具核梭杆菌感染对结肠癌转化生长因子-β1表达及肿瘤浸润淋巴细胞的影响

Effects of Fusobacterium nucleatum infection on TGF-β1 expression and tumor-infiltrating lymphocytes in colon cancer

目的探讨具核梭杆菌(Fusobacterium nucleatum,Fn)感染对结肠癌转化生长因子-β1(transforming growth factor-β1,TGF-β1)表达和肿瘤浸润淋巴细胞(tumor infiltrating lymphocytes,TIL)的影响。方法qPCR和IHC检测结肠癌组织Fn表达和TGF-β1与FoxP3表达;ELISA、qPCR、流式细胞术检测Fn感染对结肠癌细胞TGF-β1表达、TIL、及FoxP3 mRNA的影响。结果25例癌组织表达Fn,其Fn丰度高于癌旁组织,且TGF-β1与FoxP3表达正相关(P<0.05)。Fn感染使结肠癌细胞TGF-β1分泌升高及mRNA上调。活化CD4^+T细胞与Fn感染的HCT116细胞共培养,CD4^+T细胞增殖较对照组降低,加入TGF-β1抗体结果逆转;初始CD4^+T细胞与Fn感染的HCT116共培养,FoxP3^+Treg增加,FoxP3 mRNA上调,加入TGF-β1抗体结果逆转(P<0.05)。结论具核梭杆菌可能通过上调结肠癌细胞TGF-β1表达,产生局部免疫抑制效应,介导肿瘤细胞免疫逃逸。

Objective To investigate the effects of Fusobacterium nucleatum(Fn)infection on the expression of TGF-β1 and tumor infiltrating lymphocytes(TIL)s in colon cancer tissues.Methods qPCR and IHC were used to measure the expression of Fn in colon cancer tissues and determine TGF-β1 expression and Foxp3+Tregs of cancer tissues.The expression levels of TGF-β1 in 3 types of colon cancer cells were detected by ELISA and qPCR.Flow cytometry(FCM)and qPCR were respectively applied to measure CD4^+T cell proliferation and FoxP3+Tregs and FoxP3 mRNA in the conditions of Fn infection.Results Fn was found in 25 cancer tissues using qPCR.Among the 25 patients who had positive Fn expression,the abundance of Fn in cancer tissues was significantly higher than that in adjacent normal tissues.IHC demonstrated TGF-β1 expression was correlated with FoxP3 expression in cancer tissues infected with Fn(P<0.05).TGF-β1 expression excreted from colon cancer cells with Fn infection increased and TGF-β1 mRNA expression was upregulated,when compared with the control groups.FCM analysis showed that the proliferation of activated CD4^+T cells reduced in the presence of Fn-CM than that of the CM control group when in coculture with Fn-treated HCT116.The inhibitory effect of Fn-CM was partially reversed after adding anti-TGF-β1 neutralizing antibodies,and proliferation rates of activated CD4^+T cells was increased.Addition of Fn-CM to cultures resulted in a strong generation of Tregs,with CD4^+CD25^+FoxP3^+Treg and FoxP3 mRNA increased compared to the level for the CM control group,whereas neutralization of TGF-β1 in Fn-CM-treated cultures resulted in reversed results(P<0.05).Conclusion Fn infection can inhibit immune response by upregulating the expression of TGF-β1 in colon cancer,which probably facilitated tumor immune escape in colon cancer.