维替泊芬对鼻息肉上皮细胞Yes相关蛋白的作用
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摘要
目的研究Ki-67和Yes相关蛋白(YAP)在鼻息肉组织中的表达,探讨维替泊芬对人鼻息肉上皮细胞(hNPEC)增殖的影响。方法实时荧光定量PCR及蛋白免疫印迹法分别检测正常下鼻甲黏膜组织和鼻息肉组织的Ki-67和YAP的mRNA及蛋白水平。体外培养hNPEC,噻唑蓝比色法(MTT)确定维替泊芬的半数抑制浓度(IC_(50)),蛋白免疫印迹法检测维替泊芬IC_(50)处理组中Ki-67、YAP及TEAD1的蛋白表达,免疫荧光染色观察Ki-67、YAP的细胞内表达。结果在鼻息肉组织中, Ki-67和YAP的mRNA和蛋白表达均高于下鼻甲对照组,差异有统计学意义(P均<0.05)。维替泊芬作用hNPEC 24 h后IC_(50)为2.516μmol/L。维替泊芬下调hNPEC中Ki-67及YAP、TEAD1的蛋白表达水平,差异有统计学意义(P均<0.05)。免疫荧光染色显示维替泊芬组中Ki-67阳性细胞数较少,YAP核内荧光强度明显降低。结论维替泊芬可能通过抑制YAP-TEAD复合物来抑制hNPEC的增殖。
Objective To investigate the expression levels of Ki-67 and Yes-associated protein(YAP) in the nasal polyps tissues and evaluate the effect of verteporfin on the proliferation of human nasal polyps epithelial cells(hNPECs). Methods The expression of Ki-67 and YAP at the mRNA and protein levels in normal inferior turbinate mucosa(n=10) and nasal polyps tissues(n=20) were detected by quantitative PCR(qPCR) and Western blot. The hNPECs were cultured in vitro. The half maximal inhibitory concentration(IC_(50)) of verteporfin at 24 h was determined by MTT assay. The expression levels of Ki-67, YAP and TEAD1 proteins in hNPECs treated with IC_(50) of verteporfin were detected by Western blot. The expression of Ki-67 and YAP in hNPECs was observed by immunofluorescence staining. Results The expression of Ki-67 and YAP at the mRNA and protein levels in the nasal polyps tissues was significantly higher compared with those in the control group(all P<0.05). MTT assay demonstrated that the IC_(50) of verteporfin was 2.516 μmol/L in hNPECs after 24-h incubation. The expression levels of Ki-67, YAP and TEAD1 proteins in hNPECs were significantly down-regulated by verteporfin(all P<0.05). Immunofluorescence staining revealed that the quantity of Ki-67 positive cells was considerably decreased and the fluorescence intensity of nucleus YAP was significantly reduced in the verteporfin group. Conclusions Verteporfin inhibits the proliferation of hNPECs probably by suppressing the YAP-TEAD complex.
Objective To investigate the expression levels of Ki-67 and Yes-associated protein(YAP) in the nasal polyps tissues and evaluate the effect of verteporfin on the proliferation of human nasal polyps epithelial cells(hNPECs). Methods The expression of Ki-67 and YAP at the mRNA and protein levels in normal inferior turbinate mucosa(n=10) and nasal polyps tissues(n=20) were detected by quantitative PCR(qPCR) and Western blot. The hNPECs were cultured in vitro. The half maximal inhibitory concentration(IC_(50)) of verteporfin at 24 h was determined by MTT assay. The expression levels of Ki-67, YAP and TEAD1 proteins in hNPECs treated with IC_(50) of verteporfin were detected by Western blot. The expression of Ki-67 and YAP in hNPECs was observed by immunofluorescence staining. Results The expression of Ki-67 and YAP at the mRNA and protein levels in the nasal polyps tissues was significantly higher compared with those in the control group(all P<0.05). MTT assay demonstrated that the IC_(50) of verteporfin was 2.516 μmol/L in hNPECs after 24-h incubation. The expression levels of Ki-67, YAP and TEAD1 proteins in hNPECs were significantly down-regulated by verteporfin(all P<0.05). Immunofluorescence staining revealed that the quantity of Ki-67 positive cells was considerably decreased and the fluorescence intensity of nucleus YAP was significantly reduced in the verteporfin group. Conclusions Verteporfin inhibits the proliferation of hNPECs probably by suppressing the YAP-TEAD complex.
引文
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[7]Liu Y, Wang G, Yang Y, Mei Z, Liang Z, Cui A, Wu T, Liu CY, Cui L. Increased TEAD4 expression and nuclear localization in colorectal cancer promoteepithelial-mesenchymal transition and metastasis in a YAP-independent manner. Oncogene,2016,35(21):2789-2800.
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[11]Zhao L, Li YY, Li CW, Chao SS, Liu J, Nam HN, Dung NTN,Shi L, Wang DY. Increase of poorly proliferated p63(+)/Ki67(+)basal cells forming multiple layers in the aberrant remodeled epithelium in nasal polyps. Allergy,2017,72(6):975-984.
[12]邓慧仪,李美娇,王玮豪,黄雪琨,黄健聪,张革化,杨钦泰. Yes相关蛋白在慢性鼻窦炎伴鼻息肉中的表达及与细胞增殖的相关性分析.新医学,2017,48(11):779-783.
[13]Miller JW, Schmidt-Erfurth U, Sickenberg M, Pournaras CJ,Laqua H, Barbazetto I, Zografos L, Piguet B, Donati G, Lane AM, Birngruber R, van den Berg H, Strong A, Manjuris U,Gray T, Fsadni M, Bressler NM, Gragoudas ES. Photodynamic therapy with verteporfin for choroidal neovascularization caused by age-related macular degeneration:results of a single treatment in a phase 1 and2 study. Arch Ophthalmol,1999,117(9):1161-1173.
[14]Gibault F, Corvaisier M, Bailly F, Huet G, Melnyk P, Cotelle P.Non-photoinduced biological properties of verteporfin. Curr Med Chem,2016,23(11):1171-1184.
[15]Taniguchi K, Wu LW, Grivennikov SI, de Jong PR, Lian I,Yu FX, Wang K, Ho SB, Boland BS, Chang JT, Sandborn WJ, Hardiman G, Raz E, Maehara Y, Yoshimura A, ZucmanRossi J, Guan KL, Karin M. A gp130-Src-YAP module links inflammation to epithelial regeneration. Nature,2015,519(7541):57-62.
[16]Brodowska K, Al-Moujahed A, Marmalidou A, Meyer Zu Horste M, Cichy J, Miller JW, Gragoudas E, Vavvas DG. The clinically used photosensitizer Verteporfin(VP)inhibits YAPTEAD and humanretinoblastoma cell growth in vitro without light activation. Exp Eye Res,2014,124:67-73.
[17]Feng J, Gou J, Jia J, Yi T, Cui T, Li Z. Verteporfin, a suppressor of YAP-TEAD complex, presents promising antitumor properties on ovarian cancer. Onco Targets Ther,2016,9:5371-5381.
[18]Donohue E, Balgi AD, Komatsu M, Roberge M. Induction of covalently crosslinked p62 oligomers with reduced binding to polyubiquitinated proteins by the autophagy inhibitor verteporfin.PLoS One,2014,9(12):e114964.
[19]Li H, Huang Z, Gao M, Huang N, Luo Z, Shen H, Wang X,Wang T, Hu J, Feng W. Inhibition of YAP suppresses CML cell proliferation and enhances efficacy of imatinib in vitro and in vivo. J Exp Clin Cancer Res,2016,35(1):134.
[20]Li Y, Du J, Zhu E, Zhang J, Han J, Zhao W, Sun B, Tian D. Liraglutide suppresses proliferation and induces adipogenic differentiation of 3T3-L1 cells via the Hippo-YAP signaling pathway. Mol Med Rep,2018,17(3):4499-4507.
[21]Dawes LJ, Shelley EJ, Mcavoy JW, Lovicu FJ. A role for Hippo/YAP-signaling in FGF-induced lens epithelial cell proliferation and fibre differentiation. Exp Eye Res,2018,169:122-133.
[2]Sivasubramaniam R, Harvey RJ. How to assess, control, and manage uncontrolled CRS/nasal polyp patients. Curr Allergy Asthma Rep,2017,17(9):58.
[3]Willson TJ, Naclerio RM, Lee SE. Monoclonal antibodies for the treatment of nasal polyps. Immunol Allergy Clin North Am,2017,37(2):357-367.
[4]YuFX,GuanKL.TheHippopathway:regulatorsand regulations. Genes Dev,2013,27(4):355-371.
[5]Vassilev A, Kaneko KJ, Shu H, Zhao Y, DePamphilis ML.TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm.Genes Dev,2001,15(10):1229-1241.
[6]Lin C, Yao E, Zhang K, Jiang X, Croll S, Thompson-Peer K,Chuang PT. YAP is essential for mechanical force production and epithelial cell proliferation during lung branching morphogenesis.Elife,2017,6. pii:e21130.
[7]Liu Y, Wang G, Yang Y, Mei Z, Liang Z, Cui A, Wu T, Liu CY, Cui L. Increased TEAD4 expression and nuclear localization in colorectal cancer promoteepithelial-mesenchymal transition and metastasis in a YAP-independent manner. Oncogene,2016,35(21):2789-2800.
[8]Liu-Chittenden Y, Huang B, Shim JS, Chen Q, Lee SJ, Anders RA, Liu JO, Pan D. Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP. Genes Dev,2012,26(12):1300-1305.
[9]Ehmer U, Sage J. Control of proliferation and cancer growth by the hippo signaling pathway. Mol Cancer Res,2016,14(2):127-140.
[10]Hsu MC, Shun CT, Liu CM. Increased epithelial cell proliferation in nasal polyps. J Formos Med Assoc,2002,101(3):227-229.
[11]Zhao L, Li YY, Li CW, Chao SS, Liu J, Nam HN, Dung NTN,Shi L, Wang DY. Increase of poorly proliferated p63(+)/Ki67(+)basal cells forming multiple layers in the aberrant remodeled epithelium in nasal polyps. Allergy,2017,72(6):975-984.
[12]邓慧仪,李美娇,王玮豪,黄雪琨,黄健聪,张革化,杨钦泰. Yes相关蛋白在慢性鼻窦炎伴鼻息肉中的表达及与细胞增殖的相关性分析.新医学,2017,48(11):779-783.
[13]Miller JW, Schmidt-Erfurth U, Sickenberg M, Pournaras CJ,Laqua H, Barbazetto I, Zografos L, Piguet B, Donati G, Lane AM, Birngruber R, van den Berg H, Strong A, Manjuris U,Gray T, Fsadni M, Bressler NM, Gragoudas ES. Photodynamic therapy with verteporfin for choroidal neovascularization caused by age-related macular degeneration:results of a single treatment in a phase 1 and2 study. Arch Ophthalmol,1999,117(9):1161-1173.
[14]Gibault F, Corvaisier M, Bailly F, Huet G, Melnyk P, Cotelle P.Non-photoinduced biological properties of verteporfin. Curr Med Chem,2016,23(11):1171-1184.
[15]Taniguchi K, Wu LW, Grivennikov SI, de Jong PR, Lian I,Yu FX, Wang K, Ho SB, Boland BS, Chang JT, Sandborn WJ, Hardiman G, Raz E, Maehara Y, Yoshimura A, ZucmanRossi J, Guan KL, Karin M. A gp130-Src-YAP module links inflammation to epithelial regeneration. Nature,2015,519(7541):57-62.
[16]Brodowska K, Al-Moujahed A, Marmalidou A, Meyer Zu Horste M, Cichy J, Miller JW, Gragoudas E, Vavvas DG. The clinically used photosensitizer Verteporfin(VP)inhibits YAPTEAD and humanretinoblastoma cell growth in vitro without light activation. Exp Eye Res,2014,124:67-73.
[17]Feng J, Gou J, Jia J, Yi T, Cui T, Li Z. Verteporfin, a suppressor of YAP-TEAD complex, presents promising antitumor properties on ovarian cancer. Onco Targets Ther,2016,9:5371-5381.
[18]Donohue E, Balgi AD, Komatsu M, Roberge M. Induction of covalently crosslinked p62 oligomers with reduced binding to polyubiquitinated proteins by the autophagy inhibitor verteporfin.PLoS One,2014,9(12):e114964.
[19]Li H, Huang Z, Gao M, Huang N, Luo Z, Shen H, Wang X,Wang T, Hu J, Feng W. Inhibition of YAP suppresses CML cell proliferation and enhances efficacy of imatinib in vitro and in vivo. J Exp Clin Cancer Res,2016,35(1):134.
[20]Li Y, Du J, Zhu E, Zhang J, Han J, Zhao W, Sun B, Tian D. Liraglutide suppresses proliferation and induces adipogenic differentiation of 3T3-L1 cells via the Hippo-YAP signaling pathway. Mol Med Rep,2018,17(3):4499-4507.
[21]Dawes LJ, Shelley EJ, Mcavoy JW, Lovicu FJ. A role for Hippo/YAP-signaling in FGF-induced lens epithelial cell proliferation and fibre differentiation. Exp Eye Res,2018,169:122-133.
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