原发性肝癌细胞Coronin-1C基因后干扰Rac1基因激活及损伤肿瘤形成潜能的研究
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摘要
目的：研究Coronin-1C在HCC中的作用及其可能机制进行了探讨，以期为临床CCRCC的有效治疗提供新的有效靶位点。
方法：采用免疫组织化学染色和蛋白质免疫印迹的方法检测了Coronin1C在HCC中的表达情况；采用MTT分析，伤口愈合实验，小室侵袭实验检测了Coronin1C对HCC细胞增殖，迁移及侵袭能力的影响。裸鼠体外成瘤实验检测了Coronin1C对HCC成瘤能力的影响。采用免疫荧光染色和鬼笔环肽标记的方法检测了Coronin1C对HCC细胞的极性和肌动蛋白网络的影响。GST-pull down实验结合蛋白质免疫印迹检测了Coronin1C对Rac-1活化的影响。
结果：Coronin1C mRNA和Coronin1C蛋白在HCC组织中的表达普遍上调；在细胞创伤愈合实验中，Coronin1C过表达使伤口愈合速度变快，而Coronin1C敲除抑引起伤口愈合速度减缓；MTT分析中，Coronin1C的敲除引起细胞增殖抑制；小室侵袭实验中，Coronin1C敲除后穿过基膜的细胞数量明显减少；裸小鼠成瘤实验中，Coronin1A敲除的细胞体内致瘤能力下降；免疫荧光染色和鬼笔环肽标记发现Coronin1A敲除的稳定转染细胞中，应力纤维的含量明显下降，高尔基器的重排减少，同时细胞片状伪足的数量也明显减少；GST-pull down实验发现Coronin1C敲除后细胞中与GST-PKA-CD结合的Rac1的量减少。
结论：在HCC组织中Coronin1C的表达普遍增高，在HCC细胞中抑制Coronin1C的表达引起细胞的增殖，迁移以及侵袭能力下降，进一步的研究发现Coronin1C的表达是通过调节肌动蛋白骨架的装配，减少细胞的极性，激活Rac1促进肿瘤细胞的生长增殖。
Objective: To investigate roles of Coronin-1C in hepatocellular carcinoma cells, andto unclose the related mechanisms.
Methods: q-PCR, Western blotting and immunochemistry were used to determine theexpression of Coronin1C mRNA and protein in HCC; HCC cell line BEL-7402cellwith stable knock-down of Coronin1C was generated. Wound healing assay and MTTassay and transwell migration assay were performed to investigate the effects ofCoronin1C on HCC cell migration, proliferation ability and invation ability respectly.Immunofluorescence and FITC-phalloidin staining metholds were used to detect thethe effect of Coronin1C on cell polarity and stress fiber network. Rac1GST-PAKpull-down assay was performed to to determine the effect of Coronin1C on Rac1activity. The effect of Coronin1C on cell tumorigenicity was evaluted by an in vivomouse model.
Results: Coronin-1C was overexpressed in human HCC tissues compared with theadjacent non-tumor tissues. Overexpression of Coronin-1C enhanced the cellmigration in the human HCC cell line BEL-7402, whereas suppressed cell migrationand proliferation were observed in Coronin-1C-knockdown BEL-7402cells togetherwith impaired cell polarity, disrupted cytoskeleton and decreased Rac-1activation.Moreover, the Coronin-1C knockdown cells displayed a lower degree of malignancyby inducing smaller tumors in nude mice.
Conclusion: From results mentioned above we demonstrated a relationship betweenCoronin-1C overexpression and human HCC growth through enhancement of tumorcell proliferation and migration, which are correlated with Rac-1activation.
方法：采用免疫组织化学染色和蛋白质免疫印迹的方法检测了Coronin1C在HCC中的表达情况；采用MTT分析，伤口愈合实验，小室侵袭实验检测了Coronin1C对HCC细胞增殖，迁移及侵袭能力的影响。裸鼠体外成瘤实验检测了Coronin1C对HCC成瘤能力的影响。采用免疫荧光染色和鬼笔环肽标记的方法检测了Coronin1C对HCC细胞的极性和肌动蛋白网络的影响。GST-pull down实验结合蛋白质免疫印迹检测了Coronin1C对Rac-1活化的影响。
结果：Coronin1C mRNA和Coronin1C蛋白在HCC组织中的表达普遍上调；在细胞创伤愈合实验中，Coronin1C过表达使伤口愈合速度变快，而Coronin1C敲除抑引起伤口愈合速度减缓；MTT分析中，Coronin1C的敲除引起细胞增殖抑制；小室侵袭实验中，Coronin1C敲除后穿过基膜的细胞数量明显减少；裸小鼠成瘤实验中，Coronin1A敲除的细胞体内致瘤能力下降；免疫荧光染色和鬼笔环肽标记发现Coronin1A敲除的稳定转染细胞中，应力纤维的含量明显下降，高尔基器的重排减少，同时细胞片状伪足的数量也明显减少；GST-pull down实验发现Coronin1C敲除后细胞中与GST-PKA-CD结合的Rac1的量减少。
结论：在HCC组织中Coronin1C的表达普遍增高，在HCC细胞中抑制Coronin1C的表达引起细胞的增殖，迁移以及侵袭能力下降，进一步的研究发现Coronin1C的表达是通过调节肌动蛋白骨架的装配，减少细胞的极性，激活Rac1促进肿瘤细胞的生长增殖。
Objective: To investigate roles of Coronin-1C in hepatocellular carcinoma cells, andto unclose the related mechanisms.
Methods: q-PCR, Western blotting and immunochemistry were used to determine theexpression of Coronin1C mRNA and protein in HCC; HCC cell line BEL-7402cellwith stable knock-down of Coronin1C was generated. Wound healing assay and MTTassay and transwell migration assay were performed to investigate the effects ofCoronin1C on HCC cell migration, proliferation ability and invation ability respectly.Immunofluorescence and FITC-phalloidin staining metholds were used to detect thethe effect of Coronin1C on cell polarity and stress fiber network. Rac1GST-PAKpull-down assay was performed to to determine the effect of Coronin1C on Rac1activity. The effect of Coronin1C on cell tumorigenicity was evaluted by an in vivomouse model.
Results: Coronin-1C was overexpressed in human HCC tissues compared with theadjacent non-tumor tissues. Overexpression of Coronin-1C enhanced the cellmigration in the human HCC cell line BEL-7402, whereas suppressed cell migrationand proliferation were observed in Coronin-1C-knockdown BEL-7402cells togetherwith impaired cell polarity, disrupted cytoskeleton and decreased Rac-1activation.Moreover, the Coronin-1C knockdown cells displayed a lower degree of malignancyby inducing smaller tumors in nude mice.
Conclusion: From results mentioned above we demonstrated a relationship betweenCoronin-1C overexpression and human HCC growth through enhancement of tumorcell proliferation and migration, which are correlated with Rac-1activation.
引文
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