下调PTEN促进肝癌抑制性肿瘤免疫微环境形成

Downregulation of PTEN expression promotes immunosuppressive microenvironment in hepatocellular carcinoma

目的探索PTEN在肝细胞癌免疫微环境形成中的作用。方法实验小鼠C57BL/6被随机分为PTEN小干扰RNA(small interfering RNA,siRNA)组和siRNA阴性对照(negative control,NC)组,构建小鼠皮下移植瘤模型,并在小鼠皮下移植瘤中分别注射PTEN siRNA及siRNA NC。应用real-time qPCR和免疫组织化学法(immunohistochemistry,IHC)检测移植瘤中PTEN的表达。应用IHC检测移植瘤中CD8^(+)T淋巴细胞和Foxp3^(+)T淋巴细胞的浸润情况、PD-L1、IDO和ICOS的表达;应用免疫荧光法(immunofluorescence,IF)检测CD4^(+)CD25^(+)T淋巴细胞浸润情况。同时,分析癌症基因组图谱(The Cancer Genome Atlas,TCGA)中Foxp3、PD-L1、IDO、ICOS、IFNAR1和IFNAR2的表达。结果与siRNA NC组相比,PTEN siRNA组中PTEN在mRNA和蛋白水平表达明显下调(P<0.05)。PTEN siRNA组小鼠移植瘤的生长速率显著高于siRNA NC组(P<0.05)。与siRNA NC组相比,PTEN siRNA组的移植瘤中,CD8^(+)T淋巴细胞浸润明显减少(P<0.05),而Foxp3^(+)和CD4^(+)CD25^(+)T淋巴细胞明显增加(P<0.05)。此外,与siRNA NC相比,PD-L1、IDO和ICOS在PTEN siRNA组的移植瘤中表达升高(P<0.05)。最后,TCGA数据提示PTEN低表达组中PD-L1、IDO和Foxp3的表达高于PTEN高表达组,而IFNAR1和IFNAR2在PTEN低表达组中的表达显著低于PTEN高表达组(P<0.05)。结论下调PTEN促进肝癌抑制性肿瘤免疫微环境的形成。

Objective To explore the role of PTEN in immune microenvironment in hepatocellular carcinoma.Methods The C57BL/6 mice were randomly assigned to PTEN siRNA group and siRNA NC group.Allogeneic subcutaneous tumor model was established in both groups.Allogeneic subcutaneous tumors were injected with PTEN siRNA and siRNA NC,respectively.The expression of PTEN in subcutaneous tumors was detected by real-time PCR and immunohistochemistry.The infiltration of CD8^(+)T lymphocytes and Foxp3^(+)T lymphocytes,and the expression of PD-L1,IDO and ICOS in tumors in PTEN siRNA group and siRNA NC group were detect-ed by immunohistochemistry.The infiltration of CD4^(+)CD25^(+)T lymphocytes in tumors was detected in PTEN siRNA group and siRNA NC group by immunofluorescence.Meanwhile,the expression of Foxp3,PD-L1,IDO,ICOS,IFNAR1 and IFNAR2 in the Cancer Genome Atlas(TCGA)was also analyzed.Results The growth rate of subcutaneous tumors in PTEN siRNA group was statistically higher than that in siRNA NC group(P<0.05).The number of tumor-infiltrating CD8^(+)T lymphocytes was significantly lower in PTEN siRNA group than that in siRNA NC group,while the numbers of tumor-infiltrating Foxp3^(+)and CD4^(+)CD25^(+)T lymphocytes were signi-ficantly higher in subcutaneous tumors in PTEN siRNA group than those in siRNA NC group(P<0.05).Compared with siRNA NC group,the expression levels of PD-L1,IDO and ICOS were increased in subcutaneous tumors in PTEN siRNA group(P<0.05).TCGA data suggested that the expression of PD-L1,IDO,and Foxp3 was higher,while the expression of IFNAR1 and IFNAR2 was statistically lower in patients with low PTEN expression than that in patients with high PTEN expression(P<0.05).Conclusion Downregulation of PTEN expression promotes the suppressive immune microenvironment in hepatocellular carcinoma.