雌激素联合miR-20a促进卵巢癌细胞MICA/B表达并介导其免疫逃逸的机制研究

Ovarian cancer is the second most common gynecological malignancy followed by endometrial tumor in the femal population. A large number of studes have shown that ovarian cancer cells have developed strategies to successfully evade immune surveillance by secreting a variety of immune inhibitory molecule, such as soluble human MHC class I chain-related genes MICA and MICB (sMICA/B), which can directly block or downregulate NKG2D expression on NK cells and γδT cells and inhibit their anti-tumor cytotoxicity.Earlier studies have indicated that estrogen could upregulate of MICA expression on endometrial cells, but the exact mechanisms were unknown. In the present study, the E2, MICA, and MICB concentrations were measured in healthy individuals (18 cases) and ovarian cancer patients (24 cases). The MICA/B levels in ovarian cancer patients were also higher compared with those in healthy (Figure 4). No correlation was observed between MK and MICA/B expression in the healthy donors . However, the expression of MICA and MICB was positively correlated with the E2 levels in cancer patients. But, No estrogen response element was identified in the MICA / B promoter region, indicating that estrogen can not directly regulate the MICA / B expression. The microRNA microarray assay shows different microRNA expression profiles in ovarian cancer tissues and their corresponding adjacent normal tissue (Figure 2). Significant differences in microRNA expression were found, including miR-20a, which was complementary to a region in the 3' untranslated region [3'-UTR] of the MICA/B mRNA (Figure 3). Further preliminary experiments in vitro suggested that estradiol could decrease the expression of miR-20a and increase the mRNA expression of MICA / B (Figure 6) and matrix metalloproteinase protease, which the exact mechanism was unknown. Overall, these studies strongly suggest that estrogen may inhibit miR-20a expression by the estrogen receptor, increase the MICA / B expression by miR-20a, and further increase soluble MICA / B in serum to successfully evade the cytotoxicity of immune cells.

卵巢癌是妇科三大恶性肿瘤之一。卵巢癌细胞可分泌多种免疫抑制分子来逃脱免疫监视，sMICA/B就是其中之一。sMICA/B可以通过降解免疫细胞活化受体NKG2D而抑制其抗肿瘤作用。研究报道，雌激素能增加子宫内膜细胞MICA表达，但具体机制不明。我们发现，卵巢癌患者血清中雌二醇与sMICA/B正相关，与淋巴细胞NKG2D受体负相关，但MICA/B启动子区没有雌激素反应元件，提示雌激素可能不直接调控MICA/B表达。分析卵巢癌及对应癌旁组织miRNA芯片发现，癌组织中miR-20a显著下降，且可与MICA/B mRNA的3'UTR区序列互补。预实验还发现，雌二醇刺激卵巢癌细胞后，miR-20a表达下降，MICA/B表达上升，但具体机制不明。这些强烈提示，雌激素可能通过雌激素受体抑制miR-20a表达，增加MICA/B表达，在基质金属蛋白酶作用下sMICA/B升高，抑制免疫细胞活性，发生免疫逃逸。

卵巢癌是妇科三大恶性肿瘤之一。卵巢癌细胞可通过分泌多种免疫抑制分子，使机体抗瘤免疫应答处于低能状态，从而使肿瘤细胞逃逸免疫监视而发展。我们分析了恶性卵巢癌（39例）、卵巢囊肿(10例)和健康同龄段（20例）女性外周血中免疫细胞及其亚群：与对照组相比，卵巢癌患者Treg细胞水平升高，Th1水平和CD8+ T细胞表面的活化受体NKG2D均明显减少（P<0.05），患者外周血中成熟DC水平降低，而浆细胞样DC升高，且升高程度与病理分级有关（P>0.05）。且与外周血相比，腹水中NK细胞的CD16+和CD8+ T细胞的NKG2D受体水平均下降，它们免疫功能都受到明显抑制，且这种抑制可能部分与DC细胞亚群变化有关。与对照组相比，卵巢癌患者血清中miR20a、miR21、miR222和miR142-3p表达水平显著高于健康对照组，在III-IV 级卵巢癌患者血清中miR21水平高于I-II级，高级别患者血清中miR142-3p水平低于低级别的患者（p<0.05），而且，患者腹水中miR21和miR222高于外周血中（p<0.05）。.根据基因序列比对，我们发现miR20a可以与NKG2D配体MICA/B的3’端UTR区结合。随后在卵巢癌细胞株SKOV3和ES2上和荷瘤鼠模型上进一步进行了miRNA-20a对MICA/B的分子调控机制和免疫逃逸机制研究。结果验证了我们的设想：miRNA-20a可以抑制卵巢癌细胞中MICA/B的表达，从而抑制了NK细胞抗肿瘤免疫功能。研究还发现，雌激素不仅可以调节MICA/B的转录，诱导ADAM17的表达和活性，并促进癌细胞表面MICA/B的脱落，从而抑制NK细胞表面NKG2D介导的细胞杀伤活性。