着丝粒蛋白F通过mTORC1信号通路促进胶质瘤恶性进展

Centromere protein F promotes the malignant progression of glioma via the mTORC1 signaling pathway

摘要:

目的 探讨着丝粒蛋白F(CENPF)对胶质瘤细胞增殖、侵袭、周期和凋亡能力的影响及其作用机制。 方法 通过生物信息学方法分析CENPF在胶质瘤中的表达水平和预后因素,及其在胶质瘤发展中可能参与的信号通路。体外培养人正常胶质细胞和胶质瘤细胞系,通过蛋白免疫印迹筛选出CENPF表达量最高的两株细胞；分为阴性对照组(siNC组)和靶向CENPF的小干扰RNA组(siCENPF#1、siCENPF#2、siCENPF#3),选取干扰能力强的两个实验组进行后续实验；敲降CENPF后采用CCK-8实验、Transwell实验和流式细胞术检测细胞增殖、侵袭、细胞周期和凋亡的变化；采用蛋白免疫印迹检测信号通路蛋白水平的变化。 结果 生物信息学分析显示,CENPF在胶质瘤中普遍高表达,且与WHO分级呈正相关；多因素Cox回归分析显示,CENPF是胶质瘤的独立预后标志物。蛋白免疫印迹分析显示,CENPF在U251和LN229细胞株中过表达(P<0.05),故选择这两株细胞株进行后续实验。CCK-8实验和Transwell实验结果显示,敲降CENPF可导致胶质瘤细胞增殖和侵袭能力降低(P<0.05)。进一步分析发现CENPF参与细胞周期、G2/M检查点、P53信号通路和mTORC1信号通路。流式细胞术和蛋白免疫印迹结果证实,敲降CENPF可导致胶质瘤细胞停滞在G2/M期,并增加细胞凋亡(P<0.05)。最后蛋白免疫印迹结果证实,敲降CENPF能够使mTORC1信号通路蛋白磷酸化水平明显降低(P<0.05)。结论 CENPF在胶质瘤中高表达且与胶质瘤的不良预后相关,通过调控细胞周期、细胞凋亡、细胞增殖和侵袭促进胶质瘤恶性行为,其调控机制可能与促进mTORC1信号通路有关。

Abstract:

Objective To investigate the effects of centromere protein F (CENPF) on the proliferation, invasion, cell cycle and apoptosis of glioma cells and its mechanism. Methods Bioinformatics methods were used to analyze the expression level and prognostic factors of CENPF in glioma, as well as the signaling pathways that may be involved in the development of glioma. Normal human glial cells and glioma cell lines were cultured in vitro, and the two cell lines with the highest expression of CENPF were screened by western blotting; they were divided into a negative control group (siNC group) and a small interfering RNA group targeting CENPF (siCENPF#1, siCENPF#2, siCENPF#3), and the two experimental groups with strong interference ability were selected for subsequent experiments; after knocking down CENPF, CCK-8 experiment, Transwell experiment and flow cytometry were used to detect changes in cell proliferation, invasion, cell cycle and apoptosis; western blotting was used to detect changes in signaling pathway protein levels. Results Bioinformatics analysis showed that CENPF was generally highly expressed in gliomas and was positively correlated with WHO grade; multivariate Cox regression analysis showed that CENPF was an independent prognostic marker for gliomas. Western blot analysis showed that CENPF was overexpressed in U251 and LN229 cell lines (P<0.05), so these two cell lines were selected for subsequent experiments. The results of CCK-8 and Transwell experiments showed that knocking down CENPF could reduce the proliferation and invasion ability of glioma cells (P<0.05). Further analysis found that CENPF was involved in the cell cycle, G2/M checkpoint, P53 signaling pathway, and mTORC1 signaling pathway. Flow cytometry and Western blot results confirmed that knocking down CENPF could cause glioma cells to arrest in the G2/M phase and increase cell apoptosis (P<0.05). Finally, Western blot results confirmed that knocking down CENPF could significantly reduce the phosphorylation level of mTORC1 signaling pathway proteins (P<0.05). Conclusion CENPF is highly expressed in glioma and is associated with poor prognosis of glioma. It promotes the malignant behavior of glioma by regulating cell cycle, apoptosis, cell proliferation and invasion. Its regulatory mechanism may be related to promoting the mTORC1 signaling pathway.