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Kindlin-2诱导胆囊癌细胞上皮间质转化促进其侵袭转移的实验研究

Kindlin-2 promotes gallbladder cancer metastasis and invasion by inducing epithelial-mesenchymal transition
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摘要 目的 探讨黏着斑蛋白Kindlin-2对胆囊癌细胞生物学行为的影响及相关分子机制.方法 收集2012年9月至2013年5月在郑州大学第一附属医院肝胆胰外科接受手术切除的30例胆囊癌组织和癌旁组织标本.使用real-time PCR和免疫组化方法检测胆囊癌组织和癌旁组织标本中Kindlin-2表达水平;通过构建质粒和转染使胆囊癌细胞株GBC-SD、SGC-996过表达Kindlin-2,应用细胞黏附实验观察胆囊癌细胞与胞外基质的黏附能力,通过Transwell侵袭实验及细胞划痕实验观察胆囊癌细胞的侵袭迁移能力改变;采用免疫印迹法检测过表达Kindlin-2后胆囊癌细胞上皮间质转化相关标记蛋白的变化.分别使用t检验和 χ2检验对计量资料和计数资料进行统计学分析.结果real-time PCR和免疫组化检测结果显示,Kindlin-2在人胆囊癌组织中表达水平高于癌旁组织(t=4.372,P=0.001;t=7.477,P=0.000),肿瘤组织Kindlin-2表达水平与Nevin分期密切相关(χ2=5.932,P=0.035);细胞黏附实验结果显示,过表达Kindlin-2后胆囊癌细胞株GBC-SD、SGC-996与细胞外基质分子纤连蛋白Fibronectin的黏附能力较对照组增强(1. 66±0.03比1.07±0.22,t=2.710,P=0.041;2.66±0.24比1.03±0.02,t=6.610,P=0,020);Transwell侵袭实验结果显示,过表达Kindlin-2后胆囊癌细胞株GBC-SD、 SGS-996穿过Transwell小室基质的细胞数较对照组明显增多(116.1±13.9比54.7±8.4,t=3.781,P=0.0194;136.3±7.5比64.3±6.4,t=7.302, P=0.002) ;细胞划痕实验结果显示,过表达Kindlin-2后胆囊癌细胞株GBC-SD12h和24h划痕修复率高于对照组[(42.9±2.2)%比(29.7±1.7)%,t=4.690,P=0.009;(65.0±2.4)%比(40.4±2.0)%,t=7.945,P=0.001],过表达Kindlin-2后胆囊癌细胞株SGC-996l2h和24h划痕修复率高于对照组[(32.9±1.3 )%比(24.1±1.5)%,t=4.518,P=0.011;(51.3±1.1)%比(39.2±1.1)%,t=8.001,P=0.001〕;免疫印迹法检测结果显示,过表达 Kindlin-2后胆囊癌细胞间质化表型神经 Objective To investigate the effects of Kindlin-2 on malignant phenotypes of human gallbladder cancer cells and discuss the mechanisms. Methods The expression level of Kindling2 in 30 cases of gallbladder cancer tissues and adjacent non-tumoral tissues collected from the First Affiliated Hospital of Zhengzhou University between September 2012 and May 2013 was assessed by real-time PCR and immunohistochemistry. Lentivirns-mediated Kindlin-2 overexpression was used in gallbladder cancer cell lines GBC-SD and SGC-996. Transwell assay and adhesion assay were investigated to explore the functional role of Kindlin-2 on gallbladder cancer cells. Western Blot was used to test the protein change of epithelial- mesenehymal transition(EMT) characteristics. The t-test was used to analyzed results. Results The RNAand protein levels of Kindlin-2 in gallbladder cancer tissues were higher than in the non-tumoral tissues ( t = 4. 372, P = 0. 001 ;t = 7. 477, P = 0. 000). The expression level of Kindlin-2 in gallbladder cancer tissues was correlated with Nevin stage( χ2= 5. 932,P= 0. 035). Compared with control groups,the cell-matrix adhesion ability of GBC-SD and SGC-996 with Kindlin-2 overexpression was obviously promoted (1.66 ±0. 03 vs. 1.07±0.22,t=2.710,P=0.041;2.66±0.24 vs. 1. 03 ±0. 02 , t = 6. 610 , P = 0. 020 ) . The number of GBC-SD and SGC-996 cells with Kindlin-2 overexpression passing through the Transwell chamber matrix increased significantly compared with the control groups ( 116. 1 ±13.9 vs. 54. 7 ± 8.4, t = 3.781, P =- 0. 019 ; 136. 3±7.5 vs. 64. 3 ±6.4, t = 7. 302, P =0. 002) . The wound healing rate of GBC-SD with Kindlin-2 overexpression at 12-hour and 24-hour was higher than that of the group ( ( 42. 9 ±2. 2) % vs. ( 29. 7 ±1.7 )%, t = 4. 690, P = 0. 009;( 65.0± 2.4)% vs. (40. 4 ±2. 0)%, t = 7. 945, P = 0. 001 ). The wound healing rate of SGC-996 with Kindlin-2 overexpression at 12-hour and 24-hour was also higher than that of the group ((32.9±1.3)%
作者 陆旭 周闯 李仁锋 叶健文 翟文龙 Lu Xu, Zhou Chuang, Li Renfeng, Ye Jianwen, Zhai Wenlong. (Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital of Zhengzhou University, Digestive Organ Transplantation of Henan Provirtce,Zhengzhou 450052,China)
出处 《中华外科杂志》 CSCD 北大核心 2018年第8期617-622,共6页 Chinese Journal of Surgery
基金 河南省高校科技创新人才支持项目(14HASTIT030)
关键词 胆囊肿瘤 上皮细胞-间充质细胞转换 Kindlin-2 Gallbladder neoplasms Epithelial-mesenchymal transition Kindlin-2
作者简介 通信作者:翟文龙,Email:zhaiwenlong66@126.com
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