Notch1调控PI3K/AKT信号通路促进黑素瘤细胞发展的研究
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摘要
目的:分析Notch1在黑素瘤发展过程中的作用和机制。方法:采用RT-PCR检测Notch1 mRNA在黑素瘤和正常黑素细胞中的表达,采用慢病毒转染技术构建Notch1过表达的黑色素瘤细胞系,CCK法检测细胞增殖能力,Transwell检测细胞侵袭能力,划线法检测细胞迁移能力,流式细胞术检测细胞凋亡能力,Westernblot检测细胞Notch1过表达对PI3K/AKT信号通路中AKT磷酸化水平的影响。结果:黑素瘤细胞系MM200、Mel-CV、A2058和A375细胞中Notch1mRNA水平明显高于正常黑素细胞系HemN-MP(P<0.05)。黑素瘤细胞A2058细胞中Notch1水平升高后,细胞的增殖速率显著提高,在48h、72h和96h转染组细胞增殖能力明显高于对照组(P<0.05);Transwell结果显示Notch1水平升高可以显著增加穿过小室膜A2058细胞数量(P<0.05);划线法结果显示24h转染组细胞迁移率明显高于对照组(P<0.05);流式细胞检测结果显示转染组细胞早期凋亡率明显低于对照组(P<0.05)。Notch1水平增加后,AKT蛋白磷酸化水平明显提高,说明Notch1能够促进PI3K/AKT信号通路的激活。加入了Notch信号通路特异性阻断剂DAPT后,能够有效抑制Notch1过表达引起的AKT蛋白磷酸化的升高。结论:Notch1能够调节PI3K/AKT信号通路通过促进黑素瘤的发展,本研究为临床治疗黑素瘤提供了新的思路。
Objective To analyze the role and mechanism of Notch1 in the development of melanoma. Methods Notch1 mRNA expression in normal melanocytes and melanoma was detected by RT-PCR. Slow virus transfection technique was used to construct Notch1 expression of melanoma cell lines. Using CCK method to detect cell proliferation ability, Transwell to detect cell invasion ability, marking method to detect cell migration ability, flow cytometry to detect cell apoptosis, Western blot to detect cell Notch1 over expression of PI3K/AKT signal pathway of AKT phosphorylation level. Results The expression levels of Notch1 mRNA in melanoma cell lines MM200, Mel-CV, A2058 and A375 were significantly higher than that in normal melanocyte lines HemN-MP(P<0.05). When the level of Notch1 in the melanoma cell A2058 cells was increased, the cell proliferation rate was significantly increased, and the proliferation capacity of the transfection group at 48 h, 72 h and 96 h was significantly higher than that of the control group(P<0.05). Transwell results showed that increased Notch1 level significantly increased the number of A2058 cells passing through the compartment(P<0.05). The results showed that the cell migration rate in the 24 h transfection group was significantly higher than that in the control group(P<0.05). Flow cytometry test showed that the early apoptosis rate in transfection group was significantly lower than that in control group(P<0.05). After the increase of Notch1 level, AKT protein phosphorylation level was significantly increased,suggesting that Notch1 could promote the activation of PI3K/AKT signaling pathway.The addition of Notch signaling pathway specific blocking agent DAPT can effectively inhibit the increase of AKT protein phosphorylation caused by Notch1 overexpression. Conclusion Notch1 can regulate PI3K/AKT signaling pathway by promoting the development of melanoma, which provides a new idea for clinical treatment of melanoma.
Objective To analyze the role and mechanism of Notch1 in the development of melanoma. Methods Notch1 mRNA expression in normal melanocytes and melanoma was detected by RT-PCR. Slow virus transfection technique was used to construct Notch1 expression of melanoma cell lines. Using CCK method to detect cell proliferation ability, Transwell to detect cell invasion ability, marking method to detect cell migration ability, flow cytometry to detect cell apoptosis, Western blot to detect cell Notch1 over expression of PI3K/AKT signal pathway of AKT phosphorylation level. Results The expression levels of Notch1 mRNA in melanoma cell lines MM200, Mel-CV, A2058 and A375 were significantly higher than that in normal melanocyte lines HemN-MP(P<0.05). When the level of Notch1 in the melanoma cell A2058 cells was increased, the cell proliferation rate was significantly increased, and the proliferation capacity of the transfection group at 48 h, 72 h and 96 h was significantly higher than that of the control group(P<0.05). Transwell results showed that increased Notch1 level significantly increased the number of A2058 cells passing through the compartment(P<0.05). The results showed that the cell migration rate in the 24 h transfection group was significantly higher than that in the control group(P<0.05). Flow cytometry test showed that the early apoptosis rate in transfection group was significantly lower than that in control group(P<0.05). After the increase of Notch1 level, AKT protein phosphorylation level was significantly increased,suggesting that Notch1 could promote the activation of PI3K/AKT signaling pathway.The addition of Notch signaling pathway specific blocking agent DAPT can effectively inhibit the increase of AKT protein phosphorylation caused by Notch1 overexpression. Conclusion Notch1 can regulate PI3K/AKT signaling pathway by promoting the development of melanoma, which provides a new idea for clinical treatment of melanoma.
引文
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[8]Ho YJ,Anaparthy N,Molik D,et al.Single-cell RNA-seq analysis identifies markers of resistance to targeted BRAF inhibitors in melanoma cell populations[J].Genome Res,2018,28(9):1353-1363.
[9]PourquiéO.Publisher's note:eya1 controls cell polarity,spindle orientation,cell fate and Notch signaling in distal embryonic lung epithelium by El-Hashash et al.[J].Development,2017,144(3):529.
[10]Yen WC,Fischer MM,Axelrod F,et al.Targeting Notch signaling with a Notch2/Notch3 antagonist(tarextumab)inhibits tumor growth and decreases tumor-initiating cell frequency[J].Clin Cancer Res,2015,21(9):2084-2095.
[11]Massi D,Tarantini F,Franchi A,et al.Evidence for differential expression of Notch receptors and their ligands in melanocytic nevi and cutaneous malignant melanoma[J].Mod Pathol,2006,19(2):246.
[12]Golan T,Messer AR,Amitai-Lange A,et al.Interactions of melanoma cells with distal keratinocytes trigger metastasis via notch signaling inhibition of MITF[J].Mol Cell,2015,59(4):664-676.
[13]齐艳霞,康世均,刘新会,等.Notch1 siRNA对小鼠恶性黑色素瘤细胞体内外增殖的抑制作用研究[J].山东医药,2014,54(10):14-16.
[14]Bugide S,Gonugunta VK,Penugurti V,et al.HPIP promotes epithelial-mesenchymal transition and cisplatin resistance in ovarian cancer cells through PI3K/AKT pathway activation[J].Cell Oncol,2017,40(2):133-144.
[15]Wang Y,Wang Y,Chen H,et al.Endothelial cells promote formation of medulloblastoma stem-like cells via notch pathway activation[J].JMolec Neurosc,2017,63(2):152-158.
[16]Su F,Zhu S,Ruan J,et al.Combination therapy of RY10-4 with theγ-secretase inhibitor DAPT shows promise in treating HER2-amplified breast cancer[J].Oncotarget,2016,7(4):4142.
[2]Takebe N,Miele L,Harris PJ,et al.Targeting Notch,Hedgehog,and Wnt pathways in cancer stem cells:clinical update[J].Nat Rev Clin Oncol,2015,12(8):445.
[3]Mamaeva V,Niemi R,Beck M,et al.Inhibiting notch activity in breast cancer stem cells by glucose functionalized nanoparticles carryingγ-secretase inhibitors[J].Mol Ther,2016,24(5):926-936.
[4]Ibrahim SA,Gadalla R,El-Ghonaimy EA,et al.Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3,Notch and EGFR signaling pathways[J].Mol Cancer,2017,16(1):57.
[5]Negri FV,Bozzetti C,Azzoni C,et al.P-027 Cancer stem cells marker CD44 and Notch activation predict unfavorable prognosis in metastatic colon cancer patients treated with anti VEGF-therapy[J].Annals of Oncology,2016,27(suppl 2):ii8.
[6]Ciofani M,Zuniga-Pflücker JC.Notch promotes survival of pre-T cells at theβ-selection checkpoint by regulating cellular metabolism[J].Nat Immunol,2005,6(9):881.
[7]Kleffel S,Posch C,Barthel SR,et al.Melanoma cell-intrinsic PD-1 receptor functions promote tumor growth[J].Cell,2015,162(6):1242-1256.
[8]Ho YJ,Anaparthy N,Molik D,et al.Single-cell RNA-seq analysis identifies markers of resistance to targeted BRAF inhibitors in melanoma cell populations[J].Genome Res,2018,28(9):1353-1363.
[9]PourquiéO.Publisher's note:eya1 controls cell polarity,spindle orientation,cell fate and Notch signaling in distal embryonic lung epithelium by El-Hashash et al.[J].Development,2017,144(3):529.
[10]Yen WC,Fischer MM,Axelrod F,et al.Targeting Notch signaling with a Notch2/Notch3 antagonist(tarextumab)inhibits tumor growth and decreases tumor-initiating cell frequency[J].Clin Cancer Res,2015,21(9):2084-2095.
[11]Massi D,Tarantini F,Franchi A,et al.Evidence for differential expression of Notch receptors and their ligands in melanocytic nevi and cutaneous malignant melanoma[J].Mod Pathol,2006,19(2):246.
[12]Golan T,Messer AR,Amitai-Lange A,et al.Interactions of melanoma cells with distal keratinocytes trigger metastasis via notch signaling inhibition of MITF[J].Mol Cell,2015,59(4):664-676.
[13]齐艳霞,康世均,刘新会,等.Notch1 siRNA对小鼠恶性黑色素瘤细胞体内外增殖的抑制作用研究[J].山东医药,2014,54(10):14-16.
[14]Bugide S,Gonugunta VK,Penugurti V,et al.HPIP promotes epithelial-mesenchymal transition and cisplatin resistance in ovarian cancer cells through PI3K/AKT pathway activation[J].Cell Oncol,2017,40(2):133-144.
[15]Wang Y,Wang Y,Chen H,et al.Endothelial cells promote formation of medulloblastoma stem-like cells via notch pathway activation[J].JMolec Neurosc,2017,63(2):152-158.
[16]Su F,Zhu S,Ruan J,et al.Combination therapy of RY10-4 with theγ-secretase inhibitor DAPT shows promise in treating HER2-amplified breast cancer[J].Oncotarget,2016,7(4):4142.