Hippo/YAP信号通路对HPV感染宫颈癌细胞调节作用
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摘要
目的 YAP作为Hippo信号通路的主要蛋白,调控多种肿瘤细胞的增殖。本研究探讨Hippo/YAP信号通路对HPV感染宫颈癌细胞TGF-α及EGFR表达的调节作用。方法免疫组化法检测HPV-16(+)Ca Ski细胞、HPV-18(+)C4-1细胞,HPV(-)人宫颈癌细胞C33a中YAP、TGF-α及EGFR的表达。给予YAP抑制剂Vertepofin干预后,CCK-8法检测对Ca Ski及C4-1细胞增殖影响,流式细胞术检测细胞周期,蛋白质印迹法及Real time PCR法检测YAP、TGF-α、EGFR蛋白及mRNA的表达。结果 Ca Ski、C4-1细胞YAP、TGF-α、EGFR蛋白水平均高于HPV(-)组C33a细胞,差异有统计学意义,均P<0.001。给予YAP抑制剂Vertepofin后,在Ca Ski细胞中,干预后第8天减缓速度更加明显,P=0.005;在C4-1细胞中,在干预后第8天后,增殖速度较正常培养减缓明显,P=0.004。干预后CaSki及C4-1细胞处于G0-G1期,且差异有统计学意义,F值分别为463.207和65.872,均P<0.01。在抑制YAP表达的同时,TGF-α、EGFR的mRNA及蛋白随之下降。结论 Hippo/YAP信号通路对HPV感染宫颈癌细胞中TGF-α与EGFR的表达具有正调控作用。
OBJECTIVE YAP as the major protein of Hippo signaling pathway,regulates the proliferation of various tumor cells.In this study,we investigated the role of Hippo/YAP signaling in the regulation of TGF-αand EGFR expression in cervical cancer cells infected by HPV.METHODS The expressions of YAP,TGF-αand EGFR in HPV-16(+),HPV-18(+)human cervical cancer cells Ca Ski,C4-1 and HPV(-)human cervical cancer cells C33 awere detected by immunohistochemistry.The effect of YAP inhibitor Vertepofin on the proliferation of Ca Ski and C4-1 cells was detected by CCK-8 assay.Flow cytometry was used to detect cell cycle.The expression of YAP,TGF-α,EGFR protein and mRNA were detected by Western blotting and Realtime PCR.RESULTS The protein levels of YAP,TGF-αand EGFR in Ca Ski and C4-1 cells were higher than those in HPV(-)group,and the Pvalues were all<0.001,and the difference was statistically significant.After the YAP inhibitor Vertepofin was administered,the rate of mitigation was more pronounced in the Ca Ski cells on the 8 th day after the intervention(P=0.005).In the C4-1 cells,the proliferation rate was slower than the normal culture on the 8 th day after the intervention(P=0.004).CaSki and C4-1 cells were in G0-G1 phase after intervention,and the difference was statistically significant(F=463.207,P=0.00;F=65.872,P=0.01).While suppressing the expression of YAP,the mRNA and protein of TGF-αand EGFR decreased.CONCLUSION The Hippo/YAP signaling pathway has a positive regulatory effect on the expression of TGF-αand EGFR in cervical cancer cells infected by HPV.
OBJECTIVE YAP as the major protein of Hippo signaling pathway,regulates the proliferation of various tumor cells.In this study,we investigated the role of Hippo/YAP signaling in the regulation of TGF-αand EGFR expression in cervical cancer cells infected by HPV.METHODS The expressions of YAP,TGF-αand EGFR in HPV-16(+),HPV-18(+)human cervical cancer cells Ca Ski,C4-1 and HPV(-)human cervical cancer cells C33 awere detected by immunohistochemistry.The effect of YAP inhibitor Vertepofin on the proliferation of Ca Ski and C4-1 cells was detected by CCK-8 assay.Flow cytometry was used to detect cell cycle.The expression of YAP,TGF-α,EGFR protein and mRNA were detected by Western blotting and Realtime PCR.RESULTS The protein levels of YAP,TGF-αand EGFR in Ca Ski and C4-1 cells were higher than those in HPV(-)group,and the Pvalues were all<0.001,and the difference was statistically significant.After the YAP inhibitor Vertepofin was administered,the rate of mitigation was more pronounced in the Ca Ski cells on the 8 th day after the intervention(P=0.005).In the C4-1 cells,the proliferation rate was slower than the normal culture on the 8 th day after the intervention(P=0.004).CaSki and C4-1 cells were in G0-G1 phase after intervention,and the difference was statistically significant(F=463.207,P=0.00;F=65.872,P=0.01).While suppressing the expression of YAP,the mRNA and protein of TGF-αand EGFR decreased.CONCLUSION The Hippo/YAP signaling pathway has a positive regulatory effect on the expression of TGF-αand EGFR in cervical cancer cells infected by HPV.
引文
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[14]Morishita T,Hayakawa F,Sugimoto K,et al.The photosensitizer verteporfin has light-independent anti-leukemic activity for Ph-positive acute lymphoblastic leukemia and synergistically works with dasatinib[J].Oncotarget,2016,7(35):56241-56252.
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[17]Mouradian M,Kikawa KD,Johnson ED,et al.Key roles for GRB2-associated-binding protein 1,phosphatidylinositol-3-kinase,cyclooxygenase 2,prostaglandin E2and transforming growth factor alpha in linoleic acid-induced upregulation of lung and breast cancer cell growth.[J].Prostaglandins Leukot Essent Fatty Acids,2014,90(4):105-115.
[18]乔莹,黄芬,蔡鑫泽,等.HBV感染诱发AID高表达与肝细胞癌变相关性研究[J].中华肿瘤防治杂志,2015,22(1):13-18.
[19]Matsushima-Nishiwaki R,Toyoda H,Nagasawa T,et al.Phosphorylated heat shock protein 20(HSPB6)regulates transforming growth factor-α-induced migration and invasion of hepatocellular carcinoma cell[EB/OL].PLoS One,2016,11(4):e0151907[2016-04-05].https://journals.plos.org/plosone/article id=10.1371/journal.pone.0151907.
[20]刘坤,王海江,孙振强,等.胃癌组织HER-2和VEGF及EGFR表达临床意义研究[J].中华肿瘤防治杂志,2015,22(10):781-785.
[21]He C,Mao D,Hua G,et al.The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression[J].EMBO Mol Med,2015,7(11):1426-1449.
[22]Zhang J,Ji JY,Yu M.et al.YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway[J].Nat Cell Biol,2009,11(12):1444-1450.
[2]宋晓玲,蔡红兵.HPV16-E6-DNA对宫颈癌预后预测价值分析[J].中华肿瘤防治杂志,2015,22(6):416-420.
[3]Lin M,Li C,Wei L,et al.Prevalence of past-year dental visit among Us adults,1999-2010:comparison of trends and estimates between the behavioral risk factor surveillance system and three national surveys[J].J Public Health Dent,2017,77(2):105-114.
[4]Pan W,Wang Q,Zhang Y,et al.Verteporfin can reverse the paclitaxel resistance induced by YAP over-expression in HCT-8/Tcells without photoactivation through Inhibiting YAP expression[J].Cell Physiol Biochem,2016,39(2):481-490.
[5]Yap TA,Aerts JG,Popat S,et al.Novel insights into mesothelioma biology and implications for therapy[J].Nat Rev Cancer,2017,17(8):475-488.
[6]Li Y,Li L,Zhu M,et al.Upregulation of Yes associated protein and transcriptional co activator with PDZ binding motif influences the behavior of LOVO human colon adenocarcinoma cells[J].Exp Ther Med,2017,14(4):3831-3835.
[7]Yamamoto M,Xin B,Watanabe K,et al.Oncogenic determination of a broad spectrum of phenotypes of hepatocyte-derived mouse liver tumors[J].Am J Pathol,2017,187(12):2711-2725.
[8]Huang H,Zhang W,Pan Y,et al.YAP suppresses lung squamous cell carcinoma progression via deregulation of the DNp63-GPX2axis and ROS accumulation[J].Cancer Res,2017,77(21):5769-5781.
[9]Wang Y,Justilien V,Brennan KI,et al.PKCιregulates nuclear YAP1localization and ovarian cancer tumorigenesis[J].Oncogene,2017,36(4):534-545.
[10]Chen H,Chen Q,Luo Q.Expression of netrin-1by hypoxia contributes to the invasion and migration of prostate carcinoma cells by regulating YAP activity[J].Exp Cell Res,2016,349(2):302-309.
[11]Vlug EJ,Ven RAHVD,Vermeulen JF,et al.Nuclear localization of the transcriptional coactivator YAP is associated with invasive lobular breast cancer[J].Cell Oncol(Dordr),2013,36(5):375-384.
[12]Einstein MH,Baron M,Levin MJ,et al.Comparison of the immunogenicity and safety of CervarixTM and Gardasil○R human papillomavirus(HPV)cervical cancer vaccines in healthy women aged 18-45years[J].Human Vaccines,2009,5(10):705-719.
[13]Buglioni S,Vici P,Sergi D,et al.Analysis of the hippo transducers TAZ and YAP in cervical cancer and its microenvironment[EB/OL].Oncoimmunology,2016,5(6):e1160187[2016-03-28].https://www.tandfonline.com/doi/abs/10.1080/2162402X.2016.1160187.
[14]Morishita T,Hayakawa F,Sugimoto K,et al.The photosensitizer verteporfin has light-independent anti-leukemic activity for Ph-positive acute lymphoblastic leukemia and synergistically works with dasatinib[J].Oncotarget,2016,7(35):56241-56252.
[15]Singh B,Coffey RJ.From wavy hair to naked proteins:the role of transforming growth factor alpha in health and disease[J].Semin Cell Dev Bioll,2014,28:12-21.
[16]Ding L,El Zaatari M,Merchant JL.Recapitulating human gastric cancer pathogenesis:experimental models of gastric cancer[J].Adv Exp Med Biol,2016,908:441-478.
[17]Mouradian M,Kikawa KD,Johnson ED,et al.Key roles for GRB2-associated-binding protein 1,phosphatidylinositol-3-kinase,cyclooxygenase 2,prostaglandin E2and transforming growth factor alpha in linoleic acid-induced upregulation of lung and breast cancer cell growth.[J].Prostaglandins Leukot Essent Fatty Acids,2014,90(4):105-115.
[18]乔莹,黄芬,蔡鑫泽,等.HBV感染诱发AID高表达与肝细胞癌变相关性研究[J].中华肿瘤防治杂志,2015,22(1):13-18.
[19]Matsushima-Nishiwaki R,Toyoda H,Nagasawa T,et al.Phosphorylated heat shock protein 20(HSPB6)regulates transforming growth factor-α-induced migration and invasion of hepatocellular carcinoma cell[EB/OL].PLoS One,2016,11(4):e0151907[2016-04-05].https://journals.plos.org/plosone/article id=10.1371/journal.pone.0151907.
[20]刘坤,王海江,孙振强,等.胃癌组织HER-2和VEGF及EGFR表达临床意义研究[J].中华肿瘤防治杂志,2015,22(10):781-785.
[21]He C,Mao D,Hua G,et al.The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression[J].EMBO Mol Med,2015,7(11):1426-1449.
[22]Zhang J,Ji JY,Yu M.et al.YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway[J].Nat Cell Biol,2009,11(12):1444-1450.