miR-4539通过靶向作用于叉头盒转录因子1抑制胶质瘤细胞生长的实验研究

miR-4539 inhibits the growth of glioma cells by targeting Foxp1

目的 探讨miR-4539抑制胶质瘤细胞生长的效应及相关机制。方法 采用RT-PCR法检测脑胶质瘤标本和7株胶质瘤细胞株中miR-4539的表达水平。采用CCK-8法、流式细胞法、Western blot法检测miR-4539在胶质瘤细胞中的作用。采用双荧光素酶实验验证叉头盒转录因子1(Foxp1)是否是miR-4539的直接靶点。构建小鼠移植瘤模型,探讨miR-4539转染对小鼠肿瘤生长的抑制作用。结果 (1)miR-4539在胶质瘤组织中的表达水平显著低于正常脑组织(P<0.05)。miR-4539在7个常用的人脑胶质瘤细胞系(U87、U251、U373、T98G、LN18、LN229、SF295)中的表达量显著低于人正常脑胶质细胞株HEB(P<0.05)。(2)与miR-NC组相比,miR-4539转染72 h后,T98G细胞和LN18细胞中miR-4539的表达水平都显著增加(P<0.05)。CCK-8检测显示,外源性miR-4539过表达可显著抑制T98G和LN18细胞的增殖。同时可诱导T98G和LN18细胞周期阻滞,G0/G1期细胞百分率增加,S期细胞百分率降低,且细胞周期调控蛋白cyclinD1、cyclinE1和Ser780的表达水平也显著降低(P<0.05)。(3)荧光素酶报告显示,Foxp1是miR-4539的直接靶点,可被miR-4539直接调控。接种T98G细胞后6周,miR-4539转染组小鼠移植瘤平均体积、平均重量均显著小于miR-NC组(P<0.05)。(4)T98G和LN18细胞过表达miR-4539后,Foxp1mRNA和蛋白表达都显著降低,免疫荧光结果也确认,miR-4539降低了Foxp1在T98G细胞和LN18细胞中的表达。结论 miR-4539可通过靶向作用于Foxp1发挥抑癌因子效应,有望成为脑胶质瘤患者诊断和治疗的新靶点。

Objective To investigate the inhibitory effect of miR-4539 on the growth of glioma cells. Methods The expression levels of miR-4539 in glioma samples and 7 glioma cell lines were detected by RT-PCR. CCK-8 method, flow cytometry and Western blot were used to detect the role of miR-4539 in glioma cells. Dual luciflucase assay was used to verify whether Foxp1 is a direct target of miR-4539. To investigate the inhibitory effect of miR-4539 transfection on tumor growth in mice, a mouse transplanted tumor model was constructed. Results(1) The expression level of miR-4539 in glioma tissues was significantly lower than that in normal brain tissues(P<0.05). The expression level of miR-4539 in 7 commonly used human glioma cell lines(U87, U251, U373, T98 G, LN18, LN229, SF295) was significantly lower than that in human normal glioma cell line HEB(P<0.05).(2) Compared with the miR-NC group, the expression levels of miR-4539 in T98 G cells and LN18 cells were significantly increased 72 h after transfection of miR-4539(P<0.05). CCK-8 showed that the overexpression of exogenous miR-4539 significantly inhibited the proliferation of T98 G and LN18 cells. Meanwhile, T98 G and LN18 cell cycle arrest was induced, and the percentage of G0/G1 phase cells was increased, while the percentage of S phase cells was decreased, and the expression levels of cell cycle regulatory proteins cyclinD1, cyclinE1 and Ser780 were also significantly decreased(P<0.05).(3) Luciferase reports showed that Foxp1 is a direct target of miR-4539 and can be directly regulated by miR-4539. At 6 weeks after T98 G cells were inoculated, the average volume and weight of transplanted tumor in the miR-4539-transfected mice were significantly smaller than those in the miR-NC group(P<0.05).(4) After overexpression of miR-4539 in T98 G and LN18 cells, the expression of Foxp1 mrna and protein were significantly decreased. Immunofluorescence result also confirmed that miR-4539 reduced the expression of Foxp1 in T98 G and LN18 cells. Conclusions MiR-4539 acts as a tumor suppressor by targeting Foxp1, which is expected to be a new target for the diagnosis and treatment of gliomas.