[摘 要] 目的：探究肌动蛋白样蛋白6A（ACTL6A）通过调控铁死亡参与弥漫大B细胞淋巴瘤（DLBCL）细胞多柔比星（DOX） 耐药的机制。方法：培养亲本DLBCL细胞SU-DHL-4与耐药细胞SU-DHL-4/DOX，qPCR和WB法检测ACTL6A表达变化；通过 转染携带靶向 ACTL6A 干扰序列（sh-ACTL6A）及其阴性对照（sh-NC）的质粒，构建敲减 ACTL6A 的 SU-DHL-4/DOX，qPCR 和 WB法检测转染后细胞中ACTL6A、铁死亡相关蛋白谷胱甘肽过氧化酶4（GPX4）、溶质载体家族7成员11（SLC7A11）、长链酰基 辅酶A合成酶4（ACSL4）的表达水平，染色质免疫沉淀（ChIP）、双萤光素酶报告基因实验验证ACTL6A与GPX4间的靶向结合与 调控作用。将SU-DHL-4/DOX细胞分为对照组、sh-NC组、sh-ACTL6A组、sh-NC + oe-GPX4组和sh-ACTL6A + oe-GPX4组，用转 染试剂将相应质粒转染至细胞中，qPCR和WB法检测各组细胞中ACTL6A和GPX4表达水平，CCK-8法检测不同浓度DOX处理 后各组细胞存活率，FerroOrange探针检测各组细胞中亚铁离子（Fe2+ ）水平，Liperfluo探针检测各组细胞中脂质过氧化物水平， DCFH-DA探针检测各组细胞中活性氧（ROS）水平，比色法检测各组细胞中谷胱甘肽（GSH）和丙二醛（MDA）含量。结果： 与 SU-DHL-4细胞相比，SU-DHL-4/DOX细胞中ACTL6A mRNA和蛋白均呈高表达（均P < 0.05）；ACTL6A与GPX4间存在靶向结 合关系；敲减 ACTL6A 后 SU-DHL-4/DOX 细胞中 ACTL6A 和 GPX4 mRNA 及其蛋白表达水平均明显下降（均 P < 0.05），表明 ACTL6A可调控GPX4的表达；敲减ACTL6A可抑制SU-DHL-4/DOX细胞的存活率，促进细胞内Fe2+ 、脂质过氧化物、ROS、MDA 的生成，抑制GSH生成（均P < 0.05）；而在敲减ACTL6A的同时过表达GPX4可上调SU-DHL-4/DOX细胞中GPX4的mRNA及其 蛋白表达水平（均P < 0.05），并提高细胞存活率，抑制细胞内Fe2+ 、脂质过氧化物、ROS、MDA 的生成 ，并增加 GSH 生成（均 P < 0.05）。结论：ACTL6A在DOX耐药DLBCL细胞中高表达，通过调控GPX4表达抑制铁死亡，促进DLBCL细胞耐药。

[Abstract] Objective: To investigate the mechanism by which actin-like protein 6A (ACTL6A) regulates ferroptosis and contributes to doxorubicin (DOX) resistance in diffuse large B cell lymphoma (DLBCL) cells. Methods: The parental DLBCL cell line SU-DHL-4 cells and its DOX-resistant variant SU-DHL-4/DOX cells were cultured. Changes in the expression of ACTL6A were detected by qPCR and WB assay. SU-DHL-4/DOX cells with ACTL6A knockdown were constructed by transfecting plasmids carrying a short hairpin RNA targeting ACTL6A (sh-ACTL6A) or its negative control (sh-NC). The expression levels of ACTL6A and ferroptosis-related proteins, including glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and acyl-CoA synthetase longchain family member 4 (ACSL4), were measured using qPCR and WB. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were performed to verify the targeting and regulatory relationship between ACTL6A and GPX4. SU-DHL-4/DOX cells were divided into Control, sh-NC, sh-ACTL6A, sh-NC + oe-GPX4, and sh-ACTL6A + oe-GPX4 groups. Corresponding plasmids were transfected into the cells. CCK-8 assay was used to detect cell survival rates of each group under different concentrations of DOX treatment. FerroOrange, Liperfluo, and DCFH-DA probes were used to detect ferrous ion (Fe2+) levels, lipid peroxidation, and reactive oxygen species (ROS) in each group of cells, respectively. A colorimetric method was used to measure the contents of glutathione (GSH) and malondialdehyde (MDA) in each group of cells. Results: Both ACTL6A mRNA and protein were highly expressed in SU-DHL-4/DOX cells (both P < 0.05), compared to SU-DHL-4 cells. ACTL6A and GPX4 have a targeting binding relationship. Knockdown of ACTL6A significantly decreased the mRNA and protein expression of ACTL6A and GPX4 in SU-DHL-4/DOX cells (both P < 0.05), indicating that ACTL6A regulates GPX4 expression. Knockdown of ACTL6A significantly inhibited the survival of SU-DHL-4/DOX cells, increased intracellular Fe2+, lipid peroxides, ROS, and MDA levels, and inhibited GSH production (all P < 0.05). However, overexpression of GPX4 in ACTL6A-knockdown cells upregulated the mRNA and protein expression levels of GPX4 in SU-DHL-4/DOX cells (both P < 0.05), increased cell survival rate, inhibited the production of intracellular Fe2+, lipid peroxides, ROS, and MDA, and increased GSH production (all P < 0.05). Conclusion: ACTL6A is highly expressed in DOX-resistant DLBCL cells. By regulating GPX4 expression, ACTL6A inhibits ferroptosis and promotes drug resistance in DLBCL cells.

[基金项目] 国家自然科学青年基金（82303915）；中国青年创业就业基金；南通大学临床医学专项科研基金（2023JY001）