金属硫蛋白在低氧促进甲状腺乳头状癌发生侵袭转移中的作用研究

发布时间：2018-10-24 21:01

【摘要】：目的本研究旨在探讨金属硫蛋白(metallothioneins, MTs)在低氧诱导甲状腺乳头状癌(Papillary thyroid carcinoma, PTC)侵袭转移中的作用及分子机制。方法使用5%O2的低氧培养箱孵育甲状腺乳头状癌BCPAP细胞,用Western blotting及RT-PCR检测细胞中MT、HIF-1α、SIP1、 E-cadherin等相关蛋白和mRNA表达；使用Transwell观察细胞侵袭迁移的情况。采用免疫组化SP法检测72例人甲状腺乳头状癌和37例癌旁正常组织中MT、HIF-1α、SIP1和MMP-9的表达水平,分析其表达与临床病理指标的关系及表达的相关性。将BCPAP细胞分为常氧、低氧、低氧+Scrambled-siRNA、低氧+过表达空质粒、低氧+MT2AsiRNA和低氧+MT2A过表达组培养12h后检测MT、HIF-1α、SIP1、E-cadherin和MMP-9蛋白表达及侵袭迁移情况。结果与常氧组相比,低氧处理组BCPAP细胞MT、HIF-1α、SIP1蛋白质表达水平增高,E-cadherin的蛋白质表达水平下降。MT、SIP1的mRNA表达水平增高,E-cadherin的mRNA表达水平明显下降,HIF-1α无显著性差异(P0.05)。侵袭与迁移实验中,低氧组的穿膜与迁移细胞数均较常氧组显著的增多(P0.05)。在72例PTC组织中,MT、HIF-1α、SIP1和MMP-9的阳性率分别为52.78%、55.56%、51.39%和54.17%,均显著高于癌旁正常组织(P0.05)。MT、HIF-1α、SIP1和MMP-9的阳性表达与患者颈部淋巴结转移有显著相关性(P0.05),MMP-9的阳性表达与肿瘤大小有显著相关性(P0.05)。MT、HIF-1α及SIP1的表达呈正相关。用MT2A-siRNA转染BCPAP细胞后,低氧诱导的MT、HIF-1α、SIP1和MMP-9表达降低,E-cadherin表达升高。过表达MT2A后,低氧诱导的MT、HIF-1α、SIP1和MMP-9表达升高,E-cadherin表达则降低。结论低氧诱导甲状腺乳头状癌细胞MT、SIP1表达上调,E-cadherin表达下调,以及侵袭转移性增强。MT、HIF-1α、SIP1和MMP-9在PTC组织中的表达与颈部淋巴结转移密切相关,且存在相关性。MT可能对低氧诱导的PTC细胞侵袭转移具有促进作用。
[Abstract]:Objective to investigate the role and molecular mechanism of metallothionein (metallothioneins, MTs) in (Papillary thyroid carcinoma, PTC) invasion and metastasis of papillary thyroid carcinoma induced by hypoxia. Methods BCPAP cells of papillary thyroid carcinoma were incubated with 5%O2 hypoxia incubator. The expressions of MT,HIF-1 伪, SIP1, E-cadherin and mRNA were detected by Western blotting and RT-PCR, and the invasion and migration of the cells were observed by Transwell. The expression of MT,HIF-1 伪, SIP1 and MMP-9 in 72 cases of human papillary thyroid carcinoma and 37 cases of adjacent normal tissues were detected by immunohistochemical SP method. BCPAP cells were divided into normoxic and hypoxic Scrambled-siRNA, overexpression plasmids. The expression of MT,HIF-1 伪, SIP1,E-cadherin and MMP-9 protein and invasion and migration were detected in hypoxic MT2AsiRNA and hypoxic MT2A overexpression groups for 12 h. Results compared with the normoxic group, the expression of MT,HIF-1 伪 and SIP1 protein in BCPAP cells was increased, the expression of E-cadherin protein was decreased, the mRNA expression level of MT,SIP1 was increased, the mRNA expression level of E-cadherin was significantly decreased in hypoxia treatment group, and there was no significant difference in HIF-1 伪 expression between hypoxia group and normoxic group (P0.05). In invasion and migration experiment, the number of transmembrane and migration cells in hypoxia group was significantly higher than that in normal oxygen group (P0.05). In 72 cases of PTC, the positive rates of MT,HIF-1 伪, SIP1 and MMP-9 were 52.7855.56% and 54.1717%, respectively, which were significantly higher than those of adjacent normal tissues (P0.05). The positive expression of MT,HIF-1 伪, SIP1 and MMP-9 was significantly correlated with cervical lymph node metastasis (P0.05), MMP-9 positive expression was significantly correlated with tumor size (P0.05). The expression of MT,HIF-1 伪 and SIP1 was positively correlated. After transfection of MT2A-siRNA into BCPAP cells, hypoxia induced MT,HIF-1 伪, SIP1 and MMP-9 expression decreased and E-cadherin expression increased. Overexpression of MT2A increased the expression of MT,HIF-1 伪, SIP1 and MMP-9, but decreased the expression of E-cadherin. Conclusion the expression of MT,SIP1 is up-regulated, the expression of E-cadherin is down-regulated, and the invasion and metastasis are enhanced. The expression of MT,HIF-1 伪, SIP1 and MMP-9 in PTC tissue is closely related to cervical lymph node metastasis. MT may promote the invasion and metastasis of PTC cells induced by hypoxia.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R736.1

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