平分型N-糖链在卵巢癌腹腔转移中的作用机制研究

Implantation of ovarian cancer cells on peritoneal membrane is the most common form of ovarian cancer metastasis and the main couse of death. It is clear that clearance of peritoneal metastatic lesions may prolong the survival time of ovarian cancer patients.Recent findings demenstrated important roles of glycosylation on cancer metastasis. We thus investigated N-glycan profile of SKOV3 (serous ovarian cancer cell line) and its high metastatic derivative SKOV3 ip by means of MS spectrometry. We found lower bisecting N-glycan level on SKOV3 ip cells. Further qPCR analysis also verified this phenomenon by showing lower level of Mgat III in SKOV3 ip cells. Thus, it is probably that bisecting N-glycans inhibits peritoneal metastasis of ovarian cancer.To demonstrate this hypothesis, we will first construct SKOV3(Mgat III-) by interfering expression of Mgat III in SKOV3. Next, we will compare SKOV3(Mgat III-) with SKOV3 and SKOV3(vehicle) on cell shape, motility, expression of E-cadherin, N-cadherin, and Vementin to reveal EMT process; apoptosis rate after anchorage independent culture to reveal resistance to anoikis; adhesion to Matrigel; and metastasis in peritoneal of nude rat. To elucidate mechanism involved in inhibiting effects of bisecting N-glycan on ovarian cancer metastasis, we are planning to identify proteins attached with bisecting N-glycan on SKOV3 cells by means of lecting chromatography and MS spectrometry. Fuctional changes of these protein after modification by bisecting N-glycan and its impact on downstream signal pathways will be investigated as well, by means of Western Blot, cell immunochemistry, co-immunopricipitation, and RNAi. This project will provide evidence on metastatic inhibiting effects of bisecting N-glycan on ovarian cancer, and shining lights on the mechanism involve in this inhibitory effects. These data may be precious for designing of theraputic strategies targeting the metastatic process of ovarian cancer.

腹腔播散是上皮性卵巢癌最主要的转移方式。抑制腹腔转移对改善卵巢癌患者预后具有积极意义。近期研究表明细胞表面糖链结构在肿瘤粘附、转移、侵袭等方面发挥重要作用。我们前期通过质谱和qPCR比较卵巢癌SKOV3及其高转移株SKOV3ip细胞糖基化差异，发现SKOV3ip细胞中平分型N-糖链修饰减少，其特异性合成酶Mgat III明显下调。据此我们推测平分型-N糖链在卵巢癌腹腔转移中发挥抑制作用。我们拟干扰SKOV3细胞平分型N-糖链修饰，利用细胞免疫化学、Western Blot、流式细胞等技术考察细胞EMT、抗失巢凋亡、粘附以及在裸大鼠体内腹腔播散能力的改变；并利用色谱质谱鉴定携带有平分型N-糖链的细胞表面蛋白；探讨其细胞膜定位、配体结合能力以及相应信号通路活性与平分型N-糖链修饰的关系，最终阐明平分型N-糖链对卵巢癌转移能力的影响及其机制，为针对卵巢癌腹腔转移的靶向治疗策略研究提供基础。

腹腔播散是上皮性卵巢癌最主要的转移方式。抑制腹腔转移对改善卵巢癌患者预后具有积极意义。近期研究表明细胞表面糖链结构在肿瘤粘附、转移、侵袭等方面发挥重要作用。本课题前期通过质谱和qPCR比较卵巢癌SKOV3及其高转移株SKOV3ip细胞糖基化差异，发现平分型N-糖链及其特异性合成酶MGAT3在SKOV3ip细胞中明显下调。进一步我们利用Western blot和Lectin blot证实了这一发现。接着，我们通过调节卵巢癌细胞MGAT3表达而改变平分型N-糖链修饰水平，观察到细胞迁移能力与平分型N-糖链修饰水平明显负相关。因此我们认为平分型N-糖链抑制卵巢癌细胞迁移。为了探索这一抑制作用的机制，我们检测了与MGAT3密切相关的MGAT5及其催化产物—多分支N-糖链的表达水平。我们发现MGAT3能够抑制多分支N-糖链的修饰，但同时MGAT5表达水平也呈现SKOV3 ip高于SKOV3细胞的状态。因此，MGAT3抑制MGAT5催化多分支N-糖链合成可能是其抑制卵巢癌迁移的作用机制之一。MGAT5表达改变本身也参与了细胞迁移能力调节。上述发现为基于特殊N-糖链修饰的卵巢癌诊断技术和靶向治疗策略提供了基础。