Abstract：
OBJECTIVE: We aimed to explore the effects and mechanisms of action of dehydroepiandrosterone (DHEA) on immune evasion of oral squamous cell carcinoma (OSCC) to provide evidence for enhancing the effect of immunotherapy.
METHODS: A xenograft mouse model and immunohistochemistry were used to reveal the patterns of tumor-infiltrating lymphocytes (TILs). The CAL27 and SCC VII cell lines were used for the in vitro study. Western blotting, qPCR, immunofluorescence, and flow cytometry were used to evaluate the expression of B7-H4. Recombinant mouse B7-H4 protein (rmB7-H4) and PG490, an inhibitor of NF-κB p65 were used for the \"rescue study.\" Gain- and loss-of-function, luciferase reporter, and chromatin immunoprecipitation assays were performed to verify this mechanism.
RESULTS: DHEA inhibited tumor growth in an OSCC xenograft mouse model, increased CD8 + cells, and decreased FOXP3 + cells in TILs. DHEA reduced the expression of B7-H4 in CAL27 and SCC VII cells RmB7-H4 reverses the effect of DHEA on tumor growth and TIL patterns. DHEA increased the expression of miR-15b-5p and activated its transcriptional factor NF-κB p65. Further experiments demonstrated that miR-15b-5p inhibited B7-H4 expression by binding to its 3\'-UTR regions, and NF-κB p65 activated miR-15b transcription. PG490 reversed the effects of DHEA on tumor growth, antitumor immunity in the OSCC xenograft model, and the expression/phosphorylation of NF-κB p65, miR-15b-5p, and B7-H4.
CONCLUSIONS: This study indicates that DHEA attenuates the immune escape of OSCC cells by inhibiting B7-H4 expression, providing new insights for cancer immunotherapy.
METHODS: A xenograft mouse model and immunohistochemistry were used to reveal the patterns of tumor-infiltrating lymphocytes (TILs). The CAL27 and SCC VII cell lines were used for the in vitro study. Western blotting, qPCR, immunofluorescence, and flow cytometry were used to evaluate the expression of B7-H4. Recombinant mouse B7-H4 protein (rmB7-H4) and PG490, an inhibitor of NF-κB p65 were used for the \"rescue study.\" Gain- and loss-of-function, luciferase reporter, and chromatin immunoprecipitation assays were performed to verify this mechanism.
RESULTS: DHEA inhibited tumor growth in an OSCC xenograft mouse model, increased CD8 + cells, and decreased FOXP3 + cells in TILs. DHEA reduced the expression of B7-H4 in CAL27 and SCC VII cells RmB7-H4 reverses the effect of DHEA on tumor growth and TIL patterns. DHEA increased the expression of miR-15b-5p and activated its transcriptional factor NF-κB p65. Further experiments demonstrated that miR-15b-5p inhibited B7-H4 expression by binding to its 3\'-UTR regions, and NF-κB p65 activated miR-15b transcription. PG490 reversed the effects of DHEA on tumor growth, antitumor immunity in the OSCC xenograft model, and the expression/phosphorylation of NF-κB p65, miR-15b-5p, and B7-H4.
CONCLUSIONS: This study indicates that DHEA attenuates the immune escape of OSCC cells by inhibiting B7-H4 expression, providing new insights for cancer immunotherapy.
摘要：
目的：探讨脱氢表雄酮(DHEA)在口腔鳞状细胞癌(OSCC)免疫逃逸中的作用及其机制,为提高免疫治疗效果提供依据。
方法：使用异种移植小鼠模型和免疫组织化学来揭示肿瘤浸润淋巴细胞（TIL）的模式。CAL27和SCCVII细胞系用于体外研究。西方印迹,qPCR,免疫荧光，流式细胞仪检测B7-H4的表达。重组小鼠B7-H4蛋白（rmB7-H4）和NF-κBp65抑制剂PG490用于“拯救研究”。“功能的获得和丧失，荧光素酶报告基因,和染色质免疫沉淀试验来验证这种机制。
结果：DHEA在OSCC异种移植小鼠模型中抑制肿瘤生长，CD8+细胞增加，TIL中FOXP3+细胞减少。DHEA降低CAL27和SCCVII细胞中B7-H4的表达RmB7-H4逆转了DHEA对肿瘤生长和TIL模式的影响。DHEA增加miR-15b-5p的表达并激活其转录因子NF-κBp65。进一步的实验表明，miR-15b-5p通过结合其3'-UTR区抑制B7-H4的表达，和NF-κBp65激活miR-15b转录。PG490逆转了DHEA对肿瘤生长的影响，OSCC异种移植模型中的抗肿瘤免疫，和NF-κBp65，miR-15b-5p的表达/磷酸化，还有B7-H4.
结论：本研究表明，DHEA通过抑制B7-H4表达减弱OSCC细胞的免疫逃逸，为癌症免疫治疗提供新的见解。
方法：使用异种移植小鼠模型和免疫组织化学来揭示肿瘤浸润淋巴细胞（TIL）的模式。CAL27和SCCVII细胞系用于体外研究。西方印迹,qPCR,免疫荧光，流式细胞仪检测B7-H4的表达。重组小鼠B7-H4蛋白（rmB7-H4）和NF-κBp65抑制剂PG490用于“拯救研究”。“功能的获得和丧失，荧光素酶报告基因,和染色质免疫沉淀试验来验证这种机制。
结果：DHEA在OSCC异种移植小鼠模型中抑制肿瘤生长，CD8+细胞增加，TIL中FOXP3+细胞减少。DHEA降低CAL27和SCCVII细胞中B7-H4的表达RmB7-H4逆转了DHEA对肿瘤生长和TIL模式的影响。DHEA增加miR-15b-5p的表达并激活其转录因子NF-κBp65。进一步的实验表明，miR-15b-5p通过结合其3'-UTR区抑制B7-H4的表达，和NF-κBp65激活miR-15b转录。PG490逆转了DHEA对肿瘤生长的影响，OSCC异种移植模型中的抗肿瘤免疫，和NF-κBp65，miR-15b-5p的表达/磷酸化，还有B7-H4.
结论：本研究表明，DHEA通过抑制B7-H4表达减弱OSCC细胞的免疫逃逸，为癌症免疫治疗提供新的见解。
