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    上调A2AR影响JAK1/STAT3通路增强结直肠癌细胞干性并诱导巨噬细胞M2极化

    Upregulation of A2AR affecting the JAK1/STAT3 pathway to enhance the stemness of colorectal cancer cells and induce M2 polarization of macrophages

      摘要
    • 摘要:
      目的 研究腺苷受体A2A（A2AR）的表达对结直肠癌（CRC）细胞干性和巨噬细胞极化的影响及相关分子机制。
      方法 选取40例CRC病人术中切除的病理组织，通过免疫组织化学染色检测癌组织和癌旁组织中A2AR表达差异。培养人CRC细胞系SW480、HCT116、Caco–2、SW620、LoVo以及正常结肠上皮细胞系NCM460，Western blotting检测A2AR表达水平。通过慢病毒转染细胞的方法构建稳定敲低A2AR的HCT116细胞株（shA2AR–HCT116）及其阴性对照细胞株（shA2AR–NC–HCT116）、稳定过表达A2AR的SW480细胞株（A2AR–OE–SW480）及其阴性对照细胞株（A2AR–OENC–SW480），Western blotting检测各组细胞JAK1/STAT3通路蛋白、细胞干性相关蛋白（CD133、OCT4、SOX2）表达水平，采用肿瘤细胞成球实验比较各组细胞成球数量。选取人单核–巨噬细胞系THP–1，使用佛波酯诱导THP–1细胞分化为M0巨噬细胞。将A2AR–OENC组和A2AR–OE组细胞培养上清液分别与M0巨噬细胞进行共培养，M0巨噬细胞分组为A2AR–OENC–SW480上清组、A2AR–OE–SW480上清组，采用流式细胞术检测各组巨噬细胞极化情况。
      结果 CRC癌组织中A2AR表达水平明显高于癌旁组织（P ＜ 0.01）；与NCM460细胞相比，CRC细胞SW480、HCT116、Caco–2、SW620、LoVo中A2AR表达水平均明显增加（P ＜ 0.05）。与shA2AR–NC–HCT116细胞相比，shA2AR–HCT116细胞p–JAK1、p–STAT3、OCT4、SOX2表达水平均降低，成球数量减少（P ＜ 0.01）；与A2AR–OENC–SW480细胞相比，A2AR–OE–SW480细胞p–JAK1、p–STAT3、OCT4、SOX2表达水平均升高（P ＜ 0.01），成球数量增加。与A2AR–OENC–SW480上清组比较，A2AR–OE–SW480上清组巨噬细胞M1极化减少，M2极化增加（P＜0.01）。
      结论 A2AR在CRC组织和细胞系中均表达上调，并通过影响JAK1/STAT3通路增强CRC细胞干性，诱导巨噬细胞M2极化增加，促进肿瘤免疫逃逸。

       

      Abstract:
      Objective To investigate the effect of adenosine receptor A2A (A2AR) expression on the stemness and macrophage polarization of colorectal cancer(CRC) cells and its related molecular mechanisms.
      Methods The pathological tissues resected during the operation from 40 CRC patients were selected, and the expression differences of A2AR in cancer tissues and adjacent tissues were detected by immunohistochemical staining. The human CRC cell lines SW480, HCT116, Caco-2, SW620, LoVo and normal colonic epithelial cell line NCM460 were cultured, and the expression levels of A2AR were detected by Western blotting. The HCT116 cell line with knockdown of A2AR(shA2AR-HCT116) and its negative control cell line(shA2AR-NC-HCT116) and SW480 cell line with stable overexpression of A2AR(A2AR-OE-SW480) and its negative control cell line (A2AR-OENC-SW480) were constructed by lentivirus transfection of cells. Western blotting was used to detect the expression levels of JAK1/STAT3 pathway proteins and cell stemness-related proteins(CD133, OCT4, SOX2) in each group of cells. The tumor cell spheroidization experiment was used to compare the number of spheroidized cells in each group. The human mononuclear-macrophage cell line THP-1 was selected, and the phorbol ester was used to induce the differentiation of THP-1 cells into M0 macrophages. The cell culture supernatants of the A2AR-OENC group and A2AR-OE group were co-cultured with M0 macrophages, respectively. The M0 macrophages were divided into the A2AR-OENC-SW480 supernatant group and A2AR-OE-SW480 supernatant group. The polarization of macrophages in each group was detected by flow cytometry.
      Results The expression level of A2AR in CRC cancer tissue was significantly higher than that in adjacent tissues(P ＜ 0.01); Compared with NCM460 cells, the expression levels of A2AR in CRC cells SW480, HCT116, Caco-2, SW620 and LoVo significantly increased(P ＜ 0.05). Compared with shA2AR-NC-HCT1116 cells, the expression levels of p-JAK1, p-STAT3, OCT4, and SOX2 in shA2AR-HCT116 cells decreased, and the number of spheroids was reduced(P ＜ 0.01); Compared with A2AR-OENC-SW480 cells, the expression levels of p-JAK1, p-STAT3, OCT4, and SOX2 in A2AR-OE-SW480 cells increased(P ＜ 0.01), and the number of spheroids increased. Compared with the A2AR-OEC-SW480 supernatant group, the M1 polarization of macrophages in the A2AR-OEC-SW480 supernatant group decreased, while the M2 polarization increased(P ＜ 0.01).
      Conclusions A2AR is upregulated in both CRC tissues and cell lines, and enhances the stemness of CRC cells by affecting the JAK1/STAT3 pathway, induces an increase in M2 polarization of macrophages, and promotes tumor immune escape.

       

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