Expression and Clinical Significance of Stem Cells Gene Lgr5 in Esophageal Squamous Cell Carcinoma Tissues
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摘要:目的
探讨干细胞基因Lgr5在食管鳞癌组织及其成球细胞中的表达意义。
方法采用免疫组织化学法（IHC）检测280例食管鳞癌和150例癌旁组织中Lgr5蛋白的表达水平；低黏附培养法培养食管鳞癌细胞KYSE70形成成球细胞，Quantitative real-time PCR（qRT-PCR）、Western blot法检测干性相关基因SOX2、ALDH1A1、NANOG以及Lgr5在亲代细胞和成球细胞中的表达差异；Transwell实验检测成球细胞的迁移和侵袭能力、qRT-PCR法检测成球细胞中上皮间质转化（epithelial-mesenchymal transition, EMT）相关标志物Vimentin和N-cadherin的表达水平。
结果Lgr5在食管鳞癌组织的表达水平明显高于癌旁正常食管组织（P < 0.05），且与淋巴结转移、浸润深度、肿块大小、分化程度、肿瘤分期密切相关（均P < 0.05）。与亲代细胞相比，食管鳞癌成球细胞中干性相关基因SOX2、ALDH1A1、NANOG表达水平升高（均P < 0.05），Lgr5在食管鳞癌成球细胞中的表达水平也明显升高（P < 0.05），并且其迁移和侵袭能力增强（P < 0.05），EMT相关标志物Vimentin和N-cadherin的mRNA表达水平升高（均P < 0.05）。
结论Lgr5可能为食管鳞癌复发、进展、侵袭转移的标志之一，Lgr5高表达的食管鳞癌细胞可能具有干细胞样特性，并且可能为食管鳞癌细胞的一个干性调控基因。
Abstract:ObjectiveTo investigate the expression and clinical significance of stem cells gene Lgr5 in esophageal squamous cell carcinoma(ESCC) tissues and spheroid body-forming cells in ESCC.
MethodsThe expression level of Lgr5 was assessed in 280 cases of ESCC and 150 cases of normal esophageal squamous epithelia tissues by immunohistochemistry(IHC). Meanwhile, we developed spheroid body-forming cells in ESCC cell line KYSE70. The expression levels of stemness-related genes SOX2, ALDH1A1, NANOG and Lgr5 in spheroid body-forming cells and parental cells were detected via quantitative real-time PCR (qRT-PCR) and Western blot; the detection of Vimentin and N-cadherin, and Transwell experiment were performed to evaluate the invasion and migration abilities in spheroid body-forming cells.
ResultsThe expression level of Lgr5 in ESCC was significantly higher, compared with normal esophageal squamous epithelia (P < 0.05). The high expression of Lgr5 was correlated with the tumor differentiation, lymph node metastasis, invasion, tumor size and AJCC stage (all P < 0.05). Compared with parental cells, the expression levels of SOX2, ALDH1A1 and NANOG were increased in spheroid body-forming cells (all P < 0.05); Lgr5 could up-regulate the expression levels of mRNA and proteins in spheroid body-forming cells (P < 0.05); the migration and invasion abilities of spheroid body-forming cells were increased (all P < 0.05); and the mRNA expression levels of Vimentin and N-cadherin were increased (all P < 0.05).
ConclusionLgr5 may be a biomarker of recurrence, progression, invasion and migration of ESCC, and the overexpression of Lgr5 in ESCC may identify the ESCC' s stem-like properties, thus Lgr5 may be a gene of regulating stem-like properties in ESCC.
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Key words:
- Lgr5 /
- ESCC /
- Cancer stem cells /
- KYSE70 /
- Spheroid body-forming cells
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图 1 免疫组织化学法检测食管鳞癌和癌旁正常组织中Lgr5蛋白的表达(SP ×400)
Figure 1 Expression of Lgr5 in ESCC tissues and normal esophageal squamous epithelia tissues detected by immunohistochemistry (SP ×400)
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图 2 食管鳞癌细胞系KYSE70在无血清低黏附培养下形成的克隆细胞球
Figure 2 ESCC KYSE70 cells formed spheroid bodies in serum-free medium at low adherence condition
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图 3 qRT-PCR法检测Lgr5在食管鳞癌KYSE70成球细胞和亲代细胞中mRNA表达水平
Figure 3 mRNA expression levels of Lgr5 in ESCC KYSE70 spheroid body cells and parental cells evaluated by qRT-PCR
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图 4 Western blot法检测Lgr5在食管鳞癌KYSE70成球细胞和亲代细胞中蛋白表达水平
Figure 4 Protein expression levels of Lgr5 in ESCC KYSE70 spheroid body cells and parental cells evaluated by Western blot
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图 5 qRT-PCR和Western blot法检测干性相关基因SOX2、ALDH1A1、NANOG在食管鳞癌KYSE70成球细胞和亲代细胞中的mRNA和蛋白表达水平
Figure 5 mRNA and protein expression levels of stemness related genes SOX2, ALDH1A1, NANOG in ESCC KYSE70 spheroid body cells and parental cells evaluated by qRT-PCR and Western blot
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图 6 Transwell实验检测KYSE70成球细胞和亲代细胞的迁移侵袭能力
Figure 6 Migration and invasion abilities of KYSE70 spheroid body cells and parental cells evaluated by Transwell
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图 7 qRT-PCR法检测Vimentin、N-cadherin在食管鳞癌KYSE70成球细胞和亲代细胞中mRNA表达水平
Figure 7 mRNA expression levels of Vimentin and N-cadherin in KYSE70 spheroid body cells and parental cells evaluated by qRT-PCR
表 1 Lgr5在人食管鳞癌和癌旁正常组织中的表达
Table 1 Expression of Lgr5 in esophageal squamous cell carcinoma(ESCC) tissues and normal esophageal squamous epithelia tissues
下载: 导出CSV
表 2 Lgr5蛋白表达与食管鳞癌患者临床病理参数间的关系
Table 2 Association between Lgr5 expression and clinicopathological features of ESCC patients
下载: 导出CSV
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