Layered double hydroxide-loaded si-NEAT1 regulates paclitaxel resistance and tumor-associated macrophage polarization in breast cancer by targeting miR-133b/PD-L1

doi:10.12122/j.issn.1673-4254.2025.08.16

Abstract

Abstract:

Objective To study the molecular mechanisms of LDH-loaded si-NEAT1 for regulating paclitaxel resistance and tumor-associated macrophage (TAM) polarization in breast cancer. Methods 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. Results 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. Conclusion LDH@si-NEAT1 reduces paclitaxel resistance of breast cancer cells and inhibits TAM polarization by targeting the miR-133b/PD-L1 axis.

Key words: paclitaxel resistance, breast cancer, lncRNA NEAT1, miR-133b, PD-L1, layered double hydroxide, M2 macrophage polarization

Zhaojun ZHANG, Qiong WU, Miaomiao XIE, Ruyin YE, Chenchen GENG, Jiwen SHI, Qingling YANG, Wenrui WANG, Yurong SHI. Layered double hydroxide-loaded si-NEAT1 regulates paclitaxel resistance and tumor-associated macrophage polarization in breast cancer by targeting miR-133b/PD-L1[J]. Journal of Southern Medical University, 2025, 45(8): 1718-1731.

Figures/Tables 11

Tab.1 PCR primer sequences

Gene	Forward primer	Reverse primer
GAPDH	CAGCCTCAAGATCATCAGCA	TGTGGTCATGAGTCCTTCCA
NEAT1	CTTCCTCCCTTTAACTTATCCATTCAC	CTCTTCCTCCACCATTACCAACAATAC
miR-133b	TCCCCTTCAACCAGCTAA	Universal primer
PD-L1	GTAAGACCACCACCACCAATTC	AGTTGTTGTGTTGATTCTCAGTGTG
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
Arg-1	GGACCTGCCCTTTGCTGACATC	TCTTCTTGACTTCTGCCACCTTGC
CD163	ACAATGAAGATGCTGGCGTGAC	ACAATGAAGATGCTGGCGTGAC
IL-10	ACCAAGACCCAGACATCAAGGC	AGGCATTCTTCACCTGCTCCAC
CD68	CCCACCTGCTTCTCTCATTCCC	TTGTACTCCACCGCCATGTAGC

Fig.1 LncRNA NEAT1 regulates paclitaxel resistance of breast cancer cells. A, B: Western blotting and immunofluorescence detection of expression of drug resistance-related proteins in resistant breast cancer cells (scale bar=50 μm). C: qRT-PCR technology for detecting the expression of NEAT1. D: Western blotting for detecting drug resistance of the cells. E: Flow cytometry for analyzing apoptosis rate of the resistant breast cancer cells. F, G: Scratch and Transwell assays for assessing the migration and invasion capabilities of resistant cells (scale bar=200 μm). Scratch and Transwell assays for assessing migration and invasion capabilities of resistant breast cancer cells. *P<0.05, **P<0.01, ***P<0.001.

Fig.2 LncRNA NEAT1 affects drug resistance in breast cancer cells by regulating miR-133b. A, B: qRT-PCR for detecting the expression of miR-133b. C: Western blotting for detecting expressions of drug resistance proteins. D: Immunofluorescence for detecting the expression of resistance proteins. E, F: Scratch and Transwell assays for assessing the migration and invasiveness of resistant cells. G: Flow cytometry for detecting apoptosis rate of resistant cells. *P<0.05, **P<0.01, ***P<0.001.

Fig.3 LncRNA NEAT1 regulates miR-133b to target PD-L1 and affect paclitaxel resistance in breast cancer cells. A, B: qRT-PCR for detecting the expression of PD-L1. C: Bioinformatics analysis reveals a regulatory relationship between miR-133b and PD-L1. D, E: Western blotting for detecting the expression of PD-L1. F: Western blotting for detecting the expressions of MRP, BCRP, and PD-L1 in drug-resistant breast cancer cells. *P<0.05, **P<0.01, ***P<0.001.

Fig.4 LncRNA NEAT1 regulates miR-133b to target PD-L1 and affect paclitaxel resistance in breast cancer cells. A: qRT-PCR for detecting the expression of PD-L1. B: Immunofluorescence for detecting the expression of BCRP in resistant cells (scale bar=50 μm). C, D: Scratch and Transwell assays for assessing migration and invasion capabilities of resistant cells (scale bar=200 μm). E: Flow cytometry for detecting apoptosis rate of the resistant cells. *P<0.05, **P<0.01, ***P<0.001.

Fig.5 Resistance cell supernatant can regulate the polarization of tumor-associated macrophages. A: Morphology of Thp-1 and M0-type macrophages (scale bar=200 μm ). B: qRT-PCR for detecting the expression of CD68. C, D: qRT-PCR for detecting the expression of M2 macrophage polarization markers Arg-1, CD163, and IL-10. E: Immunofluorescence staining for detecting M2 polarization markers CD163 and CD206 in the macrophages (scale bar=50 μm). F: Timer2.0 website predicts that the expression of NEAT1 is positively correlated with macrophage infiltration. *P<0.05, **P<0.01, ***P<0.001.

Fig.6 LncRNA NEAT1 regulates polarization of tumor-associated macrophages in breast cancer. A, D: qRT-PCR for detecting the expressions of NEAT1, miR-133b, and PD-L1, respectively. B, C: qRT-PCR and Immunofluorescence for detecting M2 macrophage polarization markers (scale bar=50 μm). E: Western blotting for detecting the expression of PD-L1 in the macrophages. *P<0.05, **P<0.01, ***P<0.001.

Fig.7 Construction of LDH Nanoparticles Loaded with si-NEAT1. A: Synthesis and loading of LDH. B: Agarose gel electrophoresis to determine the optimal loading ratio of LDH with si-NEAT1. C, D: Malvern particle size analysis for assessing particle size distribution and zeta potential of LDH and LDH@si-NEAT1. E: Transmission electron microscopy images of LDH and LDH@si-NEAT1. F, G: X-ray diffraction and UV-Vis spectra of LDH and LDH@si-NEAT1.

Fig.8 LDH@si-NEAT1 characterization experiments. A: Uptake experiment of LDH@si-NEAT1 (Original magnification: ×200). B: Confocal microscopy to detect lysosomal escape of LDH@si-NEAT1 (×200). C: Acridine orange staining to test lysosomal membrane permeability (×600).

Fig.9 LDH@si-NEAT1 regulate paclitaxel resistance in breast cancer cells. A, B: Scratch and Transwell assays to assess the migration and invasion capabilities of drug-resistant cells. C: Flow cytometry for detecting apoptosis rate of drug-resistant cells. D: qRT-PCR for detecting expressions of miR-133b and PD-L1, respectively. E: Western blotting for detecting expressions of drug resistance proteins and PD-L1. *P<0.05, ***P<0.001.

Fig.10 LDH@si-NEAT1 regulate M2 polarization state. A: Immunofluorescence staining for detecting expressions of drug resistance proteins (scale bar=50 μm). B, C: qRT-PCR and immunofluorescence staining for detecting M2 macrophage polarization markers (scale bar=50 μm). D: qRT-PCR of the expressions of miR-133b and PD-L1. E: Western blotting of the expression of PD-L1 in macrophages. *P<0.05, **P<0.01, ***P<0.001.

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