导为胶质母细胞瘤干细胞,观察细胞球形成情况;流式细胞术检测肿瘤干细胞标志物CD133表达水平;CCK-8法检测细胞增殖情况和PI/Annexin V-FITC双染法检测细胞凋亡情况;qRCP和Western blot法分别检测IDH1、miR-191-5p及p53表达水平。(2)以NOD/SCID荷瘤小鼠作为研究对象,在体内水平考察miR-191-5p、p53表达及肿瘤生长情况。结果IDH1-R132H突变能够在体内外抑制U87 MG胶质母细胞瘤干细胞增殖(P<0.01)及分化(P<0.05),诱导其凋亡(P<0.01),下调miR-191-5p表达(体内P<0.05,体外P<0.001),上调p53(P<0.001)及其编码基因TP53(体内P<0.01,体外P<0.000 1)表达。结论 IDH1-R132H突变引起的U87 MG胶质瘤干细胞增殖及凋亡的调控可能与其作用于mi R-191-5p/p53轴,抑制胶质瘤细胞活性密切相关。

Aim To investigate the possible mechanism of IDH 1-R132H mutation regulating proliferation and apoptosis activity of U87 MG glioblastoma stem cells.Methods U87 MG cells were used as the research object,IDH 1-R132H mutant and wild-type cell lines were constructed by lentiviral transfection in vitro.The cells were induced to differentiate to glioma stem cells.The formation of cell spheres was observed,and the expression of CD133 was detected by flow cytometry.Then proliferation was investigated by CCK-8 method,the apoptosis was determined by PI/Annexin V-FITC straining.The expression levels of IDH1,miR-191-5p and p53 were detected by qPCR and Western blot methods.NOD/SCID tumor-bearing mice were employed to study the expression of miR-191-5p,p53 and tumor growth in vivo.Results IDH 1-R132H mutation inhibited proliferation(P <0.01) and formation(P <0.05)of U87 MG stem cells both in vivo and in vivo,promoted apoptosis(P <0.01),down-regulated the expression of mi R-191-5p(P <0.05 in vivo,P <0.001 in vivo),and up-regulated the expression of p53(P <0.001) and its encoding gene TP 53(P <0.01 in vivo,P <0.000 1 in vivo).Conclusion The regulation by IDH 1-R132H mutation on U87 MG glioma stem cells proliferation and apoptosis may be closely related to the mi R-191-5p/p53.