G蛋白耦联受体30与Hippo-Yes相关蛋白/转录共激活因子PDZ结合基序在甲状腺乳头状癌组织的表达及作用

Expression and role of G protein-coupled receptor 30 and Hippo-Yes associated protein/transcriptional co-activator with PDZ-binding domain in thyroid papillary carcinoma

目的探讨G蛋白耦联受体30(GPR30)与Hippo-Yes相关蛋白(YAP)/转录共激活因子PDZ结合基序(TAZ)信号通路在甲状腺乳头状癌发生、发展中的影响。方法收集福建医科大学附属第二医院2019年9月至2020年4月之间40例甲状腺乳头状癌及癌旁组织标本,分析GPR30、YAP、TAZ mRNA及蛋白表达,并分析三者mRNA表达量与甲状腺乳头状癌病理资料之间的关系。采用GPR30特异性激动剂G1及抑制剂G15处理人甲状腺乳头状癌细胞系(TPC-1),分析GPR30、YAP、TAZ表达变化。采用噻唑蓝(MTT)法、流式细胞仪等检测细胞增殖、细胞周期、细胞凋亡,细胞衰老的检测应用β-半乳糖苷酶染色法,细胞迁移侵袭采用Transwell实验。采用配对t检验分析各组数据差异。用t检验评估GPR30、YAP、TAZ表达与具体病理资料之间的关系。用皮尔森(Person)相关分析评估GPR30表达量与YAP、TAZ表达量的关系。结果40例甲状腺乳头状癌组织中GPR30、YAP、TAZ的表达在转录及翻译水平均明显高于癌旁组织(癌与癌旁mRNA相对表达量分别为3.438±0.123比2.463±0.747,t=7.786,P<0.01;3.325±0.111比2.638±0.541,t=8.285,P<0.01;2.581±0.089比2.041±0.418,t=8.068,P<0.01;癌及癌旁蛋白相对表达量分别为1.838±0.074比1.172±0.211,t=13.333,P<0.01、1.735±0.152比1.200±0.321,t=7.641,P<0.01、2.265±0.024比1.310±0.301,t=13.915,P<0.01),差异均有统计学意义。GPR30的mRNA表达与淋巴结转移(3.519±0.094比3.328±0.096,t=3.786,P<0.01)和肿瘤大小(<2 cm为3.424±0.116,≥2 cm为3.600±0.099,t=2.553,P<0.05)明显相关,与年龄、性别、腺外侵犯、病灶数量无明显相关。YAP、TAZmRNA表达与淋巴结转移(3.395±0.105比3.283±0.093,t=3.519,P<0.01;2.627±0.117比2.554±0.053,t=2.686,P<0.05)明显相关,与年龄、性别、肿瘤大小、腺外侵犯、病灶数量无明显相关。GPR30特异性激动剂G1处理TPC-1细胞后,GPR30 mRNA表达量高于对照组(1.224±0.067比0.976±0.048,t=5.212,P<0.01),YAP、TAZ表达量高于对照组(分别为1.359±0.096比0.995±0.004,t=6.562,P<0.01;1.311±0.024比0.985±0.066,t=8.040,P<0.01),促进细胞的增殖(0.381±0.002、0.523±0.003、0.754±0.004比0.359±0.004、0.476±0.003、0.665±0.003,t=8.521、19.190、30.830,P<0.01)、侵袭(422.33±7.23比210.00±3.61,t=45.510,P<0.01)、迁移(550.67±10.69比229.67±8.33,t=41.030,P<0.01),抑制细胞的衰老(0.052±0.001比0.094±0.001,t=51.440,P<0.01)、凋亡[(1.8±0.2)%比(5.8±0.3)%,t=19.220,P<0.01],差异均有统计学意义。结论GPR30、YAP、TAZ的的高表达可能与甲状腺乳头状癌淋巴结转移以及预后相关,GPR30可能通过Hippo-TAZ/YAP通路促进甲状腺乳头状癌的发生、发展。

Objective To investigate the effect of G protein-coupled receptor 30(GPR30)and Hippo-Yes associated protein(YAP)/transcriptional co-activator with PDZ-binding domain(TAZ)signaling pathway on the occurrence and development of thyroid papillary carcinoma.Methods Totally 40 cases of papillary thyroid carcinoma and paracancerous tissue specimens were collected in our hospital from September 2019 to April 2020,the expression of GPR30,YAP and TAZ mRNA and protein was detected,and the relationship between the expression and the clinicopathological characteristics of thyroid papillary carcinoma was analyzed.GPR30 specific agonist G1 and inhibitor G15 were used to treat human papillary thyroid papillary carcinoma cell line(TPC-1),and the expression changes of GPR30,YAP and TAZ were analyzed.Methyl thiazolyl tetrazolium(MTT)and flow cytometry were used to detect cell proliferation,cell cycle and cell apoptosis.Cell senescence was detected byβ-galactosidase staining kit,and Transwell assay was used to detect cell migration and invasion.Results The transcription and translation levels of GPR30,YAP,and TAZ in 40 cases of papillary thyroid carcinoma tissues were significantly higher than those in adjacent tissues(for mRNA:3.438±0.123 and 2.463±0.747,t=7.786,P<0.01;3.325±0.111 and 2.638±0.541,t=8.285,P<0.01;2.581±0.089 and 2.041±0.418,t=8.068,P<0.01.for protein:1.838±0.074 and 1.172±0.211,t=13.333,P<0.01;1.735±0.152 and 1.200±0.321,t=7.641,P<0.01;2.265±0.024 and 1.310±0.301,t=13.915,P<0.01).The GPR30 mRNA expression was significantly correlated with lymph node metastasis(3.519±0.094 and 3.328±0.096,t=3.786,P<0.01)and tumor size(3.424±0.116 and 3.600±0.099,t=2.553,P<0.05),but not significantly related to age,sex,extraglandular invasion,and number of lesions.The expression of YAP and TAZ mRNA was significantly correlated with lymph node metastasis(t=3.519,P<0.01;t=2.686,P<0.05),but not significantly correlated with age,sex,tumor size,extraglandular invasion,and number of lesions.After treating TPC-1 cells with GPR30 specific agonist G1,the GPR30 mRNA expression increased(1.224±0.067 vs.0.976±0.048,t=5.212,P<0.01),the expression of YAP and TAZ increased(1.359±0.096 vs.0.995±0.004,t=6.562,P<0.01;1.311±0.024 vs.0.985±0.066,t=8.040,P<0.01),promoted cell proliferation(0.381±0.002,0.523±0.003 and 0.754±0.004 vs.0.359±0.004,0.476±0.003 and 0.665±0.003,t=8.521,19.190,30.830,allP<0.01),invasion(422.33±7.23 vs.210.00±3.61,t=45.510,P<0.01)and migration(550.67±10.69 vs.229.67±8.33,t=41.030,P<0.01),and inhibited cell senescence(0.052±0.001 vs.0.094±0.001,t=51.440,P<0.01)and apoptosis[(1.8±0.2)%vs.(5.8±0.3)%,t=19.220,P<0.01].Conclusion The high expression of GPR30,YAP and TAZ may be related to lymph node metastasis and prognosis of papillary thyroid carcinoma.GPR30 may promote the occurrence and development of papillary thyroid carcinoma through the Hippo-TAZ/YAP pathway,and may become a potential therapeutic target.