Background: Astragaloside IV (AS-IV) is a pivotal contributor to anti-tumour effects and has garnered extensive attention in research. Tumour cell immune suppression is closely related to the increase in Programmed Death-Ligand 1 (PD-L1). Hepatocellular carcinoma (HCC) is a malignant tumour originating from hepatic epithelial tissue, and the role of AS-IV in regulating PD-L1 in anti-HCC activity remains unclear. Methods: Various concentrations of AS-IV were administered to both human liver immortalised cells (THEL2) and HCC (Huh-7 and SMMC-7721), and cell growth was assessed using the CCK-8 assay. HCC levels and cell apoptosis were examined using flow cytometry. Mice were orally administered AS-IV at different concentrations to study its effects on HCC in vivo. Immunohistochemistry was employed to evaluate PD-L1 levels. Western blotting was employed to determine PD-L1 and CNDP1 protein levels. We carried out a qRT-PCR to quantify the levels of miR-135b-3p and CNDP1. Finally, a dual-luciferase reporter assay was employed to validate the direct interaction between miR-135b-3p and the 3′UTR of CNDP1. Results: AS-IV exhibited a dose-dependent inhibition of proliferation in Huh-7 and SMMC-7721 while inhibiting PD-L1 expression induced by interferon-γ (IFN-γ), thus attenuating PD-L1-mediated immune suppression. MiR-135b-5p showed significant amplification in HCC tissues and cells. AS-IV mitigated PD-L1-mediated immune suppression through miR-135b-5p. MiR-135b-5p targeted CNDP1, and AS-IV mitigated PD-L1-induced immunosuppression by modulating the miR-135b-5p/CNDP1 pathway. Conclusion: AS-IV decreases cell surface PD-L1 levels and alleviates PD-L1-associated immune suppression via the miR-135b-5p/CNDP1 pathway. AS-IV may be a novel component for treating HCC.
背景：黄芪甲苷IV（AS-IV）是发挥抗肿瘤作用的关键成分，在研究中受到广泛关注。肿瘤细胞免疫抑制与程序性死亡配体1（PD-L1）的增加密切相关。肝细胞癌（HCC）是一种起源于肝上皮组织的恶性肿瘤，而AS-IV在抗HCC活性中调控PD-L1的作用尚不明确。方法：对人肝永生化细胞（THEL2）及HCC细胞系（Huh-7和SMMC-7721）给予不同浓度的AS-IV处理，采用CCK-8法检测细胞生长情况。通过流式细胞术检测HCC细胞凋亡水平。通过给小鼠口服不同浓度AS-IV研究其对体内HCC的影响。采用免疫组化法评估PD-L1表达水平，Western blotting检测PD-L1和CNDP1蛋白表达。通过qRT-PCR定量分析miR-135b-3p和CNDP1的表达水平。最后采用双荧光素酶报告基因实验验证miR-135b-3p与CNDP1基因3′UTR区的直接相互作用。结果：AS-IV以剂量依赖性方式抑制Huh-7和SMMC-7721细胞增殖，同时抑制干扰素-γ（IFN-γ）诱导的PD-L1表达，从而减弱PD-L1介导的免疫抑制。miR-135b-5p在HCC组织和细胞中呈现显著扩增。AS-IV通过miR-135b-5p减轻PD-L1介导的免疫抑制。miR-135b-5p靶向调控CNDP1，AS-IV通过调节miR-135b-5p/CNDP1通路减轻PD-L1诱导的免疫抑制。结论：AS-IV通过miR-135b-5p/CNDP1通路降低细胞表面PD-L1水平，缓解PD-L1相关免疫抑制。AS-IV可能成为治疗HCC的新型组分。
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