Zhang Zhaojun, Wu Qiong, Xie Miaomiao, Ye Ruyin, Geng Chenchen, Shi Jiwen, Yang Qingling, Wang Wenrui, Shi Yurong
Anhui Provincial Key Laboratory of Tumor Evolution and Intelligent Diagnosis and Treatment, Bengbu Medical University, Bengbu 233030, China.
Department of Biochemistry and Molecular Biology, Bengbu Medical University, Bengbu 233030, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2025 Aug 20;45(8):1718-1731. doi: 10.12122/j.issn.1673-4254.2025.08.16.
To study the molecular mechanisms of LDH-loaded si-NEAT1 for regulating paclitaxel resistance and tumor-associated macrophage (TAM) polarization in breast cancer.
qRT-PCR and Western blotting were used to detect the expression of lncRNA NEAT1, miR-133b, and PD-L1 in breast cancer SKBR3 cells and paclitaxel-resistant SKBR3 cells (SKBR3-PR). The effects of transfection with si-NEAT1 and miR-133b mimics on MRP, MCRP and PD-L1 expressions and cell proliferation, migration and apoptosis were investigated using qRT-PCR, Western blotting, scratch and Transwell assays, and flow cytometry. Rescue experiments were conducted using si-NEAT1 and miR-133b inhibitor. Human THP-1 macrophages were cultured in the presence of conditioned media (CM) derived from SKBR3 and SKBR3-PR cells with or with si-NEAT1 transfection for comparison of IL-4-induced macrophage polarization by detecting the surface markers. LDH@si-NEAT1 nanocarriers were constructed, and their effects on MRP, MCRP and PD-L1 expressions and cell behaviors of the tumor cells were examined. THP-1 cells were treated with the CM from LDH@si-NEAT1-treated tumor cells, and the changes in their polarization were assessed.
SKBR3-PR cells showered significantly upregulated NEAT1 and PD-L1 expressions and lowered miR-133b expression as compared with their parental cells. Transfection with si-NEAT1 and miR-133b mimics inhibited viability, promoted apoptosis and enhanced MRP and BCRP expressions in SKBR3-PR cells. NEAT1 knockdown obvious upregulated miR-133b and downregulated PD-L1, MRP and BCRP expressions. The CM from SKBR3-PR cells obviously promoted M2 polarization of THP-1 macrophages, which was significantly inhibited by CM from si-NEAT1-transfected cells. Treatment with LDH@si-NEAT1 effectively inhibited migration and invasion, promoted apoptosis, and reduced MRP, BCRP and PD-L1 expressions in the tumor cells. The CM from LDH@si-NEAT1-treated SKBR3-PR cells significantly downregulated Arg-1, CD163, IL-10, and PD-L1 and upregulated miR-133b expression in THP-1 macrophages.
LDH@si-NEAT1 reduces paclitaxel resistance of breast cancer cells and inhibits TAM polarization by targeting the miR-133b/PD-L1 axis.
研究负载乳酸脱氢酶（LDH）的小干扰RNA（si-NEAT1）调节乳腺癌中紫杉醇耐药性及肿瘤相关巨噬细胞（TAM）极化的分子机制。
采用qRT-PCR和蛋白质免疫印迹法检测lncRNA NEAT1、miR-133b和程序性死亡配体1（PD-L1）在乳腺癌SKBR3细胞及耐紫杉醇SKBR3细胞（SKBR3-PR）中的表达。运用qRT-PCR、蛋白质免疫印迹法、划痕实验、Transwell实验及流式细胞术，研究转染si-NEAT1和miR-133b模拟物对多药耐药相关蛋白（MRP）、乳腺癌耐药蛋白（MCRP）及PD-L1表达以及细胞增殖、迁移和凋亡的影响。使用si-NEAT1和miR-133b抑制剂进行挽救实验。在含有或不含有经si-NEAT1转染的SKBR3和SKBR3-PR细胞来源的条件培养基（CM）存在的情况下培养人THP-1巨噬细胞，通过检测表面标志物比较白细胞介素-4诱导的巨噬细胞极化情况。构建LDH@si-NEAT1纳米载体，并检测其对肿瘤细胞中MRP、MCRP和PD-L1表达及细胞行为的影响。用来自经LDH@si-NEAT1处理的肿瘤细胞的CM处理THP-1细胞，并评估其极化变化。
与亲本细胞相比，SKBR3-PR细胞中NEAT1和PD-L1表达显著上调，miR-133b表达降低。转染si-NEAT1和miR-133b模拟物可抑制SKBR3-PR细胞的活力，促进其凋亡，并增强MRP和BCRP表达。敲低NEAT1可明显上调miR-133b并下调PD-L1、MRP和BCRP表达。SKBR3-PR细胞来源的CM明显促进THP-1巨噬细胞向M2极化，而经si-NEAT1转染的细胞来源的CM可显著抑制这种极化。用LDH@si-NEAT1处理可有效抑制肿瘤细胞的迁移和侵袭，促进其凋亡，并降低MRP、BCRP和PD-L1表达。来自经LDH@si-NEAT1处理的SKBR3-PR细胞的CM可显著下调THP-1巨噬细胞中精氨酸酶-1（Arg-1）、CD163、白细胞介素-10（IL-10）和PD-L1的表达，并上调miR-133b表达。
LDH@si-NEAT1通过靶向miR-133b/PD-L1轴降低乳腺癌细胞的紫杉醇耐药性并抑制TAM极化。
