2型固有淋巴细胞的极化效应在乳腺癌中的研究

Polarization effect of group 2 innate lymphoid cells in breast cancer

摘要:

目的：探讨2型固有淋巴细胞（group 2 innate lymphoid cells，ILC2）及其转录调控因子和产物在乳腺癌中的作用。方法：收集正常人和乳腺癌患者（早期和晚期）的外周血。分离所有受试者的外周血单个核细胞（peripheral blood mononuclear cell，PBMC），流式细胞术检测PBMC中ILC2的比例。从PBMC中提取总RNA，检测调控ILC2发育的特异性转录因子（GATA3和RORα）和ILC2产物（IL-13和IL-4）的基因表达水平，并检测血浆中Th2型细胞因子（IL-13、IL-4、IL-33和IL-10）的蛋白表达水平。用外源性IL-13和IL-4刺激MCF-7和MDA-MB 231癌细胞，以模拟乳腺癌患者体内ILC2的极化后效应：检测癌细胞的增殖活力；检测增殖及自噬信号分子蛋白表达的改变；流式细胞术和荧光显微镜观察细胞凋亡情况。结果：乳腺癌患者外周循环中的ILC2水平增高（正常组与早期组：0.019 6% vs. 0.028 9%，P=0.008；正常组与晚期组：0.019 6% vs. 0.027 3%，P=0.032）；雌/孕激素表达状态是影响ILC2水平的重要因素（HR=0.436，P=0.038）。GATA3、RORα、IL-13、IL-4、IL-33和IL-10的表达随着肿瘤负荷的加重而增加（P<0.05）。外源性IL-13和IL-4联合刺激可通过上调ERK 1/2和pERK 1/2来增强乳腺癌细胞的增殖活力（P=0.000）；联合刺激可诱导MCF-7和MDA-MB 231癌细胞的自噬信号分子表达（P=0.000），还可诱导MDA-MB 231癌细胞的凋亡（P=0.000），但对MCF-7细胞凋亡无明显诱导作用（P>0.05）。结论：ILC2在外周循环中比例增高和ILC2上下游因子表达增高与乳腺癌的发生发展有关。

Abstract:

Objective：To investigate the role of group 2 innate lymphoid cells（ILC2） and its transcription factors and products in the breast cancer（BC）. Methods：The peripheral blood from healthy subjects and BC patients（at early and advanced stage） was collect－ed. The peripheral blood mononuclear cell（PBMC） was isolated and the level of ILC2 in PBMC was detected by flow cytometry. Total RNA was extracted from PBMC. The gene expression level of specific transcription factor（GATA3 and RORα） and ILC2 production （IL-13 and IL-4），which regulated ILC2 development，was detected. The protein expression of T-helper 2 cytokine in serum（IL-13，IL-4，IL-33 and IL-10） were detected. Exogenous IL-13 and IL-4 were used to stimulate MCF-7 and MDA-MB 231 cancer cells，so as to simulate the polarization effect of ILC2 in BC patients：detecting the proliferation activity of cancer cells，detecting changes of protein expression of proliferation and autophagy signal factors；observing cell apoptosis via flow cytometry and fluorescence micro－scope. Results：ILC2 level in peripheral circulation of BC patients was increased（normal group vs. early stage group，0.019 6% vs. 0.028 9%，P=0.008；normal group vs. advanced stage group，0.019 6% vs. 0.027 3%，P=0.032）. Estrogen/progesterone expression status was an important factor influencing ILC2 level（HR=0.436，P=0.038）. The expression of GATA3，RORα，IL-13，IL-4，IL-33 and IL-10 was increased with tumor burden aggravation（P<0.05）. Com－bined stimulation of exogenous IL-13 and IL-4 was able to en－hance the proliferation viability of BC cells by up-regulating ERK1/2 and pERK1/2（P=0.000）. Combined stimulation was able to induce the expression of autophagy signaling molecules of MCF-7 and MDA-MB 231 cells（P=0.000） and to induce the apoptosis of MDA-MB 231 cells（P=0.000），but it had no obvious induction for the apoptosis of MCF-7 cells（P>0.05）. Conclusion：The increased proportion of ILC2 in peripheral circulation and the increased expression of up-and-down-stream factors of ILC2 is re－lated to the oncogenesis and development of BC.