过表达FCHSD1对细胞增殖以及迁移行为的影响
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摘要
目的通过检测FCHSD1对细胞增殖情况以及细胞迁移行为的影响,为进一步探讨FCHSD1的生物学功能提供理论依据。方法利用G418压力筛选下构建稳定表达FCHSD1的COS7细胞株;通过MTT实验检测稳转细胞株的细胞增殖情况,利用细胞划痕实验检测其迁移能力,western blot检测细胞中AKt、p-AKt的表达情况。结果利用500μg/m L G418筛选一个稳定表达FCHSD1的单克隆细胞株;此稳转细胞株稳定表达MYC-FCHSD1蛋白;与对照组相比较,过表达FCHSD1能够明显降低细胞的增殖速率;另外,MYC-FCHSD1稳转细胞株中的Akt表达水平明显降低,并且p-Akt的表达量也明显下降;过表达FCHSD1稳转细胞株的迁移能力与对照组相比较没有显著差异。结论过表达FCHSD1可能通过调控Akt信号途径来抑制细胞增殖,但对细胞迁移能力的作用不明显。
Objective: To observe the effect of FCHSD1 on the cell proliferation and cell migration behavior,and to provide theoretical basis for studying the functions of FCHSD1. Methods: Stable FCHSD1 expression of cos7 cell line was constructed by G418. Then,MTT assay and cell scratch test were separately used to detect the cell strain proliferation and the migration ability. Besides,western blot was used to detect the expression of Akt and p-Akt in cells. Results: It was determined that the working concentration of G418 was 500μg/m L. The stable cell lines stably expressing MYC-FCHSD1 were successfully obtained. FCHSD1 significantly decreased cell proliferation rate and significantly reduced the expression levels of Akt and p-Akt in cells,but did not have the effect on cell migration ability. Conclusion: FCHSD1 can significantly inhibit cell proliferation by regulating Akt signaling pathway,but has no significant effect on cell migration.
Objective: To observe the effect of FCHSD1 on the cell proliferation and cell migration behavior,and to provide theoretical basis for studying the functions of FCHSD1. Methods: Stable FCHSD1 expression of cos7 cell line was constructed by G418. Then,MTT assay and cell scratch test were separately used to detect the cell strain proliferation and the migration ability. Besides,western blot was used to detect the expression of Akt and p-Akt in cells. Results: It was determined that the working concentration of G418 was 500μg/m L. The stable cell lines stably expressing MYC-FCHSD1 were successfully obtained. FCHSD1 significantly decreased cell proliferation rate and significantly reduced the expression levels of Akt and p-Akt in cells,but did not have the effect on cell migration ability. Conclusion: FCHSD1 can significantly inhibit cell proliferation by regulating Akt signaling pathway,but has no significant effect on cell migration.
引文
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[4]Kessels MM,Qualmann B.The syndapin protein family:linking membrane trafficking with the cytoskeleton[J].Journal of Cell Science,2004,117:3077-3086.
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[8]Modregger J,Ritter B,Witter B,et al.All three PACSIN isoforms bind to endocytic proteins and inhibit endocytosis[J].Journal of Cell Science,2000,113(24):4511-4521.
[9]Peter BJ,Kent HM,Mills IG,et al.BAR domains as sensors of membrane curvature:the amphiphysin BAR structure[J].Science,2004,303:495-499.
[10]Chitu V,Pixley FJ,Macaluso F,et al.The PCH family member MAYP/PSTPIP2 directly regulates F-actin bundling and enhances filopodia formation and motility in macrophages[J].Molecular Biology of the Cell,2005,16:2947-2959.
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[12]Soderling SH,Binns KL,Wayman GA,et al.The WRP component of the WAVE-1 complex attenuates Rac-mediated signaling[J].Nat Cell Biol,2002,4(12):970-975.
[13]Heath RJ,Insall RH.Dictyostelium MEGAPs:F-BAR domain proteins that regulate motility and membrane tubulation in contractile vacuoles[J].J Cell Sci,2008,121(7):1054-1064.
[14]Han Y,Cui J,Lu Y,et al.FCHSD2 predicts response to chemotherapy in acute myeloid leukemia patients[J].Leuk Res,2012,36(11):1339-1346.
[15]Cao H,Yin X,Cao Y,et al.FCHSD1 and FCHSD2 are expressed in hair cell stereocilia and cuticular plate and regulate actin polymerization in vitro[J].PLo S One,2013,8(2):e56516.
[16]Becalska AN,Kelley CF,Berciu C,et al.Formation of membrane ridges an-d scallops by the F-BAR protein Nervous Wreck[J].Mol Biol Cell,2013,24(15):2406-2418.
[17]Rodal AA,Motola-Barnes RN,Littleton JT.Nervous wreck and Cdc42 cooperate to regulate endocytic actin assembly during synaptic growth[J].J Neurosci,2008,28(33):8316-8325.
[18]O'Connor-Giles KM,Ho LL,Ganetzky B.Nervous wreck interacts with thickveins and the endocytic machinery to attenuate retrograde BMP signaling during synaptic growth[J].Neuron,2008,58(4):507-518.
[19]Saglam O,Garrett CR,Boulware D,et al.Activation of the serine/threonine protein kinase AKT during the progression of colorectal Neoplasia[J].Clin Colorectal Cancer,2007,6(9):652-656.
[20]孙嫣,田华,肖法嫚,等.PI3Kp85α在大肠癌癌变过程中的表达及意义[J].南方医科大学学报,2009,29(3):416-418.