Hippo信号通路在乙烯菌核利致小鼠尿道下裂中的作用
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摘要
目的 观察Hippo信号通路在乙烯菌核利致小鼠尿道下裂中的作用。方法 将8周龄ICR孕鼠随机分为实验组和对照组,每组各10只,实验组在孕12～18 d连续7天经口给予400 mg/(kg·d)乙烯菌核利,对照组给予等体积的大豆油。分别于子鼠出生后第60天随机取实验组和对照组小鼠阴茎各20条,采用实时荧光定量PCR检测Hippo信号通路的关键基因Mst1、Lats1、Taz、Yap及雄激素受体基因Ar的表达丰度,Western blot方法检测Hippo信号通路下游Yes相关蛋白(Yap)的表达及其磷酸化水平。结果 与对照组雄性子鼠相比,实验组雄性子鼠的肛门生殖器距离明显缩短[(9.2501±2.5504)vs.(16.1253±1.3562) mm,P<0.05]、阴茎质量明显较轻[(0.0293±0.0075)vs.(0.4731±0.004) g,P<0.05]、阴茎长度缩短[(5.3875±0.4524)vs.(12.4688±1.2290) mm,P<0.05],阴茎直径也较对照组小鼠小[(1.5513±0.1158)vs.(2.6013±0.1469) mm,P<0.05]。对照组小鼠阴茎中Hippo信号通路的关键基因Mst1、Lats1、Taz、Yap及雄激素受体基因Ar均表达;实验组Yap、Ar未表达,Mst1、Lats1的表达丰度高于对照组(6.6097±1.3188 vs. 0.3517±0.1524, 5.7071±2.7210 vs. 0.1235±0.0658,P<0.05),Taz的表达丰度低于对照组(0.3937±0.1519 vs. 3.2329±0.4339,P<0.05)。实验组小鼠阴茎Yap表达量降低(0.3348±0.0639 vs. 0.8023±0.4275,P<0.05)、磷酸化程度高于对照组(3.9940±0.2177 vs. 0.3128±0.0867,P<0.05)。结论 乙烯菌核利致小鼠尿道下裂模型中,Ar未表达,Hippo信号通路被激活,Yap磷酸化增加,该通路可能在乙烯菌核利致雄性小鼠尿道下裂中起重要作用。
OBJECTIVE To observe the role of Hippo signaling pathway in mice hypospadias induced by vinclozolin. METHODS After 8 weeks of ICR mice were conceived, they were divided randomly into the treatment and the control groups(10 for each group). Vinclozolin was orally administraed to the pregnant mice in the treatment group with 400 mg/(kg·d) during gestation days 12-18, while those of the control group were treated with equal volume of soybean oil. About 60 days after birth, 20 penises of the mouse offspring were taken randomly from both of the two groups separately. And furthermore, the relative expression abundance of Hippo signaling pathway key genes(Mst1,Lats1,Taz,Yap),androgen receptor gene Ar of all the samples were measured by the real-time quantity reverse transcript polymerase chain reaction(qPCR). The Yes-related protein(Yap) and its phosphorylation in the downstream of Hippo signaling pathway were measured by Western blot. RESULTS Compared with the male mice of the control group, the anal genital distances of the treatment group were significantly shortened((9.2501±2.5504) vs.(16.1253±1.3562) mm, P<0.05), the quality of the penises was significantly less than((0.0293±0.0075) vs.(0.4731±0.004) g, P<0.05), the length of the penises was shorter((5.3875±0.4524) vs.(12.4688±1.2290) mm, P<0.05), and the diameters of the penises were also less than the control((1.5513±0.1158) vs.(2.6013±0.1469) mm, P<0.05). All the genes in the penises of the control group were expressed. The Yap and Ar in the treatment group's penises were not expressed, the expression abundance of Mst1 and Lats1 was higher than that of the control(6.6097±1.3188 vs. 0.3517±0.1524,5.7071±2.7210 vs. 0.1235±0.0658, P<0.05), and the expression abundance of Taz was lower than that of the control(0.3937±0.1519 vs. 3.2329±0.4339, P<0.05). The expression of Yap was decreased in the treatment group(0.3348±0.0639 vs. 0.8023±0.4275, P<0.05), and the phosphorylation level of Yap was higher than that of the control group(3.9940±0.2177 vs. 0.3128±0.0867, P<0.05). CONCLUSION In the model of mice hypospadias induced by vinclozolin, the Ar was not expressed, the Hippo signaling pathway was activated and the phosphorylation of Yap was increased. This pathway may play an important role in the model.
OBJECTIVE To observe the role of Hippo signaling pathway in mice hypospadias induced by vinclozolin. METHODS After 8 weeks of ICR mice were conceived, they were divided randomly into the treatment and the control groups(10 for each group). Vinclozolin was orally administraed to the pregnant mice in the treatment group with 400 mg/(kg·d) during gestation days 12-18, while those of the control group were treated with equal volume of soybean oil. About 60 days after birth, 20 penises of the mouse offspring were taken randomly from both of the two groups separately. And furthermore, the relative expression abundance of Hippo signaling pathway key genes(Mst1,Lats1,Taz,Yap),androgen receptor gene Ar of all the samples were measured by the real-time quantity reverse transcript polymerase chain reaction(qPCR). The Yes-related protein(Yap) and its phosphorylation in the downstream of Hippo signaling pathway were measured by Western blot. RESULTS Compared with the male mice of the control group, the anal genital distances of the treatment group were significantly shortened((9.2501±2.5504) vs.(16.1253±1.3562) mm, P<0.05), the quality of the penises was significantly less than((0.0293±0.0075) vs.(0.4731±0.004) g, P<0.05), the length of the penises was shorter((5.3875±0.4524) vs.(12.4688±1.2290) mm, P<0.05), and the diameters of the penises were also less than the control((1.5513±0.1158) vs.(2.6013±0.1469) mm, P<0.05). All the genes in the penises of the control group were expressed. The Yap and Ar in the treatment group's penises were not expressed, the expression abundance of Mst1 and Lats1 was higher than that of the control(6.6097±1.3188 vs. 0.3517±0.1524,5.7071±2.7210 vs. 0.1235±0.0658, P<0.05), and the expression abundance of Taz was lower than that of the control(0.3937±0.1519 vs. 3.2329±0.4339, P<0.05). The expression of Yap was decreased in the treatment group(0.3348±0.0639 vs. 0.8023±0.4275, P<0.05), and the phosphorylation level of Yap was higher than that of the control group(3.9940±0.2177 vs. 0.3128±0.0867, P<0.05). CONCLUSION In the model of mice hypospadias induced by vinclozolin, the Ar was not expressed, the Hippo signaling pathway was activated and the phosphorylation of Yap was increased. This pathway may play an important role in the model.
引文
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[7] XIAO Y,HILL M C,ZHANG M,et al.Hippo signaling plays an essential role in cell state transitions during cardiac fibroblast development [J].Dev Cell,2018,45(2):153-169.
[8] WOLF C J,LEBLANC G A,OSTBY J S,et al.Characterization of the period of sensitivity of fetal male sexual development to vinclozoline [J].Toxicol Sci,2000,55(1):152-161.
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[11] CHEN S N,GURHA P,LOMBARDI R,et al.The hippo pathway is activated and is a causal mechanism for adipogenesis in arrhythmogenic cardiomyopathy [J].Circ Res,2014,114(3):454-468.
[12] SUKUMARAN S K,STUMPF M,SALAMON S,et al.CDK5RAP2 interaction with components of the hippo signaling pathway may play a role in primary microcephaly [J].Mol Genet Genomics,2017,292(2):365-383.
[13] KANG J,WANG J,YAO Z,et al.Fascin induces melanoma tumorigenesis and stemness through regulating the Hippo pathway [J].Cell Commun Signal,2018,16(1):37.
[14] SMITH S,SESSIONS R B,SHOEMARK D,et al.Anti-proliferative and anti-migratory effects of a novel YAP-TEAD interaction inhibitor identified using in silico molecular docking [J].J Med Chem,2019,62(3):1291-1305.
[15] FALLAHI E,O′DRISCOLL N A,MATALLANAS D.The Mst/Hippo pathway and cell death:a non-canonical affair [J].Genes,2016,7(6):28.
[16] SONG Y,FU J,ZHOU M,et al.Activated Hippo/Yes-associated protein pathway promotes cell proliferation and anti-apoptosis in endometrial stromal cells of endometriosis [J].J Clin Endocrinol Metab,2016,101(4):1552-1561.
[17] SHAEER O,SHAEER K.Shaeer’s corporal rotation III:shortening-free correction of congenital penile curvature-the noncorporotomy technique [J].Eur Urol,2016,69(1):129-134.
[18] TOURCHI A,SHABANINIA M,STEWART M,et al.Complete penile disassembly for repair of epispadias causes erectile tissue alteration through transforming growth factor beta 1 overexpression in a rabbit model [J].Urology,2018,111:151-156.
[19] MA L M,WANG Z,WANG H,et al.Estrogen effects on fetal penile and urethral development in organotypic mouse genital tubercle culture [J].J Urol,2009,182(5):2511-2517.
[20] KUSER-ABALI G,ALPTEKIN A,LEWIS M,et al.YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer [J].Nat Commun,2015,6:8126.
[21] HILLEBRAND A C,PIZZOLATO L S,BRANCHINI G,et al.Androgenic modulation of AR-Vs [J].Endocrine,2018,62(2):477-486.
[2] HE H G,HAN C H,ZHANG W.A mouse model of hypospadias induced by estradiol benzoate [J].Cell Biochem Biophys,2015,73(3):589-592.
[3] DEEL M D,LI J J,CROSE L E,et al.A review:molecular aberrations within hippo signaling in bone and soft-tissue sarcomas [J].Front Oncol,2015,5:190.
[4] HATA Y,TIMALSINA S,MAIMAITI S.Okadaic acid:a tool to study the hippo pathway [J].Mar Drugs,2013,11(3):896-902.
[5] CONLEY J M,LAMBRIGHT C S,EVANS N,et al.Mixed “antiandrogenic” chemicals at low individual doses produce reproductive tract malformations in the male rat [J].Toxicol Sci,2018,164(1):166-178.
[6] BEN MAAMAR M,SADLER-RIGGLEMAN I,BECK D,et al.Alterations in sperm DNA methylation,non-coding RNA expression,and histone retention mediate vinclozolin-induced epigenetic transgenerational inheritance of disease [J].Environ Epigenet,2018,4(2):dvy010.
[7] XIAO Y,HILL M C,ZHANG M,et al.Hippo signaling plays an essential role in cell state transitions during cardiac fibroblast development [J].Dev Cell,2018,45(2):153-169.
[8] WOLF C J,LEBLANC G A,OSTBY J S,et al.Characterization of the period of sensitivity of fetal male sexual development to vinclozoline [J].Toxicol Sci,2000,55(1):152-161.
[9] AQUILINO M,SANCHEZ-ARGUELLO P,MARTINEZ-GUITARTE J L.Vinclozolin alters the expression of hormonal and stress genes in the midge chironomus riparius [J].Aquat Toxicol,2016,174:179-187.
[10] AQUILINO M,SANCHEZ-ARGUELLO P,NOVO M,et al.Effects on tadpole snail gene expression after exposure to vinclozolin [J].Ecotoxicol Environ Saf,2018,170:568-577.
[11] CHEN S N,GURHA P,LOMBARDI R,et al.The hippo pathway is activated and is a causal mechanism for adipogenesis in arrhythmogenic cardiomyopathy [J].Circ Res,2014,114(3):454-468.
[12] SUKUMARAN S K,STUMPF M,SALAMON S,et al.CDK5RAP2 interaction with components of the hippo signaling pathway may play a role in primary microcephaly [J].Mol Genet Genomics,2017,292(2):365-383.
[13] KANG J,WANG J,YAO Z,et al.Fascin induces melanoma tumorigenesis and stemness through regulating the Hippo pathway [J].Cell Commun Signal,2018,16(1):37.
[14] SMITH S,SESSIONS R B,SHOEMARK D,et al.Anti-proliferative and anti-migratory effects of a novel YAP-TEAD interaction inhibitor identified using in silico molecular docking [J].J Med Chem,2019,62(3):1291-1305.
[15] FALLAHI E,O′DRISCOLL N A,MATALLANAS D.The Mst/Hippo pathway and cell death:a non-canonical affair [J].Genes,2016,7(6):28.
[16] SONG Y,FU J,ZHOU M,et al.Activated Hippo/Yes-associated protein pathway promotes cell proliferation and anti-apoptosis in endometrial stromal cells of endometriosis [J].J Clin Endocrinol Metab,2016,101(4):1552-1561.
[17] SHAEER O,SHAEER K.Shaeer’s corporal rotation III:shortening-free correction of congenital penile curvature-the noncorporotomy technique [J].Eur Urol,2016,69(1):129-134.
[18] TOURCHI A,SHABANINIA M,STEWART M,et al.Complete penile disassembly for repair of epispadias causes erectile tissue alteration through transforming growth factor beta 1 overexpression in a rabbit model [J].Urology,2018,111:151-156.
[19] MA L M,WANG Z,WANG H,et al.Estrogen effects on fetal penile and urethral development in organotypic mouse genital tubercle culture [J].J Urol,2009,182(5):2511-2517.
[20] KUSER-ABALI G,ALPTEKIN A,LEWIS M,et al.YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer [J].Nat Commun,2015,6:8126.
[21] HILLEBRAND A C,PIZZOLATO L S,BRANCHINI G,et al.Androgenic modulation of AR-Vs [J].Endocrine,2018,62(2):477-486.