RNA干扰FZD4基因表达对人胶质瘤干细胞自我更新能力的抑制作用

doi:10.13481/j.1671-587x.20150406

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (04): 701-705.doi: 10.13481/j.1671-587x.20150406

RNA干扰FZD4基因表达对人胶质瘤干细胞自我更新能力的抑制作用

高兴春, 郭娜, 闫爱丽, 冯浩, 史宏恩, 苟兴春

西安医学院基础医学研究所, 陕西西安 710021

收稿日期:2014-01-12 发布日期:2015-08-01
通讯作者: 苟兴春,副教授,硕士研究生导师(Tel:029-6177472,E-mail:gouxingchun@189.cn) E-mail:gouxingchun@189.cn
作者简介:高兴春(1981-),男,陕西省西安市人,讲师,医学博士,主要从事肿瘤发生的分子机制及治疗方面的研究。
基金资助:
国家自然科学基金资助课题(81402063);陕西省卫生厅科研基金资助课题(2014D21,2014D24);陕西省大学生创新创业训练计划资助课题(2014118401661);陕西省教育厅优势学科建设经费资助课题(陕教位[2014]3号-1001)

Inhibitory effect of RNA silencing FZD4 expression on self-renewal ability of glioma stem cells

GAO Xingchun, GUO Na, YAN Aili, FENG Hao, SHI Hongen, GOU Xingchun

Institute of Basic Medical Science, Xi'an Medical University, Xi'an 710021, China

Received:2014-01-12 Published:2015-08-01

摘要/Abstract

摘要：

目的: 探讨FZD4在人胶质瘤干细胞自我更新能力中的作用,并阐明其作用机制。方法: 针对人的FZD4设计了2条短发夹RNA(shRNA)序列,利用慢病毒包装后感染人胶质瘤U251细胞(FZD4 shRNA干扰组分为FZD4-shRNA-1#和FZD4-shRNA-2#组),经空病毒感染的U251细胞为对照组。倒置显微镜观察感染的U251细胞中绿色荧光蛋白(GFP)表达情况,通过RT-PCR和免疫印迹法检测各组细胞中FZD4 mRNA和蛋白表达水平。利用干细胞培养基筛选出胶质瘤干细胞,采用极限稀释实验观察FZD4 shRNA对肿瘤干细胞增殖及自我更新的影响。结果: 与对照组比较,FZD4 shRNA干扰组FZD4 mRNA和蛋白表达水平明显降低(P<0.01)。与对照组比较,FZD4 shRNA干扰组中干细胞形成的神经球直径明显缩小(P<0.01)。极限稀释实验,与对照组比较,FZD4 shRNA干扰组干细胞在每个孔中生成至少1个神经球所需要的细胞数明显增加(P<0.05或P<0.01)。结论: 通过shRNA下调U251细胞中FZD4的表达后可以显著抑制胶质瘤肿瘤干细胞的自我更新能力。

关键词: 胶质瘤, 卷曲蛋白4, 短发夹RNA, 肿瘤干细胞, 自我更新

Abstract:

Objective To investigate the effect of FZD4 on the self-renewal ability of glioma stem cells,and to clarify its mechanism. Methods Two shRNA sequences were designed according to FZD4 mRNA sequence,named FZD4-shRNA-1# and FZD4-shRNA-2#.Then the lentiviral vector particles were transfected into U251cells(FZD4-shRNA-1# and FZD4-shRNA-2#),The U251 cells infected with the empty vector were used as control group.The expression of green fluorescence protein (GFP) in the transfected cells were observed under inverted fluorescence microscope.The expression levels of FZD4 mRNA and protein in the U251 cells in various gorups were detected by reverse transcription (RT)-PCR and Western blotting method.The U251 cells were cultured in SFM and the glioma stem cells were selected;the proliferation and self-renewal of the tumore stem cells were observed with limiting dilution assay. Results Compared with control group,the expression levels of FZD4 mRNA and protein in FZD4 shRNA groups were significantly reduced(P<0.01).The diameters of neurospheres in FZD4 shRNA groups were significantly smaller than that in control group(P<0.01).The number of cells required to generate at least one tumor sphere/well was significantly increased in FZD4 shRNA groups than that in control group(P<0.05 or P<0.01) in limiting dilution assay. Conclusion Down-regulation of FZD4 expression that in inhibit the self-renewal ability U251 glioma stem cells.

Key words: glioma, frizzled, short hairpin RNA, tumor stem cell, self-renewal

中图分类号:

R739.41

高兴春, 郭娜, 闫爱丽, 冯浩, 史宏恩, 苟兴春. RNA干扰FZD4基因表达对人胶质瘤干细胞自我更新能力的抑制作用[J]. 吉林大学学报(医学版), 2015, 41(04): 701-705.

GAO Xingchun, GUO Na, YAN Aili, FENG Hao, SHI Hongen, GOU Xingchun. Inhibitory effect of RNA silencing FZD4 expression on self-renewal ability of glioma stem cells[J]. Journal of Jilin University Medicine Edition, 2015, 41(04): 701-705.

参考文献

[1] Wen PY,Kesari S. Malignant gliomas in adults[J].N Engl J Med,2008,359(5):492-507.

[2] Nakano I,Kornblum HI.Brain tumor stem cells[J].Pediatr Res,2006,59(4 Pt 2):54R-58R.

[3] Man J,Shoemake JD,Ma T,et al.Hyperthermia sensitizes glioma stem-like cells to radiation by inhibiting akt signaling[J].Cancer Res,2015,75(8):1760-1769.

[4] Tu Y,Gao X,Li G,et al.Microrna-218 inhibits glioma invasion,migration,proliferation,and cancer stem-like cell self-renewal by targeting the polycomb group gene bmi1[J].Cancer Res,2013,73(19):6046-6055.

[5] Fodde R,Brabletz T.Wnt/beta-catenin signaling in cancer stemness and malignant behavior[J].Curr Opin Cell Biol,2007,19(2):150-158.

[6] Gao X,Mi Y,Ma Y,et al.Lef1 regulates glioblastoma cell proliferation,migration,invasion,and cancer stem-like cell self-renewal[J].Tumour Biol,2014,35(11):11505-11511.

[7] Shih AH,Holland EC.Notch signaling enhances nestin expression in gliomas[J].Neoplasia,2006,8(12):1072-1082.

[8] Mori H,Ninomiya K,Kino-oka M,et al.Effect of neurosphere size on the growth rate of human neural stem/progenitor cells[J].J Neurosci Res,2006,84(8):1682-1691.

[9] Qiang L,Yang Y,Ma YJ,et al.Isolation and characterization of cancer stem like cells in human glioblastoma cell lines[J].Cancer Lett,2009,279(1):13-21.

[10] Teodorczyk M,Martin-Villalba A.Sensing invasion:Cell surface receptors driving spreading of glioblastoma[J].J Cell Physiol,2010,222(1):1-10.

[11] Chesler DA,Berger MS,Quinones-Hinojosa A.The potential origin of glioblastoma initiating cells[J].Front Biosci (Schol Ed),2012,4:190-205.

[12] Germano I,Swiss V,Casaccia P.Primary brain tumors,neural stem cell,and brain tumor cancer cells:Where is the link?[J].Neuropharmacology,2010,58(6):903-910.

[13] Liu Y,Yan W,Zhang W,et al.Mir-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor lef1[J].Oncol Rep,2012,28(3):1013-1021.

[14] Angeloni V,Tiberio P,Appierto V,et al.Implications of stemness-related signaling pathways in breast cancer response to therapy[J].Semin Cancer Biol,2014,31:43-51.

[15] Zhang Y,Zhang X,Huang J,et al.Wnt signaling regulation of stem-like properties in human lung adenocarcinoma cell lines[J].Med Oncol,2015,32(5):596-606.

[16] 王凤伟,牛光明,张鹏远,等.脑胶质瘤组织中Beclin-1的表达及与预后的关系[J].郑州大学学报:医学版,2014,49(4):472-475.

[17] 梁博,王新军,周少龙.胶质瘤组织中CPEB4和Bcl-xL的表达及预后影响因素分析[J].郑州大学学报:医学版,2013,48(5):605-609.

RNA干扰FZD4基因表达对人胶质瘤干细胞自我更新能力的抑制作用

Inhibitory effect of RNA silencing FZD4 expression on self-renewal ability of glioma stem cells

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