LYAR在结直肠癌中的表达及其功能研究

发布时间：2018-07-20 21:06

【摘要】：Ly-1抗体反应克隆(LYAR)基因最早是从小鼠T细胞白血病细胞系中克隆得到的。它编码一个由380个氨基酸残基组成的45kD核仁蛋白,现已被证实是一个具有特异的DNA结合基序(GGTTAT/G)的转录因子。为了研究LYAR在结直肠癌发生发展中可能的作用,我们首先对77例石蜡包埋正常大肠黏膜及结直肠癌组织的芯片进行免疫组织化学(IHC)分析,检测LYAR的表达。免疫组织化学结果表明,与相邻的正常大肠黏膜相比,大约有49.4%(38/77)的结直肠癌肿瘤组织中LYAR是显著高表达的。进一步,我们对LYAR在结直肠癌中的功能进行深入研究。结果表明,LYAR基因沉默表达对肠癌细胞HCT8和HCT15的增殖、细胞周期、凋亡、平板集落形成、细胞黏附等没有显著影响。然而,相对于HCT8、HCT15亲本和对照细胞(HCT8-NC和HCT15-NC),LYAR基因沉默表达后细胞的迁移和侵袭能力明显被抑制。我们推测,LYAR可能促进结直肠癌细胞的迁移和侵袭。为了寻找LYAR的作用靶分子,我们应用全基因组表达谱微阵列技术分析比较HCT8-NC和HCT8 LYAR-siRNA细胞中mRNA表达差异。我们发现在HCT8细胞中,抑制LYAR表达可在mRNA和蛋白水平上检测到相应的LGALS1(galectin-1)基因表达变化,在HCT116、LoVo和RKO细胞中同样观察到类似结果。这一结果表明,在结直肠癌中,LYAR可能参与调控LGALS1(galectin-1)的表达。接着我们对该基因的启动子进行了生物信息学分析,结果发现位于启动子转录起始点上游-1359 bp处含有LYAR特异结合序列(CTAACC)。为了证实LYAR能否结合到这一特异序列上,我们进行了染色质免疫沉淀(ChIp)和荧光素酶报告基因活性分析实验。综合分析实验结果表明LYAR通过直接结合在LGALS1(galectin-1)启动子转录起始点上游-1359 CTAACC-1354序列上促进半乳糖凝集素-1(galectin-1)的表达。在结直肠癌中,半乳糖凝集素-1已被证实在细胞迁移过程中发挥重要作用。为了研究半乳糖凝集素-1是否介导了 LYAR促进肿瘤细胞的侵袭和转移,我们对HCT8和HCT116的LYAR-Knock Down(LYAR-KD)稳定细胞系进行了 galectin-1异源表达的回复实验。异源表达半乳糖凝集素-1促进LYAR-KD细胞部分回复了迁移和侵袭能力。这一结果表明,在结直肠癌细胞中,半乳糖凝集素-1介导了LYAR促进细胞迁移和侵袭。综上所述,LYAR可作为一个潜在的诊断和治疗靶点用于辅助结直肠癌的预测和治疗。此外,本论文还研究了 Suv420H1的表达改变与K562细胞增殖、凋亡和周期变化的关系,结果发现Suv420H1是通过增加Cyclin D1表达促进K562细胞增殖和细胞周期进程。
[Abstract]:Ly-1 antibody response clone (LYAR) gene was first cloned from mouse T cell leukemia cell line. It encodes a 45kD nucleolar protein consisting of 380 amino acid residues and has been confirmed to be a transcription factor with a specific DNA-binding motif (GGTTAT / G). In order to study the possible role of LYAR in the occurrence and development of colorectal cancer, 77 cases of paraffin-embedded normal colorectal mucosa and colorectal cancer tissue were analyzed by immunohistochemistry (IHC) to detect the expression of LYAR. Immunohistochemical results showed that LYAR was significantly overexpressed in about 49.4% (38 / 77) of colorectal cancer tissues compared with adjacent normal colorectal mucosa. Furthermore, we studied the function of LYAR in colorectal cancer. The results showed that the silencing expression of LYAR gene had no significant effect on the proliferation, cell cycle, apoptosis, plate colony formation and cell adhesion of human intestinal cancer cell line HCT8 and HCT15. However, the cell migration and invasion ability of HCT8-NC and HCT15-NC cells were inhibited after the expression of LYAR gene was silenced compared with HCT8 and HCT15 parent and control cell lines (HCT8-NC and HCT15-NC). We speculate that LYAR may promote the migration and invasion of colorectal cancer cells. In order to find the target molecules of LYAR, we analyzed and compared the difference of mRNA expression between HCT8-NC and HCT8 LYAR-siRNA cells by microarray technique of whole genome expression profile. We found that the changes of LGALS1 (galectin-1) gene expression could be detected in HCT8 cells by inhibiting LYAR expression at both the mRNA and protein levels, and similar results were also observed in HCT116LoVo and RKO cells. These results suggest that LYAR may be involved in the regulation of LGALS1 (galectin-1) expression in colorectal cancer. Then we analyzed the promoter of the gene by bioinformatics and found that there was a LYAR specific binding sequence (CTAACC) located at the upstream of the promoter transcriptional starting point -1359bp. In order to confirm whether LYAR can bind to this specific sequence, we carried out the assay of chromatin immunoprecipitation (ChIp) and luciferase reporter gene activity analysis. The results of comprehensive analysis showed that LYAR promoted the expression of galactose agglutinin 1 (galectin-1) on the upstream of the transcription initiation site of LGALS1 (galectin-1) promoter -1359 CTAACC-1354. Galactose lectin-1 has been shown to play an important role in cell migration in colorectal cancer. In order to investigate whether galactose agglutinin 1 mediates the invasion and metastasis of tumor cells by LYAR, we carried out the galectin-1 heterologous expression recovery of HCT8 and HCT116 LYAR-Knock Down (LYAR-KD) stable cell lines. Heterologous expression of galactose agglutinin-1 (galactose lectin-1) promoted partial migration and invasion of LYAR-KD cells. These results suggest that galactose lectin-1 mediates cell migration and invasion in colorectal cancer cells. In conclusion, LYAR can be used as a potential diagnostic and therapeutic target for the prediction and treatment of colorectal cancer. In addition, the relationship between the expression of Suv420H1 and the proliferation, apoptosis and cycle changes of K562 cells was studied. The results showed that Suv420H1 promoted the proliferation and cell cycle progression of K562 cells by increasing the expression of Cyclin D1.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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