β-半乳糖苷α-2,6唾液酸轉(zhuǎn)移酶2的表達(dá)與乳腺癌的預(yù)后以及腫瘤進(jìn)展的相關(guān)性研究
發(fā)布時(shí)間：2018-11-13 20:58
【摘要】：乳腺癌是女性發(fā)病率第一位的腫瘤,也是女性中常見的腫瘤相關(guān)死因。乳腺癌的診斷治療措施還不是非常令人滿意,需要研究人員們?nèi)ヌ剿餍碌脑\斷或治療的靶點(diǎn)。β-半乳糖苷α-2,6-唾液酸轉(zhuǎn)移酶1 (ST6Gal1)是目前研究最為透徹的唾液酸轉(zhuǎn)移酶之一,在包括乳腺癌在內(nèi)的多種腫瘤中均出現(xiàn)表達(dá)上調(diào),且與腫瘤的進(jìn)展和轉(zhuǎn)移關(guān)系密切。作為ST6Gal1的同工酶,ST6Gal2在近年來被人發(fā)現(xiàn)。然而目前對(duì)ST6Gal2功能所知甚少,ST6Gal2對(duì)腫瘤進(jìn)展的作用目前仍不清楚。為了研究ST6Gal2在乳腺癌中的作用,我們收集了40例乳腺癌臨床標(biāo)本和相應(yīng)的癌旁正常乳腺組織。我們運(yùn)用RT-PCR和免疫組化染色來研究ST6Gal2在腫瘤組織中的表達(dá)是否出現(xiàn)升高。我們接下來從癌癥基因組圖譜(TCGA)數(shù)據(jù)庫下載了778例乳腺癌病人的基因表達(dá)譜和病人疾病特征的數(shù)據(jù),并根據(jù)患者腫瘤的ST6Gal2表達(dá)水平將其分為兩組：ST6Gal2高表達(dá)組和低表達(dá)組,用Fisher確切概率法比較其病理特征的差別：如病理分期,ER,PR,HER2表達(dá)水平,年齡等。此外,我們繪制了兩組病人的生存率曲線,用對(duì)數(shù)秩和檢驗(yàn)(Log-rank)統(tǒng)計(jì)了兩組生存率的差異。隨后我們選取了5珠乳腺癌細(xì)胞株,用Western Blot檢測(cè)了它們ST6Gal2的表達(dá)水平,并在其中挑選了兩組相對(duì)ST6Gal2高表達(dá)的細(xì)胞株進(jìn)行后續(xù)實(shí)驗(yàn)。我們用siRNA沉默了兩株細(xì)胞中ST6Gal2的表達(dá),用RT-PCR檢測(cè)了沉默的效果。然后用CCK-8和流式細(xì)胞儀檢測(cè)了細(xì)胞的增值和周期變化,并用纖連蛋白包被的12孔板和Transwell實(shí)驗(yàn)檢測(cè)了細(xì)胞的粘附和侵襲能力的變化。為了做進(jìn)一步的機(jī)制研究,我們用前面從TCGA下載的基因表達(dá)譜數(shù)據(jù),做基因富集分析(GSEA)來研究ST6Gal2的表達(dá)與粘附和轉(zhuǎn)移相關(guān)的基因集中基因表達(dá)的相關(guān)性。并選取了具有代表性的促進(jìn)侵襲的基因,用RT-PCR和Western Blot檢測(cè)了ST6Gal2的沉默對(duì)這些基因表達(dá)的影響。我們通過RT-PCR和免疫組化發(fā)現(xiàn),相比較正常組織,ST6Gal2的表達(dá)在腫瘤組織中升高。我們通過分析TCGA數(shù)據(jù)得出,ST6Gal2的高表達(dá)和年齡以及HER2的表達(dá)水平具有正相關(guān)性。重要的是,我們發(fā)現(xiàn),ST6Gal2的高表達(dá)與乳腺癌的預(yù)后不佳顯著相關(guān)。我們通過siRNA沉默兩株乳腺癌細(xì)胞株ST6Gal2的表達(dá),發(fā)現(xiàn)細(xì)胞的生長(zhǎng)受到抑制,細(xì)胞出現(xiàn)G0/G1期周期阻滯,且細(xì)胞與基質(zhì)的粘附和侵襲能力下降。GSEA分析結(jié)果顯示,ST6Gal2的高表達(dá)與粘附和轉(zhuǎn)移相關(guān)基因集中的基因的表達(dá)升高出現(xiàn)顯著的相關(guān)性。進(jìn)一步的Western Blot和RT-PCR表明,沉默ST6Gal2導(dǎo)致6個(gè)與轉(zhuǎn)移和侵襲有關(guān)的基因：RhoA、TGF-β3、VEGFC、CTNNB1、FUT8以及WISP-1的表達(dá)下調(diào)�？傊�,我們的研究表明ST6Gal2的高表達(dá)可能導(dǎo)致腫瘤的進(jìn)展并可能因此導(dǎo)致乳腺癌病人不佳的預(yù)后。我們的發(fā)現(xiàn)說明ST6Gal2有潛力成為一個(gè)預(yù)測(cè)乳腺癌預(yù)后的生物學(xué)標(biāo)記或作為治療的一個(gè)靶點(diǎn)。
[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 偽 -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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：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 偽 -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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9
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