肝细胞癌中PD-L1表达与STAT3/PRKDC/MYC信号通路的关系研究

Relationship between PD-L1 expression and the STAT3/PRKDC/MYC signaling pathway in hepatocellular carcinoma

摘要:

背景与目的 目前，肝细胞癌（HCC）的治疗仍面临复发和转移的严峻挑战，而肿瘤免疫逃逸是导致这些问题的关键机制之一。信号转导与转录激活因子3（STAT3）作为重要的转录因子，在多种恶性肿瘤中呈现过度激活状态，不仅参与肿瘤的发生与进展，还与肿瘤免疫逃逸密切相关。程序性死亡配体1（PD-L1）是关键的免疫检查点，其表达上调能够帮助肿瘤细胞逃避免疫监视，从而抑制抗肿瘤免疫。研究显示，STAT3可能通过与蛋白激酶DNA激活催化多肽（PRKDC）的相互作用激活骨髓细胞瘤病毒癌基因（MYC）信号通路，进而促进PD-L1的表达并诱导免疫逃逸。然而，STAT3/PRKDC/MYC轴在HCC中的具体作用机制尚不明确。本研究旨在揭示STAT3通过PRKDC/MYC信号通路调控PD-L1表达并可能诱导HCC免疫逃逸的分子机制，以期为HCC免疫治疗提供潜在靶点。方法 用qRT-PCR和Western blot检测人正常肝细胞（HL-7702）与人HCC细胞（HuH-7、HepG2）中STAT3的表达。构建敲低STAT3（si-STAT3）和过表达PRKDC（oe-PRKDC）质粒，以及各自的阴性对照（si-NC、oe-NC）质粒，按实验设计分别转染至HCC细胞（HuH-7）中，以无处理的HuH-7细胞为空白对照。采用Western blot分析各组细胞STAT3、PRKDC、PD-L1和MYC通路相关蛋白的表达。采用CCK-8、Transwell、划痕试验和流式细胞术评估HCC细胞的增殖、侵袭、迁移和凋亡。将HuH-7细胞与人外周血单个核细胞（hPBMC）共培养后，采用ELISA法检测免疫调节因子干扰素γ（IFN-γ）的含量。采用免疫共沉淀和免疫荧光共定位验证STAT3与PRKDC蛋白之间的相互作用。结果 qRT-PCR和Western blot结果显示，HCC细胞中STAT3的mRNA和蛋白表达水平均明显升高（均P<0.05）。功能实验结果显示，si-STAT3组HCC细胞的增殖、迁移和侵袭能力明显减弱，细胞凋亡明显升高；PD-L1和MYC通路相关蛋白的表达水平明显下调；与hPBMC共培养后IFN-γ的分泌水平明显升高（均P<0.05）。与oe-PRKDC质粒共培养后，STAT3敲低对HCC细胞的以上影响均被明显逆转（均P<0.05）。Scansite 4.0数据库分析结果显示，STAT3与PRKDC存在结合位点，免疫共沉淀与免疫荧光共定位实验表明STAT3与PRKDC蛋白的相互作用。结论 STAT3在HCC细胞中高表达，并可通过与PRKDC相互作用促进HCC细胞增殖、迁移和侵袭以及免疫逃逸，抑制细胞凋亡，并激活MYC通路，增加PD-L1表达，STAT3/PRKDC/MYC轴可能是HCC免疫治疗的潜在靶点。

Abstract:

Background and Aims Currently, the treatment of hepatocellular carcinoma (HCC) faces significant challenges due to recurrence and metastasis, with tumor immune evasion being one of the key mechanisms underlying these issues. Signal transducer and activator of transcription 3 (STAT3), an important transcription factor, is overactivated in many malignancies and is involved in both tumorigenesis and progression, closely associated with immune evasion. Programmed cell death ligand 1 (PD-L1), a key immune checkpoint, helps tumor cells evade immune surveillance when its expression is upregulated, thereby suppressing anti-tumor immunity. Studies have shown that STAT3 may activate the MYC signaling pathway through interaction with DNA-activated protein kinase (PRKDC), thereby promoting PD-L1 expression and inducing immune evasion. However, the specific mechanism of the STAT3/PRKDC/MYC axis in HCC remains unclear. This study aims to elucidate the molecular mechanism by which STAT3 regulates PD-L1 expression through the PRKDC/MYC signaling pathway, potentially inducing immune evasion in HCC, with the goal of providing potential targets for HCC immunotherapy.Methods The expressions of STAT3 in human normal liver cells (HL-7702) and human HCC cells (HuH-7, HepG2) were detected by qRT-PCR and Western blot. Plasmids with STAT3 knockdown (si-STAT3) and PRKDC overexpression (oe-PRKDC), along with their respective negative controls (si-NC, oe-NC), were constructed and transfected into HCC cells (HuH-7) according to the experimental design, with untreated HuH-7 cells as the blank control. Western blot was used to analyze the expression of STAT3, PRKDC, PD-L1, and MYC pathway-related proteins. Cell proliferation, invasion, migration, and apoptosis of HCC cells were assessed by CCK-8, Transwell, wound healing assay, and flow cytometry. After co-culturing HuH-7 cells with human peripheral blood mononuclear cells (hPBMCs), ELISA was used to detect the secretion of the immune regulatory factor interferon γ (IFN-γ). Co-immunoprecipitation and immunofluorescence co-localization were performed to verify the interaction between STAT3 and PRKDC proteins.Results Results of qRT-PCR and Western blot showed that the mRNA and protein levels of STAT3 were significantly elevated in HCC cells (both P<0.05). Functional experiments demonstrated that in the si-STAT3 group, HCC cell proliferation, migration, and invasion were significantly weakened, and cell apoptosis was notably increased; the expression of PD-L1 and MYC pathway-related proteins was significantly downregulated; the secretion of IFN-γ was significantly increased after co-culturing with hPBMCs (all P<0.05). After co-culturing with oe-PRKDC plasmids, the effects of STAT3 knockdown on HCC cells were significantly reversed (all P<0.05). Scansite 4.0 database analysis revealed that STAT3 and PRKDC have binding sites, and co-immunoprecipitation and immunofluorescence co-localization experiments confirmed the interaction between STAT3 and PRKDC proteins.Conclusion STAT3 is highly expressed in HCC cells and can promote HCC cell proliferation, migration, invasion, and immune evasion through interaction with PRKDC, suppress cell apoptosis, activate the MYC pathway, and increase PD-L1 expression. The STAT3/PRKDC/MYC axis may serve as a potential target for HCC immunotherapy.