PKC参与阿霉素上调PD-L1介导的免疫抑制促进骨肉瘤耐药的机制研究

Chemoresistance is the main cause of treatment failure of osteosarcoma, and immune escape and chemoresistance is closely related. Programmed death receptor 1 (PD-L1) is one of the major proteins mediating immune escape. Our previous study found that doxorubicin will significantly increase the PD-L1 expression in osteosarcoma cells, and inhibit the proliferation and function of lymphocytes through the PD-L1/PD-1 pathway, which suggested that chemotherapy can induce tumor immunosuppression microenvironment. PD-L1 antibody combined with doxorubicin can increase the sensitivity of chemotherapy in the subcutaneous tumor implant model. However, PD-L1 antibody can only block the membrane PD-L1 mediated immune related chemoresistance , and cannot eliminate the chemoresistance mediated by the upregulation of PD-L1 in the nucleus. Therefore, unveiling the mechanism of how chemotherapy could increase PD-L1 will provide new ideas and targets for the treatment of osteosarcoma. This project will demonstrate the function of doxorubicin induced PD-L1 in immune escape and chemoresistance through tibial in situ lung metastasis osteosarcoma model; and explore the mechanism that how doxorubicin induces expression of PD-L1 through protein kinase C (PKC) with the help of gene chip and gene interaction network analysis. This project will also provide a theoretical basis on the immunosuppression induced by chemotherapy, and to provide new ideas and experimental basis for the clinical treatment of osteosarcoma.

化疗耐药是骨肉瘤治疗失败的主要原因，免疫逃逸和耐药关系密切，程序性死亡受体1（PD-L1）是介导免疫逃逸的主要蛋白之一。我们前期研究发现，骨肉瘤一线化疗药物阿霉素会显著上调肿瘤细胞PD-L1的表达，并通过PD-L1/PD-1通路抑制淋巴细胞的增殖和功能，提示化疗会诱导肿瘤免疫抑制微环境的产生；PD-L1抗体联合阿霉素能够增加骨肉瘤皮下模型对化疗的敏感性。但是PD-L1抗体只能阻断细胞膜上PD-L1介导的免疫相关耐药，并不能消除细胞核内上调的PD-L1介导的耐药。因此，明确化疗药物上调PD-L1的机制，将为骨肉瘤的治疗提供新的思路和靶点。本项目将通过胫骨原位骨肉瘤模型，探讨阿霉素诱导的PD-L1在免疫逃逸和耐药中的作用；并借助基因芯片和基因互做网络分析，探索蛋白激酶C（PKC）参与阿霉素上调PD-L1的机制，为化疗诱导骨肉瘤的免疫抑制提供理论基础，并为临床骨肉瘤的治疗提供新的思路和实验依据。

化疗耐药是骨肉瘤治疗失败的主要原因，免疫逃逸和耐药关系密切，程序性死亡受体1（PD-L1）是介导免疫逃逸的主要蛋白之一。我们的研究发现，骨肉瘤一线化疗药物阿霉素会显著上调肿瘤细胞PD-L1的表达，并通过PD-L1/PD-1通路抑制CD8+ T淋巴细胞的增殖和功能，提示化疗会诱导肿瘤免疫抑制微环境的产生。在骨肉瘤的动物模型中，我们发现PD-L1抗体联合阿霉素能够抑制皮下肿瘤的生长，增加肿瘤对化疗药物的敏感性。.在机制探讨中，我们发现miRNA-200能够调控肿瘤细胞中PD-L1的表达，过表达miRNA-200能够上调PD-L1的表达，而敲低miRNA-200能够下调PD-L1的表达。我们进一步证实了，阿霉素能够影响miRNA-200的表达，且阿霉素是通过调控miRNA-200来影响PD-L1表达的。通过荧光素酶实验，我们发现miRNA-200的结合靶点是PTEN，并进一步验证了miRNA-200是通过调节PTEN来调控PD-L1的表达的。.综上所述，我们的研究发现，阿霉素能够正向调节miRNA-200的表达，miRNA-200则可以反向抑制PTEN的表达来调控PD-L1的表达，从而影响CD8+ T淋巴细胞的功能，并诱导肿瘤免疫抑制微环境的产生，产生对化疗药物的耐药。

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