ERα36介导雌激素促进甲状腺乳头状癌增殖和侵袭转移的分子机制研究

发布时间：2018-04-07 18:00

本文选题：雌激素　切入点：雌激素受体α36　出处：《重庆医科大学》2017年硕士论文

【摘要】：目的:本实验旨在研究雌激素受体α36(Estrogen receptor alpha-36,ERα36)在介导雌激素促进甲状腺乳头状癌(Papillary thyroid carcinoma,PTC)增殖和侵袭转移的分子机制及其相关信号通路。方法:用免疫组织化学SP法对PTC组织进行ERα36蛋白检测,分析其表达情况与临床病理特征的相关性,同时检测PTC组织中EGFR和HER2的表达,分析三者表达之间的相关性。Western blot方法检测PTC细胞株K-1和BCPAP中ERα36的表达情况;Western blot检测E2对K-1和BCPAP细胞ERK1/2、AKT磷酸化水平和NF-κB核转位的影响,以及ERα36-si RNA对其的抑制作用;Western blot检测NF-κB下游靶基因Cyclin Dl、Survivin和MMP2的蛋白表达情况,以及ERα36-siRNA的抑制作用;梯度浓度的E2处理BCPAP细胞,MTT法检测其细胞增殖情况;Transwell小室实验检测BCPAP细胞侵袭迁移能力。结果:在218例PTC组织中,ERα36、EGFR和HER2的阳性表达率分别为112(51.4%)、132(60.6%)和135(61.9%),明显高于正常组织和结节性增生(P0.001)。ERα36、EGFR和HER2的表达与TNM分期和颈部淋巴结转移显著相关(P0.001)。此外,ERα36与EGFR的表达呈正相关(rs=0.285,P0.001),ERα36与HER2的表达呈正相关(rs=0.352,P0.001)。两种蛋白(ERα36/EGFR、ERα36/HER2和EGFR/HER2)联合表达和仅一种蛋白表达相比,与颈部淋巴结转移和TNM分期更具相关性(P0.001)。ERα36、EGFR和HER2三种蛋白联合表达较仅一种或两种蛋白表达与颈部淋巴结转移和TNM分期更具有相关性(P0.001)。PTC细胞株K-1和BCPAP均表达ERα36;E2处理细胞的最佳条件是10-8mol/L持续24h。E2能促进BCPAP和K-1细胞ERK1/2和AKT蛋白磷酸化水平增高,且在15min时BCPAP细胞最明显,10min时K-1细胞最明显,ERα36-si RNA能抑制E2的诱导效果;随着E2处理时间的增加,核NF-κB p65表达水平逐渐升高,细胞质NF-κB p65逐渐降低,即E2促进BCPAP和K-1细胞发生NF-κB核转位,ERα36-si RNA抑制E2诱导的核转位;E2促进Cyclin Dl、Survivin和MMP2的表达,ERα36-siRNA抑制该作用;E2促进BAPAP细胞增殖,ERα36-siRNA则抑制E2的诱导效果;ERα36-siRNA抑制E2诱导的BAPAP细胞侵袭迁移能力。结论:ERα36、EGFR和HER2在PTC组织中的表达与TNM分期及颈部淋巴结转移呈显著正相关。ERα36通过ERK/AKT-NF-κB通路介导雌激素促进PTC细胞增殖和侵袭转移。
[Abstract]:Aim: to investigate the molecular mechanism of estrogen receptor 伪 36(Estrogen receptor alpha-36 (ER 伪 36) in promoting the proliferation, invasion and metastasis of papillary thyroid carcinoma of thyroid (Papillary thyroid carcinoma-PTC36).Methods: immunohistochemical SP method was used to detect ER 伪 36 protein in PTC tissues. The correlation between the expression of ER 伪 36 protein and clinicopathological features was analyzed. The expression of EGFR and HER2 in PTC tissues was also detected.Western blot was used to detect the expression of ER 伪 36 in PTC cell line K-1 and BCPAP. Western blot was used to detect the effect of E2 on phosphorylation level and NF- 魏 B nuclear translocation in K-1 and BCPAP cells.The inhibitory effect of ER 伪 36-si RNA on the expression of survivin and MMP2, as well as the inhibitory effect of ER 伪 36-siRNA on the expression of NF- 魏 B downstream target gene Cyclin Dlnsurvivin and MMP2 were detected by Western blot.The proliferation of BCPAP cells treated with E _ 2 at gradient concentration was assayed by MTT assay. Transwell chamber assay was used to detect the invasion and migration ability of BCPAP cells.Results: the positive expression rates of ER 伪 36-EGFR and HER2 in 218 cases of PTC were 11251.4 and 1352o60.6, respectively. The positive rates of ER 伪 36-EGFR and HER2 were significantly higher than those of normal tissues and nodular hyperplasia (P0.001T, ER 伪 36-EGFR and HER2), and the expression of ER 伪 36-EGFR and HER2 were significantly correlated with TNM stage and cervical lymph node metastasis.In addition, the expression of ER 伪 36 was positively correlated with the expression of EGFR. The expression of ER 伪 36 was positively correlated with the expression of HER2.Two proteins, ER 伪 36 / EGFRN ER 伪 36/HER2 and EGFR / HER2, were expressed in comparison with only one protein.There was a significant correlation between the expression of P0.001T, ER 伪 36G, EGFR and HER2 protein and the cervical lymph node metastasis and TNM stage. The expression of ER 伪 36C E2 in P0.001PTC cell line K-1 and BCPAP was more correlated with cervical lymph node metastasis and TNM stage.The best condition for cells was that 10-8mol/L continuous 24h.E2 could increase the phosphorylation of ERK1/2 and AKT protein in BCPAP and K-1 cells.The expression of nuclear NF- 魏 B p65 increased and the cytoplasm NF- 魏 B p65 decreased with the increase of E2 treatment time, and the effect of ER 伪 36-si RNA was most obvious in K-1 cells at 10 minutes after 15min treatment, and the expression level of NF- 魏 B p65 increased gradually with the increase of E2 treatment time, and the expression of NF- 魏 B p65 in cytoplasm decreased gradually with the increase of E2 treatment time.Conclusion the expression of EGFR and HER2 in PTC tissues is positively correlated with TNM stage and cervical lymph node metastasis. ER 伪 36 mediates estrogen mediated by ERK / AKT-NF- 魏 B pathway to promote the proliferation and invasion and metastasis of PTC cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R736.1

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