β-半乳糖苷α-2,6唾液酸转移酶2的表达与乳腺癌的预后以及肿瘤进展的相关性研究
发布时间：2018-11-13 20:58
【摘要】：乳腺癌是女性发病率第一位的肿瘤,也是女性中常见的肿瘤相关死因。乳腺癌的诊断治疗措施还不是非常令人满意,需要研究人员们去探索新的诊断或治疗的靶点。β-半乳糖苷α-2,6-唾液酸转移酶1 (ST6Gal1)是目前研究最为透彻的唾液酸转移酶之一,在包括乳腺癌在内的多种肿瘤中均出现表达上调,且与肿瘤的进展和转移关系密切。作为ST6Gal1的同工酶,ST6Gal2在近年来被人发现。然而目前对ST6Gal2功能所知甚少,ST6Gal2对肿瘤进展的作用目前仍不清楚。为了研究ST6Gal2在乳腺癌中的作用,我们收集了40例乳腺癌临床标本和相应的癌旁正常乳腺组织。我们运用RT-PCR和免疫组化染色来研究ST6Gal2在肿瘤组织中的表达是否出现升高。我们接下来从癌症基因组图谱(TCGA)数据库下载了778例乳腺癌病人的基因表达谱和病人疾病特征的数据,并根据患者肿瘤的ST6Gal2表达水平将其分为两组：ST6Gal2高表达组和低表达组,用Fisher确切概率法比较其病理特征的差别：如病理分期,ER,PR,HER2表达水平,年龄等。此外,我们绘制了两组病人的生存率曲线,用对数秩和检验(Log-rank)统计了两组生存率的差异。随后我们选取了5珠乳腺癌细胞株,用Western Blot检测了它们ST6Gal2的表达水平,并在其中挑选了两组相对ST6Gal2高表达的细胞株进行后续实验。我们用siRNA沉默了两株细胞中ST6Gal2的表达,用RT-PCR检测了沉默的效果。然后用CCK-8和流式细胞仪检测了细胞的增值和周期变化,并用纤连蛋白包被的12孔板和Transwell实验检测了细胞的粘附和侵袭能力的变化。为了做进一步的机制研究,我们用前面从TCGA下载的基因表达谱数据,做基因富集分析(GSEA)来研究ST6Gal2的表达与粘附和转移相关的基因集中基因表达的相关性。并选取了具有代表性的促进侵袭的基因,用RT-PCR和Western Blot检测了ST6Gal2的沉默对这些基因表达的影响。我们通过RT-PCR和免疫组化发现,相比较正常组织,ST6Gal2的表达在肿瘤组织中升高。我们通过分析TCGA数据得出,ST6Gal2的高表达和年龄以及HER2的表达水平具有正相关性。重要的是,我们发现,ST6Gal2的高表达与乳腺癌的预后不佳显著相关。我们通过siRNA沉默两株乳腺癌细胞株ST6Gal2的表达,发现细胞的生长受到抑制,细胞出现G0/G1期周期阻滞,且细胞与基质的粘附和侵袭能力下降。GSEA分析结果显示,ST6Gal2的高表达与粘附和转移相关基因集中的基因的表达升高出现显著的相关性。进一步的Western Blot和RT-PCR表明,沉默ST6Gal2导致6个与转移和侵袭有关的基因：RhoA、TGF-β3、VEGFC、CTNNB1、FUT8以及WISP-1的表达下调。总之,我们的研究表明ST6Gal2的高表达可能导致肿瘤的进展并可能因此导致乳腺癌病人不佳的预后。我们的发现说明ST6Gal2有潜力成为一个预测乳腺癌预后的生物学标记或作为治疗的一个靶点。
[Abstract]:Breast cancer is the first cancer in women and a common tumor-related cause of death in women. The diagnosis and treatment of breast cancer is not very satisfactory. It requires researchers to explore new diagnostic or therapeutic targets. 尾 -galactoside 伪 -2-sialic acid transferase 1 (ST6Gal1) is one of the most thoroughly studied sialic acid transferases. The expression is up-regulated in many tumors, including breast cancer, and is closely related to tumor progression and metastasis. As an isoenzyme of ST6Gal1, ST6Gal2 has been discovered in recent years. However, little is known about the function of ST6Gal2, and the role of ST6Gal2 in tumor progression remains unclear. In order to study the role of ST6Gal2 in breast cancer, we collected 40 clinical specimens of breast cancer and corresponding adjacent normal breast tissues. We used RT-PCR and immunohistochemical staining to study whether the expression of ST6Gal2 increased in tumor tissues. We then downloaded the gene expression profiles and disease characteristics of 778 breast cancer patients from the (TCGA) database of the cancer genome map, and divided them into two groups according to the tumor ST6Gal2 expression levels: high ST6Gal2 expression group and low expression group. Fisher exact probability method was used to compare the pathological features, such as pathological stage, ER,PR,HER2 expression level, age and so on. In addition, the survival rate curve was drawn and the difference between the two groups was calculated by logarithmic rank sum test (Log-rank). Then we selected 5 bead breast cancer cell lines, detected their ST6Gal2 expression level by Western Blot, and selected two groups of cell lines with high ST6Gal2 expression for follow-up experiments. The expression of ST6Gal2 was silenced by siRNA and the effect of silencing was detected by RT-PCR. Then the cell proliferation and cell cycle were detected by CCK-8 and flow cytometry. The adhesion and invasiveness of the cells were detected by 12 well plate coated with fibronectin and Transwell. In order to further study the mechanism, we used the previously downloaded gene expression profiles from TCGA to analyze the gene concentration of (GSEA) to study the correlation between the expression of ST6Gal2 and the gene expression in gene set related to adhesion and metastasis. RT-PCR and Western Blot were used to detect the effect of ST6Gal2 silencing on the expression of these genes. We found that the expression of ST6Gal2 was higher in tumor tissues than in normal tissues by RT-PCR and immunohistochemistry. By analyzing the TCGA data, we found that there was a positive correlation between the high expression of ST6Gal2 and age and the expression level of HER2. Importantly, we found that high expression of ST6Gal2 was significantly associated with poor prognosis in breast cancer. By silencing the expression of ST6Gal2 in two breast cancer cell lines by siRNA, we found that cell growth was inhibited, cell cycle arrest occurred in G0/G1 phase, and cell adhesion and invasion to matrix decreased. There was a significant correlation between the high expression of ST6Gal2 and the increase of gene expression in adhesion and metastasis related gene set. Further Western Blot and RT-PCR showed that silencing ST6Gal2 resulted in six genes associated with metastasis and invasion: RhoA,TGF- 尾 3VEGFNB1FUT8 and down-regulated expression of WISP-1. In summary, our study suggests that high expression of ST6Gal2 may lead to tumor progression and, consequently, poor prognosis in breast cancer patients. Our findings suggest that ST6Gal2 has the potential to be a biological marker for predicting the prognosis of breast cancer or as a therapeutic target.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
[Abstract]:Breast cancer is the first cancer in women and a common tumor-related cause of death in women. The diagnosis and treatment of breast cancer is not very satisfactory. It requires researchers to explore new diagnostic or therapeutic targets. 尾 -galactoside 伪 -2-sialic acid transferase 1 (ST6Gal1) is one of the most thoroughly studied sialic acid transferases. The expression is up-regulated in many tumors, including breast cancer, and is closely related to tumor progression and metastasis. As an isoenzyme of ST6Gal1, ST6Gal2 has been discovered in recent years. However, little is known about the function of ST6Gal2, and the role of ST6Gal2 in tumor progression remains unclear. In order to study the role of ST6Gal2 in breast cancer, we collected 40 clinical specimens of breast cancer and corresponding adjacent normal breast tissues. We used RT-PCR and immunohistochemical staining to study whether the expression of ST6Gal2 increased in tumor tissues. We then downloaded the gene expression profiles and disease characteristics of 778 breast cancer patients from the (TCGA) database of the cancer genome map, and divided them into two groups according to the tumor ST6Gal2 expression levels: high ST6Gal2 expression group and low expression group. Fisher exact probability method was used to compare the pathological features, such as pathological stage, ER,PR,HER2 expression level, age and so on. In addition, the survival rate curve was drawn and the difference between the two groups was calculated by logarithmic rank sum test (Log-rank). Then we selected 5 bead breast cancer cell lines, detected their ST6Gal2 expression level by Western Blot, and selected two groups of cell lines with high ST6Gal2 expression for follow-up experiments. The expression of ST6Gal2 was silenced by siRNA and the effect of silencing was detected by RT-PCR. Then the cell proliferation and cell cycle were detected by CCK-8 and flow cytometry. The adhesion and invasiveness of the cells were detected by 12 well plate coated with fibronectin and Transwell. In order to further study the mechanism, we used the previously downloaded gene expression profiles from TCGA to analyze the gene concentration of (GSEA) to study the correlation between the expression of ST6Gal2 and the gene expression in gene set related to adhesion and metastasis. RT-PCR and Western Blot were used to detect the effect of ST6Gal2 silencing on the expression of these genes. We found that the expression of ST6Gal2 was higher in tumor tissues than in normal tissues by RT-PCR and immunohistochemistry. By analyzing the TCGA data, we found that there was a positive correlation between the high expression of ST6Gal2 and age and the expression level of HER2. Importantly, we found that high expression of ST6Gal2 was significantly associated with poor prognosis in breast cancer. By silencing the expression of ST6Gal2 in two breast cancer cell lines by siRNA, we found that cell growth was inhibited, cell cycle arrest occurred in G0/G1 phase, and cell adhesion and invasion to matrix decreased. There was a significant correlation between the high expression of ST6Gal2 and the increase of gene expression in adhesion and metastasis related gene set. Further Western Blot and RT-PCR showed that silencing ST6Gal2 resulted in six genes associated with metastasis and invasion: RhoA,TGF- 尾 3VEGFNB1FUT8 and down-regulated expression of WISP-1. In summary, our study suggests that high expression of ST6Gal2 may lead to tumor progression and, consequently, poor prognosis in breast cancer patients. Our findings suggest that ST6Gal2 has the potential to be a biological marker for predicting the prognosis of breast cancer or as a therapeutic target.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
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