沉默CHPF基因表达对人脑胶质瘤U87细胞增殖、凋亡及侵袭的影响

发布时间：2018-03-23 21:52

本文选题：神经胶质瘤　切入点：软骨素聚合因子　出处：《肿瘤》2017年11期

【摘要】：目的 :探讨沉默软骨素聚合因子(chondroitin polymerizing factor,CHPF)基因对脑胶质瘤U87细胞增殖及侵袭的影响,及可能的作用机制。方法 :构建携带有靶向CHPF基因sh RNA(CHPF-sh RNA)的重组慢病毒并感染胶质瘤U87细胞。采用实时荧光定量PCR和蛋白质印迹法检测CHPF m RNA及蛋白表达水平的变化,CCK-8法检测细胞的增殖情况,FCM法检测细胞周期及细胞凋亡率,Transwell小室侵袭实验检测U87细胞侵袭能力的变化;蛋白质印迹法检测沉默CHPF基因表达后对单核细胞趋化蛋白1(monoeyte chemoattractant protein 1,MCP-1,又称CCL2)表达水平的影响。最后,采用脂质体法将特异性针对CCL2基因的CCL2-si RNA转入U87细胞,用蛋白质印迹法检测对CHPF蛋白表达的影响。结果 :实时荧光定量PCR和蛋白质印迹法检测结果表明,感染CHPF-sh RNA后U87细胞中CHPF m RNA和蛋白的表达水平均明显被抑制(P值均0.05),成功建立了稳定沉默CHPF基因表达的U87细胞。与空白对照组及阴性对照组比较,CHPF-sh RNA感染组细胞的增殖速度明显下降,细胞周期阻滞在G0/G1期,凋亡率明显增高(P值均0.05);CHPF-sh RNA感染组U87细胞,穿过小室膜的细胞数明显减少,侵袭能力下降(P0.05);CHPF-sh RNA感染组U87细胞中CCL2的表达水平明显下调(P0.05)。沉默CCL2基因表达后,U87细胞中CHPF m RNA的表达水平无明显变化(P0.05)。结论:下调CHPF基因表达后,可能通过调控CCL2抑制脑胶质瘤细胞的增殖及侵袭能力,并促进细胞凋亡。
[Abstract]:Aim: to investigate the effect of silencing chondroitin polymerizing factor-CHPF1 gene on the proliferation and invasion of glioma U87 cells. Methods: recombinant lentivirus carrying target CHPF gene sh RNA(CHPF-sh RNAs was constructed and infected with glioma U87 cells. The expression of CHPF m RNA and protein was detected by real-time fluorescence quantitative PCR and Western blotting. The cell cycle and apoptosis rate were detected by FCM and the invasion ability of U87 cells was detected by Transwell chamber invasion assay. The effect of silencing CHPF gene expression on the expression of monocyte chemoattractant protein 1(monoeyte chemoattractant protein 1 (also known as CCL2) was detected by Western blot. Finally, the specific CCL2-si RNA targeting CCL2 gene was transfected into U87 cells by liposome method. Results: Real-time fluorescent quantitative PCR and Western blot were used to detect the expression of CHPF protein. The expression levels of CHPF m RNA and protein in U87 cells after infection with CHPF-sh RNA were significantly inhibited (P = 0.05). U87 cells with stable silence of CHPF gene expression were successfully established. Compared with control group and negative control group, U87 cells infected with CHPF-sh RNA were successfully established. The rate of proliferation decreased significantly, The cell cycle was blocked in G0/G1 phase, the apoptosis rate was significantly increased (P = 0.05) and the number of cells passing through the ventricular membrane decreased significantly in the U87 cells infected with CHPF-sh RNA. The expression level of CCL2 in U87 cells was significantly down-regulated by P0.05 RNA infection, but the expression level of CHPF m RNA in U87 cells did not change after CCL2 gene expression was silenced. Conclusion: after down-regulating CHPF gene expression, the expression of CHPF m RNA in U87 cells was significantly decreased. CCL2 may inhibit the proliferation and invasion of glioma cells and promote apoptosis.
【作者单位】：南昌大学第二附属医院神经外科;抚州市第一人民医院神经外科;
【基金】：国家自然科学基金资助项目(编号:81760446) 江西省教育厅科学技术研究资助项目(编号:GJJ160253,GJJ160166)~~
【分类号】：R739.41

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