β-catenin蛋白发生O-GlcNAc糖基化促进绒毛膜癌细胞迁移

发布时间：2018-07-20 09:53

【摘要】：人绒毛膜癌是恶性滋养叶细胞肿瘤，常发生在正常妊娠或者异常妊娠后，病变部位主要位于子宫，具有高转移和高侵袭能力，并易通过血行转移到全身，恶性程度极高。因此，研究绒毛膜癌的转移机制并寻求有效的靶点抑制其转移将有助于肿瘤的防治。目前，对肿瘤转移能力的研究逐渐集中在蛋白翻译后修饰，尤其是糖基化修饰，它是在各种糖基转移酶的催化下，于各功能蛋白上添加相应的糖链，而不同的糖残基对蛋白功能的影响也不尽相同，从而调控各种生物学效应。本研究则主要探讨了氧连接N-乙酰葡萄糖胺（O-linkedN-acetylglucosamine，O-GlcNAc）单糖修饰对绒毛膜癌细胞迁移能力的影响及其机制。 O-G1cNAc单糖修饰是普遍存在于细胞质、核内的一种蛋白翻译后修饰方式，发生于蛋白结构的丝氨酸(Ser)及苏氨酸(Thr)残基的侧链羟基上。它在O位-β-N-乙酰葡萄糖胺基转移酶（OGT）的作用下仅将一个N-乙酰氨基葡萄糖转移到蛋白的Ser/Thr残基上，并可以通过糖苷酶OGA移除糖链，在两个酶的共同作用下保持O-G1cNAc单糖修饰的动态平衡。一些与粘附相关的功能蛋白的O糖基化修饰往往会对其自身活性和功能造成不同的影响，进而改变细胞的生物学功能，如细胞迁移、粘附、侵袭等。本研究采用人绒毛膜癌细胞（JAR）为研究模型，首先采用OGA特异性抑制剂处理细胞，建立O-G1cNAc糖基化高表达的细胞模型，随后进行细胞迁移实验（transwell方法）观察JAR细胞迁移能力是否受到影响。初步研究发现，O-G1cNAc蛋白糖基化修饰水平增加促进了JAR细胞的迁移，，进一步通过粘附实验证实JAR细胞与脐静脉血管内皮细胞EA.hy926的粘附能力也相应增强了。说明O-G1cNAc蛋白糖基化修程度与绒毛膜癌的迁移和转移有关。接着，通过siRNA干扰，建立了OGT基因部分沉默的细胞模型，同样采取transwell及粘附实验证实O-G1cNAc蛋白糖基化水平与JAR细胞迁移及粘附能力呈正相关。采用免疫共沉淀技术，发现OGA抑制剂处理JAR细胞后，伴随着胞内O-G1cNAc糖基化整体水平的增加，细胞内重要的功能蛋白β-catenin发生O-GlcNAc糖基化修饰的程度也相应上升，但并未影响到β-catenin在绒毛膜癌细胞内的蛋白表达量。此外，我们用IL-8(100ng/ml)处理绒毛膜癌细胞24小时后，通过荧光定量PCR、western blot方法检测细胞内的OGT及O-G1cNAc糖基化修饰程度，结果显示OGT无论是RNA转录水平还是蛋白翻译水平均无明显改变，胞内O-G1cNAc糖基化整体水平亦无变化，说明O-GlcNAc蛋白糖基化修饰并不参与IL-8促进绒毛膜癌细胞转移的调控过程。综上， OGT可能通过增加β-catenin的O-GlcNAc糖基化水平促进绒毛膜癌细胞的转移及与内皮细胞的粘附，这可能也是绒毛膜癌高转移性的分子机制之一。本研究为临床上绒毛膜癌的防治提供了新的靶点及研究思路。
[Abstract]:Human choriocarcinoma is a malignant trophoblastic tumor, which usually occurs in normal pregnancy or abnormal pregnancy. The lesion is mainly located in the uterus, with high metastasis and invasiveness, and is easily metastasized to the whole body through blood, and the degree of malignancy is extremely high. Therefore, studying the metastasis mechanism of choriocarcinoma and seeking effective targets to inhibit its metastasis will be helpful to the prevention and treatment of the tumor. At present, the ability of tumor metastasis is gradually focused on protein post-translational modification, especially glycosylation modification, which is catalyzed by a variety of glycosyltransferases, and the corresponding sugar chains are added to each functional protein. The effects of different sugar residues on protein function are also different, thus regulating various biological effects. In this study, we investigated the effect of O-linkedN-acetylglucosamine-O-GlcNAc on the migration of choriocarcinoma cells and its mechanism. O-G1cNAc monosaccharide modification is a kind of post-translational modification in cytoplasm and nucleus. It occurs on the side chain hydroxyl groups of serine (Ser) and threonine (Thr) residues. It transfers only one N-acetyl glucosamine to the Ser-Thr residue of the protein under the action of O-position 尾 -N-acetyl-glucosaminotransferase (OGT), and it can remove the sugar chain through the glucosidase OGA. The dynamic equilibrium of O-G1cNAc monosaccharide was maintained under the combined action of two enzymes. The O-glycosylation modification of some functional proteins associated with adhesion may have different effects on their own activities and functions, and then change the biological functions of cells, such as cell migration, adhesion, invasion and so on. In this study, human choriocarcinoma cells (jar) were used as the study model. OGA specific inhibitors were used to treat the cells and to establish a cell model with high glycosylation of O-G1cNAc. Then the cell migration assay (transwell method) was used to observe whether the migration ability of jar cells was affected. It was found that the increase of glycosylation and modification of O-G1cNAc protein promoted the migration of jar cells, and the adhesion between jar cells and umbilical vein endothelial cells (EA.hy926) was also enhanced. These results suggest that the degree of glycosylation of O-G1cNAc protein is related to migration and metastasis of choriocarcinoma. Then, the cell model of partial silencing of OGT gene was established by siRNA interference. Transwell and adhesion experiments were used to confirm that the level of glycosylation of O-G1cNAc protein was positively correlated with the migration and adhesion ability of jar cells. By using immunoprecipitation technique, it was found that the degree of O-GlcNAc glycosylation of 尾 -catenin, an important functional protein in the cells, increased with the increase of glycosylation level of O-G1cNAc in jar cells treated with OGA inhibitor. However, the expression of 尾-catenin in choriocarcinoma cells was not affected. In addition, we treated choriocarcinoma cells with IL-8 for 24 hours and detected the degree of glycosylation of OGT and O-G1cNAc by fluorescence quantitative western blot. The overall level of glycosylation of O-G1cNAc did not change, suggesting that the glycosylation modification of O-GlcNAc protein was not involved in the regulation of IL-8 promoting the metastasis of choriocarcinoma cells. In conclusion, OGT may promote the metastasis and adhesion of choriocarcinoma cells to endothelial cells by increasing the O-GlcNAc glycosylation level of 尾 -catenin, which may also be one of the molecular mechanisms of high metastasis of choriocarcinoma. This study provides a new target for the prevention and treatment of choriocarcinoma.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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